**Meet the editor**

Born in Kolkata (formerly Calcutta) in India, Satyen Saha received B.Sc. (Chemistry honors, 1994) and M.Sc. (Chemistry, 1996) from Jadavpur University, Kolkata. He did his Ph.D. (Photochemistry, 2002) from Hyderabad Central University, Hyderabad, India under the supervision of Prof. Anunay Samanta. Subsequently he moved to Department of Chemistry, University of

Tokyo, Tokyo, Japan for post doctoral research (2002–2005) in the group of Prof. Hiro-o Hamaguchi, followed by post doctoral research work with Prof. Richard Wiess, Georgetown University, Washington DC, USA. He was the recipient of JSPS post doctoral fellowship for the foreign researcher for two years. At present he is a permanent faculty member in department of Chemistry, Banaras Hindu University (central university), Varanasi, India. His present research interests are: synthesis, structure (solid and liquid phase) and interactions in ionic liquids, high energy density materials, and photophysical studies (steady state and time resolved) of fluorescent molecules in condensed phases.

Contents

**Preface IX** 

**Part 1 Photochemistry in Solution 1** 

Chapter 1 **Quinoline-Based Fluorescence Sensors 3** 

**Part 2 Photochemistry on Metal Oxides 23** 

Elim Albiter and Salvador Alfaro

Chapter 3 **Photoisomerization of Norbornadiene** 

Chapter 4 **Improved Photochemistry of TiO2** 

**Part 3 Photochemistry in Biology 87** 

Chapter 5 **Photo-Induced Proton** 

Chapter 7 **UV Light Effects on Proteins:** 

and Steffen B. Petersen

Chapter 2 **Photocatalytic Deposition of Metal** 

Xiang-Ming Meng, Shu-Xin Wang and Man-Zhou Zhu

**Oxides on Semiconductor Particles: A Review 25**  Miguel A. Valenzuela, Sergio O. Flores, Omar Ríos-Berny˜ ,

Ji-Jun Zou, Lun Pan, Xiangwen Zhang and Li Wang

Fabrizio Sordello, Valter Maurino and Claudio Minero

Takashi Kikukawa, Jun Tamogami, Kazumi Shimono, Makoto Demura, Toshifumi Nara and Naoki Kamo

**From Photochemistry to Nanomedicine 125**  Maria Teresa Neves-Petersen, Gnana Prakash Gajula

**of the Photosynthetic Oxygen-Evolving Complex 109**  Yong Li, Yukihiro Kimura, Takashi Ohno and Yasuo Yamauchi

**Inverse Opals and some Examples 63** 

**Transfers of Microbial Rhodopsins 89** 

Chapter 6 **Function of Extrinsic Proteins in Stabilization** 

**to Quadricyclane Using Ti-Containing Photocatalysts 41** 

## Contents

### **Preface XI**


	- **Part 2 Photochemistry on Metal Oxides 23**
	- **Part 3 Photochemistry in Biology 87**

### **Part 4 Computational Aspects of Photochemistry 159**

	- **Part 5 Applications of Photochemistry 193**

## Preface

There have been various comprehensive and stand-alone text books on the introduction to Molecular Photochemistry available in market. A few fantastic books to mention are: 'Modern Molecular Photochemistry of Organic Molecules' by N. J. Turro et. al. and 'Fundamentals of Photochemistry' by K. K. Rohatgi- Mukherjee. These books along with others give crystal clear idea on the common topics both in pictorial and intuitive terms. Thereby the complexity of photochemistry (mainly organic) is reduced to a set of simple paradigms. Various critical concepts like electronic and spin interaction are also nicely clarified pictorially such that student or beginner can understand it very effectively.

This book entitled "**Molecular Spectroscopy: Various aspects**" presents various *advanced topics* that inherently utilizes those core concepts/techniques to various advanced field of photochemistry which are generally not available. The purpose of publication of this book is actually an effort to bring many such topics clubbed together. The goal of this book is to familiarize both research scholars and post graduate students with recent advancement in various fields related to Photochemistry.

The book is broadly divided in five parts: the photochemistry i) in solution, ii) of metal oxides, iii) in biology, iv) the computational aspects and v) applications. Each part provides unique aspect of photochemistry.

The book starts with the chapter on *quinoline based sensors*. These kinds of sensors are especially known for Zn2+ and Cd2+, sensing with high selectivity and low detection limit. Large number of modified quinoline chemosensors are discussed by Zhu et. al. along with their mechanisms of fluorescence sensing, which includes PET, ICT and FRET and as well as potential applications.

*Deposition of metal oxides* is an important topic of Photochemistry*.* When this decomposition is on semiconductor and is targeted to exploit solar radiation, it gets further importance because of its 'green' nature. The first chapter in the part II ('*Photochemistry of metal oxides'*) is presented by Valenzuela et. al. on the semiconductor particle assisted metal oxide decompositions. Photocatalytic oxidation and reduction

### XII Preface

of various important single component (e.g., Pb2+, Hg0, MnO4- etc.) and mixed oxides (such as Rh2-yCryO3) have nicely been discussed with detail mechanisms and schemes.

Preface XI

**Satyen Saha**

Varanasi 221005,

India

Department of Chemistry Banaras Hindu University

There are four chapters included in last part of this book i.e., *application of photochemistry*. While Oliveira et. al. discussed on a burning issue of recent times; solar photochemistry for *industrial wastewater treatment,* Cambronne and co-workers have presented an interesting and well documented chapter on lesser known facts on the *high power discharge lamps.* The effect of the current pulses on the spectral radiant flux of high and medium pressure mercury vapour lamps are also discussed in it along with various potential photochemical applications of those lamps. Though many of us use mercury vapour lamps in some or other purposes in our laboratories or in home appliances, but most of us are unaware of these dependences. Chapter XI presented by Kabatc contains an interesting discusses on t*he photoinitiating ability of the dyeing photoinitiating systems.* This book concludes with a chapter on the photochemical generation of Iminyl Radicals and their synthetic application presented by Rodríguez et. al.. The interest in mechanistic studies of iminyl radical formation is on rise due to its application in preparation of polycyclic heteroaromatic compounds and natural

Last but not the least, the future of photochemistry like in any other burgeoning field, is more exciting than the past. The increase in number of young researchers and development in the instrumentations along with clearer understanding of

I thank all the contributors for their excellent chapters and the publishing process managers of InTech; Gorana Scerbe and Marina Jozipovic for keeping me on toe to publish this book on time. It has been a nice experience to work with this dynamic

photochemistry makes its future much brighter.

products.

publishing house.

Photoisomerization, an important aspect of photochemistry, is the structural change between the isomers on photo-excitation. Photoisomerization has already found potential applications in synthesis, digital data storage, recording, solar energy harvesting, and in nano-materials with photo-modulable properties. Conformation transformation, especially the *cis-trans* photoisomerization of alkenes, and Stilbenes are extensively studied both experimentally and theoretically. Here we have one chapter written by Zou et. al. devoted on the isomerisation (typical intramolecular [2+2] cycloaddition) of a very important molecules; norbornadiene (NBD) to quadricyclane (QC). The importance of this transformation is that considerable amount of energy (e.g., solar energy) can be stored in QC isomer when it is formed from NBD isomer.

Photochemistry on 3-dimensionally ordered TiO2 pores (known as *inverse opals*) has attracted considerable attentions in recent times. Minero and co-workers have presented an impressive chapter on this topic. The chapter has fine discussions on band gaps, synthesis and various improved processes on TiO2.

There are three chapters included in part III of the book under the heading of '*Photochemistry in Biology'*. Studies of Rhodopsins has attracted immense interest in recent times. The first chapter presented by Kamo et. al. on photon mediated proton transfer of Microbial Rhodopsins. Various processes of photo-induced proton transfer associated with the photocycle of microbial rhodopsins have also been discussed.

The chapter VI of this book is on *Function of Extrinsic Proteins* by Yamauchi and coworkers. The article focused on the structural and functional roles of extrinsic proteins in the plant photosynthesis-II. A very informative discussion on oxygenic phototrophs which convert photon energy of light into chemical energy through a series of lightinduced electron transfer reactions is also included. Another chapter on protein related photochemistry is presented by Petersen and coworkers. The very important aspect of *effect of UV radiation on protein* is broadly discussed in addition to the discussion on new generation *biofunctionalized nanoparticles*. Effect of UV light on various protein residues, disulphide bonds are nicely presented with reaction schemes.

A relatively uncommon chapter in any book on photochemistry is the theoretical aspect of an *application of photochemistry*. The solo chapter in part IV is on the c*omputational modeling in Photo-Dynamic Therapy (*well-known as PDT*).* PDT is of considerable interest in recent time because of its useful medical application. Though there have been huge amount of literatures on experiments and their results but detailed theoretical studies are scarce. The chapter on PDT written by Paterson and Bergendahl details the computational techniques to model the chemical pathways theoretically. In addition, the chapter contains fine descriptions on various steps involved in PDT and presents the current status and future prospect as well as.

There are four chapters included in last part of this book i.e., *application of photochemistry*. While Oliveira et. al. discussed on a burning issue of recent times; solar photochemistry for *industrial wastewater treatment,* Cambronne and co-workers have presented an interesting and well documented chapter on lesser known facts on the *high power discharge lamps.* The effect of the current pulses on the spectral radiant flux of high and medium pressure mercury vapour lamps are also discussed in it along with various potential photochemical applications of those lamps. Though many of us use mercury vapour lamps in some or other purposes in our laboratories or in home appliances, but most of us are unaware of these dependences. Chapter XI presented by Kabatc contains an interesting discusses on t*he photoinitiating ability of the dyeing photoinitiating systems.* This book concludes with a chapter on the photochemical generation of Iminyl Radicals and their synthetic application presented by Rodríguez et. al.. The interest in mechanistic studies of iminyl radical formation is on rise due to its application in preparation of polycyclic heteroaromatic compounds and natural products.

X Preface

from NBD isomer.

of various important single component (e.g., Pb2+, Hg0, MnO4- etc.) and mixed oxides (such as Rh2-yCryO3) have nicely been discussed with detail mechanisms and schemes.

Photoisomerization, an important aspect of photochemistry, is the structural change between the isomers on photo-excitation. Photoisomerization has already found potential applications in synthesis, digital data storage, recording, solar energy harvesting, and in nano-materials with photo-modulable properties. Conformation transformation, especially the *cis-trans* photoisomerization of alkenes, and Stilbenes are extensively studied both experimentally and theoretically. Here we have one chapter written by Zou et. al. devoted on the isomerisation (typical intramolecular [2+2] cycloaddition) of a very important molecules; norbornadiene (NBD) to quadricyclane (QC). The importance of this transformation is that considerable amount of energy (e.g., solar energy) can be stored in QC isomer when it is formed

Photochemistry on 3-dimensionally ordered TiO2 pores (known as *inverse opals*) has attracted considerable attentions in recent times. Minero and co-workers have presented an impressive chapter on this topic. The chapter has fine discussions on

There are three chapters included in part III of the book under the heading of '*Photochemistry in Biology'*. Studies of Rhodopsins has attracted immense interest in recent times. The first chapter presented by Kamo et. al. on photon mediated proton transfer of Microbial Rhodopsins. Various processes of photo-induced proton transfer associated with the photocycle of microbial rhodopsins have also been discussed.

The chapter VI of this book is on *Function of Extrinsic Proteins* by Yamauchi and coworkers. The article focused on the structural and functional roles of extrinsic proteins in the plant photosynthesis-II. A very informative discussion on oxygenic phototrophs which convert photon energy of light into chemical energy through a series of lightinduced electron transfer reactions is also included. Another chapter on protein related photochemistry is presented by Petersen and coworkers. The very important aspect of *effect of UV radiation on protein* is broadly discussed in addition to the discussion on new generation *biofunctionalized nanoparticles*. Effect of UV light on various protein

A relatively uncommon chapter in any book on photochemistry is the theoretical aspect of an *application of photochemistry*. The solo chapter in part IV is on the c*omputational modeling in Photo-Dynamic Therapy (*well-known as PDT*).* PDT is of considerable interest in recent time because of its useful medical application. Though there have been huge amount of literatures on experiments and their results but detailed theoretical studies are scarce. The chapter on PDT written by Paterson and Bergendahl details the computational techniques to model the chemical pathways theoretically. In addition, the chapter contains fine descriptions on various steps

involved in PDT and presents the current status and future prospect as well as.

residues, disulphide bonds are nicely presented with reaction schemes.

band gaps, synthesis and various improved processes on TiO2.

Last but not the least, the future of photochemistry like in any other burgeoning field, is more exciting than the past. The increase in number of young researchers and development in the instrumentations along with clearer understanding of photochemistry makes its future much brighter.

I thank all the contributors for their excellent chapters and the publishing process managers of InTech; Gorana Scerbe and Marina Jozipovic for keeping me on toe to publish this book on time. It has been a nice experience to work with this dynamic publishing house.

> **Satyen Saha**  Department of Chemistry Banaras Hindu University Varanasi 221005, India

**Part 1** 

**Photochemistry in Solution** 

**Part 1** 

**Photochemistry in Solution** 

**1** 

*Anhui University* 

*China* 

**Quinoline-Based Fluorescence Sensors** 

The human body is full of various ions, which play an important role in the normal physiological activities. For example, Zinc ion (Zn2+) plays a vital role in protein organism and in many biochemical processes, such as inducing apotosis, enzyme regulation, and gene expression. Also, Ferrous ion (Fe2+) is vital in the oxygen transporting. But there are some ions harmful to human body. When exposed to mercury, even at a very low concentration, they lead to kidney and neurological diseases. What's more, Cadmium (Cd2+) could damage our tissues, resulting in renal dysfunction or even cancers. So far, we have known more

We need a forceful instrument to study these mechanisms, need to know when and where ions are distributed, when ions are released, and so on.Therefore, traditional methods such as titration and electrochemistry are obviously unsuitable for in vivo detection. As a result, to accomplish the job, we need new tools and methods, among which fluorescence sensors are a good choice. So, what is a sensor? "Sensor" is a very broad concept, which accepts physical or chemical variables (input variables) information, and converts them into the same species of other kinds or converts their nature of the device output signal (Fig. 1) by

about these ions' properties in metabolism, but little is known on mechanism.

Fig. 1. Sensor structure (Once combination of ions, the output signal will change)

A chemosensor or a molecular sensor is a molecule that interacts with an analyte to produce a detectable change. Chemosenors consist of receptor and reporter, and after the receptor binds with a guest, the signal observed by the reporter will change. Fluorescent sensor is one of the most important chemosensors which uses fluorescence as the output signal, and also a powerful tool to monitor the metal ions in vivo system because of its simplicity, high sensitivity and real-time in situ imaging. In recent years, more and more chemosensors, especially the fluorescent sensors have been used to detect different ions, elbowing their

**1. Introduction** 

following certain rules.

Xiang-Ming Meng, Shu-Xin Wang and Man-Zhou Zhu

## **Quinoline-Based Fluorescence Sensors**

Xiang-Ming Meng, Shu-Xin Wang and Man-Zhou Zhu *Anhui University China* 

### **1. Introduction**

The human body is full of various ions, which play an important role in the normal physiological activities. For example, Zinc ion (Zn2+) plays a vital role in protein organism and in many biochemical processes, such as inducing apotosis, enzyme regulation, and gene expression. Also, Ferrous ion (Fe2+) is vital in the oxygen transporting. But there are some ions harmful to human body. When exposed to mercury, even at a very low concentration, they lead to kidney and neurological diseases. What's more, Cadmium (Cd2+) could damage our tissues, resulting in renal dysfunction or even cancers. So far, we have known more about these ions' properties in metabolism, but little is known on mechanism.

We need a forceful instrument to study these mechanisms, need to know when and where ions are distributed, when ions are released, and so on.Therefore, traditional methods such as titration and electrochemistry are obviously unsuitable for in vivo detection. As a result, to accomplish the job, we need new tools and methods, among which fluorescence sensors are a good choice. So, what is a sensor? "Sensor" is a very broad concept, which accepts physical or chemical variables (input variables) information, and converts them into the same species of other kinds or converts their nature of the device output signal (Fig. 1) by following certain rules.

Fig. 1. Sensor structure (Once combination of ions, the output signal will change)

A chemosensor or a molecular sensor is a molecule that interacts with an analyte to produce a detectable change. Chemosenors consist of receptor and reporter, and after the receptor binds with a guest, the signal observed by the reporter will change. Fluorescent sensor is one of the most important chemosensors which uses fluorescence as the output signal, and also a powerful tool to monitor the metal ions in vivo system because of its simplicity, high sensitivity and real-time in situ imaging. In recent years, more and more chemosensors, especially the fluorescent sensors have been used to detect different ions, elbowing their

Quinoline-Based Fluorescence Sensors 5

in the intensity ratio of the two wave bands in absorption and/or emission, would be more

Fig. 2. PET mechanism (The intensity of Fluorescence will increase after combination of ions)

The ICT mechanism (Fig. 3) has been widely used in the design of ratiometric fluorescent chemosensors. Compared to PET mechanism, this type of chemosensor doesn't have any spacer. If a receptor (usually an amino group) is directly connected with a conjugation system and forms a new conjugation system with p-electron, resulting in electron rich and electron poor terminals, then ICT from the electron donor to receptor would be enhanced upon light excitation. When a receptor, as an electron donor within the fluorophore, is bound with a metal ion (or another cation), the cation will reduce the electrondonating capacity of the receptor and a blue shift of the emission spectrum is obtained. In the same way, if a receptor is an electron receptor, the coordination of the cation will further strengthen the push–pull effect. Then a red shift in emission will be observed. For example, the coordination of Zn2+ with quinoline derivatives can induce a red-shift ratiometric

Fig. 3. ICT mechanism (Change in the intensity ratio of the two wave bands in emission)

Recently, the fluorescence resonance energy transfer (FRET Fig. 4), which involves the nonradiative transfer of excitation energy from an excited donor to a proximal ground-state acceptor, has been employed to design ratiometric sensors. The FRET-based sensors can be designed in the form of a small molecule, which usually contains two fluorophores connected by a spacer through covalent links. The following conditions must be satisfied for FRET: 1. The donor probe should have sufficient lifetime for energy transfer to occur. 2. The distance from the donor to the acceptor must be less than 10nm. 3. The absorption spectrum

favorable in increasing the signal selectivity and can be widely used in vivo.

fluorescence signal.

way to center stage in the field of molecular recognition. Series of sensors based on fluorescein, coumarin, petide, quinoline, and proteins have been used to detect intracellular ions concentration, such as Zn2+ sensors of Zinpyr Family based on fluorescein designed by Woodroofe (2004) et al., Cadmium sensor based on boradiazaindacene synthesised by Xu (2007) et al., Cu2+ sensor based on rhodamine synthesised by Dujols (1997) et al., the benzimidazole sensor described by Henary (2004) et al., the protein sensor described by van Dongen (2006) et al., Hg2+ FRET sensor described by Joshi (2010) et al., and Fe3+ sensor based on 1,8-diacridylnaphthalene and synthesized by Wolf (2004) et al..

Different fluorophores bring different optic properties of sensors. For example, sensors based on rhodamine can be excited by visible light, but they get low Stock's shift. Benzofuran-based sensors get lower dissociation constant, but UV exiting with higher energy may damage cells. These disadvantages thus bring forward potential difficulties for quantitative determination and bioimaging, so how to solve these problems is still a challenge.

Quinoline sensors, especially Zn2+ and Cd2+, have high selectivity and low detection limit (nM or pM). Modified quinoline chemosensors can also use low energy two-photon laser as the exciting source, which can reduce cell damage. Therefore, the current research of quinoline-based sensors attracts more and more attention.

Herein, the mechanism of quinoline-based fluorescence sensing, including PET (Photoinduced electron transfer), ICT (intermolecular charge transfer) and FRET (fluorescence resonance energy transfer), the synthetic strategies for functionalization of quinoline-based sensors will be reviewed, and the reasons for the choice of a particular synthetic pathway will be discussed. In order to contextualize the potential applications, a brief introduction of the photophysics property concerning quinoline-based sesnors is contained in the essay. At the same time, calculation method of sensor properties (eg, dissociation constant and quantum yield determination) is also included.

### **2. Mechanism of quinoline-based fluorescence sensing**

Quinoline-based fluorescence sensors are usually used to measure intensity changes of fluorescence and/or shift of fluorescence wavelength. Photoinduced electron transfer (PET), intermolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) are the three major mechanisms of fluorescence signal transduction in the design of quinolinebased fluorescence chemosensors (de Silva (1997) et al., Sarkar (2006) et al. and Banthia & Samanta (2006)). We will present the basic concepts of these mechanisms.

Chemosensors based on PET mechanism (Fig. 2) often use a atoms spacer less than three carbon atoms to connect a fluorescence group to a receptor containing a high-energy nonbonding electron pair, such as nitrogen or sulfur atom, which can transfer an electron to excited fluorescence group and result in fluorescence quench. But when the electron pair is coordinated by a metal ion (or other cation), the electron transfer will be prevented and the fluorescence is switched on. Most of quinoline-based fluorescence enhancement sensors can be explained by the PET type. Generally speaking, wavelengths of most PET chemosensors in Stokes shifts are less than 25 nm, which produces potential difficulties for quantitative determination and bioimaging. However, ratiometric chemosensors, which observe changes

way to center stage in the field of molecular recognition. Series of sensors based on fluorescein, coumarin, petide, quinoline, and proteins have been used to detect intracellular ions concentration, such as Zn2+ sensors of Zinpyr Family based on fluorescein designed by Woodroofe (2004) et al., Cadmium sensor based on boradiazaindacene synthesised by Xu (2007) et al., Cu2+ sensor based on rhodamine synthesised by Dujols (1997) et al., the benzimidazole sensor described by Henary (2004) et al., the protein sensor described by van Dongen (2006) et al., Hg2+ FRET sensor described by Joshi (2010) et al., and Fe3+ sensor based

Different fluorophores bring different optic properties of sensors. For example, sensors based on rhodamine can be excited by visible light, but they get low Stock's shift. Benzofuran-based sensors get lower dissociation constant, but UV exiting with higher energy may damage cells. These disadvantages thus bring forward potential difficulties for quantitative determination and bioimaging, so how to solve these problems is still a

Quinoline sensors, especially Zn2+ and Cd2+, have high selectivity and low detection limit (nM or pM). Modified quinoline chemosensors can also use low energy two-photon laser as the exciting source, which can reduce cell damage. Therefore, the current research of

Herein, the mechanism of quinoline-based fluorescence sensing, including PET (Photoinduced electron transfer), ICT (intermolecular charge transfer) and FRET (fluorescence resonance energy transfer), the synthetic strategies for functionalization of quinoline-based sensors will be reviewed, and the reasons for the choice of a particular synthetic pathway will be discussed. In order to contextualize the potential applications, a brief introduction of the photophysics property concerning quinoline-based sesnors is contained in the essay. At the same time, calculation method of sensor properties (eg,

Quinoline-based fluorescence sensors are usually used to measure intensity changes of fluorescence and/or shift of fluorescence wavelength. Photoinduced electron transfer (PET), intermolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) are the three major mechanisms of fluorescence signal transduction in the design of quinolinebased fluorescence chemosensors (de Silva (1997) et al., Sarkar (2006) et al. and Banthia &

Chemosensors based on PET mechanism (Fig. 2) often use a atoms spacer less than three carbon atoms to connect a fluorescence group to a receptor containing a high-energy nonbonding electron pair, such as nitrogen or sulfur atom, which can transfer an electron to excited fluorescence group and result in fluorescence quench. But when the electron pair is coordinated by a metal ion (or other cation), the electron transfer will be prevented and the fluorescence is switched on. Most of quinoline-based fluorescence enhancement sensors can be explained by the PET type. Generally speaking, wavelengths of most PET chemosensors in Stokes shifts are less than 25 nm, which produces potential difficulties for quantitative determination and bioimaging. However, ratiometric chemosensors, which observe changes

on 1,8-diacridylnaphthalene and synthesized by Wolf (2004) et al..

quinoline-based sensors attracts more and more attention.

dissociation constant and quantum yield determination) is also included.

Samanta (2006)). We will present the basic concepts of these mechanisms.

**2. Mechanism of quinoline-based fluorescence sensing** 

challenge.

in the intensity ratio of the two wave bands in absorption and/or emission, would be more favorable in increasing the signal selectivity and can be widely used in vivo.

Fig. 2. PET mechanism (The intensity of Fluorescence will increase after combination of ions)

The ICT mechanism (Fig. 3) has been widely used in the design of ratiometric fluorescent chemosensors. Compared to PET mechanism, this type of chemosensor doesn't have any spacer. If a receptor (usually an amino group) is directly connected with a conjugation system and forms a new conjugation system with p-electron, resulting in electron rich and electron poor terminals, then ICT from the electron donor to receptor would be enhanced upon light excitation. When a receptor, as an electron donor within the fluorophore, is bound with a metal ion (or another cation), the cation will reduce the electrondonating capacity of the receptor and a blue shift of the emission spectrum is obtained. In the same way, if a receptor is an electron receptor, the coordination of the cation will further strengthen the push–pull effect. Then a red shift in emission will be observed. For example, the coordination of Zn2+ with quinoline derivatives can induce a red-shift ratiometric fluorescence signal.

Fig. 3. ICT mechanism (Change in the intensity ratio of the two wave bands in emission)

Recently, the fluorescence resonance energy transfer (FRET Fig. 4), which involves the nonradiative transfer of excitation energy from an excited donor to a proximal ground-state acceptor, has been employed to design ratiometric sensors. The FRET-based sensors can be designed in the form of a small molecule, which usually contains two fluorophores connected by a spacer through covalent links. The following conditions must be satisfied for FRET: 1. The donor probe should have sufficient lifetime for energy transfer to occur. 2. The distance from the donor to the acceptor must be less than 10nm. 3. The absorption spectrum

Quinoline-Based Fluorescence Sensors 7

Synthesis by Doebner-Von Miller (1996): According to this method, aniline and acetaldehyde are usually used as raw material in hydrochloric acid or zinc chloride. At the beginning, condensation acetaldehyde into crotonaldehyde, then crotonaldehyde reacts with aniline molecule, the intermediate product is produced, and then dehydrogenates into dihydroquinoline, which becomes 2-methylquinoline. The reaction formula is as follows:

(1) The improved method, which can also be applied to obtain larger conjugated system in other materials, uses crotonic aldehyde instead of methanal to get a higher yield. In this project, a sensor based on ICT and FRET mechanisms is designed and synthesized, which uses 4-bromo-phenylamine as raw materials. Besides, 4-methoxy styrene is introduced into

**N**

**O**

**N**

**N**

**CH3CHO HCl CH3CH=CHCHO**

**CH3CH=CHCHO**

**12N HCl,** *<sup>p</sup>***-chloranil CH3CH=CHCHO**

**12N HCl,** *p***-chloranil CH3CH=CHCHO** *<sup>n</sup>***-BuOH, 105oC, 1h <sup>N</sup>**

(2) Matsubara (2011) et al. reported a new synthesis method of functionalized alkyl quinolines, which was based on sequential PdCl2-catalyzed cyclization reactions of substituted anilines and alkenyl ethers. High efficiency and functional-group tolerance made this procedure widely applied in synthesis of a number of substituted 2-alkylquinolines and larger

*<sup>n</sup>***-BuOH, <sup>105</sup>oC, 1h HO <sup>N</sup>**

 (3) Guerrini (2011) group reported an innovative and convenient synthetic approach for synthesizing two important genres of heterocyclic scaffolds, which use the capability of the

**OEt PdII**

Fig. 5. Molecular structure of quinoline-based chemosensors.

the quinoline platform by applying the classic Heck reaction.

**Br Br**

**NH2**

**HO NH2**

**NH2**

**NH2**

conjugated systems.

of the acceptor fluorophore must overlap with the fluorescence emission spectrum of the donor fluorophore (by approximately 30%). 4. For energy transfer, the donor and acceptor dipole orientations must be approximately parallel. Energy transfer is demonstrated by quenching of donor fluorescence with a reduction in the fluorescence lifetime, and an increase in acceptor fluorescence emission. FRET is very sensitive to the distance between fluorophores and can be used to estimate intermolecular distances. FLIM imaging can be used in association with FRET studies to identify and characterize energy transfer. Quinoline comprising another fluorophore (usually rhodamine) that will behave as FRET donor has been synthesized in order to produce FRET-based chemosensors.

Fig. 4. A FRET chemosensor reported by Zhou (2008) et al.

It is worth mentioning that the combination of PET and ICT mechanisms in the design of chemosensors would be valuable, since a wavelength shift and fluorescence intensity enhancement can amplify the recognition event to a greater extent, for example, using decorated quinoline as mother nucleus, thus oxidizing methyl on 2 position, then connecting DPA group. Thereby excellent ICT effect and fluorescent shift can be obtained after nitrogen atom on quinoline is bound. Meanwhile, the binding N-atom on DPA can obstruct PET process, thus increasing fluorescent intensity. FRET process is also considerably flexible, which can be applied widely in double fluorescence group, and at the same time can be employed in the energy transfer between a single fluorescence group and nanoparticles. By using specific acceptor to separate fluorescent group from nanoparticles, FRET process will be blocked, and fluorescence is produced. By using acceptor to connect nanoparticles with fluorescent group, which was not formerly connected with nanoparticles, fluorescence vanishes. They are particularly significant to fluorescent sensors based on nanoparticles. These methods are extremely effective.

### **3. Structure and synthesis**

The general structure of quinoline-based chemosensors is represented in Fig 5. Most quinoline-based sensors change the receptor group in the 2 (R1) and 8 (R5) positions, and the electron donating or withdrawing group in the 4 (R2), 5 (R3) and 6 (R4) positions. Depending on the substituents R1, R2, R3, R4, R5, the sensor will present different photophysical properties in solution, such as absorption and emission maxima (λmax abs, λmax em, and fluorescence quantum yield). Herein, the synthesis of different functionalized quinolinebased sensors will be discussed.

of the acceptor fluorophore must overlap with the fluorescence emission spectrum of the donor fluorophore (by approximately 30%). 4. For energy transfer, the donor and acceptor dipole orientations must be approximately parallel. Energy transfer is demonstrated by quenching of donor fluorescence with a reduction in the fluorescence lifetime, and an increase in acceptor fluorescence emission. FRET is very sensitive to the distance between fluorophores and can be used to estimate intermolecular distances. FLIM imaging can be used in association with FRET studies to identify and characterize energy transfer. Quinoline comprising another fluorophore (usually rhodamine) that will behave as FRET

It is worth mentioning that the combination of PET and ICT mechanisms in the design of chemosensors would be valuable, since a wavelength shift and fluorescence intensity enhancement can amplify the recognition event to a greater extent, for example, using decorated quinoline as mother nucleus, thus oxidizing methyl on 2 position, then connecting DPA group. Thereby excellent ICT effect and fluorescent shift can be obtained after nitrogen atom on quinoline is bound. Meanwhile, the binding N-atom on DPA can obstruct PET process, thus increasing fluorescent intensity. FRET process is also considerably flexible, which can be applied widely in double fluorescence group, and at the same time can be employed in the energy transfer between a single fluorescence group and nanoparticles. By using specific acceptor to separate fluorescent group from nanoparticles, FRET process will be blocked, and fluorescence is produced. By using acceptor to connect nanoparticles with fluorescent group, which was not formerly connected with nanoparticles, fluorescence vanishes. They are particularly significant to fluorescent sensors based on nanoparticles.

The general structure of quinoline-based chemosensors is represented in Fig 5. Most quinoline-based sensors change the receptor group in the 2 (R1) and 8 (R5) positions, and the electron donating or withdrawing group in the 4 (R2), 5 (R3) and 6 (R4) positions. Depending on the substituents R1, R2, R3, R4, R5, the sensor will present different photophysical properties in solution, such as absorption and emission maxima (λmax abs, λmax em, and fluorescence quantum yield). Herein, the synthesis of different functionalized quinoline-

donor has been synthesized in order to produce FRET-based chemosensors.

Fig. 4. A FRET chemosensor reported by Zhou (2008) et al.

These methods are extremely effective.

**3. Structure and synthesis** 

based sensors will be discussed.

Fig. 5. Molecular structure of quinoline-based chemosensors.

Synthesis by Doebner-Von Miller (1996): According to this method, aniline and acetaldehyde are usually used as raw material in hydrochloric acid or zinc chloride. At the beginning, condensation acetaldehyde into crotonaldehyde, then crotonaldehyde reacts with aniline molecule, the intermediate product is produced, and then dehydrogenates into dihydroquinoline, which becomes 2-methylquinoline. The reaction formula is as follows:

> (1) **CH3CHO HCl CH3CH=CHCHO NH2 CH3CH=CHCHO N**

The improved method, which can also be applied to obtain larger conjugated system in other materials, uses crotonic aldehyde instead of methanal to get a higher yield. In this project, a sensor based on ICT and FRET mechanisms is designed and synthesized, which uses 4-bromo-phenylamine as raw materials. Besides, 4-methoxy styrene is introduced into the quinoline platform by applying the classic Heck reaction.

Matsubara (2011) et al. reported a new synthesis method of functionalized alkyl quinolines, which was based on sequential PdCl2-catalyzed cyclization reactions of substituted anilines and alkenyl ethers. High efficiency and functional-group tolerance made this procedure widely applied in synthesis of a number of substituted 2-alkylquinolines and larger conjugated systems.

Guerrini (2011) group reported an innovative and convenient synthetic approach for synthesizing two important genres of heterocyclic scaffolds, which use the capability of the

Quinoline-Based Fluorescence Sensors 9

*S US* ( ) *<sup>U</sup> S U F A F A* 

Au is the UV absorption of unbound sensor or bound sensor, with As being the standard. Fu is integrated fluorescence emission corresponding to sensor or metal complex, and Fs is the

 Ca2+ Cd2+ Zn2+ Co2+ Cu2+ Fe3+ Hg2+ Pb2+ Mg2+ EDTA 10.61 16.36 16.44 16.26 18.70 25.0 21.5 17.88 8.83 NTA 6.39 9.78 10.66 10.38 12.94 15.9 14.6 11.34 5.47

Quinoline and its derivatives, especially 8-hydroxyquinoline and 8-aminoquinoline, are very important fluorogenic chelators for metal ions transition. Derivative of 8 aminoquinoline with an aryl sulfonamide is the first and most widely applied fluorescent chemosensor for imaging Zn2+ in biological samples. It was first reported by Toroptsev and Eshchenko. In 1987, Frederickson (1987) et al. reported a new quinoline-based sensor 1, which showed 100 folds in fluorescence enhancement after being bound with Zn2+. And it is the first high-sensitive sensor to detect Zn2+ in high concentrations of Ca2+ and Mg2+, which is very important for application in vivo. But low water solubility limits its application, so Zalewski (1994) led in a water-soluble group at the 6-position of quinoline, chemosensors 2 and 3 were synthesized. The research showed that this improvement made these two chemosensors much more water-soluble, and also showed a large increase in fluorescence upon Zn2+addition. Ca2+ and Mg2+ had little effect on the fluorescence whereas Fe2+ and Cu2+ quenched the fluorescence. Recently, Zhang (2008) et al. reported a new high-selective water-soluble and ratiometric chemosensor 4, based on 8-aminoquinoline for Zn2+ ion, which showed 8-fold increase in fluorescence quantum yield and a 75 nm red-shift fluorescence emission from 440 to 515 nm. But its excited source's energy is too high to be applied in vivo. Except the ability to be the fluorescence report group, quinoline's capability of binding Zn2+ enables it to be used as merely a binding group, so that high selectivity recognition of Zinc ion can be achieved. Nolan and Lippard (2005) et at., use ethyl 8 aminoquinoline to synthesize chemosensor 5. In addition, 5 exhibits 150-fold increase in fluorescence upon Zn2+ binding because of the low background fluorescence and high emission when binding with Zn2+. This binding is selective for Zn2+ from other biologically relevant metal cations, toxic heavy metals, and most first-row transition metals and is of appropriate affinity (Kd=41uM) to reversibly bound Zn2+ at physiological levels, and the quantum yield for the Zn2+-bound complex is 0.7 (λex=518nm). In this job, quinoline's recognition of Zinc ions is utilized. Meanwhile, rhodamine's high yield of fluorescent quantum and high sensitivity are taken full advantage of. So we can see that excellent properties such as light excitation provide superior possible ways for designing quinoline

(2)

Table 1. Log K of different metals with NTA and EDTA.

**5.1 Quinoline used for detecting Zn2+ ion** 

**5. Quinoline used for detecting different metal ions** 

standard.

sensors.

aromatic amides to rearrange photo-Fries. Quinolines from simple acetanilides derivatives have been obtained with satisfactory yield by using a single one-pot procedure.

In order to introduce functional groups on the 2-position, we often oxidize the methyl to aldehyde. Using selenium dioxide as oxidant can gain very high yield. Generally dioxane is used as the reaction solvent at 60-80 degrees. Reaction usually ends within two hours.

The synthesis of 8-aminoquinoline: The cyclization reaction can be firstly adopted to synthesize quinoline, which is replaced by nitryl and its derivatives, then reduction is used to generate 8-aminoquinoline. Classic reactions to synthesize quinoline ring include Friedlander, Skraup, Dobner-Miller and so on. The reaction equation is as follows:

### **4. Dissociation constant and quantum yield determination**

The dissociation constant is commonly used to describe the degree of affinity between sensor and metal ion. It is a key parameter used to describe the sensor's selectivity. It can be calculated by eq 1,

$$Kd = \frac{\left[\left[\boldsymbol{M}\right]^{n+}\right]/\text{free}(F\max - F)}{F - F\alpha} \tag{1}$$

where F=normalized fluorescence intensity, Kd=dissociation constant, Fmin=fluorescence intensity without metal ions (Mn+), Fmax=fluorescence intensity of bound sensor and [Mn+]free is the concentration of the free Mn+. Free metal ion concentrations are controlled by metal ion buffers (e.g., NTA (nitrilotriacetic acid), EDTA or other chelating agent.). As for log K of different metals with NTA and EDTA, see Tab. 1 (log K (ML), I=0.1mol/L, 25oC, 0.1mol/L).

Quinine sulfate is widely used as the standard in the calculation of fluorescence quantum yields of quinoline-based chemosensors (in 0.1N H2SO4, Ф =0.55, λex = 320 nm). The quantum yields are calculated by eq 2.

aromatic amides to rearrange photo-Fries. Quinolines from simple acetanilides derivatives

**N CO2Me**

**R2**

**C**

**CO2Me**

**O**

(4)

**SeO2, 60-80<sup>o</sup>**

**1,4-dioxane**

(5) In order to introduce functional groups on the 2-position, we often oxidize the methyl to aldehyde. Using selenium dioxide as oxidant can gain very high yield. Generally dioxane is used as the reaction solvent at 60-80 degrees. Reaction usually ends within two hours.

The synthesis of 8-aminoquinoline: The cyclization reaction can be firstly adopted to synthesize quinoline, which is replaced by nitryl and its derivatives, then reduction is used to generate 8-aminoquinoline. Classic reactions to synthesize quinoline ring include

(6)

**N NO2**

The dissociation constant is commonly used to describe the degree of affinity between sensor and metal ion. It is a key parameter used to describe the sensor's selectivity. It can be

[ ]( ) *<sup>n</sup> free <sup>d</sup>*

where F=normalized fluorescence intensity, Kd=dissociation constant, Fmin=fluorescence intensity without metal ions (Mn+), Fmax=fluorescence intensity of bound sensor and [Mn+]free is the concentration of the free Mn+. Free metal ion concentrations are controlled by metal ion buffers (e.g., NTA (nitrilotriacetic acid), EDTA or other chelating agent.). As for log K of different metals with NTA and EDTA, see Tab. 1 (log K (ML), I=0.1mol/L, 25oC, 0.1mol/L). Quinine sulfate is widely used as the standard in the calculation of fluorescence quantum yields of quinoline-based chemosensors (in 0.1N H2SO4, Ф =0.55, λex = 320 nm). The

*F F*

max 0

*F F*

**Fe/HCl**

*<sup>M</sup>* (1)

**N NH2**

**N CO2K**

**NH O**

**R1**

**N**

**O**

**MeOH 2.5eq K2CO3**

Friedlander, Skraup, Dobner-Miller and so on. The reaction equation is as follows:

**4. Dissociation constant and quantum yield determination** 

*K*

calculated by eq 1,

quantum yields are calculated by eq 2.

**N**

**NH2**

**NHCOR2**

**O**

**R1**

**NO2 glycerol As2O5, H2SO4**

have been obtained with satisfactory yield by using a single one-pot procedure.

**i) hv 254nm, MeCN, 20h ii) DMAD 1-5eq., 60oC, 20h**

**N**

**R1**

$$\mathfrak{d}\mathfrak{d}\iota = \frac{\mathfrak{d}\mathfrak{s}(\mathrm{F}\iota\mathrm{A}\mathrm{s})}{\mathrm{F}\mathrm{s}\mathrm{A}\iota} \tag{2}$$

Au is the UV absorption of unbound sensor or bound sensor, with As being the standard. Fu is integrated fluorescence emission corresponding to sensor or metal complex, and Fs is the standard.


Table 1. Log K of different metals with NTA and EDTA.

### **5. Quinoline used for detecting different metal ions**

### **5.1 Quinoline used for detecting Zn2+ ion**

Quinoline and its derivatives, especially 8-hydroxyquinoline and 8-aminoquinoline, are very important fluorogenic chelators for metal ions transition. Derivative of 8 aminoquinoline with an aryl sulfonamide is the first and most widely applied fluorescent chemosensor for imaging Zn2+ in biological samples. It was first reported by Toroptsev and Eshchenko. In 1987, Frederickson (1987) et al. reported a new quinoline-based sensor 1, which showed 100 folds in fluorescence enhancement after being bound with Zn2+. And it is the first high-sensitive sensor to detect Zn2+ in high concentrations of Ca2+ and Mg2+, which is very important for application in vivo. But low water solubility limits its application, so Zalewski (1994) led in a water-soluble group at the 6-position of quinoline, chemosensors 2 and 3 were synthesized. The research showed that this improvement made these two chemosensors much more water-soluble, and also showed a large increase in fluorescence upon Zn2+addition. Ca2+ and Mg2+ had little effect on the fluorescence whereas Fe2+ and Cu2+ quenched the fluorescence. Recently, Zhang (2008) et al. reported a new high-selective water-soluble and ratiometric chemosensor 4, based on 8-aminoquinoline for Zn2+ ion, which showed 8-fold increase in fluorescence quantum yield and a 75 nm red-shift fluorescence emission from 440 to 515 nm. But its excited source's energy is too high to be applied in vivo. Except the ability to be the fluorescence report group, quinoline's capability of binding Zn2+ enables it to be used as merely a binding group, so that high selectivity recognition of Zinc ion can be achieved. Nolan and Lippard (2005) et at., use ethyl 8 aminoquinoline to synthesize chemosensor 5. In addition, 5 exhibits 150-fold increase in fluorescence upon Zn2+ binding because of the low background fluorescence and high emission when binding with Zn2+. This binding is selective for Zn2+ from other biologically relevant metal cations, toxic heavy metals, and most first-row transition metals and is of appropriate affinity (Kd=41uM) to reversibly bound Zn2+ at physiological levels, and the quantum yield for the Zn2+-bound complex is 0.7 (λex=518nm). In this job, quinoline's recognition of Zinc ions is utilized. Meanwhile, rhodamine's high yield of fluorescent quantum and high sensitivity are taken full advantage of. So we can see that excellent properties such as light excitation provide superior possible ways for designing quinoline sensors.

Quinoline-Based Fluorescence Sensors 11

Fig. 6. (a) Fluorescence spectra (λex=320 nm) of 5μM 6 upon the titration of Zn2+

Zn2+ (0–1.6 equiv.), λex=405 nm.

fluorescence.

(0–1.6 equiv.) in a HEPES buffer. (b) Fluorescence response upon titration of 8 (5 mM) with

One year later, a both visual and fluorescent sensor 9 for Zn2+ was synthesized by Zhou (2010) et al., it displays high selectivity for Zn2+ and can be used as a ratiometric Zn2+ fluorescent sensor under visible light excitation. The strong coordination ability of Zn2+ with 9 leads to approximately 14-fold Zn2+ enhancement in fluorescence response and more than 7-fold increase in quantum yield (form 0.006 to 0.045) in THF-H2O solution. It is important that 9 have little or no effect on Cd2+, whereas Cu2+ and Co2+ quench the fluorescence. The quenching is not due to the heavy-atom effect, for, other heavy-atom did not quench the

In recent years, two-photon microscopy (TPM) imaging has gained much interest in biology because this method leads to less phototoxity, better three dimensional spatial localization, and greater penetration into scattering or absorbing tissues. Sensor 10 for monitoring Zn2+ was synthesized by Chen (2009) et al. based on the structure of 7-hydroxyquinoline. Its fluorescence enhancement (14-fold) and nanomolar range sensitivity (Kd=0.117 nM) were favorable in biological applications. JOB'S plot, NMR study and X-ray crystal structure indicated the binding model between sensor and Zn2+ is 1:1. Moreover, 10 also showed high selectivity for Zn2+ toward other first row transition metal ions including Fe2+, Co2+, and

8-Hydroxyquinoline, also a traditional fluorogenic agent for analyzing Zn2+ and other metal ions, was used as a reporter group in the chemosensor. Di-2-picolylamine (DPA) is a classic chelator with high selectivity for Zn2+ over other metal ions that can not be influenced by higher concentration of Ca2+, Na+ and K+ ions in biological samples. Xue (2008) et al. incorporated DPA into 8-hydroxy-2-methylquinoline at the 2-position to prepare a series of chemosensors 6, 7 and 8. The NMR studies and crystal structures of Sensor–Zn2+ complexes indicated that oxygen at the 8-position participated in the coordination of Zn2+ along with the quinoline nitrogen atom, and that DPA group endow the sensor with a high affinity (7, Kd = 0.85 pM). The fluorescence intensities of sensors showed a 4 to 6 fold enhancement and the quantum yields were also remarkably enhanced (Fig. 6a). According to the study of sensor's selectivity, the emissions of sensors showed slight enhancement upon addition of K+, Mg2+ and Ca2+ in the millimolar range, whereas the fluorescence intensities were slightly quenched by 1 equiv. of transition metals such as Mn2+, Co2+, Fe2+, Ni2+ and Cu2+, with the exception of Cd2+ showing enhanced fluorescence. Xue (2009) improved the sensor via choosing ICT process instead of PET process. By adding a cation which interacted with a receptor, the electron-withdrawing ability of the expanded conjugated system was enhanced, and 8 was designed. This results in a larger red-shift emission and Stokes shift (Fig. 6b). 8 shows a maximum emission at 545 nm with a large Stokes shift of 199 nm in the absence of Zn2+. The ratio of emission intensity (I620 nm/I540 nm) increases linearly with increased Zn2+ concentration. Ratiometric has brought about higher sensitivity, and other background disturbance. Only Zn2+ and Cd2+ show distinct ratiometric responses. Cell staining experiments demonstrate that 8 can readily reveal changes in intracellular Zn2+. Dual emissions and cell-permeable nature of 8 make it possible to study cellular Zn2+ in hippocampus in a ratiometric approach. The same problem appears here too: high-energy excitation puts cells vulnerable to harm.

8-Hydroxyquinoline, also a traditional fluorogenic agent for analyzing Zn2+ and other metal ions, was used as a reporter group in the chemosensor. Di-2-picolylamine (DPA) is a classic chelator with high selectivity for Zn2+ over other metal ions that can not be influenced by higher concentration of Ca2+, Na+ and K+ ions in biological samples. Xue (2008) et al. incorporated DPA into 8-hydroxy-2-methylquinoline at the 2-position to prepare a series of chemosensors 6, 7 and 8. The NMR studies and crystal structures of Sensor–Zn2+ complexes indicated that oxygen at the 8-position participated in the coordination of Zn2+ along with the quinoline nitrogen atom, and that DPA group endow the sensor with a high affinity (7, Kd = 0.85 pM). The fluorescence intensities of sensors showed a 4 to 6 fold enhancement and the quantum yields were also remarkably enhanced (Fig. 6a). According to the study of sensor's selectivity, the emissions of sensors showed slight enhancement upon addition of K+, Mg2+ and Ca2+ in the millimolar range, whereas the fluorescence intensities were slightly quenched by 1 equiv. of transition metals such as Mn2+, Co2+, Fe2+, Ni2+ and Cu2+, with the exception of Cd2+ showing enhanced fluorescence. Xue (2009) improved the sensor via choosing ICT process instead of PET process. By adding a cation which interacted with a receptor, the electron-withdrawing ability of the expanded conjugated system was enhanced, and 8 was designed. This results in a larger red-shift emission and Stokes shift (Fig. 6b). 8 shows a maximum emission at 545 nm with a large Stokes shift of 199 nm in the absence of Zn2+. The ratio of emission intensity (I620 nm/I540 nm) increases linearly with increased Zn2+ concentration. Ratiometric has brought about higher sensitivity, and other background disturbance. Only Zn2+ and Cd2+ show distinct ratiometric responses. Cell staining experiments demonstrate that 8 can readily reveal changes in intracellular Zn2+. Dual emissions and cell-permeable nature of 8 make it possible to study cellular Zn2+ in hippocampus in a ratiometric approach. The same problem appears here too: high-energy

excitation puts cells vulnerable to harm.

Fig. 6. (a) Fluorescence spectra (λex=320 nm) of 5μM 6 upon the titration of Zn2+ (0–1.6 equiv.) in a HEPES buffer. (b) Fluorescence response upon titration of 8 (5 mM) with Zn2+ (0–1.6 equiv.), λex=405 nm.

One year later, a both visual and fluorescent sensor 9 for Zn2+ was synthesized by Zhou (2010) et al., it displays high selectivity for Zn2+ and can be used as a ratiometric Zn2+ fluorescent sensor under visible light excitation. The strong coordination ability of Zn2+ with 9 leads to approximately 14-fold Zn2+ enhancement in fluorescence response and more than 7-fold increase in quantum yield (form 0.006 to 0.045) in THF-H2O solution. It is important that 9 have little or no effect on Cd2+, whereas Cu2+ and Co2+ quench the fluorescence. The quenching is not due to the heavy-atom effect, for, other heavy-atom did not quench the fluorescence.

In recent years, two-photon microscopy (TPM) imaging has gained much interest in biology because this method leads to less phototoxity, better three dimensional spatial localization, and greater penetration into scattering or absorbing tissues. Sensor 10 for monitoring Zn2+ was synthesized by Chen (2009) et al. based on the structure of 7-hydroxyquinoline. Its fluorescence enhancement (14-fold) and nanomolar range sensitivity (Kd=0.117 nM) were favorable in biological applications. JOB'S plot, NMR study and X-ray crystal structure indicated the binding model between sensor and Zn2+ is 1:1. Moreover, 10 also showed high selectivity for Zn2+ toward other first row transition metal ions including Fe2+, Co2+, and

Quinoline-Based Fluorescence Sensors 13

selectivity and sensitivity in water solution (Fig. 8b). Cd2+ induces slight enhancement of fluorescence emission intensity. It is a easily synthesized recognition to connect two 2 position quinoline sensors through a bridge that comprises heteroatoms (usually N, O, S atoms). In addition, the size of the cavity after bridge connection can be controlled in order to recognize specific ions. This is a very good choice to recognize sensors of different ions.

Fig. 8. (a) Fluorescence intensity of 12 (10 μM) in the presence of various metal ions (20 μM) in acetonitrile solution (λex=305 nm, λem=423 nm). (b) Fluorescence spectra and intensity (monitored at 410 nm) of 13 (50 μ M) measured with respective metal cations (1 equiv)

Although quinoline based chemosensors can serve as both the metal ligand and the fluorophore, their optical properties limit the application in vivo. The main disadvantage of these chemosensors is high-energy UV excitation which is detrimental to cells. Fluorescence at short wavelengths (most of the emission wavelengh is under 500nm) and most of the fluorescent sensors, based on quinoline with DPA as receptor, are more or less affected by

The interference of Cd2+ is a well-known problem for zinc fluorescence sensors and cadmium fluorescence sensors. Xue (2009) et al. reported an chemosensor that modulated the 8-position oxygen of the quinoline platform on sensor 14, while bound Zn2+ in 14 can be displaced by Cd2+, resulting in another ratiometric sensing signal output (Fig. 9a). 14 shows a blue-shift of 33nm in emission spectrum. 1H-NMR and optical spectra studies indicate that that 14 has higher affinity for Cd2+ than for Zn2+, which consequently incurs the ion displacement process. Recently Xue (2011) et al., synthesized a new cadmium sensor 15 based on 4-isobutoxy-6-(dimethylamino)-8-methoxyquinaldine in line with the ICT mechanism. Sensor 15 exhibits very high sensitivity for Cd2+ (Kd=51pM) and excellent selectivity response for detection Cd2+ from other heavy and transition metal ions, such as Na+, K+, Mg2+, and Ca2+ at millimolar level. They also established a single-excitation, dualemission ratiometric measurement with a large blue shift in emission (Δλ = 63 nm) and remarkable changes in the ratio (F495 nm/F558 nm) of the emission intensity (R/R0 up to 15 fold, Fig. 9b). The crystal structures data of 15 binding with Cd2+ and Zn2+ demonstrate that the DPA moiety plays the main function of grasping the metal ions, while the 8-position

at pH 7.0 (KH2PO4–NaOH buffered solution).

**5.2 Detection of Cd2+**

Cd2+. So how to improve quinoline-based sensor is still a challenge.

Cu2+, but it was slightly enhanced by Cd2+. Furthermore, 10 can be used for imaging Zn2+ in living cells with two-photon microscopy (Fig. 7). This is also one of the directions of designing fluorescence sensors, that is, using widely used low-energy 800nm laser as excitation source so as to avoid the harm to cells caused by ultraviolet rays.

Fig. 7. (A) Bright-field image of A431 cells labeled with 30 μM 10 after 30 min of incubation, λex=800 nm. (B) TP image after a 30 min treatment with zinc(II)/pyrithione (50 μM, 1:1 ratio). (C) The overlay of (A) and (B). (D) TP image of cells that are further incubated with 50 μM TPEN for 10 min.

Aoki, S. (2006) et al. have designed and synthesized new cyclen-based Zn2+ chemosensor 11 having an 8-hydroxy-5-N and N-dimethylaminosulfonylquinoline unit on the side chain. In the study, they found that using deprotonation of the hydroxyl group of 8-HQ and chelation to Zn2+ at neutral pH allows more sensitive detection of Zn2+ than dansylamide-pendant cyclen and (anthrylmethylamino) ethyl cyclen. They also introduced deprotonation behavior and fluorescence behavior, which was different by modifying the 5-position. This was very important in designing Quinoline-based chemosensors.

A space comprised of nitrogen atoms with quinoline fragments at both ends is often used in detection of Zn2+. Sensor 12 was synthesized by Liu (2010) et al, by using ethidene diamine to connect two 2-oxo-quinoline-3-carbaldehydes, thus schiff-base was composed to achieve Zn2+ detection. Compared with other metal ions, chemosensor 12 exhibits high selectivity and sensitivity for Zn2+ in acetonitrile solution compared with Cd2+ and other metal ions (Fig. 8a). The single crystal was taken for demonstrating the binding model of sensor and Zn2+. A simple-structured sensor 13 was reported by Shiraishi (2007) et al. 13 was easily synthesized by one-pot reaction in ethanol via condensation of diethylenetriamine and 2 quinolinecarbaldehyde followed by reduction with NaBH4. 13 is a new member of the water-soluble fluorescent Zn2+ sensor capable of showing linear and stoichiometrical response to Zn2+ amount without background fluorescence. 13 also shows high Zn2+

Cu2+, but it was slightly enhanced by Cd2+. Furthermore, 10 can be used for imaging Zn2+ in living cells with two-photon microscopy (Fig. 7). This is also one of the directions of designing fluorescence sensors, that is, using widely used low-energy 800nm laser as

Fig. 7. (A) Bright-field image of A431 cells labeled with 30 μM 10 after 30 min of incubation,

Aoki, S. (2006) et al. have designed and synthesized new cyclen-based Zn2+ chemosensor 11 having an 8-hydroxy-5-N and N-dimethylaminosulfonylquinoline unit on the side chain. In the study, they found that using deprotonation of the hydroxyl group of 8-HQ and chelation to Zn2+ at neutral pH allows more sensitive detection of Zn2+ than dansylamide-pendant cyclen and (anthrylmethylamino) ethyl cyclen. They also introduced deprotonation behavior and fluorescence behavior, which was different by modifying the 5-position. This

A space comprised of nitrogen atoms with quinoline fragments at both ends is often used in detection of Zn2+. Sensor 12 was synthesized by Liu (2010) et al, by using ethidene diamine to connect two 2-oxo-quinoline-3-carbaldehydes, thus schiff-base was composed to achieve Zn2+ detection. Compared with other metal ions, chemosensor 12 exhibits high selectivity and sensitivity for Zn2+ in acetonitrile solution compared with Cd2+ and other metal ions (Fig. 8a). The single crystal was taken for demonstrating the binding model of sensor and Zn2+. A simple-structured sensor 13 was reported by Shiraishi (2007) et al. 13 was easily synthesized by one-pot reaction in ethanol via condensation of diethylenetriamine and 2 quinolinecarbaldehyde followed by reduction with NaBH4. 13 is a new member of the water-soluble fluorescent Zn2+ sensor capable of showing linear and stoichiometrical response to Zn2+ amount without background fluorescence. 13 also shows high Zn2+

λex=800 nm. (B) TP image after a 30 min treatment with zinc(II)/pyrithione (50 μM, 1:1 ratio). (C) The overlay of (A) and (B). (D) TP image of cells that are

was very important in designing Quinoline-based chemosensors.

further incubated with 50 μM TPEN for 10 min.

excitation source so as to avoid the harm to cells caused by ultraviolet rays.

selectivity and sensitivity in water solution (Fig. 8b). Cd2+ induces slight enhancement of fluorescence emission intensity. It is a easily synthesized recognition to connect two 2 position quinoline sensors through a bridge that comprises heteroatoms (usually N, O, S atoms). In addition, the size of the cavity after bridge connection can be controlled in order to recognize specific ions. This is a very good choice to recognize sensors of different ions.

Fig. 8. (a) Fluorescence intensity of 12 (10 μM) in the presence of various metal ions (20 μM) in acetonitrile solution (λex=305 nm, λem=423 nm). (b) Fluorescence spectra and intensity (monitored at 410 nm) of 13 (50 μ M) measured with respective metal cations (1 equiv) at pH 7.0 (KH2PO4–NaOH buffered solution).

Although quinoline based chemosensors can serve as both the metal ligand and the fluorophore, their optical properties limit the application in vivo. The main disadvantage of these chemosensors is high-energy UV excitation which is detrimental to cells. Fluorescence at short wavelengths (most of the emission wavelengh is under 500nm) and most of the fluorescent sensors, based on quinoline with DPA as receptor, are more or less affected by Cd2+. So how to improve quinoline-based sensor is still a challenge.

### **5.2 Detection of Cd2+**

The interference of Cd2+ is a well-known problem for zinc fluorescence sensors and cadmium fluorescence sensors. Xue (2009) et al. reported an chemosensor that modulated the 8-position oxygen of the quinoline platform on sensor 14, while bound Zn2+ in 14 can be displaced by Cd2+, resulting in another ratiometric sensing signal output (Fig. 9a). 14 shows a blue-shift of 33nm in emission spectrum. 1H-NMR and optical spectra studies indicate that that 14 has higher affinity for Cd2+ than for Zn2+, which consequently incurs the ion displacement process. Recently Xue (2011) et al., synthesized a new cadmium sensor 15 based on 4-isobutoxy-6-(dimethylamino)-8-methoxyquinaldine in line with the ICT mechanism. Sensor 15 exhibits very high sensitivity for Cd2+ (Kd=51pM) and excellent selectivity response for detection Cd2+ from other heavy and transition metal ions, such as Na+, K+, Mg2+, and Ca2+ at millimolar level. They also established a single-excitation, dualemission ratiometric measurement with a large blue shift in emission (Δλ = 63 nm) and remarkable changes in the ratio (F495 nm/F558 nm) of the emission intensity (R/R0 up to 15 fold, Fig. 9b). The crystal structures data of 15 binding with Cd2+ and Zn2+ demonstrate that the DPA moiety plays the main function of grasping the metal ions, while the 8-position

Quinoline-Based Fluorescence Sensors 15

fluorescence emission was remarkably increased by the addition of Cu2+ with 1332% efficiency. In the two operations, the main function of quinoline is reflected in fluorescence changes before and after its N atom coordination. With regard to the selectivity of ions, it is decided by the cavity size formed by the middle cyclocompounds. The design is instructional, because compared with the bridge mentioned before, it produces a three-

Although quinoline moiety can be used both as the metal binding site and the fluorophore, the application of quinoline-based chemosensors in biological systems is limited by their optical properties. The main disadvantage of these chemosensors is high-energy UV excitation, which is possibly detrimental to biological tissues. It can induce autofluorescence from endogenous components and fluorescence at short wavelengths. Chemosensors 19 developed by Ballesteros (2009) et al. enlarged the conjugated system with 5 bromoindanone at the 8-position by Suzuki reaction. With this improvement, after being excited by visible light (λex=495nm), 19 showed a 5-fold increase in the intensity of emission

Receptor is a key role in the design of chemosensors, by choosing azacrown[N,S,O] instead of DPA. Wang (2010) et al. synthesized a new chemosensor 20 based on ICT mechanism. Chemosensor 20 is an effective ratiometric fluorescent sensor for silver ion and bears the features of a large Stokes shift at about 173 nm, with red-shift up to 50 nm in the emission spectra, and brings high affinity for silver ions (log K = 7.21) in ethanol in comparison with other competitive d10 metal ions. Crown compounds are all along used as highly recognized receptor groups for particular ions. As for groups which are not easily bounded, for instance, K+, 18-crown-6 can be used to recognize it. However, crown compounds have some application limits. At the beginning, crown compounds comprised of different heteoatoms can not achieve highly efficient synthesis. Then, they are considerably

dimensional bridge, and brings about better selectivity of particular ions.

centered at 650 nm after Cu2+ was added.

methoxy oxygen can be used to tune the selectivity of the sensor. Furthermore, confocal experiments in HEK 293 cells were carried out with 15, demonstrating 15 to be a ratiometric chemosensor to image intracellular, which is obviously superior to intensity-based images of the sole emission channel. This job is a guide to design Quinoline-based Cd2+ sensor.

Fig. 9. (a) Fluorescence spectra (λex=295 nm) of 10 μM 14 + 1 equiv of Zn2+ upon the titration of Cd2+ (0-3.0 equiv) in buffer solution. (b) Fluorescence spectra (λex = 405 nm) of 10 μM 15 upon titration of Cd2+ (0-20 μM) in aqueous buffer.

Tang (2008) et al. merged 8-hydroxyquinoline with oxadiazole to develop a ratiometric chemosensor 16 for Cd2+. If 1,3,4-oxadiazole subunit contained lone electron pairs on N, the semirigid ligand could effectively chelate Cd2+ according to the ionic radius and limit the geometric structure of the complex; thus 16 showed very high selectivity over other heavy and transition metal ions. This is also a designing method of bridge connection, that is, to make the detection group to form half heterocycle structure through the bridge, control the size of the heterocycle, and use the affinity of different heteroatoms to different ions, so that the selective recognition of different ions can be reached.

### **5.3 Detection of Cu2+ and Ag+**

Calixarenes are an important class of macrocyclic compounds, and they have been widely used as an ideal platform for the development of fluorescence chemosensors for alkali and alkaline-earth metal ions. Li (2008) et al. reported a turn on fluorescent sensor 17 for detecting Cu2+ based on calyx[4]arene bearing four iminoquinoline, which showed a largely enhanced fluorescent signal (1200-fold) upon addition of Cu2+ and a high selectivity toward Cu2+ over others. The 1:1 binding mode between sensor and Cu2+ was indicated by JOB's plot and mass spectrum. In Moriuchi-Kawakami's (2009) study, Cyclotriveratrylene can also act as a host analogous to calixarenes, a new C3-functionalized cyclotriveratrylene (CTV) bearing three fluorogenic quinolinyl groups. Sesnor 18 was synthesized, meanwhile, the

methoxy oxygen can be used to tune the selectivity of the sensor. Furthermore, confocal experiments in HEK 293 cells were carried out with 15, demonstrating 15 to be a ratiometric chemosensor to image intracellular, which is obviously superior to intensity-based images of the sole emission channel. This job is a guide to design Quinoline-based Cd2+ sensor.

Fig. 9. (a) Fluorescence spectra (λex=295 nm) of 10 μM 14 + 1 equiv of Zn2+ upon the titration of Cd2+ (0-3.0 equiv) in buffer solution. (b) Fluorescence spectra (λex = 405 nm) of 10 μM 15

Tang (2008) et al. merged 8-hydroxyquinoline with oxadiazole to develop a ratiometric chemosensor 16 for Cd2+. If 1,3,4-oxadiazole subunit contained lone electron pairs on N, the semirigid ligand could effectively chelate Cd2+ according to the ionic radius and limit the geometric structure of the complex; thus 16 showed very high selectivity over other heavy and transition metal ions. This is also a designing method of bridge connection, that is, to make the detection group to form half heterocycle structure through the bridge, control the size of the heterocycle, and use the affinity of different heteroatoms to different ions, so that

Calixarenes are an important class of macrocyclic compounds, and they have been widely used as an ideal platform for the development of fluorescence chemosensors for alkali and alkaline-earth metal ions. Li (2008) et al. reported a turn on fluorescent sensor 17 for detecting Cu2+ based on calyx[4]arene bearing four iminoquinoline, which showed a largely enhanced fluorescent signal (1200-fold) upon addition of Cu2+ and a high selectivity toward Cu2+ over others. The 1:1 binding mode between sensor and Cu2+ was indicated by JOB's plot and mass spectrum. In Moriuchi-Kawakami's (2009) study, Cyclotriveratrylene can also act as a host analogous to calixarenes, a new C3-functionalized cyclotriveratrylene (CTV) bearing three fluorogenic quinolinyl groups. Sesnor 18 was synthesized, meanwhile, the

upon titration of Cd2+ (0-20 μM) in aqueous buffer.

the selective recognition of different ions can be reached.

**5.3 Detection of Cu2+ and Ag+**

fluorescence emission was remarkably increased by the addition of Cu2+ with 1332% efficiency. In the two operations, the main function of quinoline is reflected in fluorescence changes before and after its N atom coordination. With regard to the selectivity of ions, it is decided by the cavity size formed by the middle cyclocompounds. The design is instructional, because compared with the bridge mentioned before, it produces a threedimensional bridge, and brings about better selectivity of particular ions.

Although quinoline moiety can be used both as the metal binding site and the fluorophore, the application of quinoline-based chemosensors in biological systems is limited by their optical properties. The main disadvantage of these chemosensors is high-energy UV excitation, which is possibly detrimental to biological tissues. It can induce autofluorescence from endogenous components and fluorescence at short wavelengths. Chemosensors 19 developed by Ballesteros (2009) et al. enlarged the conjugated system with 5 bromoindanone at the 8-position by Suzuki reaction. With this improvement, after being excited by visible light (λex=495nm), 19 showed a 5-fold increase in the intensity of emission centered at 650 nm after Cu2+ was added.

Receptor is a key role in the design of chemosensors, by choosing azacrown[N,S,O] instead of DPA. Wang (2010) et al. synthesized a new chemosensor 20 based on ICT mechanism. Chemosensor 20 is an effective ratiometric fluorescent sensor for silver ion and bears the features of a large Stokes shift at about 173 nm, with red-shift up to 50 nm in the emission spectra, and brings high affinity for silver ions (log K = 7.21) in ethanol in comparison with other competitive d10 metal ions. Crown compounds are all along used as highly recognized receptor groups for particular ions. As for groups which are not easily bounded, for instance, K+, 18-crown-6 can be used to recognize it. However, crown compounds have some application limits. At the beginning, crown compounds comprised of different heteoatoms can not achieve highly efficient synthesis. Then, they are considerably

Quinoline-Based Fluorescence Sensors 17

quinoline was partly in the range of rhodamine absorption, so 1,8-naphthalimide could be removed. And we also removed the hydroxy group at 8-position to reach a different coordination mode choice on the Fe3+ ions. 25 shows high selectively for Fe3+ over Cr3+ both in fluorescence and visible light (Fig. 10). JOB's plot indicate that, the 1:2 binding model between Fe3+ and 25, pH and cytotoxic effect also suggest that the new sensor is suitable for bioimaging. More importantly, the improved quinoline group can be excited by 800nm twophoton laser source, which is more suitable for bioimaging. We can utilize the FRET processes of quinoline and other fluorescent groups to design sensors, so as to take advantage of their respective merits. For example, we can make use of other groups' visible light changes, water solubility, and high sensitivity brought about by switch construction and so on to compensate demerits of quinoline groups. This will be one of the directions for

Fig. 10. Top: color of 25 and 25 with different metal ions. Bottom: fluorescence (λex = 365 nm)

In this review, we cover quinoline-based chemosensors for detection of different metal ions. There has been tremendous interest in improving quinoline-based chemosensors due to its easy synthesis method, high sensitivity and stability. However, there is still much room for progress in its application in vivo such as water solubility, high selectivity, and fluorescence bio-imaging capacity. Accordingly, the design of receptor for different ions is very important. For example, 15 adopted the different bonding model to distinguish between

change upon addition of different metal ions.

**6. Conclusion** 

designing sensors.

poisonous to in vivo cells. This imposes restrictions on their application in organisms to some extent.

### **5.4 Detection of Hg2+**

Modified quinoline can also become very good binding group for Hg2+ ion. Han (2009) et al. reported highly selective and highly sensitive Hg2+ chemosensor 21 based on quinoline and porphyrin ring. The 21 complexation quenches the fluorescence of porphyrin at 646 nm and induces a new fluorescent enhancement at 603 nm. The fluorescent response of 21 towards Hg2+ and 1H-NMR indicates Hg2+ ion is binding with the quinoline moiety. Yang (2007) et al. reported chemosensor 22, which connect 8-hydroxyquinoline with rhodamine and ferrocene, and recognize Hg2+ through opening and closing rhodamine ring before and after binding. At the same time, because the density of the interior electron cloud changes before and after binding, the electrochemistry signal of ferrocene varies, so that the detection accuracy is improved. Concerning Hg2+ recognition, there is another method that receives considerable attention, that is, modified thioamides on quinoline, using the sulfur addicting feature of Hg2+, will be transformed into amides by Hg2+, which will change the PET process, thus inducing the production of fluorescence.

Song (2006) et al. reported 8-hydroxyquinoline derivative chemosensor 23. In 23, the fluorescence background is very weak. 23 is demonstrated to be highly sensitive to the detection of Hg2+, because the hydrolytic conversion of thioamides into amides catalyzed by Hg2+ is very efficient. NMR, IR, and mass studies indicate the Hg2+ ion induced the transformation of thioamide into amide.

### **5.5 Detection of Cr3+ and Fe3+**

Because paramagnetic Fe3+ and Cr3+ are reported as two of the most efficient fluorescence quenchers among the transition metal ions, the development of Fluorescence chemosensors working with these inherent quenching metal ions is a challenging job. Zhou (2008) et al. reported a FRET-based Cr3+ chemosensor 24. With increased FRET from 1,8-naphthalimide (donor) to the open, colored form of rhodamine (acceptor), the intensity of the fluorescent peak at 544 nm gradually decreased and that of new fluorescent band centered at 592 nm increased, 24 showed an 7.6-fold increase in the ratio of emission intensities (F592 nm/F544 nm). We (2011) reported a turn-on fluorescent probe 25 for Fe3+ based on the rhodamine platform. An improved quinoline fluorescent group, which could be excited by about 400 nm wavelength of light, was linked to the rhodamine platform. The emission of conjugated

poisonous to in vivo cells. This imposes restrictions on their application in organisms to

Modified quinoline can also become very good binding group for Hg2+ ion. Han (2009) et al. reported highly selective and highly sensitive Hg2+ chemosensor 21 based on quinoline and porphyrin ring. The 21 complexation quenches the fluorescence of porphyrin at 646 nm and induces a new fluorescent enhancement at 603 nm. The fluorescent response of 21 towards Hg2+ and 1H-NMR indicates Hg2+ ion is binding with the quinoline moiety. Yang (2007) et al. reported chemosensor 22, which connect 8-hydroxyquinoline with rhodamine and ferrocene, and recognize Hg2+ through opening and closing rhodamine ring before and after binding. At the same time, because the density of the interior electron cloud changes before and after binding, the electrochemistry signal of ferrocene varies, so that the detection accuracy is improved. Concerning Hg2+ recognition, there is another method that receives considerable attention, that is, modified thioamides on quinoline, using the sulfur addicting feature of Hg2+, will be transformed into amides by Hg2+, which will change the PET

Song (2006) et al. reported 8-hydroxyquinoline derivative chemosensor 23. In 23, the fluorescence background is very weak. 23 is demonstrated to be highly sensitive to the detection of Hg2+, because the hydrolytic conversion of thioamides into amides catalyzed by Hg2+ is very efficient. NMR, IR, and mass studies indicate the Hg2+ ion induced the

Because paramagnetic Fe3+ and Cr3+ are reported as two of the most efficient fluorescence quenchers among the transition metal ions, the development of Fluorescence chemosensors working with these inherent quenching metal ions is a challenging job. Zhou (2008) et al. reported a FRET-based Cr3+ chemosensor 24. With increased FRET from 1,8-naphthalimide (donor) to the open, colored form of rhodamine (acceptor), the intensity of the fluorescent peak at 544 nm gradually decreased and that of new fluorescent band centered at 592 nm increased, 24 showed an 7.6-fold increase in the ratio of emission intensities (F592 nm/F544 nm). We (2011) reported a turn-on fluorescent probe 25 for Fe3+ based on the rhodamine platform. An improved quinoline fluorescent group, which could be excited by about 400 nm wavelength of light, was linked to the rhodamine platform. The emission of conjugated

some extent.

**5.4 Detection of Hg2+**

process, thus inducing the production of fluorescence.

transformation of thioamide into amide.

**5.5 Detection of Cr3+ and Fe3+**

quinoline was partly in the range of rhodamine absorption, so 1,8-naphthalimide could be removed. And we also removed the hydroxy group at 8-position to reach a different coordination mode choice on the Fe3+ ions. 25 shows high selectively for Fe3+ over Cr3+ both in fluorescence and visible light (Fig. 10). JOB's plot indicate that, the 1:2 binding model between Fe3+ and 25, pH and cytotoxic effect also suggest that the new sensor is suitable for bioimaging. More importantly, the improved quinoline group can be excited by 800nm twophoton laser source, which is more suitable for bioimaging. We can utilize the FRET processes of quinoline and other fluorescent groups to design sensors, so as to take advantage of their respective merits. For example, we can make use of other groups' visible light changes, water solubility, and high sensitivity brought about by switch construction and so on to compensate demerits of quinoline groups. This will be one of the directions for designing sensors.

Fig. 10. Top: color of 25 and 25 with different metal ions. Bottom: fluorescence (λex = 365 nm) change upon addition of different metal ions.

### **6. Conclusion**

In this review, we cover quinoline-based chemosensors for detection of different metal ions. There has been tremendous interest in improving quinoline-based chemosensors due to its easy synthesis method, high sensitivity and stability. However, there is still much room for progress in its application in vivo such as water solubility, high selectivity, and fluorescence bio-imaging capacity. Accordingly, the design of receptor for different ions is very important. For example, 15 adopted the different bonding model to distinguish between

Quinoline-Based Fluorescence Sensors 19

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### **7. Acknowledgements**

We acknowledge financial support by NSFC(21072001, 21102002), 211 Project of Anhui University for supporting the research.

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**Part 2** 

**Photochemistry on Metal Oxides** 


**Part 2** 

**Photochemistry on Metal Oxides** 

22 Molecular Photochemistry – Various Aspects

Zhang, Y.; Guo, X. F.; Si, W. X.; Jia, L. & Qian, X. H. (2008). Ratiometric and Water-Soluble

Zhou, X. Y.; Yu, B. R.; Guo, Y. L.; Tang, X. L.; Zhang, H. H. & Liu, W. S. (2010). Both Visual

Zhou, Z.; Yu, M.; Yang, H.; Huang, K.; Li, F.; Yi, T. & Huang C. (2008). FRET-based sensor for imaging chromium(III) in living cellsw. *Chemical Communications* 3387-389

Chain as Receptor. *Organic Letters* 10, 473-476

49, 4002-4007

Fluorescent Zinc Sensor of Carboxamidoquinoline with an Alkoxyethylamino

and Fluorescent Sensor for Zn2+ Based on Quinoline Platform. *Inorganic Chemistry*

**2** 

*México D.F., México* 

**Photocatalytic Deposition of Metal Oxides** 

*Lab. Catálisis y Materiales, ESIQIE-Instituto Politécnico Nacional Zacatenco,* 

As it has been well recognized in the last decade, heterogeneous photocatalysis employing UV-irradiated titanium dioxide suspensions or films in aqueous or gas media, is now a mature field [Chong et al. 2010, Ohtani B. 2010, Paz Y., 2010]. Semiconductor photocatalysis is considered as a green process that focuses basically on exploiting solar energy in many ways. Its investigations have been mainly targeted to the degradation/mineralization of organic pollutants and water splitting solar energy conversion, among others. However, there are other exciting applications such as metal photodeposition, organic synthesis, photoimaging, antibacterial materials, which have now an intense investigation [Wu et al. 2003, Chan S. & Barteau M. 2005, Litter M. 1999, Fagnoni et al. 2007, Choi W. 2006,

In particular, the photodeposition has been used since the decade of 70's by the pioneer work of Bard [Kraeutler and Bard, 1978] to prepare supported-metal catalysts and photocatalysts as well as to recover noble metals and to remove metal cations from aqueous effluents [Ohyama et al. 2011]. In this case, the reduction of each adsorbed individual metal ions occurs at the interface by acceptance of electrons from the conduction band forming a metallic cluster. A variant of metals deposition is the reductive deposition of metal oxides and a clear example of this route is the photocatalytic reduction of Cr (VI) which is transformed to Cr(III), so that in acidic environment, chromates are easily converted to

The oxidative deposition of metal oxides is less frequently reported and it has been demonstrated that proceeds via the oxidative route [Tanaka et al. 1986]. For instance, checking the electrochemical potentials of Mn and Pb (Table 1), they are more easily oxidized by the valence band holes than reduced by the conduction band electrons in

 Mn2++ 2H2O MnO2 + 4H+ (1) TiO2

**1. Introduction** 

Valenzuela et al. 2010, Zhang et al. 2010].

Cr2O3 [Lin et al. 1993 and Flores et al. 2008].

presence of TiO2 as follows [Wu et al. 2003]:

**on Semiconductor Particles: A Review** 

Miguel A. Valenzuela, Sergio O. Flores, Omar Ríos-Bernỹ,

Elim Albiter and Salvador Alfaro

## **Photocatalytic Deposition of Metal Oxides on Semiconductor Particles: A Review**

Miguel A. Valenzuela, Sergio O. Flores, Omar Ríos-Bernỹ, Elim Albiter and Salvador Alfaro *Lab. Catálisis y Materiales, ESIQIE-Instituto Politécnico Nacional Zacatenco, México D.F., México* 

### **1. Introduction**

As it has been well recognized in the last decade, heterogeneous photocatalysis employing UV-irradiated titanium dioxide suspensions or films in aqueous or gas media, is now a mature field [Chong et al. 2010, Ohtani B. 2010, Paz Y., 2010]. Semiconductor photocatalysis is considered as a green process that focuses basically on exploiting solar energy in many ways. Its investigations have been mainly targeted to the degradation/mineralization of organic pollutants and water splitting solar energy conversion, among others. However, there are other exciting applications such as metal photodeposition, organic synthesis, photoimaging, antibacterial materials, which have now an intense investigation [Wu et al. 2003, Chan S. & Barteau M. 2005, Litter M. 1999, Fagnoni et al. 2007, Choi W. 2006, Valenzuela et al. 2010, Zhang et al. 2010].

In particular, the photodeposition has been used since the decade of 70's by the pioneer work of Bard [Kraeutler and Bard, 1978] to prepare supported-metal catalysts and photocatalysts as well as to recover noble metals and to remove metal cations from aqueous effluents [Ohyama et al. 2011]. In this case, the reduction of each adsorbed individual metal ions occurs at the interface by acceptance of electrons from the conduction band forming a metallic cluster. A variant of metals deposition is the reductive deposition of metal oxides and a clear example of this route is the photocatalytic reduction of Cr (VI) which is transformed to Cr(III), so that in acidic environment, chromates are easily converted to Cr2O3 [Lin et al. 1993 and Flores et al. 2008].

The oxidative deposition of metal oxides is less frequently reported and it has been demonstrated that proceeds via the oxidative route [Tanaka et al. 1986]. For instance, checking the electrochemical potentials of Mn and Pb (Table 1), they are more easily oxidized by the valence band holes than reduced by the conduction band electrons in presence of TiO2 as follows [Wu et al. 2003]:

$$\text{TiO}\_2$$

$$\text{Mn} \ast + 2\text{H}\_2\text{O} \xrightarrow{\text{-}} \text{MnO}\_2 + 4\text{H}^\* \tag{1}$$

Photocatalytic Deposition of Metal Oxides on Semiconductor Particles: A Review 27

by EPA is 15g/l. However, it is desirable the total elimination of lead due to its extreme potential toxicity [Murruni et al., 2007]. In a first report concerning to the photodeposition of Pb2+ ions on TiO2 and metallized TiO2, it was found that the former only produces PbO, whereas the last converts efficiently Pb ions to PbO2 [Tanaka et al., 1986]. In the same work, it was proposed a reaction mechanism in two steps involving the reduction of oxygen to

Their mechanism was supported by experiments carried out in different atmospheres: nitrogen, argon and oxygen at several partial pressures. In N2 and Ar, irradiation of TiO2 suspensions did not result in lead oxide formation. It is worth noting that a high pressure

Litter et al. 1999, have proposed a different mechanism which involves two consecutive electron transfer reactions. Lead ions are oxidized by holes or by hydroxyl radicals passing

A further enhancement was achieved with platinized TiO2 by decreasing the overpotential of oxygen. In fact, the role of oxygen is crucial to carry out the photocatalytic cycle and it has found a linear dependence of oxygen partial pressure based on a Langmuir-Hinshelwood

Recently, it has been highlighted many applications of cobalt compounds deposited on semiconductors such as: catalysts for solar oxygen production, gas sensors, batteries, electrochromic devices, among others [Steinmiller & Choi, 2009; Tak & Yong, 2008]. In particular, the photodeposition of Co3O4 spinel phase on ZnO has been prepared by two routes, one consisting in the direct photo-oxidation of Co2+ ions to Co3+ ions and the other by an indirect procedure involving the reduction of Co2+ to Co° and the oxidation of metallic

By using the direct deposition route, a ZnO electrode was immersed in an aqueous solution of CoCl2 mantaining the pH constant at 7 and illuminating with UV light (= 302 nm). Due to the oxidation potential of Co2+ to form Co3O4 is 0.7 V at pH= 7 at low concentrations of Co2+ (103 M) and the valence band edge of ZnO is located at around 2.6 V vs NHE, the photogenerated holes can easily oxidize Co2+ ions to Co3+ ions. The complete photocatalytic cycle must also include the reduction of water or dissolved oxygen in the solution to have

2 Co2+ + 3H2O Co2O3 + 6H+ + 2e

cobalt to Co3O4 by means of the oxygen coming from the photo-oxidation of water.

(5)

(7)

(8)

(9)

PbO2 + O2 (6)

form the superoxide ion and the subsequent oxidation of Pb ions:

Hg lamp (500 W) was used for all their photocatalytic deposition reactions.

through the divalent to the tetravalent state, equations 7 and 8:

h+/HO• + Pb2+ Pb3+ + OH

h+/HO• + Pb3+ Pb4+ + OH

an efficient Co2+ photo-oxidation [Steinmiller & Choi, 2009]:

2O2 + 2e- → 2O2

Pb2+ + 2O2

mechanism [Torres & Cervera-March, 1992].

**2.2 Co2+** 


$$\text{Pb}^{2+} + 2\text{H}\_2\text{O} \xrightarrow{\text{TiO}\_2} \text{PbO}\_2 + 4\text{H}^+ \tag{2}$$

Table 1. Half wave potentials of different couples.

These two reactions represent a good example of the photocatalytic deposition of metal oxides in aqueous solution onto titanium dioxide. This means that the complete photocatalytic cycle should consider the photoredox couple in which one metallic ion (single component) in solution is oxidized and the oxygen of the media is reduced. The deposition is driven by particle agglomeration after reaching their zero point charge and a critical concentration to be deposited on the surface of the semiconductor. It has been reported that single component metal oxides for example, PbO2, RuO2, U3O8, SiO2, SnO2, Fe2O3, MnO2, IrO2 and Cr2O3 can be deposited on semiconductor particles following a photo-oxidative or photo-reductive route [Maeda et al. 2008].

On the other hand, when a semiconductor is irradiated with UV light in presence of aqueous solutions containing dissolved Ag+ or Pb2+ cations a redox process is undertaken giving rise to the reduction of silver ions or the oxidation of lead ions, according to the following reactions [Giocondi et al. 2003]:

$$\text{Ag}^{\bullet} + \text{e} \to \text{Ag}^{\bullet} \tag{3}$$

$$\rm Pb^{2+} + 2H\_2O + 2h^\* \rightarrow PbO\_2 + 4H^\* \tag{4}$$

Lately, it has been reported the photocatalytic deposition of mixed-oxides such as Rh2 yCryO3 dispersed on a semiconductor powder with applications in the water splitting reaction [Maeda et al. 2008]. Hence, we intend to offer the reader a condensed overview of the work done so far considering photocatalytic deposition of a single or mixed oxide on semiconductor materials by either oxidative or reductive processes.

### **2. Photocatalytic oxidation of a single component**

### **2.1 Pb2+**

In regard with the very negative impact of lead on environment and population, many efforts are conducted to remove it from water of distinct origins. It is commonly removed by precipitation as carbonate or hydroxide; besides other physicochemical methods are available to lead elimination. The maximun contaminant level in drinking water established by EPA is 15g/l. However, it is desirable the total elimination of lead due to its extreme potential toxicity [Murruni et al., 2007]. In a first report concerning to the photodeposition of Pb2+ ions on TiO2 and metallized TiO2, it was found that the former only produces PbO, whereas the last converts efficiently Pb ions to PbO2 [Tanaka et al., 1986]. In the same work, it was proposed a reaction mechanism in two steps involving the reduction of oxygen to form the superoxide ion and the subsequent oxidation of Pb ions:

$$\text{\color{red}{2}\text{O}\_2 + 2e^- \to \text{\color{red}{2}\text{O}\_2^-} \tag{5}}$$

$$\text{Pb}^{2+} + 2\text{O}\_2^{-} \rightarrow \text{PbO}\_2 + \text{O}\_2 \tag{6}$$

Their mechanism was supported by experiments carried out in different atmospheres: nitrogen, argon and oxygen at several partial pressures. In N2 and Ar, irradiation of TiO2 suspensions did not result in lead oxide formation. It is worth noting that a high pressure Hg lamp (500 W) was used for all their photocatalytic deposition reactions.

Litter et al. 1999, have proposed a different mechanism which involves two consecutive electron transfer reactions. Lead ions are oxidized by holes or by hydroxyl radicals passing through the divalent to the tetravalent state, equations 7 and 8:

$$\text{h}^{\ast}/\text{HCO}^{\ast} + \text{Pb}^{\ast \ast} \rightarrow \text{Pb}^{\geqslant \ast} + \text{OH}^{-} \tag{7}$$

$$\mathrm{h^{\*}/HCO^{\*} + Pb^{3\*} \to Pb^{4\*} + OH^{-}} \tag{8}$$

A further enhancement was achieved with platinized TiO2 by decreasing the overpotential of oxygen. In fact, the role of oxygen is crucial to carry out the photocatalytic cycle and it has found a linear dependence of oxygen partial pressure based on a Langmuir-Hinshelwood mechanism [Torres & Cervera-March, 1992].

### **2.2 Co2+**

26 Molecular Photochemistry – Various Aspects

Half cell E° (V) MnO2/Mn2+ 1.23 Mn2+/Mn -1.18 PbO2/Pb2+ 1.46 Pb2+/Pb -0.12 Tl2O3/Tl1+ 0.02 Tl1+/Tl -0.336 Co2+/Co -0.28 Cr6+ /Cr3+ 1.232 Cr3+/Cr -0.744

TiO2

These two reactions represent a good example of the photocatalytic deposition of metal oxides in aqueous solution onto titanium dioxide. This means that the complete photocatalytic cycle should consider the photoredox couple in which one metallic ion (single component) in solution is oxidized and the oxygen of the media is reduced. The deposition is driven by particle agglomeration after reaching their zero point charge and a critical concentration to be deposited on the surface of the semiconductor. It has been reported that single component metal oxides for example, PbO2, RuO2, U3O8, SiO2, SnO2, Fe2O3, MnO2, IrO2 and Cr2O3 can be deposited on semiconductor particles following a photo-oxidative or

On the other hand, when a semiconductor is irradiated with UV light in presence of aqueous solutions containing dissolved Ag+ or Pb2+ cations a redox process is undertaken giving rise to the reduction of silver ions or the oxidation of lead ions, according to the

 Pb2+ + 2H2O + 2h+ → PbO2 + 4H+ (4) Lately, it has been reported the photocatalytic deposition of mixed-oxides such as Rh2 yCryO3 dispersed on a semiconductor powder with applications in the water splitting reaction [Maeda et al. 2008]. Hence, we intend to offer the reader a condensed overview of the work done so far considering photocatalytic deposition of a single or mixed oxide on

In regard with the very negative impact of lead on environment and population, many efforts are conducted to remove it from water of distinct origins. It is commonly removed by precipitation as carbonate or hydroxide; besides other physicochemical methods are available to lead elimination. The maximun contaminant level in drinking water established

(3)

Table 1. Half wave potentials of different couples.

photo-reductive route [Maeda et al. 2008].

following reactions [Giocondi et al. 2003]:

**2.1 Pb2+**

Ag+ + e- → Ag

semiconductor materials by either oxidative or reductive processes.

**2. Photocatalytic oxidation of a single component** 

<sup>2</sup> Pb 2H O2 2 PbO 4H (2)

Recently, it has been highlighted many applications of cobalt compounds deposited on semiconductors such as: catalysts for solar oxygen production, gas sensors, batteries, electrochromic devices, among others [Steinmiller & Choi, 2009; Tak & Yong, 2008]. In particular, the photodeposition of Co3O4 spinel phase on ZnO has been prepared by two routes, one consisting in the direct photo-oxidation of Co2+ ions to Co3+ ions and the other by an indirect procedure involving the reduction of Co2+ to Co° and the oxidation of metallic cobalt to Co3O4 by means of the oxygen coming from the photo-oxidation of water.

By using the direct deposition route, a ZnO electrode was immersed in an aqueous solution of CoCl2 mantaining the pH constant at 7 and illuminating with UV light (= 302 nm). Due to the oxidation potential of Co2+ to form Co3O4 is 0.7 V at pH= 7 at low concentrations of Co2+ (103 M) and the valence band edge of ZnO is located at around 2.6 V vs NHE, the photogenerated holes can easily oxidize Co2+ ions to Co3+ ions. The complete photocatalytic cycle must also include the reduction of water or dissolved oxygen in the solution to have an efficient Co2+ photo-oxidation [Steinmiller & Choi, 2009]:

$$2\,\mathrm{Co^{2+}} + 3\,\mathrm{H\_2O} \rightarrow \mathrm{Co\_2O\_3} + 6\,\mathrm{H^\*} + 2\,\mathrm{e^-} \tag{9}$$

Photocatalytic Deposition of Metal Oxides on Semiconductor Particles: A Review 29

Fig. 2. (a) photocatalytic reaction scheme showing the reduction of Co ions to metallic Co and its oxidation to the spinel Co3O4, (b) Co3O4 deposited on the tip of ZnO, (c) Co3O4 deposited on the whole surface of ZnO. [Reproduced from Tak and Yong with permission

solution as Hg (II). Several methods have been investigated for its removal or control, such as, precipitation, ion exchange, adsorption, coagulation and reduction. However, the photocatalytic oxidation (PCO) of gaseous mercury by UVA-irradiated TiO2 surfaces has

For instance, an enhanced process including adsorption of gaseous mercury on silica-titania nanocomposites and then its photocatalytic oxidation has been published [Li and Wu, 2007]. However, some problems of reactivation of the nanocomposite as well as pore structure modification during Hg and HgO capture and deposition have to be solved. In the same work, it has been proposed the use of pellets of silica-titania composites and it was found that a decrease of contact angle was likely responsible for mercury capture for long periods. Usually, the experimental systems to evaluate the PCO of gaseous mercury include water vapor to supply the OH radicals required for the oxidation and a source of UVA irradiation (320-400 nm, 100 W Hg lamp). Figure 3 shows a typical schematic diagram for the PCO

According to the results obtained for the PCO of Hg in gas phase using a titania-silica nanocomposite [Li and Wu, 2007] , it has been proposed the following reaction mechanism:

TiO2 + hυ → e- + h+ (10)

H2O ↔ H+ + OH- (11)

h+ + OH- → •OH (12)

h+ + H2O → •OH + H+ (13)

•OH + Hgo → HgO (14)

from The Journal of Physical Chemistry; copyright 2008].

using titania-silica pellets.

been reported as a good option for its capture [Snider and Ariya, 2010].

From a thermodynamic point of view, Co3+ ions can be deposited on any semiconductor that has a valence band edge located at a more positive potential than that of the Co2+ ions, as shown in Figure 1.

Fig. 1. Schematic representation. (A) Photochemical deposition of the Co-based catalyst on ZnO and (B) relevant energy levels. [Reproduced from Steinmiller and Choi, with permission from PNAS, copyright 2009 by National Academy of Sciences].

By the second route, the ZnO nanowires were grown by ammonia solution hydrothermal method and then coated with Co3O4 using a photocatalytic reaction. This last method was selected considering that the redox reactions of aqueous chemical species on irradiated semiconductor surfaces has characteristics of site-specific growth. Briefly, the ZnO nanowire array was immersed in an aqueous solution of Co(NO3)2 and was irradiated with UV-light of 325 nm from minutes to 24 h. According to the results of this work, the morphology of the heterostructures depended on the photocatalytic reaction parameters such as the concentration of Co2+ in solution, UV irradiation time and the geometrical alignment of the ZnO nanowires. The photocatalytic process was explained in terms of redox cycle which includes the reduction of Co2+ species into Co° and the oxidation of water to produce O2. In fact, after irradiation of ZnO with photon energy larger than the band gap of ZnO (3.4 eV) generates the charge carriers (electron-hole pairs). The photogenerated electrons in the conduction band reduce Co2+ to Co° favoring the accumulation of holes in the valence band. In addition, the holes oxidizes water to molecular oxygen which carries out the partial oxidation of Co° to Co2+Co23+ O4 spinel, as outlined in Figure 2. It seemed that this simple, room temperature and selective photodeposition process can be applicable to other semiconductors (e.g. TiO2, CdS, SnO2…) or to other shapes of nanomaterials.

### **2.3 Hg°**

Mercury is a neurotoxic heavy metal frecuently found in industrial wastewaters at concentrations higher than 0.005 ppm and unfortunately it cannot be bio- or chemically degraded [Clarkson & Magos, 2006]. It is released to the environment by coal combustion and trash incineration, mainly as gaseous mercury producing methyl mercury in the aquatic ecosystem by the action of sulfate-reducing bacteria. Certainly, due to its multiple industrial applications (e.g. pesticides, paints, catalysts, electrical device etc.) it can also be found in

From a thermodynamic point of view, Co3+ ions can be deposited on any semiconductor that has a valence band edge located at a more positive potential than that of the Co2+ ions, as

Fig. 1. Schematic representation. (A) Photochemical deposition of the Co-based catalyst on

By the second route, the ZnO nanowires were grown by ammonia solution hydrothermal method and then coated with Co3O4 using a photocatalytic reaction. This last method was selected considering that the redox reactions of aqueous chemical species on irradiated semiconductor surfaces has characteristics of site-specific growth. Briefly, the ZnO nanowire array was immersed in an aqueous solution of Co(NO3)2 and was irradiated with UV-light of 325 nm from minutes to 24 h. According to the results of this work, the morphology of the heterostructures depended on the photocatalytic reaction parameters such as the concentration of Co2+ in solution, UV irradiation time and the geometrical alignment of the ZnO nanowires. The photocatalytic process was explained in terms of redox cycle which includes the reduction of Co2+ species into Co° and the oxidation of water to produce O2. In fact, after irradiation of ZnO with photon energy larger than the band gap of ZnO (3.4 eV) generates the charge carriers (electron-hole pairs). The photogenerated electrons in the conduction band reduce Co2+ to Co° favoring the accumulation of holes in the valence band. In addition, the holes oxidizes water to molecular oxygen which carries out the partial oxidation of Co° to Co2+Co23+ O4 spinel, as outlined in Figure 2. It seemed that this simple, room temperature and selective photodeposition process can be applicable to other

ZnO and (B) relevant energy levels. [Reproduced from Steinmiller and Choi, with permission from PNAS, copyright 2009 by National Academy of Sciences].

semiconductors (e.g. TiO2, CdS, SnO2…) or to other shapes of nanomaterials.

Mercury is a neurotoxic heavy metal frecuently found in industrial wastewaters at concentrations higher than 0.005 ppm and unfortunately it cannot be bio- or chemically degraded [Clarkson & Magos, 2006]. It is released to the environment by coal combustion and trash incineration, mainly as gaseous mercury producing methyl mercury in the aquatic ecosystem by the action of sulfate-reducing bacteria. Certainly, due to its multiple industrial applications (e.g. pesticides, paints, catalysts, electrical device etc.) it can also be found in

shown in Figure 1.

**2.3 Hg°**

Fig. 2. (a) photocatalytic reaction scheme showing the reduction of Co ions to metallic Co and its oxidation to the spinel Co3O4, (b) Co3O4 deposited on the tip of ZnO, (c) Co3O4 deposited on the whole surface of ZnO. [Reproduced from Tak and Yong with permission from The Journal of Physical Chemistry; copyright 2008].

solution as Hg (II). Several methods have been investigated for its removal or control, such as, precipitation, ion exchange, adsorption, coagulation and reduction. However, the photocatalytic oxidation (PCO) of gaseous mercury by UVA-irradiated TiO2 surfaces has been reported as a good option for its capture [Snider and Ariya, 2010].

For instance, an enhanced process including adsorption of gaseous mercury on silica-titania nanocomposites and then its photocatalytic oxidation has been published [Li and Wu, 2007]. However, some problems of reactivation of the nanocomposite as well as pore structure modification during Hg and HgO capture and deposition have to be solved. In the same work, it has been proposed the use of pellets of silica-titania composites and it was found that a decrease of contact angle was likely responsible for mercury capture for long periods. Usually, the experimental systems to evaluate the PCO of gaseous mercury include water vapor to supply the OH radicals required for the oxidation and a source of UVA irradiation (320-400 nm, 100 W Hg lamp). Figure 3 shows a typical schematic diagram for the PCO using titania-silica pellets.

According to the results obtained for the PCO of Hg in gas phase using a titania-silica nanocomposite [Li and Wu, 2007] , it has been proposed the following reaction mechanism:

$$\rm TiO\_2 + hp \rightarrow e^\cdot + h^\cdot \tag{10}$$

$$\rm H\_2O \leftrightarrow H^\* + OH^- \tag{11}$$

$$\text{H}^\* + \text{OH} \cdot \text{H} \to \text{\textasciicircum} \text{OH} \tag{12}$$

$$\rm{H}^\* + \rm{H}\_2\rm{O} \rightarrow \rm{\text{\textbullet{}OH}} + \rm{H}^\* \tag{13}$$

•OH + Hgo → HgO (14)

Photocatalytic Deposition of Metal Oxides on Semiconductor Particles: A Review 31

electrons move in the network of Ti4+ ions in the nanosheet. On the other hand, the photooxidation of Mn2+ ions occurred on all over the surface of the nanosheet, which is indicative of the presence of holes at the O2- ion. The photo-oxidation reaction was carried out in a diluted solution of MnSO4 (10-6 M) in air at room temperature irradiating with UV light of 265 nm. It was found that pH played an important role in the photodeposition: the amount of formed MnO2 increased at higher values of pH, and no metallic Mn was observed at lower values of pH (pH=2.1). The coupled reactions that takes place during the

Mn2+ + 2H2O + 4h+ (produced in VB) MnO2 + 4H+ (on the surface) (15)

 O2 (in air) + 4H+ + 4e- (produced in CB) 2H2O (at the edge) (16) A complete model explaining the photodeposition process and charge mobility are illustrated in Figure 4. In other words, according to the results reported by (Matsumoto et al., 2008) the photoproduced electrons move at the 3d orbital conduction band of the Ti4+ network in the nanosheet, whereas the photoproduced holes are located at the 2p orbital as O2- species at the surface. Finally, the charge carriers recombination is favored under low pH which was found as a key parameter to control the photoprocess on the oxide nanosheet.

Fig. 4. Model of the movements of the photoproduced electron and hole at the TiOx

exists at the 2p VB consisting of the O2- surface and oxidizes Mn2+ on the surface. Reproduced from Matsumoto et al., with permission from The Journal of Physical

Chemistry C, copyright 2008.

**2.5 Fe2+** 

nanosheet with a lepidocrocite-type structure. The electron moves in the 3d CB consisting of the Ti4+ network in the nanosheet and then reduces Ag+ and Cu2+ at the edge, while the hole

One of the main drawbacks to commercialize the TiO2 photocatalytic process at large scale is the use of UV light as irradiation source. Then, many efforts have been done during the past two decades to develop new photocatalysts active under visible light [Choi, 2006]. For instance, the presence of Fe3+ ions on TiO2 favors the absorption of photons in the visible region as well as accelerates the photocatalytic oxidation of organic compounds. In this case, Fe3+ ions reduce to Fe2+ by the photoelectrons of the conduction band avoiding the charge

photodeposition of MnO2 are described by eqs. (15,16):

which was successfully expressed by the Langmuir-Hinshelwood model. The rate of photooxidation of Hg was significantly inhibited by the presence of water vapor explained in terms of a competitive adsorption of water and mercury on the surface of TiO2.

Efforts to gas mercury oxidation in air are now focused by using immobilized semiconductors irradiated with visible light looking for a potentially safe, low-cost process [Snider and Ariya, 2010].

Fig. 3. Schematic diagram for the photocatalytic oxidation of mercury gas. After [Li and Wu, 2007].

### **2.4 Mn2+**

Manganese (II) in aquatic systems is a problem of environment concern due to its slow oxidation to MnO2 which is responsible for the formation of dark precipitates. The photocatalytic oxidation of Mn2+ to Mn4+ in the presence of irradiated titanium dioxide has been scarcely studied since the 80's [Tanaka et al. 1986, Lozano et al. 1992 and Tateoka et al. 2005, Matsumoto et al., 2008]. This process represents an alternative route for its removal and the resulting material could be used as supported metal oxides catalysts [Tateoka et al. 2005, Matsumoto et al., 2008]. In the first publication, it was used concentrations ranging from 10-4-10-3 mol/L aqueous solutions of Mn2+ with irradiated TiO2 and Pt/TiO2 photocatalysts using a high pressure Hg lamp of 500 W. Mn2+ conversion to Mn4+ was 98 and 78% from low to high concentrations onto Pt-loaded TiO2 in 1 h of irradiation time. In the second work published in 1992, the oxidation of Mn2+ was carried out in acidic conditions using TiO2 Degussa P-25 and irradiating with a Hg vapor lamp of 125 W at initial concentration of Mn2+ of 10-4 mol/L. One of the visual evidence of the photocatalytic oxidation of Mn2+ to Mn4+ is the appearance of a slight dark coloration over the TiO2. The overall reaction scheme for the photo-oxidation was presented as follows:

$$\mathrm{Mn^{2+}} + \mathrm{l/}\mathrm{O\_{2}} + \mathrm{H\_{2}O} \rightarrow \mathrm{MnO\_{2}} + \mathrm{2H^{+}} \tag{14}$$

In a recent work, it was studied the photodeposition of metal and metal oxide at the TiOx nanosheet to observe the photocatalytic active site (Matsumoto et al., 2008). It was investigated the photodeposition of Ag, Cu, Cu2O and MnO2 at a TiOx nanosheet with a lepidocrocite-type structure prepared from K-Ti-Li mixed oxide. As expected, the photoreduction of Ag, Cu and Cu2O, occurred mainly at edges where the photoproduced electrons move in the network of Ti4+ ions in the nanosheet. On the other hand, the photooxidation of Mn2+ ions occurred on all over the surface of the nanosheet, which is indicative of the presence of holes at the O2- ion. The photo-oxidation reaction was carried out in a diluted solution of MnSO4 (10-6 M) in air at room temperature irradiating with UV light of 265 nm. It was found that pH played an important role in the photodeposition: the amount of formed MnO2 increased at higher values of pH, and no metallic Mn was observed at lower values of pH (pH=2.1). The coupled reactions that takes place during the photodeposition of MnO2 are described by eqs. (15,16):

Mn2+ + 2H2O + 4h+ (produced in VB) MnO2 + 4H+ (on the surface) (15)

$$\text{O}\_2\text{ (in air)} + 4\text{H}^+ + 4\text{e} \text{ (produced in CB)} \rightarrow 2\text{H}\_2\text{O (at the edge)}\tag{16}$$

A complete model explaining the photodeposition process and charge mobility are illustrated in Figure 4. In other words, according to the results reported by (Matsumoto et al., 2008) the photoproduced electrons move at the 3d orbital conduction band of the Ti4+ network in the nanosheet, whereas the photoproduced holes are located at the 2p orbital as O2- species at the surface. Finally, the charge carriers recombination is favored under low pH which was found as a key parameter to control the photoprocess on the oxide nanosheet.

Fig. 4. Model of the movements of the photoproduced electron and hole at the TiOx nanosheet with a lepidocrocite-type structure. The electron moves in the 3d CB consisting of the Ti4+ network in the nanosheet and then reduces Ag+ and Cu2+ at the edge, while the hole exists at the 2p VB consisting of the O2- surface and oxidizes Mn2+ on the surface. Reproduced from Matsumoto et al., with permission from The Journal of Physical Chemistry C, copyright 2008.

### **2.5 Fe2+**

30 Molecular Photochemistry – Various Aspects

which was successfully expressed by the Langmuir-Hinshelwood model. The rate of photooxidation of Hg was significantly inhibited by the presence of water vapor explained in

Efforts to gas mercury oxidation in air are now focused by using immobilized semiconductors irradiated with visible light looking for a potentially safe, low-cost process

Manganese (II) in aquatic systems is a problem of environment concern due to its slow oxidation to MnO2 which is responsible for the formation of dark precipitates. The photocatalytic oxidation of Mn2+ to Mn4+ in the presence of irradiated titanium dioxide has been scarcely studied since the 80's [Tanaka et al. 1986, Lozano et al. 1992 and Tateoka et al. 2005, Matsumoto et al., 2008]. This process represents an alternative route for its removal and the resulting material could be used as supported metal oxides catalysts [Tateoka et al. 2005, Matsumoto et al., 2008]. In the first publication, it was used concentrations ranging from 10-4-10-3 mol/L aqueous solutions of Mn2+ with irradiated TiO2 and Pt/TiO2 photocatalysts using a high pressure Hg lamp of 500 W. Mn2+ conversion to Mn4+ was 98 and 78% from low to high concentrations onto Pt-loaded TiO2 in 1 h of irradiation time. In the second work published in 1992, the oxidation of Mn2+ was carried out in acidic conditions using TiO2 Degussa P-25 and irradiating with a Hg vapor lamp of 125 W at initial concentration of Mn2+ of 10-4 mol/L. One of the visual evidence of the photocatalytic oxidation of Mn2+ to Mn4+ is the appearance of a slight dark coloration over the TiO2. The

 Mn2+ + ½ O2 + H2O MnO2 + 2H+ (14) In a recent work, it was studied the photodeposition of metal and metal oxide at the TiOx nanosheet to observe the photocatalytic active site (Matsumoto et al., 2008). It was investigated the photodeposition of Ag, Cu, Cu2O and MnO2 at a TiOx nanosheet with a lepidocrocite-type structure prepared from K-Ti-Li mixed oxide. As expected, the photoreduction of Ag, Cu and Cu2O, occurred mainly at edges where the photoproduced

terms of a competitive adsorption of water and mercury on the surface of TiO2.

Fig. 3. Schematic diagram for the photocatalytic oxidation of mercury gas.

overall reaction scheme for the photo-oxidation was presented as follows:

[Snider and Ariya, 2010].

After [Li and Wu, 2007].

**2.4 Mn2+**

One of the main drawbacks to commercialize the TiO2 photocatalytic process at large scale is the use of UV light as irradiation source. Then, many efforts have been done during the past two decades to develop new photocatalysts active under visible light [Choi, 2006]. For instance, the presence of Fe3+ ions on TiO2 favors the absorption of photons in the visible region as well as accelerates the photocatalytic oxidation of organic compounds. In this case, Fe3+ ions reduce to Fe2+ by the photoelectrons of the conduction band avoiding the charge

Photocatalytic Deposition of Metal Oxides on Semiconductor Particles: A Review 33

Sn4+. Then Sn4+ was hydrolized to the insoluble H2SnO3, which finally is decomposed to

Fig. 5. A schematic representation of the photocatalytic oxidation of metal oxides in presence of a conductive substrate (Pt film) and dissolved oxygen. After [Kamada and Moriyasu 2011].

This particular route for depositing metal oxides on semiconductors, also called reductive deposition, has been studied intensively for its potential in environmental remediation: for instance in the partial reduction of chromates (Cr6+ are extremely toxic) to the much less toxic Cr3+ or for UO22+ to UO2 or but also in the preparation of special catalysts containing

Lately, it has been reported works devoted to the reductive deposition of Mn3O4 or RuO2 on titanium dioxide by using KMnO4 contained in waste water or pure aqueous solutions of KRuO4. The reaction mechanism involves a cathodic process where anions (e.g. CrO42-, MnO4-, etc.) having strong oxidation power effectively accept the photogenerated electrons of the conduction band of TiO2 after irradiation with UV light and the deposition of the corresponding oxide. On the other hand, in the anodic process the holes found in the valence band oxidize the sacrificial oxidant agent to produce the proton required for the

In this sense, Nishimura et al., 2008, have prepared coupled catalysts nanoparticles of MnO2/TiO2 by the photoreduction of harmful MnO4 anions in water, see Fig. 6, and applied to the decomposition of hydrogen peroxide in the dark or irradiated with UV light. This coupled semiconductors can improve the charge separation efficiency through interfacial electron transfer. In addition, it is well known the catalytic properties of MnO2 for the oxidation of organic pollutants which coupled with TiO2 could have a special synergism in conventional catalytic or photocatalytic reactions. It was used a 10-3 M aqueous solution of KMnO4 at pH 7, UV light (λ>300 nm) and inert atmosphere to carry out the photoreduction reaction of manganate ions. In a blank experiment during the irradiation of the solution of KMnO4 (without TiO2) it was only found a partial decomposition of MnO4- ions to MnO42 and O2. The photodeposition of Mn3O4 on TiO2 was confirmed by XPS and these stick-

**3. Photocatalytic reduction of single component** 

Cu2O obtained by the partial reduction of Cu2+ to Cu1+ [Wu et al., 2003].

SnO2.

**3.1 MnO4¯/MnO2**

photoreduction of the anion.

recombination and increasing the photonic efficiency. However, the reverse process, this means the photo-oxidation of Fe2+ has been scarcely studied. A photoelectrochemical oxidation of Fe2+ ions on porous nanocrystalline TiO2 electrodes was studied by using in situ EQCM (electrochemical quartz crystal microbalance) technique [Si et al., 2002]. In this work, it was found that the pH of iron precursor solution plays an important role in terms of the amount of adsorbed Fe2+ ions. The maximum value was 1.1 mmol Fe2+ at pH 4. The stability and the adsorption process was studied by the EQCM technique and it was found that the adsorption amount of Fe2+ ions on TiO2 support was not affected by bias potential drop. The above result was attributed to Fe2+ ions are coordinated with hydroxyl groups of TiO2 surface by the following reaction:

$$\text{Ti-OH} + \text{Fe^{2+}} \rightarrow \text{Ti-OH} \dots \text{Fe^{2+}} \tag{17}$$

As is well known at low pH values, TiO2 has negative surface charge favoring the electrostatic attraction of Fe2+ ions. Therefore, the adsorption-desorption behavior of Fe2+ ions on TiO2 surface is strongly affected by pH changes. After irradiation of the adsorbed Fe2+ ions on TiO2 the following photochemical reactions can be expected:

$$\rm{TiO\_2 + hv \to h^\* \,\_{vb} + e^\cdot} \,\_{cb} \tag{18}$$

$$\text{Ti-OH} \dots \text{Fe^{2+}} + \text{h}^{\*}\text{.} \text{\textbullet Ti-OH} \dots \text{Fe^{3+}} \tag{19}$$

$$(\mathrm{H\_2O})\_{\mathrm{surf}} + \mathrm{h^\*}\_{\mathrm{vb}} \rightarrow (\mathrm{\bullet OH})\_{\mathrm{surf}} + \mathrm{H^\*} \tag{20}$$

$$2\text{Fe}^{2+} + 2\text{(\bullet OH)}\_{\text{surf or aq}} + \text{H}\_2\text{O} \rightarrow \text{Fe}\_2\text{O}\_3 + 4\text{H}^\*\tag{21}$$

### **2.6 Ce3+and Sn2+**

Nowadays, the preparation of semiconductor nanoparticles with precise control of size and morphology has found new applications as ion-conducting,sun-screening, anti-corrosion and electro-catalytic properties [Kamada & Moriyasu 2011]. For instance, CeO2 and SnO2 have been synthesized as semiconducting oxide films by a photodeposition method [Kamada & Moriyasu 2011]. This method has the advantage of depositing homogeneously a thin film of the respective semiconductor by manipulating certain parameters such as concentration of the precursor, time and intensity of the irradiation, etc.

In the work reported by Kamada and Moriyasu, a photo-excited electroless deposition was carried out by the irradiation with UV light of an aqueous solution of cerium triacetate in a platinum substrate. It was observed an enhancement of the deposition of CeO2, which was explained in terms of an electron transfer local cell mechanism. In this case, Ce3+ was oxidized by dissolved oxygen through an electron transfer in the Pt substrate and then transformed in a CeO2 thin film, as shown in Figure 5. Surprisingly, the deposition rate was detrimentally affected by increasing the concentration of Ce3+ ions.

In a similar way, Sn2+ ions were anodically oxidized to Sn4+ and deposited on a Pt electrode with UV light irradiation. This process was followed through a different reaction mechanism than that of cerium. Tin oxide deposition proceeded by a photochemical reaction started with the disproportionation of Sn2+ and the further production of Sno and Sn4+. Then Sn4+ was hydrolized to the insoluble H2SnO3, which finally is decomposed to SnO2.

Fig. 5. A schematic representation of the photocatalytic oxidation of metal oxides in presence of a conductive substrate (Pt film) and dissolved oxygen. After [Kamada and Moriyasu 2011].

### **3. Photocatalytic reduction of single component**

### **3.1 MnO4¯/MnO2**

32 Molecular Photochemistry – Various Aspects

recombination and increasing the photonic efficiency. However, the reverse process, this means the photo-oxidation of Fe2+ has been scarcely studied. A photoelectrochemical oxidation of Fe2+ ions on porous nanocrystalline TiO2 electrodes was studied by using in situ EQCM (electrochemical quartz crystal microbalance) technique [Si et al., 2002]. In this work, it was found that the pH of iron precursor solution plays an important role in terms of the amount of adsorbed Fe2+ ions. The maximum value was 1.1 mmol Fe2+ at pH 4. The stability and the adsorption process was studied by the EQCM technique and it was found that the adsorption amount of Fe2+ ions on TiO2 support was not affected by bias potential drop. The above result was attributed to Fe2+ ions are coordinated with hydroxyl groups of TiO2

As is well known at low pH values, TiO2 has negative surface charge favoring the electrostatic attraction of Fe2+ ions. Therefore, the adsorption-desorption behavior of Fe2+ ions on TiO2 surface is strongly affected by pH changes. After irradiation of the adsorbed

Ti-OH Fe2+ + h+vb Ti-OH …Fe3+ (19)

(H2O)surf + h+vb (•OH)surf + H+ (20)

Nowadays, the preparation of semiconductor nanoparticles with precise control of size and morphology has found new applications as ion-conducting,sun-screening, anti-corrosion and electro-catalytic properties [Kamada & Moriyasu 2011]. For instance, CeO2 and SnO2 have been synthesized as semiconducting oxide films by a photodeposition method [Kamada & Moriyasu 2011]. This method has the advantage of depositing homogeneously a thin film of the respective semiconductor by manipulating certain parameters such as

In the work reported by Kamada and Moriyasu, a photo-excited electroless deposition was carried out by the irradiation with UV light of an aqueous solution of cerium triacetate in a platinum substrate. It was observed an enhancement of the deposition of CeO2, which was explained in terms of an electron transfer local cell mechanism. In this case, Ce3+ was oxidized by dissolved oxygen through an electron transfer in the Pt substrate and then transformed in a CeO2 thin film, as shown in Figure 5. Surprisingly, the deposition rate was

In a similar way, Sn2+ ions were anodically oxidized to Sn4+ and deposited on a Pt electrode with UV light irradiation. This process was followed through a different reaction mechanism than that of cerium. Tin oxide deposition proceeded by a photochemical reaction started with the disproportionation of Sn2+ and the further production of Sno and

Fe2+ ions on TiO2 the following photochemical reactions can be expected:

concentration of the precursor, time and intensity of the irradiation, etc.

detrimentally affected by increasing the concentration of Ce3+ ions.

TiO2 + hν h+vb + e-

Ti-OH + Fe2+ Ti-OH Fe2+ (17)

aq + 2(•OH)surf or aq + H2O Fe2O3 + 4H+ (21)

cb (18)

surface by the following reaction:

2Fe2+

**2.6 Ce3+and Sn2+**

This particular route for depositing metal oxides on semiconductors, also called reductive deposition, has been studied intensively for its potential in environmental remediation: for instance in the partial reduction of chromates (Cr6+ are extremely toxic) to the much less toxic Cr3+ or for UO22+ to UO2 or but also in the preparation of special catalysts containing Cu2O obtained by the partial reduction of Cu2+ to Cu1+ [Wu et al., 2003].

Lately, it has been reported works devoted to the reductive deposition of Mn3O4 or RuO2 on titanium dioxide by using KMnO4 contained in waste water or pure aqueous solutions of KRuO4. The reaction mechanism involves a cathodic process where anions (e.g. CrO42-, MnO4-, etc.) having strong oxidation power effectively accept the photogenerated electrons of the conduction band of TiO2 after irradiation with UV light and the deposition of the corresponding oxide. On the other hand, in the anodic process the holes found in the valence band oxidize the sacrificial oxidant agent to produce the proton required for the photoreduction of the anion.

In this sense, Nishimura et al., 2008, have prepared coupled catalysts nanoparticles of MnO2/TiO2 by the photoreduction of harmful MnO4 anions in water, see Fig. 6, and applied to the decomposition of hydrogen peroxide in the dark or irradiated with UV light. This coupled semiconductors can improve the charge separation efficiency through interfacial electron transfer. In addition, it is well known the catalytic properties of MnO2 for the oxidation of organic pollutants which coupled with TiO2 could have a special synergism in conventional catalytic or photocatalytic reactions. It was used a 10-3 M aqueous solution of KMnO4 at pH 7, UV light (λ>300 nm) and inert atmosphere to carry out the photoreduction reaction of manganate ions. In a blank experiment during the irradiation of the solution of KMnO4 (without TiO2) it was only found a partial decomposition of MnO4 - ions to MnO4 2 and O2. The photodeposition of Mn3O4 on TiO2 was confirmed by XPS and these stick-

Photocatalytic Deposition of Metal Oxides on Semiconductor Particles: A Review 35

Pt/RuO2 on single crystals of TiO2. Searching a good photocatalyst for overall water splitting, Maeda et al., 2006, developed a complex semiconductor (Ga1-xZnx)(N1-xOx) as a promising stable material active under visible light irradiation. However, this semiconductor only presented activity for water oxidation and its activity for water reduction was very low. Therefore, an effective modification of the GaN:ZnO semiconductor to promote the water reduction photoactivity was required. As is well known, noble metals or transition-metal oxides are often employed as cocatalysts to facilitate the water reduction reaction. Then, it was proposed the preparation of a noblemetal/mixed oxide (core/shell) supported on the GaN:ZnO solid solution by in situ photodeposition method [Maeda et al., 2006]. A two steps procedure was employed, Rh nanoparticles were firstly deposited on the mixed support with an aqueous precursor of Na3RhCl6.H2O and then Cr2O3 was deposited from a K2CrO4 solution, in both steps visible light irradiation was employed (λ>400 nm), as shown in Fig. 7. The authors confirmed the formation of a Rh/Cr2O3 core/shell nanoparticle with an average size of the ensemble of 12 nm and found a dramatical change in photocatalytic activity for overall water splitting

In a second similar work of Maeda et al., 2008, it was reported a method to prepare mixed oxides of rhodium and chromium on five different semiconductors. They used aqueous solutions of (NH4)RhCl6 and K2CrO4 containing dispersed semiconductor powders and irradiated them during 4 hours with wavelengths whose energy exceeded those of each

> Chromium (%wt)

Irradiation wavelength (nm)

in comparison with Rh or Cr2O3/GaN:ZnO supported systems.

(%wt)

proceeds via a band-gap transition of the semiconductor powder.

(x=0.12) 1 1.5 >400

(x=0.44) 1 1.5 >400 SrTiO3 0.5 0.75 >200 Ca2Nb2O7 0.5 0.75 >200 ß-Ga2O3 0.5 0.75 >200 Table 2. Semiconductor powders and Rh-Cr content for mixed oxide photodeposition.

Based on XPS characterization, authors concluded that photodeposited mixed oxides have the composition Rh2-yCryO3 and explained that the photoreduction of both, Rh3+ and Cr6+

Furthermore, it was found that this mixed oxide is only formed when Rh and Cr are simultaneously present in the precursor solution. The photocatalytic performance of the materials was investigated for the evolution of H2/O2 in water splitting displaying different photocatalytic activity values depending of the support employed. In particular, photocatalyst containing the mixed oxide Rh2-yCryO3 exhibited a two fold activity compared

semiconductor band gap, as shown in Table 2.

Semiconductor Rhodium

(Ga1-xZnx)(N1-xOx)

(Zn1+xGe)(N2Ox)

to that of semiconductor alone.

shaped nanoparticles were converted to cubic -MnO2 by heating a 600°C.The overall photodeposition reaction of Mn3O4 was as follows:

$$\text{3MnO}\_4^- + \text{12H} \xrightarrow[\text{hv>300 nm}]{\text{TiO}\_2} \text{Mn} \text{O}\_4 + \text{6H}\_2\text{O} + \text{O}\_2 \tag{22}$$

Fig. 6. Scheme showing the simultaneous photocatalytic reduction of permanganate anions in aqueous solution forming Mn3O4 and the photooxidation of water forming oxygen during UV illumination of TiO2. After Nishimura et al., 2008.

### **3.2 RuO4 - /RuO2**

The photocatalytic decomposition of water strongly requires the presence of effective catalysts for hydrogen and oxygen evolution. Usually, most published works are focused to the overall water splitting and a few have independently tested the water photo-oxidation reaction. In particular, the water photo-oxidation has been successfully studied with partially dehydrated RuO2. However, its loading onto substrate surfaces by the conventional thermal methods lead to deep dehydration and sintering, reducing dramatically its activity and stability. An early work of Mills et al., 2010, has achieved the photodeposition of RuO2 on titanium dioxide by a simple reaction of an aqueous solution of KRuO4 mixed with TiO2 and irradiation with a Xe or Hg lamp and Ce4+ ions as sacrificial electron donor. The following reaction scheme was proposed:

$$4\text{RuO}\_4 + 4\text{H} + 4\text{TiO}\_2 \rightarrow 4\text{TiO}\_2/\text{RuO}\_2 + 3\text{O}\_2 + 2\text{H}\_2\text{O}\tag{23}$$

The photoreduction of ruthenate ion (RuO4-) in the absence of the titania photocatalyst remain unchanged.

### **4. Photocatalytic oxidation to obtain mixed oxides**

### **4.1 Rh2-yCryO3**

The direct photodeposition of nanoparticulate mixed oxides on semiconductors was firstly reported by Maeda et al. [Maeda et al. 2008] supported in the pioneer work of Kobayashi et al. 1983, who studied the simultaneous photodeposition of Pd/PbO2 and

shaped nanoparticles were converted to cubic -MnO2 by heating a 600°C.The overall

TiO2

Fig. 6. Scheme showing the simultaneous photocatalytic reduction of permanganate anions in aqueous solution forming Mn3O4 and the photooxidation of water forming oxygen during

The photocatalytic decomposition of water strongly requires the presence of effective catalysts for hydrogen and oxygen evolution. Usually, most published works are focused to the overall water splitting and a few have independently tested the water photo-oxidation reaction. In particular, the water photo-oxidation has been successfully studied with partially dehydrated RuO2. However, its loading onto substrate surfaces by the conventional thermal methods lead to deep dehydration and sintering, reducing dramatically its activity and stability. An early work of Mills et al., 2010, has achieved the photodeposition of RuO2 on titanium dioxide by a simple reaction of an aqueous solution of KRuO4 mixed with TiO2 and irradiation with a Xe or Hg lamp and Ce4+ ions as sacrificial

 4RuO4- + 4H+ + 4TiO2 4TiO2/RuO2 + 3O2 + 2H2O (23) The photoreduction of ruthenate ion (RuO4-) in the absence of the titania photocatalyst

The direct photodeposition of nanoparticulate mixed oxides on semiconductors was firstly reported by Maeda et al. [Maeda et al. 2008] supported in the pioneer work of Kobayashi et al. 1983, who studied the simultaneous photodeposition of Pd/PbO2 and

+ 12H+ Mn3O4 + 6H2O + O2 (22)

photodeposition reaction of Mn3O4 was as follows:

h>300 nm

UV illumination of TiO2. After Nishimura et al., 2008.

electron donor. The following reaction scheme was proposed:

**4. Photocatalytic oxidation to obtain mixed oxides** 

3MnO4

**3.2 RuO4**

**- /RuO2** 

remain unchanged.

**4.1 Rh2-yCryO3**

Pt/RuO2 on single crystals of TiO2. Searching a good photocatalyst for overall water splitting, Maeda et al., 2006, developed a complex semiconductor (Ga1-xZnx)(N1-xOx) as a promising stable material active under visible light irradiation. However, this semiconductor only presented activity for water oxidation and its activity for water reduction was very low. Therefore, an effective modification of the GaN:ZnO semiconductor to promote the water reduction photoactivity was required. As is well known, noble metals or transition-metal oxides are often employed as cocatalysts to facilitate the water reduction reaction. Then, it was proposed the preparation of a noblemetal/mixed oxide (core/shell) supported on the GaN:ZnO solid solution by in situ photodeposition method [Maeda et al., 2006]. A two steps procedure was employed, Rh nanoparticles were firstly deposited on the mixed support with an aqueous precursor of Na3RhCl6.H2O and then Cr2O3 was deposited from a K2CrO4 solution, in both steps visible light irradiation was employed (λ>400 nm), as shown in Fig. 7. The authors confirmed the formation of a Rh/Cr2O3 core/shell nanoparticle with an average size of the ensemble of 12 nm and found a dramatical change in photocatalytic activity for overall water splitting in comparison with Rh or Cr2O3/GaN:ZnO supported systems.

In a second similar work of Maeda et al., 2008, it was reported a method to prepare mixed oxides of rhodium and chromium on five different semiconductors. They used aqueous solutions of (NH4)RhCl6 and K2CrO4 containing dispersed semiconductor powders and irradiated them during 4 hours with wavelengths whose energy exceeded those of each semiconductor band gap, as shown in Table 2.


Table 2. Semiconductor powders and Rh-Cr content for mixed oxide photodeposition.

Based on XPS characterization, authors concluded that photodeposited mixed oxides have the composition Rh2-yCryO3 and explained that the photoreduction of both, Rh3+ and Cr6+ proceeds via a band-gap transition of the semiconductor powder.

Furthermore, it was found that this mixed oxide is only formed when Rh and Cr are simultaneously present in the precursor solution. The photocatalytic performance of the materials was investigated for the evolution of H2/O2 in water splitting displaying different photocatalytic activity values depending of the support employed. In particular, photocatalyst containing the mixed oxide Rh2-yCryO3 exhibited a two fold activity compared to that of semiconductor alone.

Photocatalytic Deposition of Metal Oxides on Semiconductor Particles: A Review 37

method also is a promising technique to obtain composite nanomaterials with the possibility to control the structural properties. Size, morphology and structure of the deposited oxides depend of the concentration of precursor and semiconductor, pH of the solution, light intensity and wavelength, illumination time and the type of sacrificial

Although most work has been focused to the use of titanium dioxide as supporting material, other semiconductors have now been investigated (e.g. ZnO, WO3, SnO2, ZnS, GaO). So that, it is possible to design new advanced compositing materials by selecting the appropriate semiconductor and depositing pure or mixed oxides with specific applications in solar energy conversion, purification of water and air streams, metal corrosion and prevention,

In addition, this method has the main advantage of not using high pressures and temperatures and in most cases the synthesis is carried out in aqueous solution. In spite of the photodeposition methods seem to be ideal for the synthesis of catalytic materials, to date, research reports have mainly focused in the photoreduction of noble metals. Therefore, the range of metal oxides deposited by a photoxidative or photoreductive routes has been limited. Finally, the oxidative deposition of metal oxides in semiconductors requires a deep investigation from fundamental to practical application. This is of crucial importance for understanding the mechanism of simple or mixed oxides formation (core-shell or alloys) during the irradiation step and the interfacial reactions of

This work was supported by grants Nos. 153356 and 106891 from the National Council of

Buono-Core G., Tejos M., Cabello G., Guzman N., Hill R, 2006, Photochemical deposition of

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electron acceptor employed.

the process.

**7. References** 

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**6. Acknowledgements** 

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2997-3027, ISSN 0043-1354.

### **4.2 NiCoOx**

In 2006, Buono-Core et al. 2006 reported the photodeposition of NiCoOx on Si (100). Interest in this mixed oxide system regards on its antiferromagnetic characteristics. Authors synthesized NiCo(DBA)2 as a single source precursor for the preparation of NiCo mixed oxide thin films, Figure 8. A solution of precursor in chloroform was prepared and then spin coated onto Si (100) chips. The films were irradiated under a 200 W Hg-Xe lamp ( ~254 nm) until no ligand absorptions were observed in FT-IR. Characterization by AFM and XRD lead the authors to conclude about the amorphous nature of the mixed oxide films. After annealing at 600 ºC of those films, XRD evidenced individual NiO and CoO oxides confirming the metastable nature of NiCox films. Furthermore, EDAX analyses demonstrated homogeneity of Ni and Co dispersion throughout Si (100) surface. Finally, authors suggest the extendibility of this technique for rendering a wide range of binary metal oxide phases.

Fig. 7. Scheme showing the photoconversion of inorganic precursors of Rh and Cr and their deposition of particles of GaN:ZnO. After from Maeda et al., 2008

### **5. Conclusions**

Photocatalytic deposition methods have been shown to be of high potentiality for loading small-size dispersed metal oxides on powder or film semiconductors. This method also is a promising technique to obtain composite nanomaterials with the possibility to control the structural properties. Size, morphology and structure of the deposited oxides depend of the concentration of precursor and semiconductor, pH of the solution, light intensity and wavelength, illumination time and the type of sacrificial electron acceptor employed.

Although most work has been focused to the use of titanium dioxide as supporting material, other semiconductors have now been investigated (e.g. ZnO, WO3, SnO2, ZnS, GaO). So that, it is possible to design new advanced compositing materials by selecting the appropriate semiconductor and depositing pure or mixed oxides with specific applications in solar energy conversion, purification of water and air streams, metal corrosion and prevention, chemical synthesis and manufacturing, nanoelectronics, medicine, among others.

In addition, this method has the main advantage of not using high pressures and temperatures and in most cases the synthesis is carried out in aqueous solution. In spite of the photodeposition methods seem to be ideal for the synthesis of catalytic materials, to date, research reports have mainly focused in the photoreduction of noble metals. Therefore, the range of metal oxides deposited by a photoxidative or photoreductive routes has been limited. Finally, the oxidative deposition of metal oxides in semiconductors requires a deep investigation from fundamental to practical application. This is of crucial importance for understanding the mechanism of simple or mixed oxides formation (core-shell or alloys) during the irradiation step and the interfacial reactions of the process.

### **6. Acknowledgements**

This work was supported by grants Nos. 153356 and 106891 from the National Council of Science and Technology (Conacyt, México).

### **7. References**

36 Molecular Photochemistry – Various Aspects

In 2006, Buono-Core et al. 2006 reported the photodeposition of NiCoOx on Si (100). Interest in this mixed oxide system regards on its antiferromagnetic characteristics. Authors synthesized NiCo(DBA)2 as a single source precursor for the preparation of NiCo mixed oxide thin films, Figure 8. A solution of precursor in chloroform was prepared and then spin coated onto Si (100) chips. The films were irradiated under a 200 W Hg-Xe lamp ( ~254 nm) until no ligand absorptions were observed in FT-IR. Characterization by AFM and XRD lead the authors to conclude about the amorphous nature of the mixed oxide films. After annealing at 600 ºC of those films, XRD evidenced individual NiO and CoO oxides confirming the metastable nature of NiCox films. Furthermore, EDAX analyses demonstrated homogeneity of Ni and Co dispersion throughout Si (100) surface. Finally, authors suggest the extendibility of this technique for rendering a wide range of binary

Fig. 7. Scheme showing the photoconversion of inorganic precursors of Rh and Cr and their

Photocatalytic deposition methods have been shown to be of high potentiality for loading small-size dispersed metal oxides on powder or film semiconductors. This

deposition of particles of GaN:ZnO. After from Maeda et al., 2008

Fig. 8. NiCo(DBA)2, DBA stands for Dibenzoylacetone.

**4.2 NiCoOx**

metal oxide phases.

**5. Conclusions** 


Photocatalytic Deposition of Metal Oxides on Semiconductor Particles: A Review 39

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**Photoisomerization of Norbornadiene** 

*Key Laboratory for Green Chemical Technology of Ministry of Education,* 

Photoisomerization, an important aspect of photochemistry, is molecular behavior in which the structural change between isomers is caused by photoexcitation. Photoisomerization is already applied or has potential in many fields, such as the synthesis of compounds that can not be obtained by other methods, pigments in digital data storage and recording, solar energy harvesting, and nanoscale devices and materials with photo-modulable properties. Conformation transformation, especially the *trans*-*cis* photoisomerization of alkenes, see Scheme 1, is the most studied photoisomerization (Waldeck, 1991; Dou & Allen, 2003; Quenneville & Martínez, 2003; Minezawa & Gordon, 2011). Stilbene is a prototypical molecule that has been extensively investigated by both experimental and theoretical approaches. The primary mechanism of isomerization is through the excited singlet state starting from either the *cis* or the *trans* geometry. After photoexcitation, the molecule can overcome a small activation barrier and twist about its central C=C bond to form a twisted intermediate. This intermediate then decays with equal probability to either ground state *cis*-stilbene or ground state *trans*-stilbene. Similarly, the torsion around N=N bond also induces photoisomerization (Ciminelli et al., 2004; Mita et al., 1989), with azobenzene as the prototype. Moreover, compounds with photoisomerizable core have been designed for some special purposes. For example, highly branched dendrimers containing azobenzene core can be excited and converted to isomers by infrared irradiation, which represents a strategy for harvesting low-energy photons via chemical transformation (Jiang & Aida,

Geometric isomerization is another important type of photoisomerization that involves bond cleavage and creation in alkenes, see Scheme 2. One typical transformation is intramolecular cycloaddition such as [2+2] and [2+3] cycloadditions (Xu et al., 2009; Filley et al., 2001; Lu et al., 2011; Somekawa et al., 2009), which is very attractive in synthetic applications. In addition, the cycloaddition may produce strained and energy-rich products,

which has received attention as a way to store solar energy.

**1. Introduction** 

1997).

**to Quadricyclane Using** 

**Ti-Containing Photocatalysts** 

*School of Chemical Engineering and Technology,* 

*Tianjin University* 

*P.R. China* 

Ji-Jun Zou, Lun Pan, Xiangwen Zhang and Li Wang

Wu T., Li Y., Chu M., 2003, *Handbook of Photochemistry and Photobiology Vol. I, Inorganic Photochemistry*, pp. 249-282, American Science Publisher, ISBN 1-58883-004-7, **3** 

## **Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts**

Ji-Jun Zou, Lun Pan, Xiangwen Zhang and Li Wang *Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University P.R. China* 

### **1. Introduction**

40 Molecular Photochemistry – Various Aspects

Wu T., Li Y., Chu M., 2003, *Handbook of Photochemistry and Photobiology Vol. I, Inorganic* 

Photoisomerization, an important aspect of photochemistry, is molecular behavior in which the structural change between isomers is caused by photoexcitation. Photoisomerization is already applied or has potential in many fields, such as the synthesis of compounds that can not be obtained by other methods, pigments in digital data storage and recording, solar energy harvesting, and nanoscale devices and materials with photo-modulable properties.

Conformation transformation, especially the *trans*-*cis* photoisomerization of alkenes, see Scheme 1, is the most studied photoisomerization (Waldeck, 1991; Dou & Allen, 2003; Quenneville & Martínez, 2003; Minezawa & Gordon, 2011). Stilbene is a prototypical molecule that has been extensively investigated by both experimental and theoretical approaches. The primary mechanism of isomerization is through the excited singlet state starting from either the *cis* or the *trans* geometry. After photoexcitation, the molecule can overcome a small activation barrier and twist about its central C=C bond to form a twisted intermediate. This intermediate then decays with equal probability to either ground state *cis*-stilbene or ground state *trans*-stilbene. Similarly, the torsion around N=N bond also induces photoisomerization (Ciminelli et al., 2004; Mita et al., 1989), with azobenzene as the prototype. Moreover, compounds with photoisomerizable core have been designed for some special purposes. For example, highly branched dendrimers containing azobenzene core can be excited and converted to isomers by infrared irradiation, which represents a strategy for harvesting low-energy photons via chemical transformation (Jiang & Aida, 1997).

Geometric isomerization is another important type of photoisomerization that involves bond cleavage and creation in alkenes, see Scheme 2. One typical transformation is intramolecular cycloaddition such as [2+2] and [2+3] cycloadditions (Xu et al., 2009; Filley et al., 2001; Lu et al., 2011; Somekawa et al., 2009), which is very attractive in synthetic applications. In addition, the cycloaddition may produce strained and energy-rich products, which has received attention as a way to store solar energy.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 43

Generally, photoisomerization is sensitized by homogenous organics and/or metal complexes. However, solid semiconductors and even zeolites have been found to effective for these photo-induced processes. For example, CdS has been extensively studied for the *trans*-*cis* transformation of alkenes (Gao et al., 1998; Yanagida et al., 1986; Al-Ekabi & Mayo, 1985). Unfortunately, the instability of CdS under irradiation is a big problem for

Photoisomerization of norbornadiene (NBD) to quadricyclane (QC) is typical intramolecular [2+2] cycloaddition. It continues to be an interesting field as potential way for storage and conversion for solar energy (Hammond et al., 1964; Bren' et al., 1991; Dubonosov et al., 2002). The photoisomerization of NBD results in metastable structure that contains highly strained cyclobutane and two cyclopropane fragments. When one mole of NBD is transformed to QC, 89 kJ of solar energy could be stored in form of strain energy. Under some catalytic conditions, the inverse QC→NBD transformation occurs easily, accompanied with considerable thermal effect (ΔH=-89 kJ/mol). This represents an idea cycle for energy

Scheme 3. Solar energy harvesting cycle based on photoisomerization of norbornadiene.

**2. Photosensitized isomerization of norbornadiene** 

conversion and storage, see Scheme 3.

application.

Scheme 1. Examples of conformation photoisomerization of alkenes along with the prototype surface diagram of stilbene isomerization.

Scheme 2. Examples of geometric photoisomerization of alkenes

Generally, photoisomerization is sensitized by homogenous organics and/or metal complexes. However, solid semiconductors and even zeolites have been found to effective for these photo-induced processes. For example, CdS has been extensively studied for the *trans*-*cis* transformation of alkenes (Gao et al., 1998; Yanagida et al., 1986; Al-Ekabi & Mayo, 1985). Unfortunately, the instability of CdS under irradiation is a big problem for application.

### **2. Photosensitized isomerization of norbornadiene**

42 Molecular Photochemistry – Various Aspects

Scheme 1. Examples of conformation photoisomerization of alkenes along with the

prototype surface diagram of stilbene isomerization.

Scheme 2. Examples of geometric photoisomerization of alkenes

Photoisomerization of norbornadiene (NBD) to quadricyclane (QC) is typical intramolecular [2+2] cycloaddition. It continues to be an interesting field as potential way for storage and conversion for solar energy (Hammond et al., 1964; Bren' et al., 1991; Dubonosov et al., 2002). The photoisomerization of NBD results in metastable structure that contains highly strained cyclobutane and two cyclopropane fragments. When one mole of NBD is transformed to QC, 89 kJ of solar energy could be stored in form of strain energy. Under some catalytic conditions, the inverse QC→NBD transformation occurs easily, accompanied with considerable thermal effect (ΔH=-89 kJ/mol). This represents an idea cycle for energy conversion and storage, see Scheme 3.

Scheme 3. Solar energy harvesting cycle based on photoisomerization of norbornadiene.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 45

Among the photocatalysts studied, TiO2 is the most widely used material owing to its lowcost, non-toxicity, chemical and biological inertness, and photostability. Previous literatures already hint that TiO2 can facilitate the photoisomerization of NBD. Although the activity of TiO2 is relatively low due to the low optical absorbance and high charge–hole recombination rate, many methods such as doping with metal and nonmetal atoms and preparation of

Recently, we focused on the photocatalytic isomerization of NBD using Ti-containing materials including metal-doped TiO2 (Pan et al., 2010; Zou et al., 2008a), Ti-containing MCM-41 molecule sieves (Zou et al., 2008b) and metal-incorporated Ti-MCM-41 (Zou et al., 2010). These photocatalysts do show improved activity compared with pure TiO2, suggesting that the photocatalysts used in environmental photocatalysis can be applied in the photoisomerization. In the following sections, a mini review of our work will be given,

Three kinds of photocatalysts, including metal doped TiO2 (M-TiO2), Ti-substituted (Ti-MCM-41) and Ti-grafted MCM-41(TiO2-MCM-41), and metal incorporated Ti-MCM-41 (M-Ti-MCM-41) were studied. M-TiO2 materials were synthesized using sol-gel method with tetrabutyl titanate, VO(SO4), Fe(SO4)3, Cu(NO3)2, Cr(NO3)3, Ce(NO3)3 and ZnSO4 as the metal resources (Pan et al., 2010; Zou et al., 2008a). Ti-MCM-41 and M-Ti-MCM-41 materials were synthesized via hydrothermal method using cetyltrimethyl ammonium bromide and tetrathyorthosilicate as the structure director and Si resource, respectively (Zou et al., 2008b, 2010), and TiO2-MCM-41 materials were prepared through chemical grafting (Zou et al., 2008b). All the prepared materials were calcined at 500°C for 3 or 5 hours. The abbreviation of materials was suffixed with a symbol *x* in parentheses to describe the original molar

The photoisomerization reaction was conducted under UV irradiation in closed quartz reactor with magnetic stirring (Pan et al., 2010; Zou et al., 2008a, 2008b, 2010). For M-TiO2(M=V, Fe, Cu, Ce and Cr), a quartz chamber was irradiated vertically by a 300 W high-pressure xenon lamp located on the upper position. The wavelength was limited in the range of 220-420 nm by an optical filter and dimethyl sulfoxide was used as the solvent. For M-TiO2(M=Zn) and Ti-contaning MCM-41 materials, a cylindrical quartz vessel was irradiated by a 400 W high pressure mercury lamp positioned inside the vessel. In this case the wavelength was not controlled and p-xylene was used as the solvent. The composition of the resulted mixture was determined by a gas chromatograph equipped with BP-1 capillary column and flame ionization detector. The rate constant *k* for each photocatalyst was calculated via kinetics fitting, assuming that the reaction obeys the first-order law. Since the reaction conditions for different type of photocatalysts are a little different, TiO2 was used as the baseline to compare the photocatalytic activity of all materials. Therefore, the reaction constant *k* of one material was divided by that of TiO2 (*k0*) under identical reaction conditions, and the obtained relative reaction rate constant,

**3. Photoisomerization of NBD over Ti-containing photocatalysts** 

highly dispersed Ti-O species have been established to overcome this problem.

with the aim to show a new and promising way for photoisomerization.

**3.1 Synthesis of materials and evaluation of activity**

Ti/M or Si/M ratio in starting synthetic mixtures.

i.e. *k/k0*, was used in this chapter.

Recently, QC has been identified as a very promising high-energy compound as replacement for, or additive to, current hydrocarbon-based rocket propellants, because the extraordinary high strain energy offers a very high specific impulse (Kokan et al., 2009; Striebich & Lawrence, 2003). It is reported that QC-based fuels provide more propulsion than most of the hydrocarbon fuels like rocket propellant RP-1. QC is also designed for satellite propulsion system to replace highly toxic fuels like hydrazine and dinitrogen tetroxide. Moreover, QC is thermally and chemically stable, which means that it can be easily stored and transported like other hydrocarbon fuels.

The quantum yield of pure NBD photoisomerization is extremely low because the absorption edge of NBD is less than 300nm. Many efforts have been done to drive this photoisomerization using longer light and improve the quantum yield, which can be categorized into three directions: use of sensitizer, modification of NBD molecule and use of NBD-containing compounds. Dubonosov *et al* already presented two comprehensive reviews on the photoisomerization of NBD and its derivatives in 1991 and 2001 (Bren' et al., 1991; Dubonosov et al., 2002). This chapter focuses on the synthesis of QC from NBD, so only a brief summary is given to the direct photoisomerization of NBD, i.e. the first direction. The photosensitized isomerization of NBD occurs via triplet, so many carbonyl compounds like acetophenone, benzophenone and Michlers' ketone were used as triplet sensitizers. Actually, a recent patent claimed a solution phase photoisomerization process of NBD based on substituted Michlers' ketone (Cahill & Steppel, 2004). However, since the energy of the triplet state of NBD (3NBD) is very high (~257 kJ/mol), only small amount of sensitizers are qualified. Then, metal complexes and derivatives of carbonyl compounds were studied. In this case, the isomerization proceeds through the formation of sensitizer-NBD complexes in electron-excited states, with or without the formation of 3NBD.

However, the photosensitized reaction suffers from many drawbacks. First, homogenous reaction brings some difficulties in product purification and sensitizer recycling. Second, sensitizer tends to decompose under UV irradiation and induces some side-reactions like polymerization of NBD. In fact, in the past decade, work on the direct photoisomerization of NBD is very scare, and only some NBD derivatives were synthesized to prepare photoresponsive materials (Chen et al., 2007; Vlaar et al., 2001).

Heterogeneous semiconductors are extensively used in photocatalytic processes such as degradation of pollutants, hydrogen generation, and solar cell. They are also attractive for photoisomerization when considering the easy purification of product and reuse of catalyst. In fact, zeolites and semiconductors were already found to be active for the photoisomerization of NBD. In a brief communication, Lahiry and Haldar firstly reported that NBD can be isomerized over semiconductors like ZnO, ZnS and CdS (Lahiry & Haldar, 1986). Then Gandi et al. reported that Y-zeolites exchanged with K+, Cs+ and Tl+ ions can sensitize the intramolecular addition of some dienes like NBD and afford the corresponding triplet products through heavy atom effect (Ghandi, 2006). In this case the reactant is preadsorbed in the micropores. Similarly, Gu and Liu compared La-, Cs-, Zn- and K-exchanged Y zeolites for the photoisomerization of NBD in liquid phase, and found LaY shows relatively high activity (Gu & Liu, 2008). They postulated that the heavy atom effect and Brönsted acid account for the result.

### **3. Photoisomerization of NBD over Ti-containing photocatalysts**

Among the photocatalysts studied, TiO2 is the most widely used material owing to its lowcost, non-toxicity, chemical and biological inertness, and photostability. Previous literatures already hint that TiO2 can facilitate the photoisomerization of NBD. Although the activity of TiO2 is relatively low due to the low optical absorbance and high charge–hole recombination rate, many methods such as doping with metal and nonmetal atoms and preparation of highly dispersed Ti-O species have been established to overcome this problem.

Recently, we focused on the photocatalytic isomerization of NBD using Ti-containing materials including metal-doped TiO2 (Pan et al., 2010; Zou et al., 2008a), Ti-containing MCM-41 molecule sieves (Zou et al., 2008b) and metal-incorporated Ti-MCM-41 (Zou et al., 2010). These photocatalysts do show improved activity compared with pure TiO2, suggesting that the photocatalysts used in environmental photocatalysis can be applied in the photoisomerization. In the following sections, a mini review of our work will be given, with the aim to show a new and promising way for photoisomerization.

### **3.1 Synthesis of materials and evaluation of activity**

44 Molecular Photochemistry – Various Aspects

Recently, QC has been identified as a very promising high-energy compound as replacement for, or additive to, current hydrocarbon-based rocket propellants, because the extraordinary high strain energy offers a very high specific impulse (Kokan et al., 2009; Striebich & Lawrence, 2003). It is reported that QC-based fuels provide more propulsion than most of the hydrocarbon fuels like rocket propellant RP-1. QC is also designed for satellite propulsion system to replace highly toxic fuels like hydrazine and dinitrogen tetroxide. Moreover, QC is thermally and chemically stable, which means that it can be

The quantum yield of pure NBD photoisomerization is extremely low because the absorption edge of NBD is less than 300nm. Many efforts have been done to drive this photoisomerization using longer light and improve the quantum yield, which can be categorized into three directions: use of sensitizer, modification of NBD molecule and use of NBD-containing compounds. Dubonosov *et al* already presented two comprehensive reviews on the photoisomerization of NBD and its derivatives in 1991 and 2001 (Bren' et al., 1991; Dubonosov et al., 2002). This chapter focuses on the synthesis of QC from NBD, so only a brief summary is given to the direct photoisomerization of NBD, i.e. the first direction. The photosensitized isomerization of NBD occurs via triplet, so many carbonyl compounds like acetophenone, benzophenone and Michlers' ketone were used as triplet sensitizers. Actually, a recent patent claimed a solution phase photoisomerization process of NBD based on substituted Michlers' ketone (Cahill & Steppel, 2004). However, since the energy of the triplet state of NBD (3NBD) is very high (~257 kJ/mol), only small amount of sensitizers are qualified. Then, metal complexes and derivatives of carbonyl compounds were studied. In this case, the isomerization proceeds through the formation of sensitizer-

NBD complexes in electron-excited states, with or without the formation of 3NBD.

However, the photosensitized reaction suffers from many drawbacks. First, homogenous reaction brings some difficulties in product purification and sensitizer recycling. Second, sensitizer tends to decompose under UV irradiation and induces some side-reactions like polymerization of NBD. In fact, in the past decade, work on the direct photoisomerization of NBD is very scare, and only some NBD derivatives were synthesized to prepare photo-

Heterogeneous semiconductors are extensively used in photocatalytic processes such as degradation of pollutants, hydrogen generation, and solar cell. They are also attractive for photoisomerization when considering the easy purification of product and reuse of catalyst. In fact, zeolites and semiconductors were already found to be active for the photoisomerization of NBD. In a brief communication, Lahiry and Haldar firstly reported that NBD can be isomerized over semiconductors like ZnO, ZnS and CdS (Lahiry & Haldar, 1986). Then Gandi et al. reported that Y-zeolites exchanged with K+, Cs+ and Tl+ ions can sensitize the intramolecular addition of some dienes like NBD and afford the corresponding triplet products through heavy atom effect (Ghandi, 2006). In this case the reactant is preadsorbed in the micropores. Similarly, Gu and Liu compared La-, Cs-, Zn- and K-exchanged Y zeolites for the photoisomerization of NBD in liquid phase, and found LaY shows relatively high activity (Gu & Liu, 2008). They postulated that the heavy atom effect and

easily stored and transported like other hydrocarbon fuels.

responsive materials (Chen et al., 2007; Vlaar et al., 2001).

Brönsted acid account for the result.

Three kinds of photocatalysts, including metal doped TiO2 (M-TiO2), Ti-substituted (Ti-MCM-41) and Ti-grafted MCM-41(TiO2-MCM-41), and metal incorporated Ti-MCM-41 (M-Ti-MCM-41) were studied. M-TiO2 materials were synthesized using sol-gel method with tetrabutyl titanate, VO(SO4), Fe(SO4)3, Cu(NO3)2, Cr(NO3)3, Ce(NO3)3 and ZnSO4 as the metal resources (Pan et al., 2010; Zou et al., 2008a). Ti-MCM-41 and M-Ti-MCM-41 materials were synthesized via hydrothermal method using cetyltrimethyl ammonium bromide and tetrathyorthosilicate as the structure director and Si resource, respectively (Zou et al., 2008b, 2010), and TiO2-MCM-41 materials were prepared through chemical grafting (Zou et al., 2008b). All the prepared materials were calcined at 500°C for 3 or 5 hours. The abbreviation of materials was suffixed with a symbol *x* in parentheses to describe the original molar Ti/M or Si/M ratio in starting synthetic mixtures.

The photoisomerization reaction was conducted under UV irradiation in closed quartz reactor with magnetic stirring (Pan et al., 2010; Zou et al., 2008a, 2008b, 2010). For M-TiO2(M=V, Fe, Cu, Ce and Cr), a quartz chamber was irradiated vertically by a 300 W high-pressure xenon lamp located on the upper position. The wavelength was limited in the range of 220-420 nm by an optical filter and dimethyl sulfoxide was used as the solvent. For M-TiO2(M=Zn) and Ti-contaning MCM-41 materials, a cylindrical quartz vessel was irradiated by a 400 W high pressure mercury lamp positioned inside the vessel. In this case the wavelength was not controlled and p-xylene was used as the solvent. The composition of the resulted mixture was determined by a gas chromatograph equipped with BP-1 capillary column and flame ionization detector. The rate constant *k* for each photocatalyst was calculated via kinetics fitting, assuming that the reaction obeys the first-order law. Since the reaction conditions for different type of photocatalysts are a little different, TiO2 was used as the baseline to compare the photocatalytic activity of all materials. Therefore, the reaction constant *k* of one material was divided by that of TiO2 (*k0*) under identical reaction conditions, and the obtained relative reaction rate constant, i.e. *k/k0*, was used in this chapter.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 47

458.3

464.0

c/s

V-TiO2 Fe-TiO2 Cu-TiO2 Cr-TiO2 Ce-TiO2

pp. 8526-8531. Copyright @ 2010 American Chemical Society.

TiO2

470 465 460 455

Binding energy (eV)

Fig. 1. Ti2p XPS spectra of metal-doped TiO2. Reprinted with permission from Pan, L.; Zou, J.-J; Zhang, X. & Wang, L. (2010), *Industrial* & *Engineering Chemistry Research,* Vol.49, No.18,

Fig. 2. TEM images of (a) pure TiO2, (b) Fe-TiO2(15), (c) V-TiO2(15) and (d) Zn-TiO2(100). (a) & (d) reprinted with permission from Zou, J.-J.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z.

### **3.2 Photoisomerization of NBD over metal-doped TiO2: Effect of metal dopants**

TiO2 is widely used in photocatalytic reactions due to its low cost and chemical stability, but suffers from the fast recombination of photoinduced electron-hole pairs. Doping with metal ions is regarded as an effective method to improve the efficiency of TiO2 (Yang et al., 2007; Adán et al., 2007). So metal (Cu, Cr, Ce, V, Fe, Zn)-doped TiO2 was studied firstly for the photoisomerization of NBD.

The structural parameters of prepared materials characterized using XRD, EDX, XPS and N2-adsorption are shown in Table 1. According to the bulk composition from EDX data and surface composition from XPS data, V, Fe and Ce are dispersed in the inner part of prepared materials whereas Cu, Cr and Zn ions are enriched on the particle surface. Specifically, only a small amount of Cu is introduced into the material. Generally, there are three possible dispersion modes for dopants, namely substitutional, interstitial and surface positions. The local structure of dopants ions can be deduced based on their ionic radii, that is, Fe and V ions with radii close to Ti ions in substitutional sites, large Ce ions in interstitial positions, whereas Cu ions with largest radii on the surface. The surface enrichment of relatively small Cr and Zn ions that have comparable radii with Ti ions is a little surprising because they could enter the lattice, but consistent with results reported by other researchers (Zhu et al., 2010; Jing et al., 2006). The reason may be that these ions are originally inside the lattice but diffuse to the surface through oxygen vacancies during the calcination process, or the hydrolysis rate of these ions is much slower than that of Ti ions.


Table 1. Structural characteristics of metal-doped TiO2 (Pan et al., 2010; Zou et al., 2008a).

When metal dopants are dispersed in the substitutional site, some Ti-O-M structures are expected to form, which will cause a shift in the binding energy of Ti species because the difference in Pauling electronegativity can induce electron transfer from Ti to M ions. As shown in Fig. 1, the XPS signal (binding energy) of Ti is shifted to higher values after doping with V and Fe, while for other doping the shift is not so obvious because the metals are not located in the substitutional sites with no, or only a few, M-O-Ti structures formed.

Doping can restrain the growth of particle to some degree no mater what the doping mode is, but the mechanism may be different. Fe and Zn-doping produces considerably small particles, see Table 1 and Fig. 2. For the substitutional doping like Fe- and V-doping, dopants in the lattice can destroy the crystal structure and restrain its growth. For the surface deposition or interstitial mode, like Ce- and Zn-doping, dopants may prevent the direct contact of TiO2 crystallites and retard them agglomerating into big particle.

TiO2 is widely used in photocatalytic reactions due to its low cost and chemical stability, but suffers from the fast recombination of photoinduced electron-hole pairs. Doping with metal ions is regarded as an effective method to improve the efficiency of TiO2 (Yang et al., 2007; Adán et al., 2007). So metal (Cu, Cr, Ce, V, Fe, Zn)-doped TiO2 was studied firstly for the

The structural parameters of prepared materials characterized using XRD, EDX, XPS and N2-adsorption are shown in Table 1. According to the bulk composition from EDX data and surface composition from XPS data, V, Fe and Ce are dispersed in the inner part of prepared materials whereas Cu, Cr and Zn ions are enriched on the particle surface. Specifically, only a small amount of Cu is introduced into the material. Generally, there are three possible dispersion modes for dopants, namely substitutional, interstitial and surface positions. The local structure of dopants ions can be deduced based on their ionic radii, that is, Fe and V ions with radii close to Ti ions in substitutional sites, large Ce ions in interstitial positions, whereas Cu ions with largest radii on the surface. The surface enrichment of relatively small Cr and Zn ions that have comparable radii with Ti ions is a little surprising because they could enter the lattice, but consistent with results reported by other researchers (Zhu et al., 2010; Jing et al., 2006). The reason may be that these ions are originally inside the lattice but diffuse to the surface through oxygen vacancies during the calcination process, or the

> *SBET* (m2·g-1)

Ti/M ratio EDX XPS

**3.2 Photoisomerization of NBD over metal-doped TiO2: Effect of metal dopants** 

hydrolysis rate of these ions is much slower than that of Ti ions.

Table 1. Structural characteristics of metal-doped TiO2

(nm)

TiO2 21.5 21.5 - - Cu-TiO2(15) 19.9 13.1 90.4 3.8 Cr-TiO2(15) 14.7 40.9 20.0 3.0 Ce-TiO2(15) 11.4 64.3 16. 9 19.8 V-TiO2(15) 9.9 102.7 19.0 15.6 Fe-TiO2(15) 7.0 120.6 18.5 19.8 Zn-TiO2(100) 8.1 84.9 - 7.1

When metal dopants are dispersed in the substitutional site, some Ti-O-M structures are expected to form, which will cause a shift in the binding energy of Ti species because the difference in Pauling electronegativity can induce electron transfer from Ti to M ions. As shown in Fig. 1, the XPS signal (binding energy) of Ti is shifted to higher values after doping with V and Fe, while for other doping the shift is not so obvious because the metals are not

Doping can restrain the growth of particle to some degree no mater what the doping mode is, but the mechanism may be different. Fe and Zn-doping produces considerably small particles, see Table 1 and Fig. 2. For the substitutional doping like Fe- and V-doping, dopants in the lattice can destroy the crystal structure and restrain its growth. For the surface deposition or interstitial mode, like Ce- and Zn-doping, dopants may prevent the

located in the substitutional sites with no, or only a few, M-O-Ti structures formed.

direct contact of TiO2 crystallites and retard them agglomerating into big particle.

Materials Grain size

(Pan et al., 2010; Zou et al., 2008a).

photoisomerization of NBD.

Fig. 1. Ti2p XPS spectra of metal-doped TiO2. Reprinted with permission from Pan, L.; Zou, J.-J; Zhang, X. & Wang, L. (2010), *Industrial* & *Engineering Chemistry Research,* Vol.49, No.18, pp. 8526-8531. Copyright @ 2010 American Chemical Society.

Fig. 2. TEM images of (a) pure TiO2, (b) Fe-TiO2(15), (c) V-TiO2(15) and (d) Zn-TiO2(100). (a) & (d) reprinted with permission from Zou, J.-J.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 49

42 3 3 42 2 4 *Ti O Fe O Ti Ti O Fe O Ti*

42 3 3 42 42 3 *Ti O Fe O Ti Ti O Fe O Ti*

(2) The trapped charges are transferred to sideward Ti-O species, resulting in separated

42 2 4 32 3 4 *Ti O Fe O Ti Ti O Fe O Ti*

42 42 3 4 32 3 *Ti O Fe O Ti Ti O Fe O Ti*

In this way, the charge induced in one Ti-O moiety is quickly transferred to another Ti-O moiety through the Fe-O-Ti structure, thus effectively separating the charge and retarding

Fig. 4. Relationship of activity for the photoisomerization of norbornadiene and the relative surface OH concentration of Zn-TiO2 (Zou et al., 2008a). OH, the content of surface OH;

charges:

the recombination.

OH0, the OH content of pure TiO2.

(2008), *Journal of Molecular Catalysis A:Chemical,* Vol.286, No.1-2, pp. 63-69. Copyright @ 2008 Elsevier. (b) & (c) Reprinted with permission from Pan, L.; Zou, J.-J; Zhang, X. & Wang, L. (2010), *Industrial* & *Engineering Chemistry Research,* Vol.49, No.18, pp. 8526-8531. Copyright @ 2010 American Chemical Society.

The relative photocatalytic activity of doped TiO2 (*k/k0*) is also shown in Fig. 3. Except Cu, doping metal ions show positive effect on the photoisomerization of NBD, among which Zn-TiO2 and Fe-TiO2 are specifically active. The photoisomerization reaction is a complex process, and the physicochemical properties of photocatalyst such as grain size, type of dopant ions as well as their local structure are very important. Small particle is of course desired because it provides large active surface. It has been reported that the surface doping of Zn ions produces many surface OH groups that greatly enhance the intensity of surface photovoltage spectrum and photoluminescence and improve the photoactivity (Jing et al., 2006). As shown in Fig. 4, the activity of NBD photoisomerization is also closely relative to the concentration of surface OH.

Fig. 3. Activity of metal-doped TiO2 for the photoisomerization of norbornadiene (Pan et al., 2010; Zou et al., 2008a).

However, the role of surface OH seems invalid for the materials with substitutional doping. As shown in Fig. 5, the activity of Fe- and V-doped TiO2 and their lattice oxygen concentration, not the surface OH, change in identical manner, strongly suggesting there is an inherent correlation between the photoisomerization and lattice oxygen. It is still not clear why two doping modes induce contrary result, probably because the reactant molecule is adsorbed on different site that will be discussed in section 4. As to the role of substitutional dopants, it has been reported that metal ions in substitutional sites can improve the photoinduced charge transfer and separation (Wang et al., 2009). It is believed that this process is very likely to occur through the M-O-Ti structure in which the metal dopants mainly serve as charge trapping and transferring center. Taking Fe-TiO2 as example, the role of Fe is shown as follows: (1) Fe ions temporarily trap photoinduced charges in the neighboring Ti-O moiety:

(2008), *Journal of Molecular Catalysis A:Chemical,* Vol.286, No.1-2, pp. 63-69. Copyright @ 2008 Elsevier. (b) & (c) Reprinted with permission from Pan, L.; Zou, J.-J; Zhang, X. & Wang, L. (2010), *Industrial* & *Engineering Chemistry Research,* Vol.49, No.18, pp. 8526-8531. Copyright @

The relative photocatalytic activity of doped TiO2 (*k/k0*) is also shown in Fig. 3. Except Cu, doping metal ions show positive effect on the photoisomerization of NBD, among which Zn-TiO2 and Fe-TiO2 are specifically active. The photoisomerization reaction is a complex process, and the physicochemical properties of photocatalyst such as grain size, type of dopant ions as well as their local structure are very important. Small particle is of course desired because it provides large active surface. It has been reported that the surface doping of Zn ions produces many surface OH groups that greatly enhance the intensity of surface photovoltage spectrum and photoluminescence and improve the photoactivity (Jing et al., 2006). As shown in Fig. 4, the activity of NBD photoisomerization is also closely relative to

Fig. 3. Activity of metal-doped TiO2 for the photoisomerization of norbornadiene (Pan et al.,

However, the role of surface OH seems invalid for the materials with substitutional doping. As shown in Fig. 5, the activity of Fe- and V-doped TiO2 and their lattice oxygen concentration, not the surface OH, change in identical manner, strongly suggesting there is an inherent correlation between the photoisomerization and lattice oxygen. It is still not clear why two doping modes induce contrary result, probably because the reactant molecule is adsorbed on different site that will be discussed in section 4. As to the role of substitutional dopants, it has been reported that metal ions in substitutional sites can improve the photoinduced charge transfer and separation (Wang et al., 2009). It is believed that this process is very likely to occur through the M-O-Ti structure in which the metal dopants mainly serve as charge trapping and transferring center. Taking Fe-TiO2 as example, the role of Fe is shown as follows: (1) Fe ions temporarily trap photoinduced

2010 American Chemical Society.

the concentration of surface OH.

2010; Zou et al., 2008a).

charges in the neighboring Ti-O moiety:

$$\begin{aligned} \text{Ti}^{4+}-\text{O}^{2-}-\text{Fe}^{3+}-\text{O}^{-}-\text{Ti}^{3+} & \rightarrow \text{Ti}^{4+}-\text{O}^{2-}-\text{Fe}^{2+}-\text{O}^{-}-\text{Ti}^{4+} \\\\ \text{Ti}^{4+}-\text{O}^{2-}-\text{Fe}^{3+}-\text{O}^{-}-\text{Ti}^{3+} & \rightarrow \text{Ti}^{4+}-\text{O}^{2-}-\text{Fe}^{4+}-\text{O}^{2-}-\text{Ti}^{3+} \end{aligned}$$

(2) The trapped charges are transferred to sideward Ti-O species, resulting in separated charges:

$$\begin{aligned} \text{Ti}^{4+}-\text{O}^{2-}-\text{Fe}^{2+}-\text{O}^{-}-\text{Ti}^{4+} & \rightarrow \text{Ti}^{3+}-\text{O}^{2-}-\text{Fe}^{3+}-\text{O}^{-}-\text{Ti}^{4+} \\\\ \text{Ti}^{4+}-\text{O}^{2-}-\text{Fe}^{4+}-\text{O}^{2-}-\text{Ti}^{3+} & \rightarrow \text{Ti}^{4+}-\text{O}^{-}-\text{Fe}^{3+}-\text{O}^{2-}-\text{Ti}^{3+} \end{aligned}$$

In this way, the charge induced in one Ti-O moiety is quickly transferred to another Ti-O moiety through the Fe-O-Ti structure, thus effectively separating the charge and retarding the recombination.

Fig. 4. Relationship of activity for the photoisomerization of norbornadiene and the relative surface OH concentration of Zn-TiO2 (Zou et al., 2008a). OH, the content of surface OH; OH0, the OH content of pure TiO2.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 51

Fig. 7. TEM images of (a) MCM-41, (b) TiO2-MCM-41 and (c) Ti-MCM-41(50). Reprinted with permission from Zou, J.-J.; Zhang, M.-Y.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z. (2008), *Catalysis Letters*, Vol.124, No.12, pp. 139-145, Copyright @ 2008 Springer Netherlands.

pores.

with bulk TiO2.

activity.

Grafting TiO2 in the pore of MCM-41 does not influence the ordered hexagonal structure of support as its XRD patterns in the low-angle region are identical to MCM-41, see Fig. 6. An additional peak corresponding to the (101) reflex of anatase TiO2 is observed at 25.5° but the intensity is extremely weak, so TiO2 crystallites are highly dispersed in the pore of MCM-41. Incorporating Ti ions in the MCM-41 framework slightly impairs the structural integrity of MCM-41 but the ordered structure is well retained, shown by the weakened but obvious diffractive peaks. Also, the cell unit of Ti-MCM-41 is enlarged because the Ti-O bond distance is longer than the Si-O bond distance. TEM images in Fig. 7 further confirm the XRD result. No TiO2 nanoparticles are observed for TiO2-MCM-41 and its pore structure is identical to MCM-41, but some linear tubular pores of Ti-MCM-41 collapse into irregular

The nature and coordination of Ti4+ ions was deduced according to the UV-vis diffuse reflectance spectra shown in Fig. 8. The absorption peak at 220 nm is ascribed to tetracoordinated Ti whereas the peak at ~270 nm represents species in higher coordination environments (penta- or hexa-coordinated species). For Ti-MCM-41, most of the Ti species are dispersed in the framework (Ti-O-Si) when Ti content is low, but polymerized Ti species (Ti-O-Ti) present in case of higher Ti content. TiO2-MCM-41 contains highly dispersed quantum-size TiO2 nanodomains, see the blue-shifted absorption compared

The overall activity for the photoisomerization of NBD is Ti-MCM-41(30) > Ti-MCM-41(50) > TiO2-MCM-41 > Ti-MCM-41(70) >TiO2, see Fig. 9a. Since the amount of Ti species is different in these materials, the activity based on TiO2 was also calculated to compare the inherent activity of different Ti species, with the order of Ti-MCM-41(50) ≈ Ti-MCM-41(70) > Ti-MCM-41(30) > TiO2-MCM-41 > TiO2, see Fig.9b. Considering the local structure of Ti, it can be seen that framework Ti species are most active in the photoisomerization of NBD, polymerized species follows and bulk TiO2 has the lowest

Fig. 5. Relationship of activity for the photoisomerization of norbornadiene and the relative lattice oxygen concentration of (a) Fe-TiO2 and (b) V-TiO2 (Pan et al., 2010).

### **3.3 Photoisomerization of NBD over Ti-containing MCM-41: Effect of Ti coordination**

MCM-41 has uniform hexagonal mesopores with large internal surface area, exhibiting great potential as the supporting materials of TiO2. It has been reported that incorporating Ti ions into framework or loading them on the wall of MCM-41 gives unique photocatalytic activity (Hu et al., 2003, 2006). So both Ti-incorporated and Ti-grafted MCM-41 materials were prepared for the photoisomerization of NBD.

Fig. 6. XRD patterns of Ti-MCM-41 and TiO2-MCM-41. Reprinted with permission from Zou, J.-J.; Zhang, M.-Y.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z. (2008), *Catalysis Letters*, Vol.124, No.12, pp. 139-145, Copyright @ 2008 Springer Netherlands.

Fig. 5. Relationship of activity for the photoisomerization of norbornadiene and the relative

**3.3 Photoisomerization of NBD over Ti-containing MCM-41: Effect of Ti coordination**  MCM-41 has uniform hexagonal mesopores with large internal surface area, exhibiting great potential as the supporting materials of TiO2. It has been reported that incorporating Ti ions into framework or loading them on the wall of MCM-41 gives unique photocatalytic activity (Hu et al., 2003, 2006). So both Ti-incorporated and Ti-grafted MCM-41 materials were

2 4 6 810

2 Theta (deg)

Fig. 6. XRD patterns of Ti-MCM-41 and TiO2-MCM-41. Reprinted with permission from Zou, J.-J.; Zhang, M.-Y.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z. (2008), *Catalysis Letters*,

Vol.124, No.12, pp. 139-145, Copyright @ 2008 Springer Netherlands.

10 20 30 40 50 60 70 80

MCM-41 (200) (110)

TiO2

Ti-MCM-41(30)

Ti-MCM-41(50) Ti-MCM-41(70)


lattice oxygen concentration of (a) Fe-TiO2 and (b) V-TiO2 (Pan et al., 2010).

prepared for the photoisomerization of NBD.

(100)

Intensity (a.u.)

Fig. 7. TEM images of (a) MCM-41, (b) TiO2-MCM-41 and (c) Ti-MCM-41(50). Reprinted with permission from Zou, J.-J.; Zhang, M.-Y.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z. (2008), *Catalysis Letters*, Vol.124, No.12, pp. 139-145, Copyright @ 2008 Springer Netherlands.

Grafting TiO2 in the pore of MCM-41 does not influence the ordered hexagonal structure of support as its XRD patterns in the low-angle region are identical to MCM-41, see Fig. 6. An additional peak corresponding to the (101) reflex of anatase TiO2 is observed at 25.5° but the intensity is extremely weak, so TiO2 crystallites are highly dispersed in the pore of MCM-41. Incorporating Ti ions in the MCM-41 framework slightly impairs the structural integrity of MCM-41 but the ordered structure is well retained, shown by the weakened but obvious diffractive peaks. Also, the cell unit of Ti-MCM-41 is enlarged because the Ti-O bond distance is longer than the Si-O bond distance. TEM images in Fig. 7 further confirm the XRD result. No TiO2 nanoparticles are observed for TiO2-MCM-41 and its pore structure is identical to MCM-41, but some linear tubular pores of Ti-MCM-41 collapse into irregular pores.

The nature and coordination of Ti4+ ions was deduced according to the UV-vis diffuse reflectance spectra shown in Fig. 8. The absorption peak at 220 nm is ascribed to tetracoordinated Ti whereas the peak at ~270 nm represents species in higher coordination environments (penta- or hexa-coordinated species). For Ti-MCM-41, most of the Ti species are dispersed in the framework (Ti-O-Si) when Ti content is low, but polymerized Ti species (Ti-O-Ti) present in case of higher Ti content. TiO2-MCM-41 contains highly dispersed quantum-size TiO2 nanodomains, see the blue-shifted absorption compared with bulk TiO2.

The overall activity for the photoisomerization of NBD is Ti-MCM-41(30) > Ti-MCM-41(50) > TiO2-MCM-41 > Ti-MCM-41(70) >TiO2, see Fig. 9a. Since the amount of Ti species is different in these materials, the activity based on TiO2 was also calculated to compare the inherent activity of different Ti species, with the order of Ti-MCM-41(50) ≈ Ti-MCM-41(70) > Ti-MCM-41(30) > TiO2-MCM-41 > TiO2, see Fig.9b. Considering the local structure of Ti, it can be seen that framework Ti species are most active in the photoisomerization of NBD, polymerized species follows and bulk TiO2 has the lowest activity.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 53

**3.4 Photoisomerization of NBD over M-Ti-MCM-41: Combination of metal doping and** 

Transition-metal-incorporated MCM-41 generally shows high photocatalytic activity due to the high dispersion of photoactive sites and effective separation of electrons and holes (Hu et al., 2007; Yamashita et al., 2001; Matsuoka & Ampo, 2003; Davydov et al., 2001). Since Ti-MCM-41 produces highly active photocatalysts for the photoisomerization of NBD, it is expected that introducing second transition metal ion into Ti-MCM-41 may further enhance the activity. So series of transition-metal-incorporated (V, Fe and Cr) Ti-MCM-41 were

According to the UV-vis spectra in Fig. 10, V and Fe ions are well dispersed in the materials whereas the dispersion of Cr ions is very poor. For V-Ti-MCM-41(150), V ions are highly dispersed in MCM-41 framework at atomic level with tetrahedral coordination, with some species in 6-fold (absorption around 370 nm) and higher coordination or even polymerized environments (absorption in >400 nm region) formed with the increase of V content. This tendency is also observed for Fe-Ti-MCM-41. However, for Cr-Ti-MCM-41, the absorption at

Absorbance (a.u.)

200 300 400 500 600 700 800

a b c d e

Wavelength (nm)

Fig. 10. UV-Vis diffuse reflectance spectra of M(V, Fe and Cr)-Ti-MCM-41 (a: Si/M=10, b: Si/M=33, c: Si/M=75, d: Si/M=100, e: Si/M=150, f: Ti-MCM-41). Reprinted with permission

from Zou, J.-J.; Liu, Y.; Pan, L.; Wang, L. & Zhang, X. (2010), *Applied Catalysis B:* 

*Environmental*, Vol.95, No.3-4, pp. 439-445. Copyright @ 2010 Elsevier.

200 300 400 500 600 700 800

Fe-Ti-MCM-41

Wavelength (nm)

a

b c d e

Cr-Ti-MCM-41

synthesized for the photoisomerization of NBD, with Si/Ti ratio of 30.

**V-Ti-MCM-41**

200 300 400 500 600 700 800

Wavelength (nm)

Absorbance (a.u.)

**framework Ti species** 

Absorbance (a.u.)

c

a b

e d

f

Fig. 8. UV-Vis diffuse reflectance spectra of Ti-MCM-41 and TiO2-MCM-41. Reprinted with permission from Zou, J.-J.; Zhang, M.-Y.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z. (2008), *Catalysis Letters*, Vol.124, No.12, pp. 139-145, Copyright @ 2008 Springer Netherlands.

Fig. 9. Activity of Ti-MCM-41 and TiO2-MCM-41 for the photoisomerization of norbornadiene (Zou et al., 2008b).

Ti-MCM-41(70)

Ti-MCM-41(30)

TiO2

TiO2

Absorbance (a.u.)

norbornadiene (Zou et al., 2008b).


200 300 400 500 600 700 800

Fig. 8. UV-Vis diffuse reflectance spectra of Ti-MCM-41 and TiO2-MCM-41. Reprinted with permission from Zou, J.-J.; Zhang, M.-Y.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z. (2008), *Catalysis Letters*, Vol.124, No.12, pp. 139-145, Copyright @ 2008 Springer Netherlands.

Fig. 9. Activity of Ti-MCM-41 and TiO2-MCM-41 for the photoisomerization of

Wavelength (nm)

Ti-MCM-41(50)

### **3.4 Photoisomerization of NBD over M-Ti-MCM-41: Combination of metal doping and framework Ti species**

Transition-metal-incorporated MCM-41 generally shows high photocatalytic activity due to the high dispersion of photoactive sites and effective separation of electrons and holes (Hu et al., 2007; Yamashita et al., 2001; Matsuoka & Ampo, 2003; Davydov et al., 2001). Since Ti-MCM-41 produces highly active photocatalysts for the photoisomerization of NBD, it is expected that introducing second transition metal ion into Ti-MCM-41 may further enhance the activity. So series of transition-metal-incorporated (V, Fe and Cr) Ti-MCM-41 were synthesized for the photoisomerization of NBD, with Si/Ti ratio of 30.

According to the UV-vis spectra in Fig. 10, V and Fe ions are well dispersed in the materials whereas the dispersion of Cr ions is very poor. For V-Ti-MCM-41(150), V ions are highly dispersed in MCM-41 framework at atomic level with tetrahedral coordination, with some species in 6-fold (absorption around 370 nm) and higher coordination or even polymerized environments (absorption in >400 nm region) formed with the increase of V content. This tendency is also observed for Fe-Ti-MCM-41. However, for Cr-Ti-MCM-41, the absorption at

Fig. 10. UV-Vis diffuse reflectance spectra of M(V, Fe and Cr)-Ti-MCM-41 (a: Si/M=10, b: Si/M=33, c: Si/M=75, d: Si/M=100, e: Si/M=150, f: Ti-MCM-41). Reprinted with permission from Zou, J.-J.; Liu, Y.; Pan, L.; Wang, L. & Zhang, X. (2010), *Applied Catalysis B: Environmental*, Vol.95, No.3-4, pp. 439-445. Copyright @ 2010 Elsevier.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 55

**c**

Fig. 12. XRD patterns of (a) V-Ti-MCM-41 and (b) Cr-Ti-MCM-41, and (c) TEM image of Cr-Ti-MCM-41(10). Reprinted with permission from Zou, J.-J.; Liu, Y.; Pan, L.; Wang, L. & Zhang, X. (2010), *Applied Catalysis B: Environmental*, Vol.95, No.3-4, pp. 439-445. Copyright @

All the materials exhibit higher activity than Ti-MCM-41, see Fig. 13, indicating that introducing second metal is beneficial to the photoisomerization. Among the three metals, V-incorporation is most effective, Fe-incorporation follows, and Cr- incorporation is the least. The photocatalytic activity has nothing to do with the concentration of second transition metal ions, and the improvement in activity should be related to their state of dispersion and local structure. It has been reported that tetrahedrally coordinated M-oxide moieties dispersed in mesoporous materials can be easily excited under UV and/or visiblelight irradiation to form corresponding charge-transfer excited states (Yamashita et al., 2001;

2 \* ( 1) [ ][ ] *MO M O <sup>n</sup> hv <sup>n</sup>* (M=V, Cr, Fe)

Then M species can donate an electron to surrounding Ti-O moieties and O- can scavenge an electron from surrounding Ti-O moieties, inducing charge separation in Ti-O species (Davydov et al., 2001). Therefore, two different excitation mechanisms exist in M-Ti-MCM-41. One is direct excitation of Ti-O moieties by UV irradiation, and the other is indirect excitation via charge transition from ( 1) \* [ ] *M O <sup>n</sup>* species. The second process should be responsible for the high photocatalytic activity of M-Ti-MCM-41 because of its high

V-Ti-MCM-41(150) shows specifically high activity because majority of V ions are highly dispersed in 4-fold coordination, which brings up highly efficient excitation of Ti-O species. In addition, the well retained ordered structure and high surface area can enhance the adsorption of NBD molecules and provide more active sites. With the increase of V content, the activity is decreased because some 4-fold ions are transformed into undesirable highlycoordinated species and the damaged structure and small surface area may suppress the adsorption of reactants. The low activity of Cr-Ti-MCM-41 is due to poorly dispersed

2010 Elsevier.

Matsuoka & Anpo, 2003):

efficiency in charge formation and separation.

chromium ions and dramatically destroyed textural structure.

470 nm and 610 nm ascribed to poly- and bulk Cr2O3 is very intensive. The local structure of Cr ions are also testified by the IR spectra in Fig. 11. All Cr-Ti-MCM-41 samples show a shoulder band at 880-900 cm-1 assigned to Cr6+ species, according to the literature (Awate et al., 2005; Zhu et al., 1999). Specifically, Cr-Ti-MCM-41(10) has two bands at 630 and 570 cm-1 belonging to extra-framework Cr2O3 oxides.

Fig. 11. IR spectra of Cr-Ti-MCM-41 (a: Si/M=10, b: Si/M=33, c: Si/M=75, d: Si/M=100, e: Si/M=150, f: Ti-MCM-41). Reprinted with permission from Zou, J.-J.; Liu, Y.; Pan, L.; Wang, L. & Zhang, X. (2010), *Applied Catalysis B: Environmental*, Vol.95, No.3-4, pp. 439-445. Copyright @ 2010 Elsevier.

The well dispersed V and Fe species show no obvious influence on the ordered structure of prepared materials, but the polymerized Cr species obviously impose negative effect on the structure, see Fig. 12. An extreme is observed for Cr-Ti-MCM-41(10), in which the characteristic diffractive peaks of ordered structure completely disappear, and a peak of bulk Cr2O3 appears. In TEM image, this material no longer possess hexagonal mesoporous structure, but agglomerate of many crystallites.

470 nm and 610 nm ascribed to poly- and bulk Cr2O3 is very intensive. The local structure of Cr ions are also testified by the IR spectra in Fig. 11. All Cr-Ti-MCM-41 samples show a shoulder band at 880-900 cm-1 assigned to Cr6+ species, according to the literature (Awate et al., 2005; Zhu et al., 1999). Specifically, Cr-Ti-MCM-41(10) has two bands at 630 and 570 cm-1

<sup>570</sup> <sup>898</sup> <sup>630</sup>

1500 1200 900 600

Wavemunbers (cm-1

Fig. 11. IR spectra of Cr-Ti-MCM-41 (a: Si/M=10, b: Si/M=33, c: Si/M=75, d: Si/M=100, e: Si/M=150, f: Ti-MCM-41). Reprinted with permission from Zou, J.-J.; Liu, Y.; Pan, L.; Wang, L. & Zhang, X. (2010), *Applied Catalysis B: Environmental*, Vol.95, No.3-4, pp. 439-445.

The well dispersed V and Fe species show no obvious influence on the ordered structure of prepared materials, but the polymerized Cr species obviously impose negative effect on the structure, see Fig. 12. An extreme is observed for Cr-Ti-MCM-41(10), in which the characteristic diffractive peaks of ordered structure completely disappear, and a peak of bulk Cr2O3 appears. In TEM image, this material no longer possess hexagonal mesoporous

Intensity (a.u.)

)

2 4 6 810

2 Theta (deg)

**20 40 60 80 2 Theta / degree**

Si/Cr=75

Si/Cr=100 Si/Cr=150

Si/Cr=10 Si/Cr=33

b

belonging to extra-framework Cr2O3 oxides.

Transmittance (%, a.u.)

Copyright @ 2010 Elsevier.

(100)

Intensity (a.u.)

structure, but agglomerate of many crystallites.

2 4 6 810

(200) Si/V=10 (110)

2 Theta (deg)

MCM-41 Ti-MCM-41 Si/V=150 Si/V=100 Si/V=75 Si/V=33

a

c b a

d

e

Fig. 12. XRD patterns of (a) V-Ti-MCM-41 and (b) Cr-Ti-MCM-41, and (c) TEM image of Cr-Ti-MCM-41(10). Reprinted with permission from Zou, J.-J.; Liu, Y.; Pan, L.; Wang, L. & Zhang, X. (2010), *Applied Catalysis B: Environmental*, Vol.95, No.3-4, pp. 439-445. Copyright @ 2010 Elsevier.

All the materials exhibit higher activity than Ti-MCM-41, see Fig. 13, indicating that introducing second metal is beneficial to the photoisomerization. Among the three metals, V-incorporation is most effective, Fe-incorporation follows, and Cr- incorporation is the least. The photocatalytic activity has nothing to do with the concentration of second transition metal ions, and the improvement in activity should be related to their state of dispersion and local structure. It has been reported that tetrahedrally coordinated M-oxide moieties dispersed in mesoporous materials can be easily excited under UV and/or visiblelight irradiation to form corresponding charge-transfer excited states (Yamashita et al., 2001; Matsuoka & Anpo, 2003):

$$\left[M^{n+} - O^{2-}\right] \xrightarrow{hv} \left[M^{(n-1)+} - O^{-}\right]^{\*} \text{ (M=V, Cr, Fe)}$$

Then M species can donate an electron to surrounding Ti-O moieties and O- can scavenge an electron from surrounding Ti-O moieties, inducing charge separation in Ti-O species (Davydov et al., 2001). Therefore, two different excitation mechanisms exist in M-Ti-MCM-41. One is direct excitation of Ti-O moieties by UV irradiation, and the other is indirect excitation via charge transition from ( 1) \* [ ] *M O <sup>n</sup>* species. The second process should be responsible for the high photocatalytic activity of M-Ti-MCM-41 because of its high efficiency in charge formation and separation.

V-Ti-MCM-41(150) shows specifically high activity because majority of V ions are highly dispersed in 4-fold coordination, which brings up highly efficient excitation of Ti-O species. In addition, the well retained ordered structure and high surface area can enhance the adsorption of NBD molecules and provide more active sites. With the increase of V content, the activity is decreased because some 4-fold ions are transformed into undesirable highlycoordinated species and the damaged structure and small surface area may suppress the adsorption of reactants. The low activity of Cr-Ti-MCM-41 is due to poorly dispersed chromium ions and dramatically destroyed textural structure.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 57

Scheme 4. Triplet sensitized photoisomerization of norbornadiene.

framework Ti species is to retard the undesired recombination.

be*TiO OH NBD* <sup>2</sup>

However, with the presence of Ti-containing photocatalyst, this mechanism is not suitable because the vertical triplet energy transfer from Ti-oxide species to NBD is very difficult. NBD molecules have to be firstly positively charged by photoinduced holes, but the free radical ion isomerization mechanism is ruled out because the energy of free NBD·+ is significantly lower than free QC·+. In fact, the transformation of QC to NBD is through the QC·+→NBD·+ free radical route (Ikezawa & Kutal, 1987). So the photoisomerization of NBD over semiconductors should be an adsorption-photoexcited process, which is very likely through the exciplex (charge-transfer intermediate), see Scheme 5. First, NBD molecule is adsorbed on the photoexcited Ti-oxides. Then surface-trapped hole is transferred to adsorbed molecule and a complex with NBD positively charged is formed. Subsequently the complex is transformed to structure with QC skeleton. Finally, QC is released into the liquid phase and the charge is recombined through reverse electron transfer. In this case the adsorption and charge transfer are two critical steps. The adsorptive site on different Ticontaining materials may be different. For Zn-TiO2, surface OH very likely serves as the site because it plays an important role in the reaction, and the excited complex may

. For Fe-TiO2 and V-TiO2, however, the lattice oxygen may work as

the adsorbing site with the complex of 4 2 42 *Ti O Ti O NBD* [ ] . Any charge recombination process can deactivate the complex, so the function of dopants and

Fig. 13. Activity of M(V, Fe and Cr)-Ti-MCM-41 for the photoisomerization of norbornadiene (Zou et al., 2010).

Since some photocatalysts show absorption in visible-light region, one may wonder whether they can catalyze the isomerization under visible-light irradiation. However, there is no any observable conversion when the experiment was conducted using visible irradiation (>420 nm). This is different from the case of H2 generation and organic degradation, where Cr-Ti-MCM-41 is reported to exhibit visible-light activity (Yamashita et al., 2001; Davydov et al., 2001; Chen & Mao, 2007). These results suggest that the reaction mechanism between the photoisomerization and other photocatalytic reactions may be very different.

### **4. Mechanism for NBD photoisomerization**

Photoisomerization of NBD in the presence of sensitizers generally proceeds via triplet state mechanism (Bren' et al., 1991; Dubonosov et al., 2002), see Scheme 4. Under irradiation, the sensitizer is excited to triplet state (3S) via single state (1S), that subsequently transfers energy to NBD molecules and excites it to triplet state (3NBD). Then 3NBD undergoes adiabatic isomerization and forms triplet state of QC (3QC) that rapidly decays to its ground state and produces QC.

Ti-MCM-41

Since some photocatalysts show absorption in visible-light region, one may wonder whether they can catalyze the isomerization under visible-light irradiation. However, there is no any observable conversion when the experiment was conducted using visible irradiation (>420 nm). This is different from the case of H2 generation and organic degradation, where Cr-Ti-MCM-41 is reported to exhibit visible-light activity (Yamashita et al., 2001; Davydov et al., 2001; Chen & Mao, 2007). These results suggest that the reaction mechanism between the photoisomerization and other photocatalytic reactions

Photoisomerization of NBD in the presence of sensitizers generally proceeds via triplet state mechanism (Bren' et al., 1991; Dubonosov et al., 2002), see Scheme 4. Under irradiation, the sensitizer is excited to triplet state (3S) via single state (1S), that subsequently transfers energy to NBD molecules and excites it to triplet state (3NBD). Then 3NBD undergoes adiabatic isomerization and forms triplet state of QC (3QC) that rapidly decays to its ground

Cr

Si/M

**4. Mechanism for NBD photoisomerization** 

100

150

norbornadiene (Zou et al., 2010).

may be very different.

state and produces QC.

ratio

33 10

Fig. 13. Activity of M(V, Fe and Cr)-Ti-MCM-41 for the photoisomerization of

75

0.0

V

Fe

0.5

1.0

1.5

*k/k0*

2.0

2.5

Scheme 4. Triplet sensitized photoisomerization of norbornadiene.

However, with the presence of Ti-containing photocatalyst, this mechanism is not suitable because the vertical triplet energy transfer from Ti-oxide species to NBD is very difficult. NBD molecules have to be firstly positively charged by photoinduced holes, but the free radical ion isomerization mechanism is ruled out because the energy of free NBD·+ is significantly lower than free QC·+. In fact, the transformation of QC to NBD is through the QC·+→NBD·+ free radical route (Ikezawa & Kutal, 1987). So the photoisomerization of NBD over semiconductors should be an adsorption-photoexcited process, which is very likely through the exciplex (charge-transfer intermediate), see Scheme 5. First, NBD molecule is adsorbed on the photoexcited Ti-oxides. Then surface-trapped hole is transferred to adsorbed molecule and a complex with NBD positively charged is formed. Subsequently the complex is transformed to structure with QC skeleton. Finally, QC is released into the liquid phase and the charge is recombined through reverse electron transfer. In this case the adsorption and charge transfer are two critical steps. The adsorptive site on different Ticontaining materials may be different. For Zn-TiO2, surface OH very likely serves as the site because it plays an important role in the reaction, and the excited complex may be*TiO OH NBD* <sup>2</sup> . For Fe-TiO2 and V-TiO2, however, the lattice oxygen may work as the adsorbing site with the complex of 4 2 42 *Ti O Ti O NBD* [ ] . Any charge recombination process can deactivate the complex, so the function of dopants and framework Ti species is to retard the undesired recombination.

Photoisomerization of Norbornadiene to Quadricyclane Using Ti-Containing Photocatalysts 59

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Scheme 5. Photoisomerization of norbornadiene via adsorption-photoexcitation over semiconductor.

### **5. Summary**

The transform of norbornadiene is typical photoisomerization and of great importance for both solar energy harvesting and aerospace fuel synthesis. Our recent work shows that the heterogeneous Ti-containing materials show activity comparable to homogeneous sensitizers, along with many additional advantages in manipulation and scale-up. Ticontaining photocatalysts are extensively used in environmental and energy science and show many exciting and rapid progress, which will undoubtedly benefit the photoisomerization of alkenes like NBD. Specially, surface modulation may be very helpful because it can tune the adsorption and even charge transfer between reactant and catalyst. Even though, the photoisomerization shows some unique characteristics and further work is necessary to understand the mechanism and substantively improve the efficiency. It is expected that the heterogeneous photocatalysis may provide a new and promising pathway for photoisomerization of alkenes.

### **6. Acknowledgements**

The authors greatly appreciate the supports from the Natural Science Foundation of China (20906069), the Foundation for the Author of National Excellent Doctoral Dissertation of China (200955), the Program for New Century Excellent Talents in University, and the Research Fund for the Doctoral Program of Higher Education of China (200800561011).

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Scheme 5. Photoisomerization of norbornadiene via adsorption-photoexcitation over

The transform of norbornadiene is typical photoisomerization and of great importance for both solar energy harvesting and aerospace fuel synthesis. Our recent work shows that the heterogeneous Ti-containing materials show activity comparable to homogeneous sensitizers, along with many additional advantages in manipulation and scale-up. Ticontaining photocatalysts are extensively used in environmental and energy science and show many exciting and rapid progress, which will undoubtedly benefit the photoisomerization of alkenes like NBD. Specially, surface modulation may be very helpful because it can tune the adsorption and even charge transfer between reactant and catalyst. Even though, the photoisomerization shows some unique characteristics and further work is necessary to understand the mechanism and substantively improve the efficiency. It is expected that the heterogeneous photocatalysis may provide a new and promising pathway

The authors greatly appreciate the supports from the Natural Science Foundation of China (20906069), the Foundation for the Author of National Excellent Doctoral Dissertation of China (200955), the Program for New Century Excellent Talents in University, and the Research Fund for the Doctoral Program of Higher Education of China (200800561011).

Adán, C.; Bahamonde, A.; Fernández-García, M. & Martínez-Arias, A. (2007). Structure and

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**4** 

 *Italy* 

**Improved Photochemistry of TiO2** 

**Inverse Opals and some Examples** 

Fabrizio Sordello, Valter Maurino and Claudio Minero

*Università degli Studi di Torino, Dipartimento di Chimica Analitica, Torino,* 

TiO2 inverse opals are porous TiO2 structures in which the pore arrangement is well ordered in three dimensions. Frequently the pores are arranged in a fcc or hcp structure and each pore is connected to the twelve nearest neighbours. TiO2 commonly occupies about 25% of the volume of the material, while the pores, which can be filled with gaseous or liquid

The ordered arrangement of pores of the same size can be seen as a periodic modulation of the refractive index in the space, and therefore TiO2 inverse opals are by definition photonic crystals (John 1987; Yablonovitch 1987). Photonic crystals are very useful in controlling the propagation of light, and they can represent for photonics the same improvement semiconductors represented in electronics. Hence properly designed TiO2 inverse opals find application in solar energy recovery (Nishimura et al. 2003; Mihi et al. 2008; Chutinan et al. 2009) and photocatalysis (J. I. L. Chen et al. 2006; Y. Li et al. 2006; Ren et al. 2006; Srinivasan

Fig. 1. SEM micrographs of TiO2 inverse opals at different magnification: (a) 20 000x magnification, (b) 100 000x magnification (Reprinted from Waterhouse & Waterland 2007,

This chapter reviews the literature to give a complete picture of the state of the art of the photochemistry on TiO2 inverse opals and outlines the more promising perspectives of the

solutions, account for the remaining 75% of the volume (Fig. 1).

& White 2007; J. I. L. Chen et al. 2008; Sordello et al. 2011a).

Copyright (2007), with permission from Elsevier)

field in the near future.

**1. Introduction** 


## **Improved Photochemistry of TiO2 Inverse Opals and some Examples**

Fabrizio Sordello, Valter Maurino and Claudio Minero *Università degli Studi di Torino, Dipartimento di Chimica Analitica, Torino, Italy* 

### **1. Introduction**

62 Molecular Photochemistry – Various Aspects

Zou, J.-J.; Zhang, M.-Y.; Zhu, B.; Wang, L.; Zhang, X. & Mi, Z. (2008). Isomerization of

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1169

norbornadiene to quadricyclane using Ti-containing MCM-41 as photocatalysts.

photocatalysts for the isomerization of norbornadiene to quadricyclane. *Journal of Molecular Catalysis A: Chemical*, Vol.286, No.1-2 (May 2008), pp. 63-69, ISSN 1381-

> TiO2 inverse opals are porous TiO2 structures in which the pore arrangement is well ordered in three dimensions. Frequently the pores are arranged in a fcc or hcp structure and each pore is connected to the twelve nearest neighbours. TiO2 commonly occupies about 25% of the volume of the material, while the pores, which can be filled with gaseous or liquid solutions, account for the remaining 75% of the volume (Fig. 1).

> The ordered arrangement of pores of the same size can be seen as a periodic modulation of the refractive index in the space, and therefore TiO2 inverse opals are by definition photonic crystals (John 1987; Yablonovitch 1987). Photonic crystals are very useful in controlling the propagation of light, and they can represent for photonics the same improvement semiconductors represented in electronics. Hence properly designed TiO2 inverse opals find application in solar energy recovery (Nishimura et al. 2003; Mihi et al. 2008; Chutinan et al. 2009) and photocatalysis (J. I. L. Chen et al. 2006; Y. Li et al. 2006; Ren et al. 2006; Srinivasan & White 2007; J. I. L. Chen et al. 2008; Sordello et al. 2011a).

Fig. 1. SEM micrographs of TiO2 inverse opals at different magnification: (a) 20 000x magnification, (b) 100 000x magnification (Reprinted from Waterhouse & Waterland 2007, Copyright (2007), with permission from Elsevier)

This chapter reviews the literature to give a complete picture of the state of the art of the photochemistry on TiO2 inverse opals and outlines the more promising perspectives of the field in the near future.

Improved Photochemistry of TiO2 Inverse Opals and some Examples 65

TiO2 inverse opals can especially improve the light absorption, allowing also a fast mass transfer of solution species due to the large-pore structure. This approach can be combined with other strategies, new or already employed to improve the efficiency of the photocatalytic process on TiO2 surfaces, for example synthesizing doped or dye sensitized TiO2 inverse opals, or realizing structures with controlled exposed surfaces. In this case the achievable structures are limited only by the creativity of the researchers and the synthetic

In the next section the origin of the better absorption of light of TiO2 inverse opals will be

As we mentioned in section 1 TiO2 inverse opals are photonic crystals, that is materials characterized by a periodic modulation in the space of the refractive index. The variation of the dielectric constant can be periodic in one dimension, two dimensions or three dimensions. Inverse opals are three dimensional photonic crystals, but for simplicity in the following we will consider the interaction of light with a monodimensional photonic crystal.

Fig. 3. The multilayer film is a monodimensional photonic crystal. Monodimensional means that the dielectric constant varies only along one direction (z). The system is composed by alternating layers of different materials with different refractive indexes and with spatial period a. Every layer is uniform and extends to infinity along the xy plane. Also the

A monodimensional photonic crystal is a multilayer film formed by alternating layers of constant thickness, with different refractive index and constant spacing among them (Fig. 3). Referring to Fig. 3 it can be noticed that light propagating in the z direction encounters on its path several interfaces between the two dielectrics. At each interface light is reflected and refracted following the Snell law. If the wavelength of light propagating along the z direction matches perfectly the periodicity of the one-dimensional photonic crystal, the reflected waves will be in phase, and as a consequence i) light will be reflected by the photonic crystal and ii) its propagation inside the material will be forbidden (Yablonovitch 2001). We say that the wavelength (or the frequency) of the incident light falls inside the

y

z

x

procedures, which can become very complicated and difficult to implement.

**3. Photonic band gap and slow photons** 

periodicity in the z direction extends to infinity.

discussed.

a

photonic band gap (Fig. 4).

### **2. The context: Light driven processes on TiO2**

TiO2 is used in heterogeneous photocatalysis as photocatalyst in water photosplitting and hydrogen production, in solar cells for the production of electricity, and in other applications that do not require light, such as lithium ion batteries, bone implants and gate insulator for MOSFETs. When TiO2 is irradiated with light with energy higher than the band gap an electron-hole couple is generated. The charge carriers can separate and migrate towards the surface where they can be trapped or react with solution species, as it can be seen in Fig. 2 (Diebold 2003). Reactive oxygen species are formed, the degradation of organic molecules and pollutants occurs, and the complete mineralization to CO2, H2O and inorganic ions has been reported (Pelizzetti et al. 1989). From the environmental point of view this process can be very useful and effective, since the harmful pollutant is not displaced into another phase, but ultimately decomposed to non harmful inorganic compounds. In the presence of a metallic cocatalyst, platinum for example, and in the absence of oxygen, the photogenerated electrons can reduce H3O+ to hydrogen, and in the absence of an effective hole scavenger, the photogenerated holes can oxidize water to oxygen, leading to water photosplitting (Ekambaram 2008; Yun et al. 2011). Recently, research focused its attention on the photocatalytic productions of value added chemicals starting from glycerol (Maurino et al. 2008) or directly from CO2 in artificial photosynthesis (Benniston & Harriman 2008; Roy et al. 2010).

The technological and commercial affirmation of these light driven processes is delayed because (Fujishima et al. 2008; Gaya & Abdullah 2008):

1. TiO2 is an indirect band gap semiconductor, and therefore its light absorption is limited.

Fig. 2. Processes occurring on TiO2 under UV irradiation: (a) light absorption and charge carriers formation, (b) electron-hole recombination at bulk trapping sites with release of heat, (c) electron-hole recombination at surface trapping sites, (d) trapped electron reacts with acceptor (e) trapped hole reacts with donor.


TiO2 is used in heterogeneous photocatalysis as photocatalyst in water photosplitting and hydrogen production, in solar cells for the production of electricity, and in other applications that do not require light, such as lithium ion batteries, bone implants and gate insulator for MOSFETs. When TiO2 is irradiated with light with energy higher than the band gap an electron-hole couple is generated. The charge carriers can separate and migrate towards the surface where they can be trapped or react with solution species, as it can be seen in Fig. 2 (Diebold 2003). Reactive oxygen species are formed, the degradation of organic molecules and pollutants occurs, and the complete mineralization to CO2, H2O and inorganic ions has been reported (Pelizzetti et al. 1989). From the environmental point of view this process can be very useful and effective, since the harmful pollutant is not displaced into another phase, but ultimately decomposed to non harmful inorganic compounds. In the presence of a metallic cocatalyst, platinum for example, and in the absence of oxygen, the photogenerated electrons can reduce H3O+ to hydrogen, and in the absence of an effective hole scavenger, the photogenerated holes can oxidize water to oxygen, leading to water photosplitting (Ekambaram 2008; Yun et al. 2011). Recently, research focused its attention on the photocatalytic productions of value added chemicals starting from glycerol (Maurino et al. 2008) or directly from CO2 in artificial photosynthesis

The technological and commercial affirmation of these light driven processes is delayed

1. TiO2 is an indirect band gap semiconductor, and therefore its light absorption is limited.

h+

h+ tr

D+

Fig. 2. Processes occurring on TiO2 under UV irradiation: (a) light absorption and charge carriers formation, (b) electron-hole recombination at bulk trapping sites with release of heat, (c) electron-hole recombination at surface trapping sites, (d) trapped electron reacts

2. TiO2 is a wide band gap semiconductor and absorbs only the UV fraction of the solar

3. TiO2 has a quite high refractive index and light absorption is limited also by reflection. 4. The efficiencies of the above mentioned processes are quite low, because the charge

(a)

(e)

D

e-

etr

A A- (d)

(b)

(c)

**2. The context: Light driven processes on TiO2**

(Benniston & Harriman 2008; Roy et al. 2010).

h

with acceptor (e) trapped hole reacts with donor.

carriers recombination is fast.

spectrum.

because (Fujishima et al. 2008; Gaya & Abdullah 2008):

TiO2 inverse opals can especially improve the light absorption, allowing also a fast mass transfer of solution species due to the large-pore structure. This approach can be combined with other strategies, new or already employed to improve the efficiency of the photocatalytic process on TiO2 surfaces, for example synthesizing doped or dye sensitized TiO2 inverse opals, or realizing structures with controlled exposed surfaces. In this case the achievable structures are limited only by the creativity of the researchers and the synthetic procedures, which can become very complicated and difficult to implement.

In the next section the origin of the better absorption of light of TiO2 inverse opals will be discussed.

### **3. Photonic band gap and slow photons**

As we mentioned in section 1 TiO2 inverse opals are photonic crystals, that is materials characterized by a periodic modulation in the space of the refractive index. The variation of the dielectric constant can be periodic in one dimension, two dimensions or three dimensions. Inverse opals are three dimensional photonic crystals, but for simplicity in the following we will consider the interaction of light with a monodimensional photonic crystal.

Fig. 3. The multilayer film is a monodimensional photonic crystal. Monodimensional means that the dielectric constant varies only along one direction (z). The system is composed by alternating layers of different materials with different refractive indexes and with spatial period a. Every layer is uniform and extends to infinity along the xy plane. Also the periodicity in the z direction extends to infinity.

A monodimensional photonic crystal is a multilayer film formed by alternating layers of constant thickness, with different refractive index and constant spacing among them (Fig. 3). Referring to Fig. 3 it can be noticed that light propagating in the z direction encounters on its path several interfaces between the two dielectrics. At each interface light is reflected and refracted following the Snell law. If the wavelength of light propagating along the z direction matches perfectly the periodicity of the one-dimensional photonic crystal, the reflected waves will be in phase, and as a consequence i) light will be reflected by the photonic crystal and ii) its propagation inside the material will be forbidden (Yablonovitch 2001). We say that the wavelength (or the frequency) of the incident light falls inside the photonic band gap (Fig. 4).

Improved Photochemistry of TiO2 Inverse Opals and some Examples 67

Fig. 6. The photonic band structure of the multilayer film depicted in Fig. 3 calculated with a freely available software (S. G. Johnson & Joannopoulos 2001). The dielectric constants of the

A more rigorous approach starts from the treatment of the electromagnetic problem in mixed dielectric media, where the dielectric constant (**r**) becomes a function of the spatial coordinate **r**. For this case the Maxwell equations have the form (Joannopoulos et

where E and H are the macroscopic electric and magnetic fields, ε<sup>0</sup> ≈ 8.854×10−12 F/m is the vacuum permittivity, μ0 = 4π × 10−7 H/m is the vacuum permeability and ε(**r**) is the scalar dielectric function. It can be demonstrated that Maxwell equations can be rearranged to

where the electromagnetic problem takes the form of an eigenvalue problem. It can be demonstrated that the operator working on the magnetic field is linear and hermitian. In a mixed periodic medium the eigenfuctions that satisfy equation 5 are in the form of Bloch waves in which the expression of a plane wave is multiplied by a periodic function that

where **u**(**r**) is a periodic function of the type **u**(**r**) = **u**(**r**+**R**) for every lattice vector **R** (Joannopoulos et al. 2008). In the case of the multilayer film in Fig. 3 the periodic function u

accounts for the periodicity of (**r**) in the photonic crystal:

depends only on the z coordinate.

·**H**(**r**,t) = 0 (1)

·[(**r**) **E**(**r**,t)] = 0 (2)

×**H**(**r**,t) – 0(**r**) ∂**E**(**r**,t)/∂t = 0 (3)

×**E**(**r**,t) + <sup>0</sup> ∂**H**(**r**,t)/∂t = 0 (4)

×{[1/(**r**)] ×**H**(**r**)} = (/c)2 **H**(**r**) (5)

**H**(**r**) = ei**k·r u**(**r**) (6)

layers are ε=5 and ε=2. The photonic band gap frequencies are highlighted in yellow.

al. 2008):

yield equation 5:

Fig. 4. Schematic representation of an electromagnetic wave impinging a photonic band gap material (a), partial reflection occurs at every interface and, since the frequency of the incident wave falls inside the photonic band gap the reflected waves are all in phase (b); as a result the light cannot travel through the material (c) (adapted from Yablonovitch 2001)

Fig. 5. Schematic representation of an electromagnetic wave with frequency outside the photonic band gap propagating into a photonic crystal (a), in this case the reflected waves are out of phase (b), and the light propagates inside the material only slightly attenuated (c) (adapted from Yablonovitch 2001)

On the contrary, if the wavelength of the incident light does not match the periodicity of the photonic crystal lattice, the waves reflected at each interface will be out of phase, and they will cancel out each other (Fig. 5). As a result the light will be able to propagate inside the material. In this case the wavelength (or the frequency) of the incident light is said to fall outside the photonic band gap.

This approach is useful from a qualitative point of view and gives a general idea of physical phenomena involved, but it cannot be extended to the bidimensional and tridimensional cases. Moreover, it lacks of quantitative understanding of the phenomenon, impeding, for example, the evaluation of the magnitude of the photonic band gap.

(a)

(b)

(c)

(b)

(c)

Incident wave

Reflected waves

Resultant wave

Reflected waves

Resultant wave

(adapted from Yablonovitch 2001)

outside the photonic band gap.

Fig. 5. Schematic representation of an electromagnetic wave with frequency outside the photonic band gap propagating into a photonic crystal (a), in this case the reflected waves are out of phase (b), and the light propagates inside the material only slightly attenuated (c)

On the contrary, if the wavelength of the incident light does not match the periodicity of the photonic crystal lattice, the waves reflected at each interface will be out of phase, and they will cancel out each other (Fig. 5). As a result the light will be able to propagate inside the material. In this case the wavelength (or the frequency) of the incident light is said to fall

This approach is useful from a qualitative point of view and gives a general idea of physical phenomena involved, but it cannot be extended to the bidimensional and tridimensional cases. Moreover, it lacks of quantitative understanding of the phenomenon, impeding, for

example, the evaluation of the magnitude of the photonic band gap.

Fig. 4. Schematic representation of an electromagnetic wave impinging a photonic band gap material (a), partial reflection occurs at every interface and, since the frequency of the incident wave falls inside the photonic band gap the reflected waves are all in phase (b); as a result the light cannot travel through the material (c) (adapted from Yablonovitch 2001)

Incident wave (a)

Fig. 6. The photonic band structure of the multilayer film depicted in Fig. 3 calculated with a freely available software (S. G. Johnson & Joannopoulos 2001). The dielectric constants of the layers are ε=5 and ε=2. The photonic band gap frequencies are highlighted in yellow.

A more rigorous approach starts from the treatment of the electromagnetic problem in mixed dielectric media, where the dielectric constant (**r**) becomes a function of the spatial coordinate **r**. For this case the Maxwell equations have the form (Joannopoulos et al. 2008):

$$\nabla \cdot \mathbf{H}(\mathbf{r}, \mathbf{t}) = 0 \tag{1}$$

$$\nabla \cdot \left[ \varepsilon(\mathbf{r}) \, \mathbf{E}(\mathbf{r}, \mathbf{t}) \right] = 0 \tag{2}$$

$$
\nabla \times \mathbf{H}(\mathbf{r}, \mathbf{t}) - \varepsilon \alpha \varepsilon(\mathbf{r}) \,\,\partial \mathbf{E}(\mathbf{r}, \mathbf{t}) / \partial \mathbf{t} = 0 \tag{3}
$$

$$
\nabla \times \mathbf{E}(\mathbf{r}, \mathbf{t}) + \mu\_0 \,\partial \mathbf{H}(\mathbf{r}, \mathbf{t}) / \partial \mathbf{t} = 0 \tag{4}
$$

where E and H are the macroscopic electric and magnetic fields, ε<sup>0</sup> ≈ 8.854×10−12 F/m is the vacuum permittivity, μ0 = 4π × 10−7 H/m is the vacuum permeability and ε(**r**) is the scalar dielectric function. It can be demonstrated that Maxwell equations can be rearranged to yield equation 5:

$$
\nabla \times \left[ \left[ 1 \right/ \left. \varepsilon(\mathbf{r}) \right] \nabla \times \mathbf{H}(\mathbf{r}) \right] = (\alpha/c)^2 \left. \mathbf{H}(\mathbf{r}) \right| \tag{5}
$$

where the electromagnetic problem takes the form of an eigenvalue problem. It can be demonstrated that the operator working on the magnetic field is linear and hermitian. In a mixed periodic medium the eigenfuctions that satisfy equation 5 are in the form of Bloch waves in which the expression of a plane wave is multiplied by a periodic function that accounts for the periodicity of (**r**) in the photonic crystal:

$$\mathbf{H}(\mathbf{r}) \equiv \mathbf{e}^{i\mathbf{k}\cdot\mathbf{r}} \mathbf{u}(\mathbf{r}) \tag{6}$$

where **u**(**r**) is a periodic function of the type **u**(**r**) = **u**(**r**+**R**) for every lattice vector **R** (Joannopoulos et al. 2008). In the case of the multilayer film in Fig. 3 the periodic function u depends only on the z coordinate.

Improved Photochemistry of TiO2 Inverse Opals and some Examples 69

from equation 7 we can argue that a flat trend of a photonic mode in the frequency vs. wavevector **k** plot is indicative of low group velocity. Looking at Fig. 7, we can observe that almost flat photonic bands are present for different frequency ranges. At those frequencies light will be able to travel inside the TiO2 inverse opal, but its group velocity will be strongly

When light with low group velocity (or slow light or slow photons) can be exploited, the optical absorption of the material can be improved as if the optical path inside the material would be lengthened. An elegant experimental demonstration of this phenomenon is the change of the absorbance spectrum of an adsorbed dye on different TiO2 inverse opals

Fig. 8. Absorption spectra of crystal violet adsorbed on different TiO2 films: (a) reference TiO2 flat film, (b) and (c) TiO2 inverse opals slowing photons outside the 450-650 nm range, (d) and (e) TiO2 inverse opals slowing photons in the 600-650 nm range Reprinted with

When the crystal violet dye is adsorbed on a TiO2 inverse opal, which can slow down the photons possibly absorbed by the dye, its absorbance is increased with respect to flat TiO2 or TiO2 inverse opals with not properly tuned periodicity (Y. Li et al. 2006). Absorption spectra reported in Fig. 8 show that also in the case of TiO2 inverse opals with not properly tuned photonic band gap (slow photons outside the 450-650 nm range) there is a higher absorption that can be explained in terms of porosity and larger amount of adsorbed dye. In the case of TiO2 inverse opals slowing photons in the 600-650 nm range, higher absorption cannot be explained only in terms of porosity and higher amount of adsorbed dye, because not only the intensity of the spectra is different from the reference film, but also because the shape of the spectra changes, as the absorption at 600-625 nm becomes predominant over that at 500 nm.

This important feature, together with porosity and indeed high surface area, makes TiO2 inverse opals very good materials for application in semiconductor photocatalysis, solar energy recovery (dye sensitized solar cells) and artificial photosynthesis (Ren & Valsaraj 2009). Owing to this, they have drawn and they are drawing a lot of research into this field.

permission from Y. Li et al. 2006 Copyright (2006) American Chemical Society.

reduced. Hence the light interaction with the material will be incremented.

(Fig. 8).

Introducing the Bloch waves of equation 6 in equation 5 the eigenproblem can be solved and the photonic band structure of the photonic crystal of interest can be studied. If in the photonic band diagram there are frequencies for which there are no photonic modes allowed for every wavevector k, a photonic band gap is present (Fig. 6). At those frequencies light cannot propagate inside the material. In such cases the photonic crystals can find application in lasing cavity, optical filter and dielectric mirrors.

If equation 5 is solved for the TiO2 inverse opal (Fig. 7) no photonic band gaps are present. To have a photonic band gap in the inverse opal structure the dielectric contrast (the difference between the dielectric constants of the two media) has to be at least 9, whereas TiO2 anatase has a dielectric constant higher than ten only for photon energies above 3.8 – 4.0 eV (310 – 325 nm) (Jellison et al. 2003). Therefore in the visible and UVA TiO2 will not have a complete photonic band gap. Nevertheless, at certain frequencies light will not be able to propagate in some direction (Fig. 7). For example in the -L direction there is a pseudo photonic gap that forbids the propagation of light at a value of the reduced frequency around 0.55. This feature is not so unsuitable for photosynthetic or photocatalytic application, since in those cases light has to propagate inside the catalyst to be absorbed and create charge carriers.

The photonic band diagram of TiO2 inverse opal shows that for some photonic bands the behaviour of the frequency as a function of the wavevector presents an almost flat trend. As the group velocity of light **vg** is defined as:

$$\mathbf{v\_{g}(k)} = \nabla\_{\mathbf{k}} \alpha \tag{7}$$

Introducing the Bloch waves of equation 6 in equation 5 the eigenproblem can be solved and the photonic band structure of the photonic crystal of interest can be studied. If in the photonic band diagram there are frequencies for which there are no photonic modes allowed for every wavevector k, a photonic band gap is present (Fig. 6). At those frequencies light cannot propagate inside the material. In such cases the photonic crystals can find

Fig. 7. Photonic band diagram of a TiO2 inverse opal with the pores filled with water. The frequencies associated with light with low group velocity are highlighted. The band structure has been calculated with a freely available software

If equation 5 is solved for the TiO2 inverse opal (Fig. 7) no photonic band gaps are present. To have a photonic band gap in the inverse opal structure the dielectric contrast (the difference between the dielectric constants of the two media) has to be at least 9, whereas TiO2 anatase has a dielectric constant higher than ten only for photon energies above 3.8 – 4.0 eV (310 – 325 nm) (Jellison et al. 2003). Therefore in the visible and UVA TiO2 will not have a complete photonic band gap. Nevertheless, at certain frequencies light will not be able to propagate in some direction (Fig. 7). For example in the -L direction there is a pseudo photonic gap that forbids the propagation of light at a value of the reduced frequency around 0.55. This feature is not so unsuitable for photosynthetic or photocatalytic application, since in those cases light has to propagate inside the catalyst to be absorbed and

The photonic band diagram of TiO2 inverse opal shows that for some photonic bands the behaviour of the frequency as a function of the wavevector presents an almost flat trend. As

**vg**(**k**) = **k** (7)

(S. G. Johnson & Joannopoulos 2001) assuming for the dielectric constants of TiO2

and water the values 4.4 and 1.7, respectively.

the group velocity of light **vg** is defined as:

create charge carriers.

application in lasing cavity, optical filter and dielectric mirrors.

from equation 7 we can argue that a flat trend of a photonic mode in the frequency vs. wavevector **k** plot is indicative of low group velocity. Looking at Fig. 7, we can observe that almost flat photonic bands are present for different frequency ranges. At those frequencies light will be able to travel inside the TiO2 inverse opal, but its group velocity will be strongly reduced. Hence the light interaction with the material will be incremented.

When light with low group velocity (or slow light or slow photons) can be exploited, the optical absorption of the material can be improved as if the optical path inside the material would be lengthened. An elegant experimental demonstration of this phenomenon is the change of the absorbance spectrum of an adsorbed dye on different TiO2 inverse opals (Fig. 8).

Fig. 8. Absorption spectra of crystal violet adsorbed on different TiO2 films: (a) reference TiO2 flat film, (b) and (c) TiO2 inverse opals slowing photons outside the 450-650 nm range, (d) and (e) TiO2 inverse opals slowing photons in the 600-650 nm range Reprinted with permission from Y. Li et al. 2006 Copyright (2006) American Chemical Society.

When the crystal violet dye is adsorbed on a TiO2 inverse opal, which can slow down the photons possibly absorbed by the dye, its absorbance is increased with respect to flat TiO2 or TiO2 inverse opals with not properly tuned periodicity (Y. Li et al. 2006). Absorption spectra reported in Fig. 8 show that also in the case of TiO2 inverse opals with not properly tuned photonic band gap (slow photons outside the 450-650 nm range) there is a higher absorption that can be explained in terms of porosity and larger amount of adsorbed dye. In the case of TiO2 inverse opals slowing photons in the 600-650 nm range, higher absorption cannot be explained only in terms of porosity and higher amount of adsorbed dye, because not only the intensity of the spectra is different from the reference film, but also because the shape of the spectra changes, as the absorption at 600-625 nm becomes predominant over that at 500 nm.

This important feature, together with porosity and indeed high surface area, makes TiO2 inverse opals very good materials for application in semiconductor photocatalysis, solar energy recovery (dye sensitized solar cells) and artificial photosynthesis (Ren & Valsaraj 2009). Owing to this, they have drawn and they are drawing a lot of research into this field.

Improved Photochemistry of TiO2 Inverse Opals and some Examples 71

replicated in the TiO2 inverse opal. For the TiO2 inverse opal preparation a TiO2 precursor solution (titanium(IV) isopropoxide, titanium(IV) butoxide, other titanium(IV) species) or a dispersion of TiO2 nanoparticles is used to fill the voids of the synthetic opal, a process normally called infiltration. Once the infiltration process is completed and TiO2 backbone is formed, the template is removed by etching with NaOH or HF in the case of silica templates, with toluene in the case of polystyrene templates, or by calcination if a cross linked

In the literature many methods to synthesize monodisperse colloids are described. For the successive colloidal crystal preparation the most suitable method to obtain monodisperse polymeric colloids is the emulsion polymerization without emulsifier in water. To synthesize monodisperse polystyrene colloids the emulsion water-styrene is heated and vigorously stirred, the addition of the initiator makes the reaction start, and the relative amounts of monomer/initiator/ionic strength affect the size of the final polymer spheres (Goodwin et al. 1974). It is also possible to synthesize positively charged polystyrene colloids (Reese et al. 2000) or functionalized colloids (X. Chen et al. 2002) to ease the colloidal crystal formation and the infiltration of TiO2. The preparation of monodisperse polymethylmethacrylate colloids is very similar (Waterhouse & Waterland 2007), and

Monodisperse silica spheres can be synthesized in ethanol with determined amount of

Many different strategies to obtain colloidal crystals are available depending on the final goal, because they can be obtained as powders, thick films, and also thin films containing

a. Direct centrifugation of the monodisperse colloid leads to the formation of a colloidal crystal. Although the method is simple, it is not suitable if a film of controlled thickness has to be obtained (Wijnhoven & Vos 1998; Waterhouse & Waterland 2007). Another drawback is the slowness, if small colloidal particles (size < 150 nm) are involved,

c. The most popular method used to synthesize colloidal crystals is based on the vertical, rather than horizontal, position of the substrate on which the monodisperse colloid is deposited. When the substrate is dipped into the colloidal dispersion of monodisperse spheres the particles self-assembly in an opaline structure in the meniscus region (Fig.

because more than one hour of centrifugation is needed (Sordello et al. 2011a). b. A related method is the sedimentation. A drop of the monodisperse colloid is deposited on a clean substrate, where the drop can broaden due to the hydrophilicity of the substrate with contact angle close to zero. The evaporation of the solvent leads to the formation of the colloidal crystal (Denkov et al. 1993). To avoid thickness non uniformity the crystallization can be carried out under silicone liquid (Fudouzi 2004). Since in most cases the evaporation of water is necessary for the synthesis of the colloidal crystal, relative humidity plays an important role (Liau & Huang 2008), and the deposition is quite slow. To accelerate the process ethanol can be used instead of

examples of synthesis in non polar solvents are also reported (Klein et al. 2003).

water in the presence of ammonia as shape controller (Jiang et al. 1999).

polymeric template has been used.

**4.2 Colloidal crystal synthesis** 

one, two or at least three layers.

water (Shin et al. 2011).

**4.1 Preparation of monodisperse colloids** 

### **4. Synthesis**

TiO2 inverse opals can be prepared in several ways and, probably, new synthetic routes will be discovered in the next years. Different approaches are possible because three dimensional ordered porous structures can be obtained exploiting many different physical principles. The invention of new methods is only limited by physics and chemistry and by the creativity of the researchers. An ideal method would be fast, requiring only standard procedures and instruments, it would be cheap and it should be able to produce homogeneous TiO2 inverse opals over large surfaces with the possibility to control the thickness of the synthesized material. A good method would be also able to produce TiO2 inverse opals with few defects both at microscopic (vacancies, dislocations, grain boundaries, stacking faults, ...) and macroscopic level (cracks, thickness inhomogeneity).

Fig. 9. Schematics of the fabrication procedure of a TiO2 inverse opal. (adapted from L. Liu et al. 2011)

In general TiO2 inverse opals are synthesized in a two steps procedure (Fig. 9). Firstly, a well monodisperse colloidal suspension of SiO2 or polymer spheres has to be prepared. The monodisperse colloid is then conveniently deposited onto a clean substrate to form a colloidal crystal, that is a solid characterized by an ordered disposition of silica or polymer spheres in three dimensions. A colloidal crystal can be described also as a superlattice of closely packed colloidal particles. If the colloidal particles are spherical the resulting colloidal crystal is called opal or synthetic opal, in analogy with natural opals characterized by an ordered arrangement of silica spheres in three dimensions. Most of the time colloidal crystals obtained in this way are synthetic opals and can find application as sensors (Endo et al. 2007; Shi et al. 2008) and as model systems in the study of crystals, phase transitions (Bosma et al. 2002) and the interaction of photonic crystals with light (Miguez et al. 2004; Pavarini et al. 2005; M. Ishii et al. 2007). Beyond these applications, colloidal crystals serve as sacrificial templates for the synthesis of TiO2 inverse opals. In this case the quality of the colloidal-crystal template is very important, because every defect in the structure will be replicated in the TiO2 inverse opal. For the TiO2 inverse opal preparation a TiO2 precursor solution (titanium(IV) isopropoxide, titanium(IV) butoxide, other titanium(IV) species) or a dispersion of TiO2 nanoparticles is used to fill the voids of the synthetic opal, a process normally called infiltration. Once the infiltration process is completed and TiO2 backbone is formed, the template is removed by etching with NaOH or HF in the case of silica templates, with toluene in the case of polystyrene templates, or by calcination if a cross linked polymeric template has been used.

### **4.1 Preparation of monodisperse colloids**

70 Molecular Photochemistry – Various Aspects

TiO2 inverse opals can be prepared in several ways and, probably, new synthetic routes will be discovered in the next years. Different approaches are possible because three dimensional ordered porous structures can be obtained exploiting many different physical principles. The invention of new methods is only limited by physics and chemistry and by the creativity of the researchers. An ideal method would be fast, requiring only standard procedures and instruments, it would be cheap and it should be able to produce homogeneous TiO2 inverse opals over large surfaces with the possibility to control the thickness of the synthesized material. A good method would be also able to produce TiO2 inverse opals with few defects both at microscopic (vacancies, dislocations, grain boundaries, stacking faults, ...) and macroscopic level (cracks, thickness inhomogeneity).

Self-assembly

Template removal

Inverse opal Infiltrated opal

In general TiO2 inverse opals are synthesized in a two steps procedure (Fig. 9). Firstly, a well monodisperse colloidal suspension of SiO2 or polymer spheres has to be prepared. The monodisperse colloid is then conveniently deposited onto a clean substrate to form a colloidal crystal, that is a solid characterized by an ordered disposition of silica or polymer spheres in three dimensions. A colloidal crystal can be described also as a superlattice of closely packed colloidal particles. If the colloidal particles are spherical the resulting colloidal crystal is called opal or synthetic opal, in analogy with natural opals characterized by an ordered arrangement of silica spheres in three dimensions. Most of the time colloidal crystals obtained in this way are synthetic opals and can find application as sensors (Endo et al. 2007; Shi et al. 2008) and as model systems in the study of crystals, phase transitions (Bosma et al. 2002) and the interaction of photonic crystals with light (Miguez et al. 2004; Pavarini et al. 2005; M. Ishii et al. 2007). Beyond these applications, colloidal crystals serve as sacrificial templates for the synthesis of TiO2 inverse opals. In this case the quality of the colloidal-crystal template is very important, because every defect in the structure will be

Fig. 9. Schematics of the fabrication procedure of a TiO2 inverse opal.

(adapted from L. Liu et al. 2011)

TiO2 infiltration

Opal

**4. Synthesis** 

Monodisperse colloid Substrate

In the literature many methods to synthesize monodisperse colloids are described. For the successive colloidal crystal preparation the most suitable method to obtain monodisperse polymeric colloids is the emulsion polymerization without emulsifier in water. To synthesize monodisperse polystyrene colloids the emulsion water-styrene is heated and vigorously stirred, the addition of the initiator makes the reaction start, and the relative amounts of monomer/initiator/ionic strength affect the size of the final polymer spheres (Goodwin et al. 1974). It is also possible to synthesize positively charged polystyrene colloids (Reese et al. 2000) or functionalized colloids (X. Chen et al. 2002) to ease the colloidal crystal formation and the infiltration of TiO2. The preparation of monodisperse polymethylmethacrylate colloids is very similar (Waterhouse & Waterland 2007), and examples of synthesis in non polar solvents are also reported (Klein et al. 2003).

Monodisperse silica spheres can be synthesized in ethanol with determined amount of water in the presence of ammonia as shape controller (Jiang et al. 1999).

### **4.2 Colloidal crystal synthesis**

Many different strategies to obtain colloidal crystals are available depending on the final goal, because they can be obtained as powders, thick films, and also thin films containing one, two or at least three layers.


Improved Photochemistry of TiO2 Inverse Opals and some Examples 73

structure in a polymeric gel that will prevent any shrinking during the drying process (Kanai & Sawada 2009). These procedures allow the synthesis of almost perfect colloidal crystals over large areas. Nevertheless the subsequent TiO2 infiltration will be considerably

f. Colloidal crystals are also produced by confinement between two flat surfaces (Fig. 11). The colloidal dispersion of monodisperse spheres can enter the cell because of capillarity forces (M. Ishii et al. 2005; X. Chen et al. 2008) or by means of a hole in the cell (Park et al. 1998; Lu et al. 2001). Evaporation or removal of the solvent with a gas

Growing colloidal crystal

Solvent evaporation

Fig. 11. Schematic representation of the formation of a colloidal crystal in a confinement cell

g. Spin coating can be a valuable and rapid method to produce thin film colloidal crystals. Even if many grain boundaries are produced, the distinctive feature of the method is the ability to produce very thin films. The synthesis of monolayered colloidal crystals

h. Finally, polymeric photonic crystal can also be prepared by mixing immiscible polymers (C. W. Wang & Moffitt 2005) and by laser holographic lithography (Moon et

The infiltration in the sacrificial template is the crucial step in the TiO2 inverse opal synthesis, because this phase is responsible for the major production of defects and macroscopic imperfections. If the infiltrated solution or colloid cannot form a stable network before the template is removed, the structure will collapse with the destruction of the three dimensional lattice. Nowadays several infiltration methods exist and in the following we

The sol-gel infiltration is probably the most popular and widespread method, because it is simple and low cost. Nevertheless, it has to be implemented with great care especially if TiO2

stream leaves behind large area colloidal crystals slightly fractured.

Monodisperse colloid

al. 2006; Y. Xu et al. 2008; Lin et al. 2009; Miyake et al. 2009).

will give the reader a general picture of the available techniques.

hindered.

Confinement cell

(adapted from X. Chen et al. 2008)

has been demonstrated (Mihi et al. 2006).

**4.3 Infiltration and TiO2 inverse opal synthesis** 

10). With the evaporation of the solution the meniscus moves downwards and the opal film grows in the same direction (Jiang et al. 1999; Z. Z. Gu et al. 2002; Norris et al. 2004; Shimmin et al. 2006). The method allows the formation of well ordered opals, but the process is slow and the thickness of the film is not uniform, because the colloid concentrates during the process. Moreover, it has been reported that in the vertical deposition method the thickness of the film has a sinusoidal trend in the length scale of the order of 100 m. The oscillations are more pronounced with increasing particle concentration and with decreasing temperature (Lozano & Miguez 2007).


Fig. 10. Schematic representation of a colloidal crystal formed in the meniscus region by slow evaporation of the suspension (adapted from Norris et al. 2004).

The shear flow method can also produce colloidal crystals without cracks over large domains. Normally, once the colloidal crystal is deposited the drying process causes the shrinking of the colloidal particles that produce macroscopic fractures in the structure (Sun et al. 2011). To avoid the formation of these imperfections core-shell spheres can be used. The colloidal crystal is assembled under high external pressure, and during the drying the soft shells will expand, compensating the volume shrinkage of the whole structure (Ruhl et al. 2004; Pursiainen et al. 2008). A further possibility is the use of a photosensitive monomer dissolved in the slurry. The irradiation after colloidal crystal formation will freeze the

concentration and with decreasing temperature (Lozano & Miguez 2007).

Meniscus

Growing colloidal crystal

poor.

al. 2006).

d. In the dip-drawing technique (Z. Liu et al. 2006) the colloidal crystal is formed by the downwards movement of the meniscus. The substrate is immersed vertical in the colloidal suspension and the meniscus is moved downwards withdrawing the suspension by means of a peristaltic pump. The rapidity of the method can be tuned, but if suspension withdrawal is too fast the quality of the resulting colloidal crystal is

e. Polymer or silica spheres can self assembly in ordered structures in the liquid phase (Reese et al. 2000) forming single crystals over large areas (cm2) by simply applying a shear flow if the colloidal particles are charged and the ionic strength of the medium is sufficiently low (Amos et al. 2000; Sawada et al. 2001). This method is fast, allows the formation of large single crystals without grain boundaries and, if colloidal particles with opposed charge are employed, colloidal crystals with particle packing different from the usual fcc or hcp geometry are obtained (Leunissen et al. 2005; Shevchenko et

Monodisperse colloid

Fig. 10. Schematic representation of a colloidal crystal formed in the meniscus region by

The shear flow method can also produce colloidal crystals without cracks over large domains. Normally, once the colloidal crystal is deposited the drying process causes the shrinking of the colloidal particles that produce macroscopic fractures in the structure (Sun et al. 2011). To avoid the formation of these imperfections core-shell spheres can be used. The colloidal crystal is assembled under high external pressure, and during the drying the soft shells will expand, compensating the volume shrinkage of the whole structure (Ruhl et al. 2004; Pursiainen et al. 2008). A further possibility is the use of a photosensitive monomer dissolved in the slurry. The irradiation after colloidal crystal formation will freeze the

slow evaporation of the suspension (adapted from Norris et al. 2004).

Substrate

Colloidal crystal

Solvent evaporation

10). With the evaporation of the solution the meniscus moves downwards and the opal film grows in the same direction (Jiang et al. 1999; Z. Z. Gu et al. 2002; Norris et al. 2004; Shimmin et al. 2006). The method allows the formation of well ordered opals, but the process is slow and the thickness of the film is not uniform, because the colloid concentrates during the process. Moreover, it has been reported that in the vertical deposition method the thickness of the film has a sinusoidal trend in the length scale of the order of 100 m. The oscillations are more pronounced with increasing particle structure in a polymeric gel that will prevent any shrinking during the drying process (Kanai & Sawada 2009). These procedures allow the synthesis of almost perfect colloidal crystals over large areas. Nevertheless the subsequent TiO2 infiltration will be considerably hindered.

f. Colloidal crystals are also produced by confinement between two flat surfaces (Fig. 11). The colloidal dispersion of monodisperse spheres can enter the cell because of capillarity forces (M. Ishii et al. 2005; X. Chen et al. 2008) or by means of a hole in the cell (Park et al. 1998; Lu et al. 2001). Evaporation or removal of the solvent with a gas stream leaves behind large area colloidal crystals slightly fractured.

Fig. 11. Schematic representation of the formation of a colloidal crystal in a confinement cell (adapted from X. Chen et al. 2008)


### **4.3 Infiltration and TiO2 inverse opal synthesis**

The infiltration in the sacrificial template is the crucial step in the TiO2 inverse opal synthesis, because this phase is responsible for the major production of defects and macroscopic imperfections. If the infiltrated solution or colloid cannot form a stable network before the template is removed, the structure will collapse with the destruction of the three dimensional lattice. Nowadays several infiltration methods exist and in the following we will give the reader a general picture of the available techniques.

The sol-gel infiltration is probably the most popular and widespread method, because it is simple and low cost. Nevertheless, it has to be implemented with great care especially if TiO2

Improved Photochemistry of TiO2 Inverse Opals and some Examples 75

Spectacular structures (Fig. 13) can be obtained with infiltration by atomic layer deposition. The technique allows a fine control of the amount of deposited TiO2, the filling of the pores of the template is optimal and, as a consequence, the synthesized TiO2 inverse opals are very resistant. The drawbacks are the slowness and the cost of the needed equipment, which cannot be considered a standard facility of every laboratory (King et al. 2005; L. Liu et al.

Infiltration can also be carried out by spin-coating, the method is fast and the produced TiO2 inverse opals are regular and characterized by smooth surfaces (Matsushita et al. 2007).

Fig. 13. SEM micrograph of the fractured surface of a TiO2 inverse opal produced by atomic layer deposition (Copyright (2005) Wiley. Used with permission from King at al. 2005)

TiO2 inverse opals can be obtained also in only one synthetic step, in which the silica or polymeric colloid, that will build the colloidal crystal template, is mixed together with a TiO2 colloid that will occupy the interstices of the colloidal crystal structure (Meng et al. 2002). For this reason TiO2 particles have to be one or two orders of magnitude smaller than the silica or polymer particles. The presence of smaller TiO2 particles forces the larger particles to self assembly in close packing. In this way the volume available for the diffusion of the small particles increases, with an overall gain in entropy for the entire system (Yodh

Beyond the classic filling of TiO2 inverse opal, in which TiO2 occupies the residual volume of the former opal template, a structure also called shell structure or residual volume structure, there are synthetic procedures that can build TiO2 inverse opals with a skeleton structure (Dong et al. 2003) or with a fractal distribution of the pores (Ramiro-Manzano et al.

Although the unique properties of TiO2 inverse opals and their possible applications for improved photochemistry are many, surprisingly the first demonstration of better use of light on TiO2 inverse opals dates back only in 2006 (Ren et al. 2006). Before they received attention as back reflector in dye sensitized solar cells (Nishimura et al. 2003; Halaoui et al. 2005; Somani et al. 2005), or in fundamental studies (N. P. Johnson et al. 2001; Schroden et al. 2002; Dong et al. 2003). In that seminal work Ren et al. demonstrated that TiO2 inverse opal

**5. Improved photochemistry on TiO2 inverse opals** 

2011).

et al. 2001).

2007).

precursors with large tendency to hydrolyze rapidly are used, because the amount of deposited TiO2 is difficult to control (Wei et al. 2011). In some cases the deposition of an extra layer of bulk TiO2 over the inverse opal can be turned into an advantage with the lift-off/turnover method (Fig. 12). Depending on the application both a self standing TiO2 inverse opal or a Bragg mirror behind a TiO2 active layer can be created (Galusha et al. 2008). To avoid dealing with violent hydrolysis it is possible to introduce TiO2 in the template in the form of nanoparticle already hydrolyzed (Yip et al. 2008; Shin et al. 2011). In this case the difficulty is the preparation of sufficiently small particles capable to penetrate into the pores of the opal template, because the resulting infiltration will be poor if the nanoparticles are too big, and the inverse opal structure will collapse after template removal.

Fig. 12. Schematic representation of the "lift-off/turn-over" technique. (a) polystyrene opal template. (b) Infiltrated opal with TiO2. (c) Infiltrated opal with adhesive copper tape placed on top. (d) Lift off of the infiltrated opal from the substrate. (e) The infiltrated opal is turned over so that the flat opal-terminated surface is on top. (f) Calcined TiO2 inverse opal with porous surface (adapted from Galusha et al. 2008).

Electrodeposition is a more complicated method requiring that the sacrificial template is deposited onto a conductive substrate, but it allows a better control of Ti4+ hydrolysis and a superior filling of the pores of the template. The potential of the conductive glass that supports the template is brought to negative potential to reduce nitrate present in solution according to the reaction:

$$\mathrm{NO}\_3^\cdot + 6\,\mathrm{H\_2O} + 8\,\mathrm{e} \to \mathrm{NH\_3} + 9\,\mathrm{OH^\cdot} \tag{8}$$

The OH- produced causes a local increase of pH and the precipitation of TiO2+ from the precursor solution. In this way the TiO2 deposition is compact and finely tunable varying the concentration of NO3- , the applied current and the deposition time (Y. Xu et al. 2008). A similar technique is electrophoresis. The method can infiltrate a TiO2 colloid constituted by charged particles, and according to Gu et al. (Z.-Z. Gu et al. 2001), the deposition can be fast and uniform.

precursors with large tendency to hydrolyze rapidly are used, because the amount of deposited TiO2 is difficult to control (Wei et al. 2011). In some cases the deposition of an extra layer of bulk TiO2 over the inverse opal can be turned into an advantage with the lift-off/turnover method (Fig. 12). Depending on the application both a self standing TiO2 inverse opal or a Bragg mirror behind a TiO2 active layer can be created (Galusha et al. 2008). To avoid dealing with violent hydrolysis it is possible to introduce TiO2 in the template in the form of nanoparticle already hydrolyzed (Yip et al. 2008; Shin et al. 2011). In this case the difficulty is the preparation of sufficiently small particles capable to penetrate into the pores of the opal template, because the resulting infiltration will be poor if the nanoparticles are too big, and the

Turn over

Calcination

e)

f)

inverse opal structure will collapse after template removal.

c) Copper tape

with porous surface (adapted from Galusha et al. 2008).


according to the reaction:

the concentration of NO3

and uniform.

a)

b)

Infiltration

Lift off

d)

Fig. 12. Schematic representation of the "lift-off/turn-over" technique. (a) polystyrene opal template. (b) Infiltrated opal with TiO2. (c) Infiltrated opal with adhesive copper tape placed on top. (d) Lift off of the infiltrated opal from the substrate. (e) The infiltrated opal is turned over so that the flat opal-terminated surface is on top. (f) Calcined TiO2 inverse opal

Electrodeposition is a more complicated method requiring that the sacrificial template is deposited onto a conductive substrate, but it allows a better control of Ti4+ hydrolysis and a superior filling of the pores of the template. The potential of the conductive glass that supports the template is brought to negative potential to reduce nitrate present in solution

 NO3- + 6 H2O + 8 e-→ NH3 + 9 OH- (8) The OH- produced causes a local increase of pH and the precipitation of TiO2+ from the precursor solution. In this way the TiO2 deposition is compact and finely tunable varying

similar technique is electrophoresis. The method can infiltrate a TiO2 colloid constituted by charged particles, and according to Gu et al. (Z.-Z. Gu et al. 2001), the deposition can be fast

, the applied current and the deposition time (Y. Xu et al. 2008). A

Spectacular structures (Fig. 13) can be obtained with infiltration by atomic layer deposition. The technique allows a fine control of the amount of deposited TiO2, the filling of the pores of the template is optimal and, as a consequence, the synthesized TiO2 inverse opals are very resistant. The drawbacks are the slowness and the cost of the needed equipment, which cannot be considered a standard facility of every laboratory (King et al. 2005; L. Liu et al. 2011).

Infiltration can also be carried out by spin-coating, the method is fast and the produced TiO2 inverse opals are regular and characterized by smooth surfaces (Matsushita et al. 2007).

Fig. 13. SEM micrograph of the fractured surface of a TiO2 inverse opal produced by atomic layer deposition (Copyright (2005) Wiley. Used with permission from King at al. 2005)

TiO2 inverse opals can be obtained also in only one synthetic step, in which the silica or polymeric colloid, that will build the colloidal crystal template, is mixed together with a TiO2 colloid that will occupy the interstices of the colloidal crystal structure (Meng et al. 2002). For this reason TiO2 particles have to be one or two orders of magnitude smaller than the silica or polymer particles. The presence of smaller TiO2 particles forces the larger particles to self assembly in close packing. In this way the volume available for the diffusion of the small particles increases, with an overall gain in entropy for the entire system (Yodh et al. 2001).

Beyond the classic filling of TiO2 inverse opal, in which TiO2 occupies the residual volume of the former opal template, a structure also called shell structure or residual volume structure, there are synthetic procedures that can build TiO2 inverse opals with a skeleton structure (Dong et al. 2003) or with a fractal distribution of the pores (Ramiro-Manzano et al. 2007).

## **5. Improved photochemistry on TiO2 inverse opals**

Although the unique properties of TiO2 inverse opals and their possible applications for improved photochemistry are many, surprisingly the first demonstration of better use of light on TiO2 inverse opals dates back only in 2006 (Ren et al. 2006). Before they received attention as back reflector in dye sensitized solar cells (Nishimura et al. 2003; Halaoui et al. 2005; Somani et al. 2005), or in fundamental studies (N. P. Johnson et al. 2001; Schroden et al. 2002; Dong et al. 2003). In that seminal work Ren et al. demonstrated that TiO2 inverse opal

Improved Photochemistry of TiO2 Inverse Opals and some Examples 77

To elucidate the reason of this behaviour Chen et al. (J. I. L. Chen et al. 2006) synthesized TiO2 inverse opals with different pore size, and hence with different position of the photonic pseudo gaps (Fig. 15). The authors observed that with narrow spectrum irradiation (370 ± 10 nm) at the red edge of the photonic pseudo gap the degradation of methylene blue was significantly accelerated with respect to nanocrystalline TiO2, whereas when irradiation was carried out at wavelengths of the photonic pseudo gap the degradation was even slower than in the presence of the reference nanocrystalline TiO2 photocatalyst (Fig. 15). These evidences suggest that the better activity arises from the exploitation of slow photons, which is maximized when the wavelength used for the irradiation falls at the red edge of the photonic pseudo gap, whereas porosity and the improved mass transfer of the species cannot explain such important variations in activity as a consequence of the small difference

The same authors studied also the effect of the disorder on the activity of TiO2 inverse opals (J. I. L. Chen et al. 2007). They found that such systems can tolerate a certain disorder, but the addition in the polymer template of up to 40% of particles with a different diameter (up

Fig. 16. SEM micrographs of TiO2 inverse opals obtained from PS templates with particle dimensions (1-x)150-x180 nm, with x=0.13 (a and b), 0.37 (c), and 0.57 (d) Reprinted with permission from J.I.L. Chen et al. 2007 Copyright (2007) American Chemical Society.

Nevertheless, partially disordered inverse opals keep an enhancement factor of 1.6, calculated as the activity ratio between TiO2 inverse opal and nanocrystalline TiO2, while the well monodispersed material can attain an enhancement factor of 2.3. This is an

to 20% different, Fig. 16) leads to a significant loss in activity.

in the pore sizes used.

with photonic pseudo gap in the UV is more efficient than P25 TiO2 in the photodegradation of 1,2-dichlorobenzene in the gas phase (Fig. 14). They also found that the rate constant for the degradation of the pollutant is proportional to the radiant flux intensity even at intensities higher than 25 mW cm-2, whereas usually for TiO2 the rate constant is proportional to the square root of the radiant flux intensity (Minero 1999).

Fig. 14. Normalized concentrations of 1,2-dichlorobenzene as a function of the irradiation time in the presence of P25 TiO2 powder (closed circles) and TiO2 inverse opal (PBG TiO2, open circles) Reprinted with permission from Ren et al. 2006 Copyright (2006) American Chemical Society.

Fig. 15. Left: Reflectance spectra of TiO2 inverse opals with photonic pseudo gaps located at 500, 430, 370, 345, 325, 300, and 280 nm (solid lines), and extinction spectra of nanocrystalline TiO2 (black dashed line) and methylene blue (gray dashed line). For clarity the spectra have been displaced in the vertical axis. The highlighted region indicates the wavelengths used during irradiation. Right: Logarithmic plot of the photodegradation of methylene blue showing the first-order decay rate for nanocrystalline TiO2 (nc-TiO2) and TiO2 inverse opals with photonic pseudo gaps at 345, 370, and 500 nm. Mesoporous SiO2 (meso-SiO2) was used as the blank (Copyright (2006) Wiley. Used with permission from J.I.L. Chen et al. 2006)

with photonic pseudo gap in the UV is more efficient than P25 TiO2 in the photodegradation of 1,2-dichlorobenzene in the gas phase (Fig. 14). They also found that the rate constant for the degradation of the pollutant is proportional to the radiant flux intensity even at intensities higher than 25 mW cm-2, whereas usually for TiO2 the rate constant is

Fig. 14. Normalized concentrations of 1,2-dichlorobenzene as a function of the irradiation time in the presence of P25 TiO2 powder (closed circles) and TiO2 inverse opal (PBG TiO2, open circles) Reprinted with permission from Ren et al. 2006 Copyright (2006) American

Fig. 15. Left: Reflectance spectra of TiO2 inverse opals with photonic pseudo gaps located at

nanocrystalline TiO2 (black dashed line) and methylene blue (gray dashed line). For clarity the spectra have been displaced in the vertical axis. The highlighted region indicates the wavelengths used during irradiation. Right: Logarithmic plot of the photodegradation of methylene blue showing the first-order decay rate for nanocrystalline TiO2 (nc-TiO2) and TiO2 inverse opals with photonic pseudo gaps at 345, 370, and 500 nm. Mesoporous SiO2 (meso-SiO2) was used as the blank (Copyright (2006) Wiley. Used with permission from

500, 430, 370, 345, 325, 300, and 280 nm (solid lines), and extinction spectra of

Chemical Society.

J.I.L. Chen et al. 2006)

proportional to the square root of the radiant flux intensity (Minero 1999).

To elucidate the reason of this behaviour Chen et al. (J. I. L. Chen et al. 2006) synthesized TiO2 inverse opals with different pore size, and hence with different position of the photonic pseudo gaps (Fig. 15). The authors observed that with narrow spectrum irradiation (370 ± 10 nm) at the red edge of the photonic pseudo gap the degradation of methylene blue was significantly accelerated with respect to nanocrystalline TiO2, whereas when irradiation was carried out at wavelengths of the photonic pseudo gap the degradation was even slower than in the presence of the reference nanocrystalline TiO2 photocatalyst (Fig. 15). These evidences suggest that the better activity arises from the exploitation of slow photons, which is maximized when the wavelength used for the irradiation falls at the red edge of the photonic pseudo gap, whereas porosity and the improved mass transfer of the species cannot explain such important variations in activity as a consequence of the small difference in the pore sizes used.

The same authors studied also the effect of the disorder on the activity of TiO2 inverse opals (J. I. L. Chen et al. 2007). They found that such systems can tolerate a certain disorder, but the addition in the polymer template of up to 40% of particles with a different diameter (up to 20% different, Fig. 16) leads to a significant loss in activity.

Fig. 16. SEM micrographs of TiO2 inverse opals obtained from PS templates with particle dimensions (1-x)150-x180 nm, with x=0.13 (a and b), 0.37 (c), and 0.57 (d) Reprinted with permission from J.I.L. Chen et al. 2007 Copyright (2007) American Chemical Society.

Nevertheless, partially disordered inverse opals keep an enhancement factor of 1.6, calculated as the activity ratio between TiO2 inverse opal and nanocrystalline TiO2, while the well monodispersed material can attain an enhancement factor of 2.3. This is an

Improved Photochemistry of TiO2 Inverse Opals and some Examples 79

chance to harvest slow light Pt/TiO2 inverse opals have the same characteristics of the

An alternative strategy used to improve photochemistry on TiO2 is the doping with transition metals, in order to extend the absorption of light to the visible and to allow practical applications with solar light. Wang et al. (C. Wang et al. 2006) were successful in synthesizing TiO2, ZrO2, Ta2O5 and Zr or Ta doped TiO2 inverse opals which were tested in the photocatalytic degradation of 4-nitrophenol and Rhodamine B in aqueous solution. They demonstrated the red shift of the absorption edge of doped TiO2, and measured higher photoactivity for the doped TiO2 inverse opal samples. The better activity was attributed to the improved absorption of light due to the doping and to the porosity of the structures constituted by interconnected macropores and mesopores that allow faster migration of

> > 50

MB Residue (%)

40

0 0.5 1.0 1.5 2.0 Illumination time (h)

 Sol-gel film Inverse mixture Inverse opal

macroporous disordered Pt/TiO2 material (H. Chen et al. 2010).

0 20 40 60 80 100 Visible light irradiation time (min)

Fig. 17. Left: Methylene blue concentration as a function of the irradiation time for N,Fdoped TiO2 inverse opals with different pore dimensions (e.g. TIO201-250 refers to TiO2 inverse opal with macropore radius of 201 nm and calcined at 250°C), for a N,F doped TiO2 macroporous disordered sample (TIOMIX-250), and for N,F doped TiO2 sol–gel thin film Reprinted with permission from J.A. Xu et al. 2010 Copyright (2010) American Chemical Society. Right: Percent residual methylene blue as a function of the irradiation time (>400 nm) for nitrogen-doped TiO2 inverse opal (macropore radius ≈ 280 nm), nitrogendoped TiO2 macroporous disordered structure (inverse mixture), and nitrogen-doped TiO2 sol–gel thin film (Copyright (2008) Wiley. Used with permission from Q. Li & Shang 2008)

To extend the absorption of TiO2 into the visible doping with nitrogen, carbon, sulphur or fluorine is usually adopted. In this case the absorption in the visible is due to colour centres and not to a narrowing of valence and conduction band (Serpone 2006). The doping with nitrogen (Q. Li & Shang 2008) and with nitrogen and fluorine (J. A. Xu et al. 2010) of TiO2 inverse opal led to an improved photocatalytic degradation of methylene blue in water with visible light, even if in the case of nitrogen and fluorine codoping the activity of the TiO2 inverse opals is only slightly higher than in the case of a macroporous disordered TiO2. This is probably due to a non optimal choice of the photonic band gap position, although in the case of nitrogen doping the improvement from disordered to ordered structure is significant (Fig. 17). The photodegradation of methylene blue with visible light alone is not sufficient to demonstrate that doped TiO2 can effectively absorb visible photons with the consequent

photogenerated electrons and holes.

 Blank TF-250 TIO201-250 TIO228-250 TIO315-250 TIO228-350 TIOMIX-250

1.0

0.8

C/C0

0.6

0.4

important result because it shows that the light scattering is not crucial in increasing the efficiency of these materials, and that the unavoidable imperfections in the periodic structure do not prevent the practical applications in environmental remediation or water purification.

An improved degradation of methylene blue on TiO2 inverse opals under UV irradiation was also observed (Srinivasan & White 2007), but in this case the better efficiency was explained in terms of better diffusion of the species due to the porosity of the material and to the large surface area. This interpretation was supported also by Chen & Ozin in a 2009 paper (J. I. L. Chen & Ozin 2009), where they partially revised the conclusions of their previous works, limiting the effectiveness of slow light only in the case of TiO2 inverse opals with high fill factors.

To clarify the relative importance of slow light, light scattering and improved mass transfer due to the porosity Sordello et al. performed the photocatalytic degradation of phenol at two different wavelengths on TiO2 inverse opal and TiO2 disordered macroporous powders (Sordello et al. 2011a). The wavelengths used were 365 nm, where, for the TiO2 inverse opal employed, the slow photon effect was maximized, and 254 nm, where, on the contrary, the slow light was negligible. At 365 nm the inverse opal is four times more active than the disordered macroporous structure. The key experiment showed that the pristine inverse opal powder is four times more active than the inverse opal crushed in a mortar, which has lost its periodicity in three dimensions. These differences vanish irradiating at 254 nm, as at that wavelength the three powders show almost the same activity. These evidences suggest that slow light plays an important role in increasing the absorption of light of TiO2 inverse opals and hence in improving their photoactivity. The photoelectrochemical study of TiO2 inverse opals confirmed the better light absorption of these materials when slow photons are involved. Furthermore, it was evidenced a faster electron transfer to the oxygen present in solution with respect to disordered macroporous TiO2 (Sordello et al. 2011b).

The better absorption of light, the porosity and the high surface area derive from the structuration of these materials and can be coupled with other physical and chemical modifications to boost efficiency of a great variety of photoreactions. A frequent modification is the addition of metallic platinum to improve the separation of the photogenerated charge carriers and reduce recombination (Kraeutler & Bard 1978). The addition of platinum to TiO2 inverse opals leads to a significant improvement of the photoactivity (J. I. L. Chen et al. 2008). The activity ratio between platinised inverse opal and nanocrystalline TiO2 is four, whereas a lower value (around 2.5) should be expected considering the activity ratios of TiO2 inverse opal (1.7) and platinised nanocrystalline TiO2 (1.8) alone, evidencing that there is a cooperation between slow light and platinisation. In summary the total effect is not merely the sum of the two contributions (J. I. L. Chen et al. 2008). A different approach consists in synthesizing the metallic platinum inverse opal first, and coating it with TiO2 in a second step (H. Chen et al. 2010). The recombination of photogenerated charge carriers is effectively reduced by the Schottky junction Pt/TiO2 as the platinised samples show higher photocurrents and faster photodegradation rate for aqueous phenol. Moreover, Pt/TiO2 inverse opal with properly tuned photonic pseudo gap has even higher photocurrents and photocatalytic activity with respect to inverse opals that cannot exploit slow light because the irradiation wavelength does not correspond to photonic modes with slow group velocity. It is also interesting to note that without the

important result because it shows that the light scattering is not crucial in increasing the efficiency of these materials, and that the unavoidable imperfections in the periodic structure do not prevent the practical applications in environmental remediation or water

An improved degradation of methylene blue on TiO2 inverse opals under UV irradiation was also observed (Srinivasan & White 2007), but in this case the better efficiency was explained in terms of better diffusion of the species due to the porosity of the material and to the large surface area. This interpretation was supported also by Chen & Ozin in a 2009 paper (J. I. L. Chen & Ozin 2009), where they partially revised the conclusions of their previous works, limiting the effectiveness of slow light only in the case of TiO2 inverse opals

To clarify the relative importance of slow light, light scattering and improved mass transfer due to the porosity Sordello et al. performed the photocatalytic degradation of phenol at two different wavelengths on TiO2 inverse opal and TiO2 disordered macroporous powders (Sordello et al. 2011a). The wavelengths used were 365 nm, where, for the TiO2 inverse opal employed, the slow photon effect was maximized, and 254 nm, where, on the contrary, the slow light was negligible. At 365 nm the inverse opal is four times more active than the disordered macroporous structure. The key experiment showed that the pristine inverse opal powder is four times more active than the inverse opal crushed in a mortar, which has lost its periodicity in three dimensions. These differences vanish irradiating at 254 nm, as at that wavelength the three powders show almost the same activity. These evidences suggest that slow light plays an important role in increasing the absorption of light of TiO2 inverse opals and hence in improving their photoactivity. The photoelectrochemical study of TiO2 inverse opals confirmed the better light absorption of these materials when slow photons are involved. Furthermore, it was evidenced a faster electron transfer to the oxygen present

in solution with respect to disordered macroporous TiO2 (Sordello et al. 2011b).

The better absorption of light, the porosity and the high surface area derive from the structuration of these materials and can be coupled with other physical and chemical modifications to boost efficiency of a great variety of photoreactions. A frequent modification is the addition of metallic platinum to improve the separation of the photogenerated charge carriers and reduce recombination (Kraeutler & Bard 1978). The addition of platinum to TiO2 inverse opals leads to a significant improvement of the photoactivity (J. I. L. Chen et al. 2008). The activity ratio between platinised inverse opal and nanocrystalline TiO2 is four, whereas a lower value (around 2.5) should be expected considering the activity ratios of TiO2 inverse opal (1.7) and platinised nanocrystalline TiO2 (1.8) alone, evidencing that there is a cooperation between slow light and platinisation. In summary the total effect is not merely the sum of the two contributions (J. I. L. Chen et al. 2008). A different approach consists in synthesizing the metallic platinum inverse opal first, and coating it with TiO2 in a second step (H. Chen et al. 2010). The recombination of photogenerated charge carriers is effectively reduced by the Schottky junction Pt/TiO2 as the platinised samples show higher photocurrents and faster photodegradation rate for aqueous phenol. Moreover, Pt/TiO2 inverse opal with properly tuned photonic pseudo gap has even higher photocurrents and photocatalytic activity with respect to inverse opals that cannot exploit slow light because the irradiation wavelength does not correspond to photonic modes with slow group velocity. It is also interesting to note that without the

purification.

with high fill factors.

chance to harvest slow light Pt/TiO2 inverse opals have the same characteristics of the macroporous disordered Pt/TiO2 material (H. Chen et al. 2010).

An alternative strategy used to improve photochemistry on TiO2 is the doping with transition metals, in order to extend the absorption of light to the visible and to allow practical applications with solar light. Wang et al. (C. Wang et al. 2006) were successful in synthesizing TiO2, ZrO2, Ta2O5 and Zr or Ta doped TiO2 inverse opals which were tested in the photocatalytic degradation of 4-nitrophenol and Rhodamine B in aqueous solution. They demonstrated the red shift of the absorption edge of doped TiO2, and measured higher photoactivity for the doped TiO2 inverse opal samples. The better activity was attributed to the improved absorption of light due to the doping and to the porosity of the structures constituted by interconnected macropores and mesopores that allow faster migration of photogenerated electrons and holes.

Fig. 17. Left: Methylene blue concentration as a function of the irradiation time for N,Fdoped TiO2 inverse opals with different pore dimensions (e.g. TIO201-250 refers to TiO2 inverse opal with macropore radius of 201 nm and calcined at 250°C), for a N,F doped TiO2 macroporous disordered sample (TIOMIX-250), and for N,F doped TiO2 sol–gel thin film Reprinted with permission from J.A. Xu et al. 2010 Copyright (2010) American Chemical Society. Right: Percent residual methylene blue as a function of the irradiation time (>400 nm) for nitrogen-doped TiO2 inverse opal (macropore radius ≈ 280 nm), nitrogendoped TiO2 macroporous disordered structure (inverse mixture), and nitrogen-doped TiO2 sol–gel thin film (Copyright (2008) Wiley. Used with permission from Q. Li & Shang 2008)

To extend the absorption of TiO2 into the visible doping with nitrogen, carbon, sulphur or fluorine is usually adopted. In this case the absorption in the visible is due to colour centres and not to a narrowing of valence and conduction band (Serpone 2006). The doping with nitrogen (Q. Li & Shang 2008) and with nitrogen and fluorine (J. A. Xu et al. 2010) of TiO2 inverse opal led to an improved photocatalytic degradation of methylene blue in water with visible light, even if in the case of nitrogen and fluorine codoping the activity of the TiO2 inverse opals is only slightly higher than in the case of a macroporous disordered TiO2. This is probably due to a non optimal choice of the photonic band gap position, although in the case of nitrogen doping the improvement from disordered to ordered structure is significant (Fig. 17). The photodegradation of methylene blue with visible light alone is not sufficient to demonstrate that doped TiO2 can effectively absorb visible photons with the consequent

Improved Photochemistry of TiO2 Inverse Opals and some Examples 81

synthesis of chemicals. The possibility of better light absorption of inverse opals is very promising to improve the efficiency of light driven reactions for a great variety of implementations, provided that knowledge and competences are transferred among different fields. This is a necessary condition to produce complex systems that take

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advantage of cooperative effects.

**7. References** 

production of holes and electrons that can respectively react with the organic dye and molecular oxygen. According to Zhao et al. (Zhao et al. 2002) the disappearance of the dye is possible under visible light irradiation, without the need of photon absorption by TiO2. In this case a dye sensitization occurs due to the electron injection from the excited dye (S\*) to the TiO2 conduction band (equations 9 and 10):

$$\mathbf{S} + \mathbf{h}\mathbf{v} \to \mathbf{S}^\* \tag{9}$$

$$\mathbf{S}^\* \rightharpoonup \mathbf{e}^\* \mathbf{c}\_{\mathbf{C}\mathbf{B}} + \mathbf{S}^\* \tag{10}$$

The oxidized dye S+ can directly react with oxygen:

$$\rm S^\* \star O\_2 \rightarrow SO\_2^\* \tag{11}$$

whereas the conduction band electron can react with the dissolved oxygen to yield several reactive oxygen species (Minero & Vione 2006 and references therein):

$$\rm O\_2 + e^\cdot \\ \rm C\_{CB} \to O\_2^\cdot \tag{12}$$

$$\rm O\_2{}^+ + H^+ \to HO\_2{}^{\cdot} \tag{13}$$

$$\text{2 HO}\_2\text{}\rightarrow\text{O}\_2\text{} + \text{H}\_2\text{O}\_2\tag{14}$$

$$\rm O\_2{}^+ + HO\_2{}^- \rightarrow O\_2 + HO\_2{}^- \tag{15}$$

$$\text{H}\_2\text{O}\_2 + \text{e}\_\text{CB} \rightarrow \text{HO}^\cdot + \text{HO}^\cdot \tag{16}$$

$$\rm H\_2O\_2 + HO \rightarrow HO\_2 + H\_2O \tag{17}$$

These species can react with the adsorbed dye and carry out its degradation up to CO2 and water. In the case of dye molecules adsorbed on the internal surfaces of a TiO2 inverse opal with properly tuned photonic band gap, dye absorption is enhanced and its degradation improved. This mechanism has been hypothesized to account for the degradation of crystal violet on undoped TiO2 inverse opal under visible irradiation (Y. Li et al. 2006). The role of the inverse opal is to slow down the photons absorbed by the dye to increase the interaction of light with organic compounds.

TiO2 inverse opals were also coupled to metallic copper in the photoreduction of CO2 to methanol in the presence of water vapour and UV light (Ren & Valsaraj 2009). With respect to nanocrystalline TiO2, on inverse opals the reaction proceeded also at lower light intensities, with a rate dependence on light intensity I0.74 with respect to nanocrystalline TiO2 that showed a dependence I0.20.

### **6. Conclusion**

TiO2 inverse opals and their role in improving the efficiency of photochemical reactions have been presented. The physical origin of the photonic band gap and of slow light has been discussed together with the synthetic routes to obtain such structures. Some applications and practical examples of improved photochemistry on TiO2 inverse opals have been reviewed, demonstrating how such materials can help photocatalysis to be competitive in solar energy recovery, environmental remediation, water purification and also in the synthesis of chemicals. The possibility of better light absorption of inverse opals is very promising to improve the efficiency of light driven reactions for a great variety of implementations, provided that knowledge and competences are transferred among different fields. This is a necessary condition to produce complex systems that take advantage of cooperative effects.

### **7. References**

80 Molecular Photochemistry – Various Aspects

production of holes and electrons that can respectively react with the organic dye and molecular oxygen. According to Zhao et al. (Zhao et al. 2002) the disappearance of the dye is possible under visible light irradiation, without the need of photon absorption by TiO2. In this case a dye sensitization occurs due to the electron injection from the excited dye (S\*) to

whereas the conduction band electron can react with the dissolved oxygen to yield several

CB → HO·

These species can react with the adsorbed dye and carry out its degradation up to CO2 and water. In the case of dye molecules adsorbed on the internal surfaces of a TiO2 inverse opal with properly tuned photonic band gap, dye absorption is enhanced and its degradation improved. This mechanism has been hypothesized to account for the degradation of crystal violet on undoped TiO2 inverse opal under visible irradiation (Y. Li et al. 2006). The role of the inverse opal is to slow down the photons absorbed by the dye to increase the interaction

TiO2 inverse opals were also coupled to metallic copper in the photoreduction of CO2 to methanol in the presence of water vapour and UV light (Ren & Valsaraj 2009). With respect to nanocrystalline TiO2, on inverse opals the reaction proceeded also at lower light intensities, with a rate dependence on light intensity I0.74 with respect to nanocrystalline

TiO2 inverse opals and their role in improving the efficiency of photochemical reactions have been presented. The physical origin of the photonic band gap and of slow light has been discussed together with the synthetic routes to obtain such structures. Some applications and practical examples of improved photochemistry on TiO2 inverse opals have been reviewed, demonstrating how such materials can help photocatalysis to be competitive in solar energy recovery, environmental remediation, water purification and also in the

S + h → S\* (9)

CB + S+ (10)

CB → O2·- (12)

2 HO2· → O2 + H2O2 (14)

+ (11)

(13)

(15)

+ HO- (16)

+ H2O (17)

the TiO2 conduction band (equations 9 and 10):

S\* → e-

O2 + e-

H2O2 + e-

of light with organic compounds.

TiO2 that showed a dependence I0.20.

**6. Conclusion** 

The oxidized dye S+ can directly react with oxygen:

S+ + O2 → SO2

O2·- + H+ → HO2·

O2·- + HO2· → O2 + HO2-

H2O2 + HO· → HO2·

reactive oxygen species (Minero & Vione 2006 and references therein):


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**Part 3** 

**Photochemistry in Biology** 

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**5** 

*Japan* 

**Photo-Induced Proton Transfers** 

*1Faculty of Advanced Life Science, Hokkaido University, 2College of Pharmaceutical Sciences, Matsuyama University,* 

Takashi Kikukawa1, Jun Tamogami2, Kazumi Shimono2, Makoto Demura1, Toshifumi Nara2 and Naoki Kamo2

Microbial rhodopsins are photoactive membrane proteins that are widely distributed over the microbial world. They commonly consist of seven transmembrane helices forming an internal pocket for a chromophore retinal, whose photo-induced isomerization triggers the respective photochemical reactions of these proteins. They are generally classified into photosensors or ion-pumps, but the members of both classes have individualities. In the case of photosensors, there exist a variety of signal-transduction modes, including interaction with other membrane proteins, interaction with cytoplasmic proteins, and lightgated ion channel activity. For ion-pumps, there exist outwardly directed H+ pumps and inwardly directed Cl- pumps. In spite of these functional diversities, most microbial rhodopsins show photo-induced proton-transfer reactions among amino acid residues and the external medium. These reactions reflect the p*K*a changes of some residues induced by the protein conformational changes during the respective photochemical reactions. To analyze these reactions, it is indispensable to detect the small pH changes of the external medium due to the proton release/uptake during the photoreaction cycles. Electrochemical cells using indium-tin oxide (ITO) or tin oxide (SnO2) transparent electrodes are a powerful and convenient tool that enables such measurement in external media under a variety of pH conditions (Robertson & Lukashev, 1995; Wang et al., 1997; Koyama et al., 1998a; Tamogami et al., 2009; Wu et al., 2009). Here, we will describe the rapidly expanding family of the

microbial rhodopsins and the application of the ITO method to their photoresponses.

Rhodopsin is a protein in the retina of animals that works as a light sensor. Rhodopsin contains retinal (vitamin A aldehyde) as a chromophore and its absorption maximum is ~500 nm. Retinal binds with a specific lysine residue *via* a protonated Schiff base. Absorption of photons induces the isomerization of 11-*cis* to all-*trans* retinal and subsequent conformational changes including the deprotonation of the Schiff base (J.L. Spudich et al., 2000). Rhodopsin had been considered to be confined to animals until the findings from

**2. Visual rhodopsins and archaeal rhodopsins** 

**1. Introduction** 

haloarchaea.

**of Microbial Rhodopsins** 

## **Photo-Induced Proton Transfers of Microbial Rhodopsins**

Takashi Kikukawa1, Jun Tamogami2, Kazumi Shimono2, Makoto Demura1, Toshifumi Nara2 and Naoki Kamo2 *1Faculty of Advanced Life Science, Hokkaido University, 2College of Pharmaceutical Sciences, Matsuyama University, Japan* 

### **1. Introduction**

Microbial rhodopsins are photoactive membrane proteins that are widely distributed over the microbial world. They commonly consist of seven transmembrane helices forming an internal pocket for a chromophore retinal, whose photo-induced isomerization triggers the respective photochemical reactions of these proteins. They are generally classified into photosensors or ion-pumps, but the members of both classes have individualities. In the case of photosensors, there exist a variety of signal-transduction modes, including interaction with other membrane proteins, interaction with cytoplasmic proteins, and lightgated ion channel activity. For ion-pumps, there exist outwardly directed H+ pumps and inwardly directed Cl pumps. In spite of these functional diversities, most microbial rhodopsins show photo-induced proton-transfer reactions among amino acid residues and the external medium. These reactions reflect the p*K*a changes of some residues induced by the protein conformational changes during the respective photochemical reactions. To analyze these reactions, it is indispensable to detect the small pH changes of the external medium due to the proton release/uptake during the photoreaction cycles. Electrochemical cells using indium-tin oxide (ITO) or tin oxide (SnO2) transparent electrodes are a powerful and convenient tool that enables such measurement in external media under a variety of pH conditions (Robertson & Lukashev, 1995; Wang et al., 1997; Koyama et al., 1998a; Tamogami et al., 2009; Wu et al., 2009). Here, we will describe the rapidly expanding family of the microbial rhodopsins and the application of the ITO method to their photoresponses.

### **2. Visual rhodopsins and archaeal rhodopsins**

Rhodopsin is a protein in the retina of animals that works as a light sensor. Rhodopsin contains retinal (vitamin A aldehyde) as a chromophore and its absorption maximum is ~500 nm. Retinal binds with a specific lysine residue *via* a protonated Schiff base. Absorption of photons induces the isomerization of 11-*cis* to all-*trans* retinal and subsequent conformational changes including the deprotonation of the Schiff base (J.L. Spudich et al., 2000). Rhodopsin had been considered to be confined to animals until the findings from haloarchaea.

Photo-Induced Proton Transfers of Microbial Rhodopsins 91

To distinguish them from rhodopsins in the microbial world, the rhodopsins in animals are

Studies on BR, HR, SRI and SRII have shown that two amino acid residues corresponding to Asp85 and Asp96 in BR are key residues for the functional difference among these archaeal rhodopsins. As mentioned above, the Schiff bases of BR and two SRs become deprotonated during the early halves of the photocycles. Asp85BR and the corresponding aspartates of SRs function as the proton acceptors from the Schiff bases. For HR, on the other hand, Asp85BR is

the protonated Schiff base. The other residue, Asp96BR, functions as a proton donor for the reprotonation process of the Schiff base and contributes to acceleration of the turnover rate of the photocycle. This residue is not conserved in the two SRs and consequently their photocycles are much slower than that of BR. HR also undergoes a fast photocycle in the manner of BR but does not conserve this aspartate, because HR undergoes its photocycle

On the basis of these findings, the physiological functions of newly found microbial rhodopsins have been deduced from the conservation states of the residues corresponding to Asp85BR and Asp96BR. In addition to these clues, the functions of some proteins have been confirmed experimentally by using the purified proteins and/or by observation of their photo-induced behaviors. From these analyses, the newly found microbial rhodopsins are now categorized as either ion-pumps (H+ or Cl-) or photoreceptors. In paticular, many newly found pigments are categorized as H+ pumps. This fact suggests that the utilization of light energy *via* H+ pumps is widely adopted in the microbial world. For many H+ pumps, the pumping activities have been experimentally confirmed. Representative examples include proteorhodopsin (PR) from proteobacteria living throughout the world's oceans (Béjà et al., 2000); xanthorhodopsin (XR) from *Salinibacter ruber*, a highly halophilic eubacterium (Balashov et al., 2005); *Leptosphaeria* rhodopsin (LR) from *Leptosphaeria maculans,* a fungal pathogen to a plant (Waschuk et al., 2005); *Gloeobacter* rhodopsin (GR) from *Gloeobacter violaceus,* a cyanobacterium living in fresh water (Miranda et al., 2009); and *Acetabularia* rhodopsin (AR) from a gigantic unicellular marine algae, *Acetabularia acetabulum,* which reaches to 10 cm in height (Tsunoda et al., 2006). Recently, two clones of AR, named ARI and ARII, were isolated from the same organism (Lee et al., 2010). These were somewhat different from the original AR. For ARII, the H+-pumping activity was confirmed using *Xenopus* oocytes and the detailed photochemistry was examined with the help of a cell-free expression system (Wada et al., 2011; Kikukawa et al., 2011). On the other hand, photosensing rhodopsins have also been found in bacteria and eukarya. Although these represent only a minority of the newly found microbial rhodopsins, they exhibit a variety of signal-transduction mechanisms. Two SRs in the archaeal membrane relay the photosignals to the cognate transducer proteins embedded in the membrane. From the eubacterium *S. ruber*, SRI itself was found and has been extensively characterized (Kitajima-Ihara et al., 2008). Unlike these SRs, the photosensor called *Anabaena* sensory rhodopsin (ASR) from *Anabaena sp.* PCC7120, a cyanobacterium living in fresh water, is considered to relay the signal to the soluble protein (Jung et al., 2003). *Anabaena* does not have a flagellum and so does not show the phototaxis. Instead, *Anabaena* shows a photoresponse called chromatic adaptation. Thus, it is considered that ASR controls the biosynthesis of chromoproteins forming the light-harvesting complex. In addition to these, a new type of

, a transportable ion, to the vicinity of the Thr and

called type 2 rhodopsins.

replaced by Thr, and thus HR can bind Cl-

without the deprotonation of the Schiff base.

In the early 1970s, however, a retinal protein was discovered in the membrane of the highly halophilic archaeon *Halobacterium salinarum* (formally *halobium*). The natural habitats of *H. salinarum* are the Dead Sea, the Great Salt Lake and salt ponds. The newly discovered rhodopsin was named bacteriorhodopsin (BR), and it was shown to act as a light-driven proton pump (Oesterhelt & Stoeckenius, 1971). By using light energy, BR transports a proton from a cytoplasmic to an extracellular space, which produces the protonelectrochemical potential difference across the membranes, which in turn drives the synthesis of ATP *via* H+-ATPase. Light irradiation to BR induces the retinal isomerization from all-*trans* to 13-*cis*, while the isomerization of visual rhodopsins is from 11-*cis* to all*trans*. In addition, the most significant difference from the visual rhodopsins is the existence of a so-called photocycle: light absorption leads to the excitation of the pigment, which decays to the original pigment *via* a variety of photo-intermediates. This linear cyclic photochemistry is called a photocycle. On the other hand, for most visual rhodopsins, the Schiff base linkage with the retinal is disrupted as a consequence of the photochemical reaction. The proton-transfer mechanism of BR has been intensively investigated so far. During the photocycle, the protonated Schiff base affords its proton to the extracellular space and receives another proton from the other side, *i.e.*, the cytoplasmic space. Thus, this cycle involves the alternation between the protonated and deprotonated states of the Schiff base.

Later, three additional retinal proteins were discovered in *H. salinarum.* These were halorhodopsin (HR) (Matsuno-Yagi & Mukohata, 1977; Schobert & Lanyi, 1982; Mukohata et al., 1999; Váró et al., 2000; Essen, 2002), sensory rhodopsin I (SRI) (Bogomolni & J.L Spudich, 1982; Hazemoto et al., 1983; J.L Spudich & Bogomolni, 1984) and sensory rhodopsin II (SRII, also called phoborhodopsin) (Takahashi et al., 1985; Tomioka et al., 1986; Wolff et al., 1986; E.N. Spudich et al., 1986; Marwan & Oesterhelt, 1987). BR and HR are light-driven ion pumps: HR is an inwardly directed Cl- -pump and BR is an outwardly directed H+-pump as described above. On the other hand, SRI and SRII act as receptors of phototaxis. In the cell membranes, these receptors form firm complexes with their cognate transducers. By utilization of these sensing systems, the cell moves toward light of the preferred wavelength (λ > 520 nm) where BR and HR can work, and escapes from the shorter wavelength light (λ < 520 nm) which may contain dangerous UV light. These retinal proteins also exhibit their own functions during the respective photocycles. Under the physiological states, HR and two SRs do not exhibit the proton-pumping activities. However, the photocycles of two SRs involve alterations between the protonated and deprotonated states of the Schiff bases, resulting in the proton releases and uptakes at the extracellular sides (Bogomolni et al., 1994; Sasaki & J.L. Spudich, 1999, 2000).

### **3. Microbial rhodopsins**

About 30 years after the discovery of BR, archaeal rhodopsin homologues began to be identified in various microorganisms, including proteobacteria, cyanobacteria, fungi, dinoflagellates, and alga (J.L. Spudich & Jung, 2005). Thus, the microbial species containing the retinal protein genes inhabit a broad range of environments. At present, these rhodopsin homologues are called type 1 rhodopsins or microbial rhodopsins, and they define a large phylogenetic class spreading to all three domains of life, *i.e.,* archaea, bacteria and eukarya.

In the early 1970s, however, a retinal protein was discovered in the membrane of the highly halophilic archaeon *Halobacterium salinarum* (formally *halobium*). The natural habitats of *H. salinarum* are the Dead Sea, the Great Salt Lake and salt ponds. The newly discovered rhodopsin was named bacteriorhodopsin (BR), and it was shown to act as a light-driven proton pump (Oesterhelt & Stoeckenius, 1971). By using light energy, BR transports a proton from a cytoplasmic to an extracellular space, which produces the protonelectrochemical potential difference across the membranes, which in turn drives the synthesis of ATP *via* H+-ATPase. Light irradiation to BR induces the retinal isomerization from all-*trans* to 13-*cis*, while the isomerization of visual rhodopsins is from 11-*cis* to all*trans*. In addition, the most significant difference from the visual rhodopsins is the existence of a so-called photocycle: light absorption leads to the excitation of the pigment, which decays to the original pigment *via* a variety of photo-intermediates. This linear cyclic photochemistry is called a photocycle. On the other hand, for most visual rhodopsins, the Schiff base linkage with the retinal is disrupted as a consequence of the photochemical reaction. The proton-transfer mechanism of BR has been intensively investigated so far. During the photocycle, the protonated Schiff base affords its proton to the extracellular space and receives another proton from the other side, *i.e.*, the cytoplasmic space. Thus, this cycle involves the alternation between the protonated and deprotonated states of the Schiff

Later, three additional retinal proteins were discovered in *H. salinarum.* These were halorhodopsin (HR) (Matsuno-Yagi & Mukohata, 1977; Schobert & Lanyi, 1982; Mukohata et al., 1999; Váró et al., 2000; Essen, 2002), sensory rhodopsin I (SRI) (Bogomolni & J.L Spudich, 1982; Hazemoto et al., 1983; J.L Spudich & Bogomolni, 1984) and sensory rhodopsin II (SRII, also called phoborhodopsin) (Takahashi et al., 1985; Tomioka et al., 1986; Wolff et al., 1986; E.N. Spudich et al., 1986; Marwan & Oesterhelt, 1987). BR and HR are light-driven ion

described above. On the other hand, SRI and SRII act as receptors of phototaxis. In the cell membranes, these receptors form firm complexes with their cognate transducers. By utilization of these sensing systems, the cell moves toward light of the preferred wavelength (λ > 520 nm) where BR and HR can work, and escapes from the shorter wavelength light (λ < 520 nm) which may contain dangerous UV light. These retinal proteins also exhibit their own functions during the respective photocycles. Under the physiological states, HR and two SRs do not exhibit the proton-pumping activities. However, the photocycles of two SRs involve alterations between the protonated and deprotonated states of the Schiff bases, resulting in the proton releases and uptakes at the extracellular sides (Bogomolni et al., 1994;

About 30 years after the discovery of BR, archaeal rhodopsin homologues began to be identified in various microorganisms, including proteobacteria, cyanobacteria, fungi, dinoflagellates, and alga (J.L. Spudich & Jung, 2005). Thus, the microbial species containing the retinal protein genes inhabit a broad range of environments. At present, these rhodopsin homologues are called type 1 rhodopsins or microbial rhodopsins, and they define a large phylogenetic class spreading to all three domains of life, *i.e.,* archaea, bacteria and eukarya.


base.

pumps: HR is an inwardly directed Cl-

Sasaki & J.L. Spudich, 1999, 2000).

**3. Microbial rhodopsins** 

To distinguish them from rhodopsins in the microbial world, the rhodopsins in animals are called type 2 rhodopsins.

Studies on BR, HR, SRI and SRII have shown that two amino acid residues corresponding to Asp85 and Asp96 in BR are key residues for the functional difference among these archaeal rhodopsins. As mentioned above, the Schiff bases of BR and two SRs become deprotonated during the early halves of the photocycles. Asp85BR and the corresponding aspartates of SRs function as the proton acceptors from the Schiff bases. For HR, on the other hand, Asp85BR is replaced by Thr, and thus HR can bind Cl- , a transportable ion, to the vicinity of the Thr and the protonated Schiff base. The other residue, Asp96BR, functions as a proton donor for the reprotonation process of the Schiff base and contributes to acceleration of the turnover rate of the photocycle. This residue is not conserved in the two SRs and consequently their photocycles are much slower than that of BR. HR also undergoes a fast photocycle in the manner of BR but does not conserve this aspartate, because HR undergoes its photocycle without the deprotonation of the Schiff base.

On the basis of these findings, the physiological functions of newly found microbial rhodopsins have been deduced from the conservation states of the residues corresponding to Asp85BR and Asp96BR. In addition to these clues, the functions of some proteins have been confirmed experimentally by using the purified proteins and/or by observation of their photo-induced behaviors. From these analyses, the newly found microbial rhodopsins are now categorized as either ion-pumps (H+ or Cl- ) or photoreceptors. In paticular, many newly found pigments are categorized as H+ pumps. This fact suggests that the utilization of light energy *via* H+ pumps is widely adopted in the microbial world. For many H+ pumps, the pumping activities have been experimentally confirmed. Representative examples include proteorhodopsin (PR) from proteobacteria living throughout the world's oceans (Béjà et al., 2000); xanthorhodopsin (XR) from *Salinibacter ruber*, a highly halophilic eubacterium (Balashov et al., 2005); *Leptosphaeria* rhodopsin (LR) from *Leptosphaeria maculans,* a fungal pathogen to a plant (Waschuk et al., 2005); *Gloeobacter* rhodopsin (GR) from *Gloeobacter violaceus,* a cyanobacterium living in fresh water (Miranda et al., 2009); and *Acetabularia* rhodopsin (AR) from a gigantic unicellular marine algae, *Acetabularia acetabulum,* which reaches to 10 cm in height (Tsunoda et al., 2006). Recently, two clones of AR, named ARI and ARII, were isolated from the same organism (Lee et al., 2010). These were somewhat different from the original AR. For ARII, the H+-pumping activity was confirmed using *Xenopus* oocytes and the detailed photochemistry was examined with the help of a cell-free expression system (Wada et al., 2011; Kikukawa et al., 2011). On the other hand, photosensing rhodopsins have also been found in bacteria and eukarya. Although these represent only a minority of the newly found microbial rhodopsins, they exhibit a variety of signal-transduction mechanisms. Two SRs in the archaeal membrane relay the photosignals to the cognate transducer proteins embedded in the membrane. From the eubacterium *S. ruber*, SRI itself was found and has been extensively characterized (Kitajima-Ihara et al., 2008). Unlike these SRs, the photosensor called *Anabaena* sensory rhodopsin (ASR) from *Anabaena sp.* PCC7120, a cyanobacterium living in fresh water, is considered to relay the signal to the soluble protein (Jung et al., 2003). *Anabaena* does not have a flagellum and so does not show the phototaxis. Instead, *Anabaena* shows a photoresponse called chromatic adaptation. Thus, it is considered that ASR controls the biosynthesis of chromoproteins forming the light-harvesting complex. In addition to these, a new type of

Photo-Induced Proton Transfers of Microbial Rhodopsins 93

2003). However, the proton movement from the proton donor residue (Glu108PR corresponding to Asp96BR) to the Schiff base and its subsequent proton uptake from the CP space occur simultaneously in the M-N transition (Dioumaev et al., 2002). For BR, these two proton movements occur separately in the M-N and N-O transitions. (2) For ARII from marine algae, the photocycle includes much larger reverse reactions between L, M, N and O than are seen in BR (Kikukawa et al., 2011). Although the reverse reactions are also present in BR, their rates in ARII appear to be much larger than in BR. A rapid reverse reaction might be disadvantageous for the unidirectional ion pumping. (3) For all microbial rhodopsins, the proton conduction channel is bisected by retinal. For BR, these two channels are severely isolated and, in the dark state, the CP half channel is kept under a highly hydrophobic condition. This asymmetric structure had been believed to be important for the ion pumping function. For several proton pumps such as XR and GR, however, this characteristic structure of BR is not conserved (Luecke et al., 2008; Miranda et al., 2009). Therefore, the newly found proton pumps seem to adopt a mechanism that is at least partly different from that in BR. Thus, it would be an interesting subject to clarify the essential

Fig. 1. The photocycle scheme (A) and the structure of BR (B) with the important residues

decay. The structure in (B) was drawn from the PDB coordinate file 1C3W.

In (A), BR, K, L, M, N and O represent the unphotolyzed state and intermediates, respectively. Their λmax's are given in the subscripts, and the lifetimes of the intermediates are also shown. The photoisomerization of the retinal by illumination triggers the stepwise photoreactions accompanied with the proton movements. In (B), these proton transfers at respective steps are indicated with arrows (see section 4.1 for the details). The timing of proton transfer depends on the pH of the medium. Above pH 5, the proton release occurs in L-decay while below at about pH 5, the proton release occurs in O-decay (arrow with a broken line) instead of L-

During the photocycles of almost all photosensing rhodopsins examined so far, the deprotonations of the Schiff bases occur much as for the H+-pumping rhodopsins. In many

mechanisms for the respective proton pumps.

involved in the proton-transfer reactions.

**4.2 Photosensing rhodopsins** 

photosensing rhodopsin called channelrhodopsin was found in *Chlamydomonas reinhardtii,* a green flagellate alga (Sineshchekov et al., 2002; Nagel et al., 2002; Suzuki et al., 2003). This is a light-gated ion channel and induces the photomotile behavior of the cell. Thus, the world of microbial rhodopsins is now rapidly expanding.

### **4. The photo-induced proton transfer associated with the photocycle of microbial rhodopsins**

Microbial rhodopsins have linear cyclic photochemical reactions called photocycles. For all microbial rhodopsins examined so far, the retinal Schiff bases are protonated in their dark states under physiological conditions. Except in the case of HR, a Cl ion pump, the Schiff bases become deprotonated during the photocycles independent of H+-pumping or photosensing rhodopsins. As described below, these primary and the subsequent protontransfer reactions are closely related to the functions of both microbial rhodopsins.

### **4.1 H<sup>+</sup> -pumping rhodopsins**

The illumination of the pigment protein leads to the excited state, which is relaxed thermally to the original pigment *via* various photochemical intermediates (Fig. 1A). The best-studied rhodopsin is BR (Haupts, 1999; Balashov, 2000; Heberle, 2000; Lanyi, 2004, 2006), and the H+-pumping mechanism of BR is described below. BR at the ground state and the intermediates K, L, M, N and O have been investigated with various biophysical methods. The photocycle comprises stepwise reactions of the thermal reisomerization of the photoisomerized 13-*cis* retinal to the initial all-*trans*, and the proton is transferred toward the higher p*K*a residue accompanied with p*K*a changes during the photocycle. Reflecting the differences in protein conformation and protonation states of some residues, the intermediates assume the respective absorption spectra. The photoisomerization from initial all-*trans* to 13-*cis* retinal is completed until the formation of K-intermediate. The subsequent proton transfer observed at around neutral pH occurs as the following sequence (see Fig. 1A and B). First, the deprotonation of the protonated Schiff base occurs in the formation of Mintermediate. The proton from the Schiff base is transferred to its counterion Asp85BR and the subsequent proton release to the extracellular (EC) space occurs from the protonreleasing complex (PRC) consisting of Glu194BR, Glu204BR, Arg82BR and water molecules. Next, the proton of protonated Asp96BR locating at the cytoplasmic (CP) channel transfers to the Schiff base in the M-N transition, which lead to the reprotonation of the Schiff base. Then the deprotonated Asp96BR uptakes a proton from the CP space in the N-O transition, which is accompanied by the reisomerization of retinal to the initial all-*trans* state. Finally, the proton of protonated Asp85BR is transferred to PRC during the decay of O-intermediate, and then the protein returns to the original state. This series of proton-transfer reactions accomplishes the net proton transport from the CP to EC side.

As mentioned above, a light-driven proton pump has been found in many microorganisms belonging to bacteria and eukarya. These rhodopsins also undergo the photocycle including the intermediates similar to those of BR. However, there are several differences in the photocycles and proton transfers between BR and other H+-pumping rhodopsins. Examples are as follows. (1) PR from marine bacteria also goes through K, L, M, N and O (or PR') intermediates at around neutral pH (Dioumaev et al., 2002; Friedrich et al., 2002; Váró et al.,

photosensing rhodopsin called channelrhodopsin was found in *Chlamydomonas reinhardtii,* a green flagellate alga (Sineshchekov et al., 2002; Nagel et al., 2002; Suzuki et al., 2003). This is a light-gated ion channel and induces the photomotile behavior of the cell. Thus, the world

Microbial rhodopsins have linear cyclic photochemical reactions called photocycles. For all microbial rhodopsins examined so far, the retinal Schiff bases are protonated in their dark

bases become deprotonated during the photocycles independent of H+-pumping or photosensing rhodopsins. As described below, these primary and the subsequent proton-

The illumination of the pigment protein leads to the excited state, which is relaxed thermally to the original pigment *via* various photochemical intermediates (Fig. 1A). The best-studied rhodopsin is BR (Haupts, 1999; Balashov, 2000; Heberle, 2000; Lanyi, 2004, 2006), and the H+-pumping mechanism of BR is described below. BR at the ground state and the intermediates K, L, M, N and O have been investigated with various biophysical methods. The photocycle comprises stepwise reactions of the thermal reisomerization of the photoisomerized 13-*cis* retinal to the initial all-*trans*, and the proton is transferred toward the higher p*K*a residue accompanied with p*K*a changes during the photocycle. Reflecting the differences in protein conformation and protonation states of some residues, the intermediates assume the respective absorption spectra. The photoisomerization from initial all-*trans* to 13-*cis* retinal is completed until the formation of K-intermediate. The subsequent proton transfer observed at around neutral pH occurs as the following sequence (see Fig. 1A and B). First, the deprotonation of the protonated Schiff base occurs in the formation of Mintermediate. The proton from the Schiff base is transferred to its counterion Asp85BR and the subsequent proton release to the extracellular (EC) space occurs from the protonreleasing complex (PRC) consisting of Glu194BR, Glu204BR, Arg82BR and water molecules. Next, the proton of protonated Asp96BR locating at the cytoplasmic (CP) channel transfers to the Schiff base in the M-N transition, which lead to the reprotonation of the Schiff base. Then the deprotonated Asp96BR uptakes a proton from the CP space in the N-O transition, which is accompanied by the reisomerization of retinal to the initial all-*trans* state. Finally, the proton of protonated Asp85BR is transferred to PRC during the decay of O-intermediate, and then the protein returns to the original state. This series of proton-transfer reactions

As mentioned above, a light-driven proton pump has been found in many microorganisms belonging to bacteria and eukarya. These rhodopsins also undergo the photocycle including the intermediates similar to those of BR. However, there are several differences in the photocycles and proton transfers between BR and other H+-pumping rhodopsins. Examples are as follows. (1) PR from marine bacteria also goes through K, L, M, N and O (or PR') intermediates at around neutral pH (Dioumaev et al., 2002; Friedrich et al., 2002; Váró et al.,

ion pump, the Schiff

**4. The photo-induced proton transfer associated with the photocycle of** 

transfer reactions are closely related to the functions of both microbial rhodopsins.

states under physiological conditions. Except in the case of HR, a Cl-

accomplishes the net proton transport from the CP to EC side.

of microbial rhodopsins is now rapidly expanding.

**microbial rhodopsins** 

**-pumping rhodopsins** 

**4.1 H<sup>+</sup>**

2003). However, the proton movement from the proton donor residue (Glu108PR corresponding to Asp96BR) to the Schiff base and its subsequent proton uptake from the CP space occur simultaneously in the M-N transition (Dioumaev et al., 2002). For BR, these two proton movements occur separately in the M-N and N-O transitions. (2) For ARII from marine algae, the photocycle includes much larger reverse reactions between L, M, N and O than are seen in BR (Kikukawa et al., 2011). Although the reverse reactions are also present in BR, their rates in ARII appear to be much larger than in BR. A rapid reverse reaction might be disadvantageous for the unidirectional ion pumping. (3) For all microbial rhodopsins, the proton conduction channel is bisected by retinal. For BR, these two channels are severely isolated and, in the dark state, the CP half channel is kept under a highly hydrophobic condition. This asymmetric structure had been believed to be important for the ion pumping function. For several proton pumps such as XR and GR, however, this characteristic structure of BR is not conserved (Luecke et al., 2008; Miranda et al., 2009). Therefore, the newly found proton pumps seem to adopt a mechanism that is at least partly different from that in BR. Thus, it would be an interesting subject to clarify the essential mechanisms for the respective proton pumps.

Fig. 1. The photocycle scheme (A) and the structure of BR (B) with the important residues involved in the proton-transfer reactions.

In (A), BR, K, L, M, N and O represent the unphotolyzed state and intermediates, respectively. Their λmax's are given in the subscripts, and the lifetimes of the intermediates are also shown. The photoisomerization of the retinal by illumination triggers the stepwise photoreactions accompanied with the proton movements. In (B), these proton transfers at respective steps are indicated with arrows (see section 4.1 for the details). The timing of proton transfer depends on the pH of the medium. Above pH 5, the proton release occurs in L-decay while below at about pH 5, the proton release occurs in O-decay (arrow with a broken line) instead of Ldecay. The structure in (B) was drawn from the PDB coordinate file 1C3W.

### **4.2 Photosensing rhodopsins**

During the photocycles of almost all photosensing rhodopsins examined so far, the deprotonations of the Schiff bases occur much as for the H+-pumping rhodopsins. In many

Photo-Induced Proton Transfers of Microbial Rhodopsins 95

transfer reaction, a proton moves from an amino acid residue having smaller p*K*a to one having higher p*K*a. Then, if the p*K*a of the residue from which the proton is released to the external medium is larger than the pH in the medium, the proton cannot be released. For such a case, a subsequent proton movement (*e.g.,* a proton uptake from the medium) occurs prior to the release. This "traffic jam" of the proton movement actually occurs in the photocycle of BR (Balashov, 2000). The p*K*a of its PRC at the proton-releasing state is about 6 (Zimányi et al., 1992; Balashov, 2000). Under an acidic pH sufficiently lower than pH 6, proton uptake is observed prior to the release (see Figs. 1A and 2B). Therefore, the extent of the pH-dependence of the proton release/uptake could be utilized to estimate the p*K*a value of a residue that is important for the proton-transfer reaction. Similarly, the proton-transfer rate may afford information about some important amino acid residues. To analyze these reactions, it is necessary to detect a small pH change in the external medium due to the

**6. Necessity of a device to measure rapid pH changes or proton-transfer** 

device for these measurements due to its high sensitivity and rapid time-resolution.

Koyama and his coworkers first developed this method using BR (Miyasaka & Koyama, 1991; Miyasaka et al., 1992). They constructed a photo-electrochemical cell in which BRs were absorbed on a SnO2-coated transparent glass electrode, and detected the electric current evoked by constant illumination by using another SnO2 electrode as a reference electrode. The origin of this electric current was assumed to be the charge displacement by BR (Koyama et al., 1994). On the other hand, Robertson and Lukashev suggested that the origin of this signal is the medium pH change caused by the photoinduced proton release and uptake in BR (Robertson & Lukashev, 1995). Actually, our group confirmed this suggestion by the following results of three experiments (Tamogami et al., 2009). 1) The equilibrium potential of an ITO electrode showed a linear relationship to pH (see Fig. 2A). 2) The amplitudes of photoelectrical signals decreased with increasing buffer concentration (see Fig. 2B). 3) HR from *N. pharaonis* (NpHR), an inwardly directed Cl- pump, did not cause

**7. An ITO (or SnO2) electrode works as a pH-sensitive electrode** 

The photocycle of the ion-pumping rhodopsins completes in ~50-100 ms, and many photosensing rhodopsins have slower photocycle of ~sec. A pH glass electrode is too slow to respond to such pH changes. For such a rapid reaction induced by a flash, the pH change is usually obtained using pH-sensitive dyes whose absorbance depends on pH (Heberle, 2000). This is a convenient method and the rapid change is measurable. However, there is a weak point in that the medium pH should be restricted at pH near the p*K*a values of the dyes. In other words, the measurements under various medium pH values cannot be performed with a single dye. In addition, the subtraction of the signal in the co-presence of a dye and a rhodopsin from that of the rhodopsin alone should be performed. If one wants to estimate the p*K*a of an important amino acid residue, the pH profile of the magnitude and/or the rate of the proton transfer are indispensable. Hence, a device is needed for the detection of pH changes or proton-transfer rates at any pH. In the following sections, we will describe an electrochemical cell using an indium-tin oxide (ITO) or tin oxide (SnO2) electrode and its application to the microbial rhodopsins. This electrochemical cell is a useful

proton movements caused during a single photocycle.

**rates at any pH** 

cases, these deprotonations result in the proton release/uptake reactions with the external medium. Thus, the proton-transfer reactions also control the decay rates of some intermediates of photosensing rhodopsins.

The best characterized photosensing rhodopsins are SRI and SRII from archaea and bacteria. Some homologues of these SRs show the outwardly directed proton pumping activities when they exist alone in the membrane (Bogomolni et al., 1994; Sasaki & J.L. Spudich, 2000; Sudo et al., 2001; Schmies et al., 2001). Upon the complex formations with the cognate transducers, both the proton release and uptake occur at only the extracellular side (this is so-called proton circulation) instead of the vectorial proton transport. These facts mean that SRs possess proton-transfer machinery like BR and this machinery is probably sensitive to the protein conformational change relating with the signal transduction mechanism. Like the H+-pumping rhodopsins, the states having deprotonated Schiff bases are also called Mintermediates, and are the putative signaling states for SRs. The longer lifetimes of Mintermediates are considered to increase the signaling efficiencies. Thus, the reprotonations of the Schiff bases influence the signaling efficiencies.

Studies on two SRIIs from *Natronomonas pharaonis* (NpSRII) (Kamo et al., 2001; J.L. Spudich & Luecke, 2002; Pebay-Peyroula et al., 2002; Klare et al., 2004) and *H. salinarum* (HsSRII) (Sasaki & J.L. Spudich, 1998, 1999) have revealed the differences in the proton-transfer reactions associated with their M-intermediate decays. For NpSRII, the reprotonation of the Schiff base occurs by uptaking a proton directly from the bulk due to the lack of a proton donor to the Schiff base (corresponding to Asp96BR). Therefore the M-decay in NpSRII is very slow as compared with BR and depends on the pH of the medium (Miyazaki et al., 1992). For HsSRII, on the other hand, there are two proton-transfer pathways in the decay of the M-intermediate. One is the pathway in which the proton comes directly from the bulk to the Schiff base, and the other is the pathway in which the proton comes from an unidentified X-H residue. Which proton pathway becomes the major component is dependent on the pH of the medium (Sasaki & J.L. Spudich, 1999; Tamogami et al., 2010). In addition, it has been reported that NpSRII possesses the H+-pumping activity (Sudo et al., 2001; Schmies et al., 2001) but HsSRII does not (Sasaki & J.L. Spudich, 1999, 2000). Thus, despite their identical physiological functions, NpSRII and HsSRII have several differences with respect to their photocycles and proton transfers. For these photosensing rhodopsins, therefore, it would be of interest to investigate the photo-induced proton-transfer mechanisms as well as their relations with the photosignaling transductions to the cognate transducers.

### **5. Importance of measurements of the photo-induced proton transfer of microbial rhodopsins**

As described above, the proton movements between the residues inside the protein as well as between the residue and the external space occur during the photocycles of most microbial rhodopsins. The protein conformational changes during the photocycles alter the p*K*a of the residues and thereby cause the proton-transfer reactions. For H+-pumping rhodopsins, these proton movements directly couple with their functional mechanisms, and for the photosensing rhodopsins, these movements affect the signal transduction efficiencies by controlling the decay rates of some intermediates and reflect the conformational alterations by the complex formation with the cognate transducers. For respective proton-

cases, these deprotonations result in the proton release/uptake reactions with the external medium. Thus, the proton-transfer reactions also control the decay rates of some

The best characterized photosensing rhodopsins are SRI and SRII from archaea and bacteria. Some homologues of these SRs show the outwardly directed proton pumping activities when they exist alone in the membrane (Bogomolni et al., 1994; Sasaki & J.L. Spudich, 2000; Sudo et al., 2001; Schmies et al., 2001). Upon the complex formations with the cognate transducers, both the proton release and uptake occur at only the extracellular side (this is so-called proton circulation) instead of the vectorial proton transport. These facts mean that SRs possess proton-transfer machinery like BR and this machinery is probably sensitive to the protein conformational change relating with the signal transduction mechanism. Like the H+-pumping rhodopsins, the states having deprotonated Schiff bases are also called Mintermediates, and are the putative signaling states for SRs. The longer lifetimes of Mintermediates are considered to increase the signaling efficiencies. Thus, the reprotonations

Studies on two SRIIs from *Natronomonas pharaonis* (NpSRII) (Kamo et al., 2001; J.L. Spudich & Luecke, 2002; Pebay-Peyroula et al., 2002; Klare et al., 2004) and *H. salinarum* (HsSRII) (Sasaki & J.L. Spudich, 1998, 1999) have revealed the differences in the proton-transfer reactions associated with their M-intermediate decays. For NpSRII, the reprotonation of the Schiff base occurs by uptaking a proton directly from the bulk due to the lack of a proton donor to the Schiff base (corresponding to Asp96BR). Therefore the M-decay in NpSRII is very slow as compared with BR and depends on the pH of the medium (Miyazaki et al., 1992). For HsSRII, on the other hand, there are two proton-transfer pathways in the decay of the M-intermediate. One is the pathway in which the proton comes directly from the bulk to the Schiff base, and the other is the pathway in which the proton comes from an unidentified X-H residue. Which proton pathway becomes the major component is dependent on the pH of the medium (Sasaki & J.L. Spudich, 1999; Tamogami et al., 2010). In addition, it has been reported that NpSRII possesses the H+-pumping activity (Sudo et al., 2001; Schmies et al., 2001) but HsSRII does not (Sasaki & J.L. Spudich, 1999, 2000). Thus, despite their identical physiological functions, NpSRII and HsSRII have several differences with respect to their photocycles and proton transfers. For these photosensing rhodopsins, therefore, it would be of interest to investigate the photo-induced proton-transfer mechanisms as well as their relations with the photosignaling transductions to the cognate

**5. Importance of measurements of the photo-induced proton transfer of** 

As described above, the proton movements between the residues inside the protein as well as between the residue and the external space occur during the photocycles of most microbial rhodopsins. The protein conformational changes during the photocycles alter the p*K*a of the residues and thereby cause the proton-transfer reactions. For H+-pumping rhodopsins, these proton movements directly couple with their functional mechanisms, and for the photosensing rhodopsins, these movements affect the signal transduction efficiencies by controlling the decay rates of some intermediates and reflect the conformational alterations by the complex formation with the cognate transducers. For respective proton-

intermediates of photosensing rhodopsins.

of the Schiff bases influence the signaling efficiencies.

transducers.

**microbial rhodopsins** 

transfer reaction, a proton moves from an amino acid residue having smaller p*K*a to one having higher p*K*a. Then, if the p*K*a of the residue from which the proton is released to the external medium is larger than the pH in the medium, the proton cannot be released. For such a case, a subsequent proton movement (*e.g.,* a proton uptake from the medium) occurs prior to the release. This "traffic jam" of the proton movement actually occurs in the photocycle of BR (Balashov, 2000). The p*K*a of its PRC at the proton-releasing state is about 6 (Zimányi et al., 1992; Balashov, 2000). Under an acidic pH sufficiently lower than pH 6, proton uptake is observed prior to the release (see Figs. 1A and 2B). Therefore, the extent of the pH-dependence of the proton release/uptake could be utilized to estimate the p*K*a value of a residue that is important for the proton-transfer reaction. Similarly, the proton-transfer rate may afford information about some important amino acid residues. To analyze these reactions, it is necessary to detect a small pH change in the external medium due to the proton movements caused during a single photocycle.

### **6. Necessity of a device to measure rapid pH changes or proton-transfer rates at any pH**

The photocycle of the ion-pumping rhodopsins completes in ~50-100 ms, and many photosensing rhodopsins have slower photocycle of ~sec. A pH glass electrode is too slow to respond to such pH changes. For such a rapid reaction induced by a flash, the pH change is usually obtained using pH-sensitive dyes whose absorbance depends on pH (Heberle, 2000). This is a convenient method and the rapid change is measurable. However, there is a weak point in that the medium pH should be restricted at pH near the p*K*a values of the dyes. In other words, the measurements under various medium pH values cannot be performed with a single dye. In addition, the subtraction of the signal in the co-presence of a dye and a rhodopsin from that of the rhodopsin alone should be performed. If one wants to estimate the p*K*a of an important amino acid residue, the pH profile of the magnitude and/or the rate of the proton transfer are indispensable. Hence, a device is needed for the detection of pH changes or proton-transfer rates at any pH. In the following sections, we will describe an electrochemical cell using an indium-tin oxide (ITO) or tin oxide (SnO2) electrode and its application to the microbial rhodopsins. This electrochemical cell is a useful device for these measurements due to its high sensitivity and rapid time-resolution.

### **7. An ITO (or SnO2) electrode works as a pH-sensitive electrode**

Koyama and his coworkers first developed this method using BR (Miyasaka & Koyama, 1991; Miyasaka et al., 1992). They constructed a photo-electrochemical cell in which BRs were absorbed on a SnO2-coated transparent glass electrode, and detected the electric current evoked by constant illumination by using another SnO2 electrode as a reference electrode. The origin of this electric current was assumed to be the charge displacement by BR (Koyama et al., 1994). On the other hand, Robertson and Lukashev suggested that the origin of this signal is the medium pH change caused by the photoinduced proton release and uptake in BR (Robertson & Lukashev, 1995). Actually, our group confirmed this suggestion by the following results of three experiments (Tamogami et al., 2009). 1) The equilibrium potential of an ITO electrode showed a linear relationship to pH (see Fig. 2A). 2) The amplitudes of photoelectrical signals decreased with increasing buffer concentration (see Fig. 2B). 3) HR from *N. pharaonis* (NpHR), an inwardly directed Cl- pump, did not cause

Photo-Induced Proton Transfers of Microbial Rhodopsins 97

pH 3.0, the proton uptake is followed by release. This may be interpreted as meaning that the proton cannot be released from PRC under this pH condition because the medium pH is lower than the p*K*a of PRC in the proton-releasing state (M-intermediate). Thus, the proton uptake in the decay of N is observed first and the release occurs in the decay of O concomitantly with the deprotonation of Asp85BR (see the arrow with a broken line in Fig. 1A). The same proton-transfer sequence was also observed in the PRC-lacking mutants, as shown in Fig. 4, where the data for a mutant of E194Q/E204QBR are summarized. For these PRC-lacking mutants, the proton uptake occurred first even at the neutral pH (see the trace labeled with "ITO signal" in Fig. 4). For wild-type BR at pH 4.5 (see Fig. 3B), the proton uptake occurred first and then overshoot of the signal was observed at around 35 ms. This may be interpreted as the mixture of the two populations of BR. The molecules exhibiting proton uptake first constitute the major population. Other molecules constituting a minor population exhibit proton release first. The final proton transfer corresponding to proton uptake (decay of the positive signal) caused by the minor population is slower than the proton release of the major population. Thus, the signal amplitude of this system can reflect

the ratio of two kinds of molecules having different proton-transfer sequence.

utilized to identify the intermediate accompanying the proton-transfer reaction.

Fig. 3. The measurement of the photo-induced proton transfer in BR by the ITO transparent

electrode.

In this measurement system, the time course of the photo-induced proton transfer can also be determined. Figure 4 shows the comparison of three types of signals measured for the E194Q/E204QBR mutant. These are (1) flash-induced absorbance changes of the mutant itself measured at three typical wavelengths; (2) a photo-induced pH change measured by the ITO system and; (3) the corresponding signal measured by a pH-sensitive dye, pyranine. The sign of the pyranine signal is opposite that of ITO. For pyranine, the upward shift signifies the proton uptake, while the downward shift signifies the proton release. As shown in this figure, the proton uptake and release agree well with the formation and decay of O, which are represented by the absorbance change at 660 nm. This is the typical proton-transfer sequence of the PRC-disabled mutant (Brown et al., 1995; Balashov et al., 1997; Dioumaev et al., 1998, Koyama et al., 1998a). The response of the dye is very fast and completely follows the pH change within this time range. The time course of the dye signal almost coincides with that of ITO signal. Thus, our current ITO measurement system can monitor the pH change in the time range of *ca.* 10 ms to several hundred milliseconds (Tamogami et al., 2009). This could be

a photoelectrical signal. HR is known to be converted into an H+ pump in the presence of azide (Váró et al., 1996). In accordance with this, a photoelectrical signal was evoked from the HR-adsorbed electrode by the addition of azide (see Fig. 2C and Koyama et al., 1998b). Thus it was established that an ITO (or SnO2) electrode works as a pH-sensitive electrode.

Fig. 2. An ITO (or SnO2) electrode works as a pH-sensitive electrode.

A) The relationship between the equilibrium potential of ITO and pH. B) The effect of buffer on the photo-induced signals in BR by the ITO electrode. The inset shows the plot of the reciprocal of the amplitudes of ITO signals against buffer concentrations. The experimental medium contains 400 mM NaCl and HEPES of each concentration (1, 3, 10, 100 and 500 mM) at pH 7.5. The excitation light (2 ms duration) was > 520 nm. C) The photo-induced ITO signals in NpHR in the presence and absence of azide. Experiments were performed in medium containing 133 mM Na2SO4, azide at each concentration (0, 50 and 200 mM) and 1 mM HEPES at pH 7.0. The excitation light (2 ms duration) was > 440 nm. NpHR was expressed in the *E. coli* expression system, and then was reconstituted with Egg L-αphosphatidylcholine. Other experimental setups are described in Fig. 3 and previous report (Tamogami et al., 2009). Panels A and B were adapted with permission from Tamogami et al., 2009, *Photochem. Photobiol.* Copyright 2009 The authors, Journal Compilation, The American Society of Photobiology.

### **8. Measurements of proton release/uptake by BR with a rapid time resolution**

BR has been investigated in great detail. Thus, BR and its mutants are good references to show the relevance of our measurements using this electrochemical cell. Figure 3A shows a schemata of our electrochemical cell. This cell is essentially the same as that previously constructed by Koyama and coworkers with some modifications (Miyasaka et al., 1992). Illumination on the electrode-attached proteins induces the electrochemical potential change between the working and the counter ITO electrodes. Figure 3B indicates the flash-induced signals in BR. In this figure, the upward shift signifies the acidification of the medium near the working electrode. Thus, this shift signified the proton release from proteins to the bulk, while the downward shift signifies the proton uptake from the bulk to the protein. For BR, as described above, the sequence of the proton release and uptake can be altered depending on the external medium pH. For example, at pH 6.0 and 9.0, the proton release is followed by uptake, since the proton can be released from PRC at the early step of the photocycle (in the formation of M). The subsequent uptake occurs in the decay of N. On the other hand, at

a photoelectrical signal. HR is known to be converted into an H+ pump in the presence of azide (Váró et al., 1996). In accordance with this, a photoelectrical signal was evoked from the HR-adsorbed electrode by the addition of azide (see Fig. 2C and Koyama et al., 1998b). Thus it was established that an ITO (or SnO2) electrode works as a pH-sensitive electrode.

A) The relationship between the equilibrium potential of ITO and pH. B) The effect of buffer on the photo-induced signals in BR by the ITO electrode. The inset shows the plot of the reciprocal of the amplitudes of ITO signals against buffer concentrations. The experimental medium contains 400 mM NaCl and HEPES of each concentration (1, 3, 10, 100 and 500 mM) at pH 7.5. The excitation light (2 ms duration) was > 520 nm. C) The photo-induced ITO signals in NpHR in the presence and absence of azide. Experiments were performed in medium containing 133 mM Na2SO4, azide at each concentration (0, 50 and 200 mM) and 1 mM HEPES at pH 7.0. The excitation light (2 ms duration) was > 440 nm. NpHR was expressed in the *E. coli* expression system, and then was reconstituted with Egg L-αphosphatidylcholine. Other experimental setups are described in Fig. 3 and previous report (Tamogami et al., 2009). Panels A and B were adapted with permission from Tamogami et al., 2009, *Photochem. Photobiol.* Copyright 2009 The authors, Journal Compilation, The

**8. Measurements of proton release/uptake by BR with a rapid time resolution**  BR has been investigated in great detail. Thus, BR and its mutants are good references to show the relevance of our measurements using this electrochemical cell. Figure 3A shows a schemata of our electrochemical cell. This cell is essentially the same as that previously constructed by Koyama and coworkers with some modifications (Miyasaka et al., 1992). Illumination on the electrode-attached proteins induces the electrochemical potential change between the working and the counter ITO electrodes. Figure 3B indicates the flash-induced signals in BR. In this figure, the upward shift signifies the acidification of the medium near the working electrode. Thus, this shift signified the proton release from proteins to the bulk, while the downward shift signifies the proton uptake from the bulk to the protein. For BR, as described above, the sequence of the proton release and uptake can be altered depending on the external medium pH. For example, at pH 6.0 and 9.0, the proton release is followed by uptake, since the proton can be released from PRC at the early step of the photocycle (in the formation of M). The subsequent uptake occurs in the decay of N. On the other hand, at

Fig. 2. An ITO (or SnO2) electrode works as a pH-sensitive electrode.

American Society of Photobiology.

pH 3.0, the proton uptake is followed by release. This may be interpreted as meaning that the proton cannot be released from PRC under this pH condition because the medium pH is lower than the p*K*a of PRC in the proton-releasing state (M-intermediate). Thus, the proton uptake in the decay of N is observed first and the release occurs in the decay of O concomitantly with the deprotonation of Asp85BR (see the arrow with a broken line in Fig. 1A). The same proton-transfer sequence was also observed in the PRC-lacking mutants, as shown in Fig. 4, where the data for a mutant of E194Q/E204QBR are summarized. For these PRC-lacking mutants, the proton uptake occurred first even at the neutral pH (see the trace labeled with "ITO signal" in Fig. 4). For wild-type BR at pH 4.5 (see Fig. 3B), the proton uptake occurred first and then overshoot of the signal was observed at around 35 ms. This may be interpreted as the mixture of the two populations of BR. The molecules exhibiting proton uptake first constitute the major population. Other molecules constituting a minor population exhibit proton release first. The final proton transfer corresponding to proton uptake (decay of the positive signal) caused by the minor population is slower than the proton release of the major population. Thus, the signal amplitude of this system can reflect the ratio of two kinds of molecules having different proton-transfer sequence.

In this measurement system, the time course of the photo-induced proton transfer can also be determined. Figure 4 shows the comparison of three types of signals measured for the E194Q/E204QBR mutant. These are (1) flash-induced absorbance changes of the mutant itself measured at three typical wavelengths; (2) a photo-induced pH change measured by the ITO system and; (3) the corresponding signal measured by a pH-sensitive dye, pyranine. The sign of the pyranine signal is opposite that of ITO. For pyranine, the upward shift signifies the proton uptake, while the downward shift signifies the proton release. As shown in this figure, the proton uptake and release agree well with the formation and decay of O, which are represented by the absorbance change at 660 nm. This is the typical proton-transfer sequence of the PRC-disabled mutant (Brown et al., 1995; Balashov et al., 1997; Dioumaev et al., 1998, Koyama et al., 1998a). The response of the dye is very fast and completely follows the pH change within this time range. The time course of the dye signal almost coincides with that of ITO signal. Thus, our current ITO measurement system can monitor the pH change in the time range of *ca.* 10 ms to several hundred milliseconds (Tamogami et al., 2009). This could be utilized to identify the intermediate accompanying the proton-transfer reaction.

Fig. 3. The measurement of the photo-induced proton transfer in BR by the ITO transparent electrode.

Photo-Induced Proton Transfers of Microbial Rhodopsins 99

are proportional to the numbers of protons moved by the photo-induced transfer. By analyzing these voltage changes, the p*K*a values concerned with the proton transfer can be estimated as described below. Figure 5A shows the peak magnitude of the voltage changes by BR (peak values in Fig. 3B) measured under different medium pH values. As shown in this figure, below pH ~ 5, the proton uptake was followed by the proton release. On the other hand, above pH ~ 5, the proton release was followed by the proton uptake. Corresponding to these two pH ranges, the pH profile consists of two bell-shaped functions having opposite signs. This suggests the contribution of the four p*K*a's of residues involved in the proton-transfer reactions. Then, this pH profile can be expressed by the following

> 

(1)

1

 

1

 

1 10

 

1

<sup>1</sup> <sup>2</sup> <sup>3</sup> <sup>4</sup> <sup>1</sup> <sup>10</sup>

*pKa pH pH pKa pKa pH pH pKa Voltage A B*

where A and B are constants used to adjust the magnitude of the response, and the four p*K*a values from p*K*a1 to p*K*a4 are assumed to increase in this order. A fitting analysis using Eq. 1 gave the following p*K*a values: p*K*a1 = 2.6, p*K*a2 = 4.1, p*K*a3 = 6.1 and p*K*a4 = 9.0. For BR, the p*K*a values involved in the photo-induced proton transfer had been estimated mainly by various spectroscopic measurements (Balashov, 2000). The p*K*a's determined by the ITO method agreed well with those previously reported. Therefore, the origins of the estimated p*K*a's were verified on the basis of previous reports. As a result, the p*K*a's from p*K*a1 to p*K*a4 were identified as the p*K*a's of Asp85BR in the dark, PRC in the O-intermediate, PRC in the M-intermediate and PRC in the dark, respectively. In addition to this analysis using the peak voltages, the rates of the voltage changes were also informative. The proton uptake by Asp96BR from the CP space, which coincides with N decay, becomes slow as the medium pH increases (see the traces at pH 6 and pH 9 in Fig. 3B). The rate constants of the proton uptake obeyed the Henderson-Hasselbalch equation with a single p*K*a, and the value of Asp96BR during N decay was estimated at 7.8 (Tamogami et al., 2009). This value also agreed with the previously reported value (Balashov, 2000). These p*K*a values determined by the ITO method are considered those of key amino acid residues for the proton pumping function of BR. Therefore this method is useful for detecting the proton transfer directly and deducing

**-pumping rhodopsins**  This ITO method has been successfully applied to the newly found H+-pumping rhodopsins (Tamogami et al., 2009; Kikukawa et al, 2011). The panels B and C in Fig. 5 are the results for ARII from marine algae and PR from marine bacteria, respectively. Interestingly, the pH profiles of these three proton pumps are quite different. The prominent differences are as follows: (1) The pH profiles of the three rhodopsins commonly have bell-shaped negative peak areas, indicating that the proton uptake occurs prior to the release. However, the negative peak of PR is located at a quite higher pH (pH~8) than the other two rhodopsins (pH 3~4). This reflects the difference of the most acidic p*K*a's, governing the pH where the proton uptake starts to occur. These p*K*a's correspond to those of aspartates (Asp85BR, Asp81ARII and Asp97PR), the counterions of the respective protonated Schiff bases. This p*K*a for PR is about 7, which is much higher than the p*K*a's of about 2.6 of the other two rhodopsins. This high p*K*a for PR reported by using spectroscopic methods (Dioumaev et

 

1 10

1

 

the important p*K*a values.

**10. Application to other H<sup>+</sup>**

1 10

equation:

(A) The structure of the photoelectrochemical cell constructed by using ITO electrodes. (1) A silicon sheet; (2) electrolyte plus buffer; (3) a counter ITO electrode; (4) a working ITO electrode; (5) a thin layer of the sample dried on the electrode surface; (6) a Lucite chamber. The emf change between the two ITO electrodes was picked up by an AC amplifier with a low-cut filter of 0.08 Hz which eliminated the baseline fluctuation. This limited the duration of observation time. (B) Flash light (2 ms)-induced signals in BR under varying pH values. Measurements were carried out in a solution containing 400 mM NaCl and 1 mM 6-mixed buffer (citrate/MES/MOPS/HEPES/CHES/CAPS) adjusted to the desired pH with HCl or NaOH. BR indicates the purple membrane prepared by a standard method (Becher & Cassim, 1975). The 6-mixed buffer was used because of its almost constant buffer capacities in a wide pH range. Panel B was adapted with permission from Tamogami et al., 2009, *Photochem. Photobiol..* Copyright 2009 The authors, Journal Compilation, The American Society of Photobiology.

Fig. 4. Comparison between the flash-induced absorbance changes and proton-transfer signals in E194Q/E204QBR.

The red, blue and green lines represent absorbance changes of BR at 410, 570 and 660 nm, where the M-intermediate, unphotolyzed state and O-intermediate are mainly monitored, respectively. These traces were obtained by the flash photolysis spectroscopy performed by the procedure as described previously (Sato et al., 2003). The black broken and solid lines are the pyranine's signal (monitored at 450 nm) and ITO signal, respectively. Measurements of the absorbance changes of BR and pyranine were performed in the solution containing 400 mM NaCl plus 0.5 mM HEPES at pH 7.1 as described elsewhere (Tamogami et al, 2009). On the other hand, the ITO experiment was performed as shown in Fig. 3 in a solution containing 400 mM NaCl plus 1 mM 6-mixed buffer at pH 7.1. The protein sample was the purple membrane isolated from *H. salinarum* expressing this mutant.

### **9. Estimation of the p***K***a values of important residues involved in the photoinduced proton transfer of BR**

It is a pronounced advantage of the ITO electrode method that the measurements can be performed over a wide pH region. Since the buffer capacity is kept constant for the measuring pH range by mixing the buffering agents and the detected pH changes are quite small due to the very faint amount of the adsorbed protein, the measured voltage changes 98 Molecular Photochemistry – Various Aspects

(A) The structure of the photoelectrochemical cell constructed by using ITO electrodes. (1) A silicon sheet; (2) electrolyte plus buffer; (3) a counter ITO electrode; (4) a working ITO electrode; (5) a thin layer of the sample dried on the electrode surface; (6) a Lucite chamber. The emf change between the two ITO electrodes was picked up by an AC amplifier with a low-cut filter of 0.08 Hz which eliminated the baseline fluctuation. This limited the duration of observation time. (B) Flash light (2 ms)-induced signals in BR under varying pH values. Measurements were carried out in a solution containing 400 mM NaCl and 1 mM 6-mixed buffer (citrate/MES/MOPS/HEPES/CHES/CAPS) adjusted to the desired pH with HCl or NaOH. BR indicates the purple membrane prepared by a standard method (Becher & Cassim, 1975). The 6-mixed buffer was used because of its almost constant buffer capacities in a wide pH range. Panel B was adapted with permission from Tamogami et al., 2009, *Photochem. Photobiol..* Copyright 2009 The authors, Journal Compilation, The American

Fig. 4. Comparison between the flash-induced absorbance changes and proton-transfer

purple membrane isolated from *H. salinarum* expressing this mutant.

The red, blue and green lines represent absorbance changes of BR at 410, 570 and 660 nm, where the M-intermediate, unphotolyzed state and O-intermediate are mainly monitored, respectively. These traces were obtained by the flash photolysis spectroscopy performed by the procedure as described previously (Sato et al., 2003). The black broken and solid lines are the pyranine's signal (monitored at 450 nm) and ITO signal, respectively. Measurements of the absorbance changes of BR and pyranine were performed in the solution containing 400 mM NaCl plus 0.5 mM HEPES at pH 7.1 as described elsewhere (Tamogami et al, 2009). On the other hand, the ITO experiment was performed as shown in Fig. 3 in a solution containing 400 mM NaCl plus 1 mM 6-mixed buffer at pH 7.1. The protein sample was the

**9. Estimation of the p***K***a values of important residues involved in the photo-**

It is a pronounced advantage of the ITO electrode method that the measurements can be performed over a wide pH region. Since the buffer capacity is kept constant for the measuring pH range by mixing the buffering agents and the detected pH changes are quite small due to the very faint amount of the adsorbed protein, the measured voltage changes

Society of Photobiology.

signals in E194Q/E204QBR.

**induced proton transfer of BR** 

are proportional to the numbers of protons moved by the photo-induced transfer. By analyzing these voltage changes, the p*K*a values concerned with the proton transfer can be estimated as described below. Figure 5A shows the peak magnitude of the voltage changes by BR (peak values in Fig. 3B) measured under different medium pH values. As shown in this figure, below pH ~ 5, the proton uptake was followed by the proton release. On the other hand, above pH ~ 5, the proton release was followed by the proton uptake. Corresponding to these two pH ranges, the pH profile consists of two bell-shaped functions having opposite signs. This suggests the contribution of the four p*K*a's of residues involved in the proton-transfer reactions. Then, this pH profile can be expressed by the following equation:

$$\Delta Voltage = -A \left( \frac{1}{1 + 10^{pKa\_1 - pH}} \right) \left( \frac{1}{1 + 10^{pH - pK\_2}} \right) + B \left( \frac{1}{1 + 10^{pKa\_3 - pH}} \right) \left( \frac{1}{1 + 10^{pH - pK\_4}} \right) \tag{1}$$

where A and B are constants used to adjust the magnitude of the response, and the four p*K*a values from p*K*a1 to p*K*a4 are assumed to increase in this order. A fitting analysis using Eq. 1 gave the following p*K*a values: p*K*a1 = 2.6, p*K*a2 = 4.1, p*K*a3 = 6.1 and p*K*a4 = 9.0. For BR, the p*K*a values involved in the photo-induced proton transfer had been estimated mainly by various spectroscopic measurements (Balashov, 2000). The p*K*a's determined by the ITO method agreed well with those previously reported. Therefore, the origins of the estimated p*K*a's were verified on the basis of previous reports. As a result, the p*K*a's from p*K*a1 to p*K*a4 were identified as the p*K*a's of Asp85BR in the dark, PRC in the O-intermediate, PRC in the M-intermediate and PRC in the dark, respectively. In addition to this analysis using the peak voltages, the rates of the voltage changes were also informative. The proton uptake by Asp96BR from the CP space, which coincides with N decay, becomes slow as the medium pH increases (see the traces at pH 6 and pH 9 in Fig. 3B). The rate constants of the proton uptake obeyed the Henderson-Hasselbalch equation with a single p*K*a, and the value of Asp96BR during N decay was estimated at 7.8 (Tamogami et al., 2009). This value also agreed with the previously reported value (Balashov, 2000). These p*K*a values determined by the ITO method are considered those of key amino acid residues for the proton pumping function of BR. Therefore this method is useful for detecting the proton transfer directly and deducing the important p*K*a values.

### **10. Application to other H<sup>+</sup> -pumping rhodopsins**

This ITO method has been successfully applied to the newly found H+-pumping rhodopsins (Tamogami et al., 2009; Kikukawa et al, 2011). The panels B and C in Fig. 5 are the results for ARII from marine algae and PR from marine bacteria, respectively. Interestingly, the pH profiles of these three proton pumps are quite different. The prominent differences are as follows: (1) The pH profiles of the three rhodopsins commonly have bell-shaped negative peak areas, indicating that the proton uptake occurs prior to the release. However, the negative peak of PR is located at a quite higher pH (pH~8) than the other two rhodopsins (pH 3~4). This reflects the difference of the most acidic p*K*a's, governing the pH where the proton uptake starts to occur. These p*K*a's correspond to those of aspartates (Asp85BR, Asp81ARII and Asp97PR), the counterions of the respective protonated Schiff bases. This p*K*a for PR is about 7, which is much higher than the p*K*a's of about 2.6 of the other two rhodopsins. This high p*K*a for PR reported by using spectroscopic methods (Dioumaev et

Photo-Induced Proton Transfers of Microbial Rhodopsins 101

The peak values of the photo-induced signals were plotted against the medium pH. The experimental conditions were identical to those described in Fig. 3. For BR, the purple membrane prepared by a standard method (Becher & Cassim, 1975) was used. ARII and PR were expressed in the cell-free system and *E. coli* expression system, respectively, and then they were reconstituted with Egg L-α-phosphatidylcholine. Panels A and C was adapted with permission from Tamogami et al., 2009, *Photochem. Photobiol.* Copyright 2009 The authors, Journal Compilation, The American Society of Photobiology. Panel B was adapted with permission from Kikukawa et al., 2011, *Biochemistry.* Copyright 2011 American

This ITO method is also applicable to the proton transfers of photosensing rhodopsins. We have adopted this method for the archaeal sensory rhodopsins, NpSRII (Iwamoto et al., 1999), HsSRII (Tamogami et al., 2010) and a putative new class of photosensing rhodopsin called sensory rhodopsin III from *Haloarcular marismortui* (HmSRIII) (Nakao et al., 2011).

Consequently, we successfully determined the timings of the proton uptake/release during their respective photocycles. These results are attributed to the high sensitivity of this method as compared with an alternative method using a pH-sensitive dye. Most photosensing rhodopsins have slow photocycles (~sec). This slow turnover rate of the photocycle makes it difficult to adopt the pH-sensitive dye method for this measurement. The absorbance change due to the pH-sensitive dye is very small, and so a slight baseline fluctuation of the absorbance change results in a significant artifact. Especially for a longterm measurement corresponding to the slow photocycle, this baseline fluctuation becomes

Fig. 6. The pH profiles of the photo-induced proton transfer in NpSRII in the presence and

The values of the data points at 10 ms after flash light (the duration of 4 ms) excitation were plotted against pH. The closed and open symbols are the plots in the absence and presence of NpHtrII1-159, respectively. The NpSRIIs reconstituted with L-α-phosphatidylcholine were employed for the measurements. Added NpHtrIIs were truncated from the 1st to the 159th amino acid. The other experimental conditions were identical to those described in Fig. 3.

Chemical Society.

prominent.

absence of NpHtrII1-159.

**11. Application to photosensing rhodopsins** 

al., 2002; Friedrich et al., 2002; Lakatos et al., 2003; Imasheva et al., 2004; Partha et al, 2005). Thus, we obtained the same result *via* direct measurements of the proton-transfer reactions. (2) As the pH increases, all three rhodopsins begin their proton releases prior to their proton uptakes.

The positive areas of the pH profiles correspond to this proton-transfer sequence. However, the starting pH's, which reflect the p*K*a's of the proton-releasing residues, are different. These p*K*a values are about 6.1 for BR, 8 for ARII and 10 for PR, respectively. The higher p*K*a's of ARII and PR might reflect the absence of residues constituting the PRC of BR. ARII lacks a residue corresponding to Glu194BR, one of two glutamates constituting the PRC. By using the mutant of ARII, we confirmed that another glutamate, Glu199ARII, which corresponds to Glu204BR, functions as the proton-releasing residue (unpublished data). On the other hand, PR lacks both glutamates. For PR, therefore, an unknown residue works as the proton-releasing residue. (3) The pH profile of ARII has a surprising feature: the magnitude of the proton release again increases with a further increase in pH above 10. This indicates that a certain residue, other than Glu199ARII, starts to work as the proton-releasing residue at this pH range. These observations suggest that, despite the identical function, these proton pumps possess partially different mechanisms. Thus various interesting phenomena have been discovered by the experiments using the ITO method.

Fig. 5. Comparison of the pH profile of the photo-induced proton transfer among various microbial rhodopsins: (A) BR; (B) ARII; (C) PR.

al., 2002; Friedrich et al., 2002; Lakatos et al., 2003; Imasheva et al., 2004; Partha et al, 2005). Thus, we obtained the same result *via* direct measurements of the proton-transfer reactions. (2) As the pH increases, all three rhodopsins begin their proton releases prior to their proton

The positive areas of the pH profiles correspond to this proton-transfer sequence. However, the starting pH's, which reflect the p*K*a's of the proton-releasing residues, are different. These p*K*a values are about 6.1 for BR, 8 for ARII and 10 for PR, respectively. The higher p*K*a's of ARII and PR might reflect the absence of residues constituting the PRC of BR. ARII lacks a residue corresponding to Glu194BR, one of two glutamates constituting the PRC. By using the mutant of ARII, we confirmed that another glutamate, Glu199ARII, which corresponds to Glu204BR, functions as the proton-releasing residue (unpublished data). On the other hand, PR lacks both glutamates. For PR, therefore, an unknown residue works as the proton-releasing residue. (3) The pH profile of ARII has a surprising feature: the magnitude of the proton release again increases with a further increase in pH above 10. This indicates that a certain residue, other than Glu199ARII, starts to work as the proton-releasing residue at this pH range. These observations suggest that, despite the identical function, these proton pumps possess partially different mechanisms. Thus various interesting phenomena have been discovered by the experiments using the

Fig. 5. Comparison of the pH profile of the photo-induced proton transfer among various

microbial rhodopsins: (A) BR; (B) ARII; (C) PR.

uptakes.

ITO method.

The peak values of the photo-induced signals were plotted against the medium pH. The experimental conditions were identical to those described in Fig. 3. For BR, the purple membrane prepared by a standard method (Becher & Cassim, 1975) was used. ARII and PR were expressed in the cell-free system and *E. coli* expression system, respectively, and then they were reconstituted with Egg L-α-phosphatidylcholine. Panels A and C was adapted with permission from Tamogami et al., 2009, *Photochem. Photobiol.* Copyright 2009 The authors, Journal Compilation, The American Society of Photobiology. Panel B was adapted with permission from Kikukawa et al., 2011, *Biochemistry.* Copyright 2011 American Chemical Society.

### **11. Application to photosensing rhodopsins**

This ITO method is also applicable to the proton transfers of photosensing rhodopsins. We have adopted this method for the archaeal sensory rhodopsins, NpSRII (Iwamoto et al., 1999), HsSRII (Tamogami et al., 2010) and a putative new class of photosensing rhodopsin called sensory rhodopsin III from *Haloarcular marismortui* (HmSRIII) (Nakao et al., 2011).

Consequently, we successfully determined the timings of the proton uptake/release during their respective photocycles. These results are attributed to the high sensitivity of this method as compared with an alternative method using a pH-sensitive dye. Most photosensing rhodopsins have slow photocycles (~sec). This slow turnover rate of the photocycle makes it difficult to adopt the pH-sensitive dye method for this measurement. The absorbance change due to the pH-sensitive dye is very small, and so a slight baseline fluctuation of the absorbance change results in a significant artifact. Especially for a longterm measurement corresponding to the slow photocycle, this baseline fluctuation becomes prominent.

Fig. 6. The pH profiles of the photo-induced proton transfer in NpSRII in the presence and absence of NpHtrII1-159.

The values of the data points at 10 ms after flash light (the duration of 4 ms) excitation were plotted against pH. The closed and open symbols are the plots in the absence and presence of NpHtrII1-159, respectively. The NpSRIIs reconstituted with L-α-phosphatidylcholine were employed for the measurements. Added NpHtrIIs were truncated from the 1st to the 159th amino acid. The other experimental conditions were identical to those described in Fig. 3.

Photo-Induced Proton Transfers of Microbial Rhodopsins 103

consider that the newly found rhodopsins acquired their original mechanisms to adapt to their living environments. In this report, we showed the pH-dependent proton movements of BR, ARII, PR and NpSRII (Figs. 5 and 6). Even though only four rhodopsins were considered, their pH dependences were quite different. This might reflect the mechanical divergence of microbial rhodopsins. In the future, a detailed analysis of each rhodopsin will certainly be important. This should be achieved by using various amino acid mutants. Moreover, the proton movements of many more microbial rhodopsins should be examined using this electrochemical cell. From these studies, we could obtain deeper insights into the

Balashov, S.P.; Imasheva, E.S.; Ebrey, T.G.; Chen, N.; Menick, D.R. & Crouch, R.K. (1997).

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Becher, B. & Cassim, J.T. (1975). Improved isolation procedures for the purple membrane of *Halobacterium halobium*. *Prep. Biochem*., Vol.5, No.2, pp. 161-178, ISSN 0032-7484 Béjà, O.; Aravind, L.; Koonin, E.V.; Suzuki, M.T.; Hadd, A.; Nguyen, L.P.; Jovanovich, S.B.;

Bogomolni, R.A. & Spudich J.L. (1982). Identification of a third rhodopsin-like pigment in

Bogomolni, R.A.; Stoeckenius, W.; Szundi, I.; Perozo, E.; Olson, K.D. & Spudich, J.L. (1994).

Brown, L.S.; Sasaki, J.; Kandori, H.; Maeda, A.; Needleman, R. & Lanyi, J.K. (1995). Glutamic

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Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna.

Gates, C.M.; Feldman, R.A.; Spudich, J.L.; Spudich, E.N. & DeLong, E.F. (2000). Bacterial rhodopsin: Evidence for a new type of phototrophy in the sea. *Science*,

phototactic *Halobacterium halobium*. *Proc. Natl. Acad. Sci. USA*, Vol.79, No.20, pp.

Removal of transducer HtrI allows electrogenic proton translocation by sensory rhodopsin I. *Proc. Natl. Acad. Sci. USA*, Vol.91, No.21, pp. 10188-10192, ISSN 0027-

acid 204 is the terminal proton release group at the extracellular surface of bacteriorhodopsin. *J. Biol. Chem*., Vol.270, No.45, pp. 27122-27126, ISSN 0021-9258 Dioumaev, A.K.; Richter, H-T.; Brown, L.S.; Tanio, M.; Tuzi, S.; Saito, H.; Kimura, Y.;

Needleman, R. & Lanyi, J.K. (1998). Existence of a proton transfer chain in bacteriorhodopsin: Participation of Glu-194 in the release of protons to the extracellular surface. *Biochemistry*, Vol.37, No.8, pp. 2496-2506, ISSN 0006-2960 Dioumaev, A.K.; Brown, L.S.; Shih, J.; Spudich, E.N.; Spudich, J.L. & Lanyi, J.K. (2002).

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mechanistic principles of individual rhodopsins.

**13. References** 

Adapted with permission from Iwamoto et al., 2004, *Biochemistry*. Copyright 2004 American Chemical Society.

For the three sensory rhodopsins examined so far, we confirmed that the rates of proton uptake reactions determined the decay of their respective M-intermediates, the putative signaling states. In addition to these, we also detected the alterations in the proton-transfer reactions of NpSRII by the complex formation with its cognate transducer, called NpHtrII (Iwamoto et al., 2004). Figure 6 shows the pH profile of the photo-induced proton transfer in NpSRII in the presence and absence of NpHtrII. As shown here, NpSRII releases a proton prior to the uptake at this pH range. From the mutation analyses, the proton-releasing residue was identified as Asp193NpSRII (corresponding to Glu204BR), which is located at the end of the EC channel. Then, the lower and higher p*K*a's governing the bell-shaped pH profile were attributed to the p*K*a's of Asp193NpSRII at the M-intermediate for release and at the dark state, respectively. As shown in this figure, the pH profile in the presence of NpHtrII shifts to a lower pH compared to that in the absence of NpHtrII, implying that the complex formation induces the conformational change of NpSRII and leads to the p*K*a changes of Asp193NpSRII. In the crystal structures, the significant conformational change around Asp193NpSRII was not observed by the complex formation (Gordeliy et al., 2002). Thus, the p*K*a's of the proton-transfer reactions could be responsive to a small perturbation of the conformation.

The bell-shaped pH profile of NpSRII, showing the first proton release, is shifted to acidic pH regions as compared with the positive peaks of the proton pumps shown in Fig. 5. This reflects the lower p*K*a's of Asp193NpSRII, the proton-releasing residue. For NpSRII, the binding of Cl- to the vicinity of Asp193NpSRII was suggested by the ITO method (Iwamoto et al., 2004), ATR-FTIR measurement (Kitade et al., 2009) and the crystal structure (Royant et al., 2001), while this Cl- binding is not known for the proton pumps examined so far. The physiological meaning of the much lower p*K*a of Asp193NpSRII should be examined in a future study.

### **12. Conclusion and future perspectives**

The electrochemical cell using ITO (or SnO2) electrodes is a powerful and convenient device to detect the proton movements associated with photo-induced reactions of microbial rhodopsins and probably other photoactive pigments. Due to the high sensitivity and rapid response, this system enables us to follow the proton movements during a single photocycle under various buffer conditions. As described above, our current system cannot follow a reaction faster than 10 ms. The first proton movements of microbial rhodopsins appear to occur within 0.1-1 ms. Thus, the system response should be improved. On this point, we have already confirmed that the combination of nsec laser pulse with a homemade amplifier can accelerate the response to about 20 μs.

Early studies on the microbial rhodopsins concerned exclusively four rhodopsins in *H. salinarum*. BR in particular attracted much interest and was investigated in great detail. Thus, BR has been considered a kind of prototype of microbial rhodopsins. However, newly found microbial rhodopsins would seem to challenge the prototype status of BR, since they possess features not seen in BR, as described above. Microbial rhodopsins have been found in a wide variety of microorganisms living in various environments. Thus, it is reasonable to consider that the newly found rhodopsins acquired their original mechanisms to adapt to their living environments. In this report, we showed the pH-dependent proton movements of BR, ARII, PR and NpSRII (Figs. 5 and 6). Even though only four rhodopsins were considered, their pH dependences were quite different. This might reflect the mechanical divergence of microbial rhodopsins. In the future, a detailed analysis of each rhodopsin will certainly be important. This should be achieved by using various amino acid mutants. Moreover, the proton movements of many more microbial rhodopsins should be examined using this electrochemical cell. From these studies, we could obtain deeper insights into the mechanistic principles of individual rhodopsins.

### **13. References**

102 Molecular Photochemistry – Various Aspects

Adapted with permission from Iwamoto et al., 2004, *Biochemistry*. Copyright 2004 American

For the three sensory rhodopsins examined so far, we confirmed that the rates of proton uptake reactions determined the decay of their respective M-intermediates, the putative signaling states. In addition to these, we also detected the alterations in the proton-transfer reactions of NpSRII by the complex formation with its cognate transducer, called NpHtrII (Iwamoto et al., 2004). Figure 6 shows the pH profile of the photo-induced proton transfer in NpSRII in the presence and absence of NpHtrII. As shown here, NpSRII releases a proton prior to the uptake at this pH range. From the mutation analyses, the proton-releasing residue was identified as Asp193NpSRII (corresponding to Glu204BR), which is located at the end of the EC channel. Then, the lower and higher p*K*a's governing the bell-shaped pH profile were attributed to the p*K*a's of Asp193NpSRII at the M-intermediate for release and at the dark state, respectively. As shown in this figure, the pH profile in the presence of NpHtrII shifts to a lower pH compared to that in the absence of NpHtrII, implying that the complex formation induces the conformational change of NpSRII and leads to the p*K*a changes of Asp193NpSRII. In the crystal structures, the significant conformational change around Asp193NpSRII was not observed by the complex formation (Gordeliy et al., 2002). Thus, the p*K*a's of the proton-transfer reactions could be responsive to a small perturbation

The bell-shaped pH profile of NpSRII, showing the first proton release, is shifted to acidic pH regions as compared with the positive peaks of the proton pumps shown in Fig. 5. This reflects the lower p*K*a's of Asp193NpSRII, the proton-releasing residue. For NpSRII, the binding of Cl- to the vicinity of Asp193NpSRII was suggested by the ITO method (Iwamoto et al., 2004), ATR-FTIR measurement (Kitade et al., 2009) and the crystal structure (Royant et al., 2001), while this Cl- binding is not known for the proton pumps examined so far. The physiological meaning of the much lower p*K*a of Asp193NpSRII should be examined in a

The electrochemical cell using ITO (or SnO2) electrodes is a powerful and convenient device to detect the proton movements associated with photo-induced reactions of microbial rhodopsins and probably other photoactive pigments. Due to the high sensitivity and rapid response, this system enables us to follow the proton movements during a single photocycle under various buffer conditions. As described above, our current system cannot follow a reaction faster than 10 ms. The first proton movements of microbial rhodopsins appear to occur within 0.1-1 ms. Thus, the system response should be improved. On this point, we have already confirmed that the combination of nsec laser pulse with a homemade amplifier

Early studies on the microbial rhodopsins concerned exclusively four rhodopsins in *H. salinarum*. BR in particular attracted much interest and was investigated in great detail. Thus, BR has been considered a kind of prototype of microbial rhodopsins. However, newly found microbial rhodopsins would seem to challenge the prototype status of BR, since they possess features not seen in BR, as described above. Microbial rhodopsins have been found in a wide variety of microorganisms living in various environments. Thus, it is reasonable to

Chemical Society.

of the conformation.

future study.

**12. Conclusion and future perspectives** 

can accelerate the response to about 20 μs.


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**6** 

*Japan* 

**Function of Extrinsic Proteins in Stabilization** 

Yong Li1, Yukihiro Kimura1,2, Takashi Ohno1 and Yasuo Yamauchi1

*1Graduate School of Agricultural Science, Kobe University* 

*2Organization of Advanced Science and Technology, Kobe University* 

**of the Photosynthetic Oxygen-Evolving Complex** 

Oxygenic phototrophs convert photon energy into chemical energy through a series of lightinduced electron transfer reactions initiated with charge separation of chlorophyll (Chl) special pairs located in the central part of photosystem I and II (PSI and PSII) (Fig. 1). The reducing power is transferred from PSII to PSI through cytochrome *b*6*f*, and finally utilized for reduction of NADP+ to assimilate CO2. The oxidized equivalents accumulated on the PSII donor side are neutralized by substrate water molecules to release protons for driving ATP synthase and O2 molecules as a by-product. This water oxidation takes place in the oxygen-evolving complex (OEC) of PSII [McEvoy & Brudvig, 2006, Renger & Renger, 2008]. The OEC assembly is largely similar between cyanobacteria and higher plants, except for a critical difference in the composition of extrinsic proteins [Roose *et al.*, 2007]. In cyanobacteria, PsbO, PsbV, and PsbU residing on the lumenal side of PSII play significant roles in the regulation and stabilization of the water oxidation machinery. Higher plants possess major nuclear gene-encoded extrinsic proteins named PsbO, PsbP, and PsbQ. PsbO is a common extrinsic protein highly conserved among the oxygenic phototrophs. PsbP and PsbQ are indigenous to plant PSII and have been proposed as the functional equivalents of PsbV and PsbU in bacterial PSII, having replaced them during the course of evolution from ancestral cyanobacteria to higher plants. These proteins play significant roles in the regulation and stabilization of the photosynthetic water oxidation [Roose *et al.*, 2007, Seidler, 1996, Williamson, 2008] although the details of their function(s) are still a matter of debate.

Fig. 1. Reaction scheme of photosynthesis in oxygenic phototrophs.

**1. Introduction** 

under pulsed and CW laser excitations. *J. Phys. Chem.B*, Vol.101, No.49, pp. 10599- 10604, ISSN 1520-6106


## **Function of Extrinsic Proteins in Stabilization of the Photosynthetic Oxygen-Evolving Complex**

Yong Li1, Yukihiro Kimura1,2, Takashi Ohno1 and Yasuo Yamauchi1 *1Graduate School of Agricultural Science, Kobe University 2Organization of Advanced Science and Technology, Kobe University Japan* 

### **1. Introduction**

108 Molecular Photochemistry – Various Aspects

Waschuk, S.A.; Bezerra, A.G.; Shi, Jr.L. & Brown, L.S. (2005). *Leptosphaeria* rhodopsin:

Wolff, E.K.; Bogomolni, R.A.; Scherrer P.; Hess B. & Stoeckenius W. (1986). Color

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under pulsed and CW laser excitations. *J. Phys. Chem.B*, Vol.101, No.49, pp. 10599-

Bacteriorhodopsin-like proton pump from a eukaryote. *Proc. Natl. Acad. Sci. USA*,

discrimination in halobacteria: Spectroscopic characterization of a second sensory receptor covering the blue-green region of the spectrum. *Proc. Natl. Acad. Sci. USA*,

determine the p*K*a of the proton release complex in the photocycle of retinal

proton release in the bacteriorhodopsin photocycle. *Biochemistry*, Vol.31, No.36, pp.

Oxygenic phototrophs convert photon energy into chemical energy through a series of lightinduced electron transfer reactions initiated with charge separation of chlorophyll (Chl) special pairs located in the central part of photosystem I and II (PSI and PSII) (Fig. 1). The reducing power is transferred from PSII to PSI through cytochrome *b*6*f*, and finally utilized for reduction of NADP+ to assimilate CO2. The oxidized equivalents accumulated on the PSII donor side are neutralized by substrate water molecules to release protons for driving ATP synthase and O2 molecules as a by-product. This water oxidation takes place in the oxygen-evolving complex (OEC) of PSII [McEvoy & Brudvig, 2006, Renger & Renger, 2008]. The OEC assembly is largely similar between cyanobacteria and higher plants, except for a critical difference in the composition of extrinsic proteins [Roose *et al.*, 2007]. In cyanobacteria, PsbO, PsbV, and PsbU residing on the lumenal side of PSII play significant roles in the regulation and stabilization of the water oxidation machinery. Higher plants possess major nuclear gene-encoded extrinsic proteins named PsbO, PsbP, and PsbQ. PsbO is a common extrinsic protein highly conserved among the oxygenic phototrophs. PsbP and PsbQ are indigenous to plant PSII and have been proposed as the functional equivalents of PsbV and PsbU in bacterial PSII, having replaced them during the course of evolution from ancestral cyanobacteria to higher plants. These proteins play significant roles in the regulation and stabilization of the photosynthetic water oxidation [Roose *et al.*, 2007, Seidler, 1996, Williamson, 2008] although the details of their function(s) are still a matter of debate.

Fig. 1. Reaction scheme of photosynthesis in oxygenic phototrophs.

Function of Extrinsic Proteins in Stabilization of the Photosynthetic Oxygen-Evolving Complex 111

has been limited to electron micrographs at low resolutions [Nield *et al.*, 2002, Nield & Barber, 2006]. Yet the findings to date strongly indicate that the structure and function of the PSII core assembly are almost identical to those of its prokaryotic counterparts, except for a critical difference in the composition of extrinsic proteins, which may provide valuable insights into the evolution of photosynthetic organisms [De Las Rivas *et al.*, 2004]. Based on the crystallographic structure of the OEC, several possible pathways for water, proton, and O2 channels were proposed [Gabdulkhakov *et al.*, 2009, Guskov *et al.*, 2009]. However, photosynthetic water oxidation is a complex process that involves S-state cycling with five intermediate states, and therefore, the reaction mechanisms are not yet

Higher plants possess gene-encoded extrinsic proteins, including PsbP, PsbQ, and PsbR, as well as PsbO, which commonly exists in all oxygenic phototrophs. These proteins play a key role in maintaining oxygen-evolving activity at physiological rates [Roose *et al.*, 2007, Williamson, 2008]. PsbO independently associates with the PSII core [Miyao & Murata, 1983, Miyao & Murata, 1989], and with PsbP through electrostatic interactions with PsbO [Miyao & Murata, 1983, Tohri *et al.*, 2004]. PsbQ requires both PsbO and PsbP for its binding [Miyao & Murata, 1983, Miyao & Murata, 1989]. In contrast, PsbO and PsbV independently bind to the PSII core, which lacks extrinsic proteins [Shen & Inoue, 1993], and the full binding of

PsbP and PsbQ are thought to be the respective functional equivalents of PsbV and PsbU in the bacterial PSII [Enami *et al.*, 2005, Shen & Inoue, 1993], despite their low structural homology between PsbP(Q) and PsbV(U) [Balsera *et al.*, 2005, Ifuku *et al.*, 2004]. A phylogenetic study indicated that PsbP and PsbQ in the plant PSII were derived from PsbP and PsbQ homologues, respecively, in bacterial PSII [Thornton *et al.*, 2004], through intensive genetic modification during endosymbiosis and subsequent gene transfer to the

PsbO is the most important protein for stabilization of the Mn4Ca cluster, and therefore, it is common in all oxygenic phototrophs. The release of PsbO induces release of Mn ions from the cluster, resulting in the loss of O2-evolving activity. The PsbO protein is common in every oxygenic phototroph but in varying proportions: one PsbO per PSII in cyanobacteria and two PsbO per PSII in higher plants [Williamson, 2008, Xu & Bricker, 1992]. Highresolution X-ray crystallographs of the PsbO protein associated with the PSII core are available for *Thermosynechococcus elongatus* [Ferreira *et al.*, 2004, Guskov *et al.*, 2009] and *Thermosynechococcus vulcanus* [Kawakami *et al.*, 2009, Umena *et al.*, 2011], in which PsbO is

analysis of plant PsbO has been delayed and is limited to low-resolution images [Nield & Barber, 2006]. PsbO proteins are believed to play significant roles in protecting and stabilizing the catalytic center, however, none of the amino acid residue from PsbO serves as


PsbU requires both PsbO and PsbV [Shen *et al.*, 1995, Shen & Inoue, 1993].

host nucleus [De Las Rivas & Roman, 2005, Ifuku *et al.*, 2008, Ishihara *et al.*, 2007].


fully understood.

**2.2.1 PsbO** 

comprised of a

a direct ligand for the Mn4Ca cluster.

**2.2 Extrinsic proteins in PSII** 

In this chapter, we describe the structural-functional roles of extrinsic proteins in the plant PSII. The effects of extrinsic proteins on the photosynthetic function of the Mn4Ca cluster and the structural stability of the OEC core complex were investigated by spectroscopic and biochemical analyses. Based on the results presented here and reported previously, the structural and functional roles of extrinsic proteins in the regulation and stabilization of photosynthetic functions are discussed.

### **2. General structure and function of oxygenic phototrophs**

### **2.1 Oxygen-evolving complex**

Photosynthetic oxygen evolution occurs in the PSII OEC which is composed of a heterodimer of D1 (psbA) and D2 (psbD) proteins associated with two chlorophyll proteins (CP), CP47 (psbB) and CP43 (psbC), and involves a catalytic Mn4Ca cluster located on the lumenal side of PSII (Fig. 2). These are highly conserved from cyanobacteria to higher plants to preserve the essential function of oxygenic phototrophs. The oxidized equivalents accumulated on the cluster and/or its ligands are reduced by electrons provided from a splitting reaction of substrate water molecules through a light-driven S-state cycle with five intermediate states Sn (n = 0 – 4), where n denotes the number of oxidizing equivalents stored. The OEC advances from the thermally stable S1 state to the next oxidation state in a stepwise manner by absorbing each photon and attains the highest oxidation state S4, followed by relaxation to the lowest oxidation state S0 concurrent with a release of one oxygen molecule [Joliot *et al.*, 1969, Kok *et al.*, 1970]. Two water molecules are converted to one oxygen molecule by the OEC concurrent with release of four protons. Calcium and chloride ions are indispensable inorganic cofactors for playing functional and structural roles in the OEC [Debus, 1992, Yocum, 2008].

Fig. 2. OEC assemblies of cyanobacteria (left) and higher plants (right).

In the past decade, X-ray crystallography has revealed the structures of cyanobacterial PSII at resolutions of 3.8 Å to 2.9 Å [Ferreira *et al.*, 2004, Guskov *et al.*, 2009, Kamiya & Shen, 2003, Loll *et al.*, 2005, Zouni *et al.*, 2001]. A very recent structural model at the atomic resolution level has revealed details of the ligation structure of the Mn4Ca cluster [Umena *et al.*, 2011]. The Mn ions are bridged by several oxygen atoms and coordinated by water molecules as well as by Asp, Glu, Ala, and His residues from PsbA and/or PsbC proteins. In contrast, high-resolution structural analysis of higher plants has been delayed due to the instability of the membrane protein complex. The visualization of plant PSII structures has been limited to electron micrographs at low resolutions [Nield *et al.*, 2002, Nield & Barber, 2006]. Yet the findings to date strongly indicate that the structure and function of the PSII core assembly are almost identical to those of its prokaryotic counterparts, except for a critical difference in the composition of extrinsic proteins, which may provide valuable insights into the evolution of photosynthetic organisms [De Las Rivas *et al.*, 2004]. Based on the crystallographic structure of the OEC, several possible pathways for water, proton, and O2 channels were proposed [Gabdulkhakov *et al.*, 2009, Guskov *et al.*, 2009]. However, photosynthetic water oxidation is a complex process that involves S-state cycling with five intermediate states, and therefore, the reaction mechanisms are not yet fully understood.

### **2.2 Extrinsic proteins in PSII**

110 Molecular Photochemistry – Various Aspects

In this chapter, we describe the structural-functional roles of extrinsic proteins in the plant PSII. The effects of extrinsic proteins on the photosynthetic function of the Mn4Ca cluster and the structural stability of the OEC core complex were investigated by spectroscopic and biochemical analyses. Based on the results presented here and reported previously, the structural and functional roles of extrinsic proteins in the regulation and stabilization of

Photosynthetic oxygen evolution occurs in the PSII OEC which is composed of a heterodimer of D1 (psbA) and D2 (psbD) proteins associated with two chlorophyll proteins (CP), CP47 (psbB) and CP43 (psbC), and involves a catalytic Mn4Ca cluster located on the lumenal side of PSII (Fig. 2). These are highly conserved from cyanobacteria to higher plants to preserve the essential function of oxygenic phototrophs. The oxidized equivalents accumulated on the cluster and/or its ligands are reduced by electrons provided from a splitting reaction of substrate water molecules through a light-driven S-state cycle with five intermediate states Sn (n = 0 – 4), where n denotes the number of oxidizing equivalents stored. The OEC advances from the thermally stable S1 state to the next oxidation state in a stepwise manner by absorbing each photon and attains the highest oxidation state S4, followed by relaxation to the lowest oxidation state S0 concurrent with a release of one oxygen molecule [Joliot *et al.*, 1969, Kok *et al.*, 1970]. Two water molecules are converted to one oxygen molecule by the OEC concurrent with release of four protons. Calcium and chloride ions are indispensable inorganic cofactors for playing functional and structural

**2. General structure and function of oxygenic phototrophs** 

Fig. 2. OEC assemblies of cyanobacteria (left) and higher plants (right).

In the past decade, X-ray crystallography has revealed the structures of cyanobacterial PSII at resolutions of 3.8 Å to 2.9 Å [Ferreira *et al.*, 2004, Guskov *et al.*, 2009, Kamiya & Shen, 2003, Loll *et al.*, 2005, Zouni *et al.*, 2001]. A very recent structural model at the atomic resolution level has revealed details of the ligation structure of the Mn4Ca cluster [Umena *et al.*, 2011]. The Mn ions are bridged by several oxygen atoms and coordinated by water molecules as well as by Asp, Glu, Ala, and His residues from PsbA and/or PsbC proteins. In contrast, high-resolution structural analysis of higher plants has been delayed due to the instability of the membrane protein complex. The visualization of plant PSII structures

photosynthetic functions are discussed.

roles in the OEC [Debus, 1992, Yocum, 2008].

**2.1 Oxygen-evolving complex** 

Higher plants possess gene-encoded extrinsic proteins, including PsbP, PsbQ, and PsbR, as well as PsbO, which commonly exists in all oxygenic phototrophs. These proteins play a key role in maintaining oxygen-evolving activity at physiological rates [Roose *et al.*, 2007, Williamson, 2008]. PsbO independently associates with the PSII core [Miyao & Murata, 1983, Miyao & Murata, 1989], and with PsbP through electrostatic interactions with PsbO [Miyao & Murata, 1983, Tohri *et al.*, 2004]. PsbQ requires both PsbO and PsbP for its binding [Miyao & Murata, 1983, Miyao & Murata, 1989]. In contrast, PsbO and PsbV independently bind to the PSII core, which lacks extrinsic proteins [Shen & Inoue, 1993], and the full binding of PsbU requires both PsbO and PsbV [Shen *et al.*, 1995, Shen & Inoue, 1993].

PsbP and PsbQ are thought to be the respective functional equivalents of PsbV and PsbU in the bacterial PSII [Enami *et al.*, 2005, Shen & Inoue, 1993], despite their low structural homology between PsbP(Q) and PsbV(U) [Balsera *et al.*, 2005, Ifuku *et al.*, 2004]. A phylogenetic study indicated that PsbP and PsbQ in the plant PSII were derived from PsbP and PsbQ homologues, respecively, in bacterial PSII [Thornton *et al.*, 2004], through intensive genetic modification during endosymbiosis and subsequent gene transfer to the host nucleus [De Las Rivas & Roman, 2005, Ifuku *et al.*, 2008, Ishihara *et al.*, 2007].

### **2.2.1 PsbO**

PsbO is the most important protein for stabilization of the Mn4Ca cluster, and therefore, it is common in all oxygenic phototrophs. The release of PsbO induces release of Mn ions from the cluster, resulting in the loss of O2-evolving activity. The PsbO protein is common in every oxygenic phototroph but in varying proportions: one PsbO per PSII in cyanobacteria and two PsbO per PSII in higher plants [Williamson, 2008, Xu & Bricker, 1992]. Highresolution X-ray crystallographs of the PsbO protein associated with the PSII core are available for *Thermosynechococcus elongatus* [Ferreira *et al.*, 2004, Guskov *et al.*, 2009] and *Thermosynechococcus vulcanus* [Kawakami *et al.*, 2009, Umena *et al.*, 2011], in which PsbO is comprised of a -barrel core with an extended -helix domain. In contrast, the structural analysis of plant PsbO has been delayed and is limited to low-resolution images [Nield & Barber, 2006]. PsbO proteins are believed to play significant roles in protecting and stabilizing the catalytic center, however, none of the amino acid residue from PsbO serves as a direct ligand for the Mn4Ca cluster.

Function of Extrinsic Proteins in Stabilization of the Photosynthetic Oxygen-Evolving Complex 113

In this study, we used two types of PSII membranes lacking functional Ca2+ with or without PsbP and PsbQ. The sample preparation method is shown in Fig. 3. Berthold-Babcock-Yocum (BBY)-type PS II membranes (untreated PSII, A) were prepared from spinach according to the method described previously [Ono *et al.*, 2001]. The O2-evolving activity was ~550 *μ*moles of O2/mgChl/h. For depletion of Ca2+, PsbP, and PsbQ, the membranes were suspended in medium A (2 M NaCl, 10 mM Mes/NaOH, and pH 6.5) at 0.5 mg of Chl per ml and gently stirred on ice under weak light(10 *μ*mol/s/m2)for 30 min. Next, the following procedures were carried out in complete darkness or dim green light unless otherwise noted: EDTA was added to the suspension to achieve a final concentration of 1 mM, followed by 10-min incubation in the dark. The suspension was centrifuged and extensively washed with Chelex-treated medium B (400 mM sucrose, 20 mM NaCl, 20 mM Mes/NaOH, and pH 6.5) to yield PSII membranes depleted of Ca2+, PsbP, and PsbQ (ExCa2+-depleted PSII, B). For depletion of PsbP and PsbQ proteins, PS II membranes were suspended in medium A at 0.5 mg of Chl per ml, and gently stirred on ice in darkness for 30 min. The extracted PsbP and PsbQ proteins were reconstituted into the NaCl/EDTA-treated

Alternatively, the PSII membranes were washed with medium C (400 mM sucrose, 20 mM NaCl, 0.1 mM Mes-NaOH, and pH 6.5) and then treated with medium D (400 mM sucrose, 20 mM NaCl, 40 mM citrate-NaOH , pH 3.0) at 2 mg of Chl per ml. After 5 min incubation on ice in darkness, the suspension was diluted with medium D (400 mM sucrose, 20 mM NaCl, 500 mM Mops-NaOH, pH 7.5), and incubated for 10 min to facilitate the rebinding of extrinsic proteins. Then, the sample was washed with medium E (400 mM sucrose, 20 mM NaCl, 40 mM Mes/NaOH, 0.5 mM EDTA, pH6.5) to obtain PSII membranes depleted of only Ca2+ (lowpH-treated PSII ,D). Finally, the resulting low-pH-treated PSII membranes were treated with medium A to produce PSII membranes depleted of both Ca2+ and

**3. Interaction of extrinsic proteins with the Mn4Ca cluster in the OEC** 

**3.1.1 Depletion/reconstitution of extrinsic proteins and functional Ca2+**

**3.1 PsbP and PsbQ** 

PSII to obtain Ca2+-depleted PSII (C).

extrinsic proteins (ExCa2+-depleted PSII, E).

Fig. 3. Schematic representation for each sample preparation.

Findings to date strongly indicate another significant role for PsbO: it is thought to modulate Ca2+ and Cl- requirements for O2 evolution [Seidler, 1996, Williamson, 2008]. However, the Ca2+-binding properties of PsbO proteins are somewhat different between higher plants and cyanobacteria. It has been reported that plant PsbO induces structural changes upon the binding of Ca2+ [Heredia & De Las Rivas, 2003, Kruk *et al.*, 2003], which is not the functional Ca2+ necessary for the water oxidation [Seidler & Rutherford, 1996]. In contrast, no significant Ca2+-induced stuctural change was found in cyanobacterial PsbO [Loll *et al.*, 2005], although it has been speculated that this protein serves as a lowaffinity binding site for functional Ca2+ [Murray & Barber, 2006, Rutherford & Faller, 2001].

### **2.2.2 PsbP and PsbV**

PsbP is also indispensable for the regulation and stabilization of PSII in higher plants [Ifuku *et al.*, 2008]. Deletion of this protein disables the normal functions of the plant PSII [Ifuku *et al.*, 2005]. This protein is related to the stability of the Mn4Ca cluster as well as to the binding affinity of Ca2+ and Cl– ions, which are essential cofactors for water oxidation reactions [Seidler, 1996]. Although it is unclear whether PsbP proteins directly interact with the Mn4Ca cluster, the binding of this protein to the PSII core in the absence of Ca2+ is known to cause modification of its physicochemical properties, including redox potentials, magnetic structures, and ligation geometries of the Mn4Ca cluster. It has also been speculated that PsbP is a metal-binding protein which reserves Mn2+ or Ca2+ to keep or deliver it to the apo-PSII [Bondarava *et al.*, 2005].

PsbV is also thought to be involved with the binding of inorganic factors. PsbV-lacking mutants were unable to grow photoautotrophically in the absence of Ca2+ or Cl- [Shen *et al.*, 1998]. In contrast to PsbP, PsbV was capable of supporting water oxidation even in the absence of PsbO [Shen *et al.*, 1995], suggesting that it serves to maintain a proper ion environment within the OEC for optimal oxygen-evolving activity [Nishiyama *et al.*, 1994, Shen *et al.*, 1995, Shen & Inoue, 1993, Shen *et al.*, 1998, Shen *et al.*, 1995]. Although none of residue from PsbV serves as a direct ligand for the Mn4Ca cluster, it has been suggested that this protein participates in stabilizing PsbA through electrostatic interactions [Sugiura *et al.*, 2010].

### **2.2.3 PsbQ and PsbU**

The roles of PsbQ protein in photosynthetic functions are not yet fully understood. The PsbQ protein is not necessary for normal growth in higher plants [Ifuku *et al.*, 2005, Yi *et al.*, 2006]. However, this protein is required for photoautotrophic growth under low-light conditions [Yi *et al.*, 2006] and it is involved in the binding of functional Cl ions [Balsera *et al.*, 2005]. In cyanobacteria, PsbU-lacking mutants were capable of photoautotrophic growth in the absence of Ca2+ or Cl- , but at reduced rates [Shen *et al.*, 1998]. The oxygen-evolving ability was reduced by the removal of PsbU and restored in part by the addition of Cl- but not Ca2+, indicating that PsbU regulates Cl requirement [Inoue-Kashino *et al.*, 2005, Shen *et al.*, 1997]. Another of its functions is the suppression of light-induced D1 degradation [Inoue-Kashino *et al.*, 2005]. Protection of the PSII core from reactive oxygen species (ROS) [Balint *et al.*, 2006] has also been proposed.

## **3. Interaction of extrinsic proteins with the Mn4Ca cluster in the OEC**

### **3.1 PsbP and PsbQ**

112 Molecular Photochemistry – Various Aspects

Findings to date strongly indicate another significant role for PsbO: it is thought to modulate Ca2+ and Cl- requirements for O2 evolution [Seidler, 1996, Williamson, 2008]. However, the Ca2+-binding properties of PsbO proteins are somewhat different between higher plants and cyanobacteria. It has been reported that plant PsbO induces structural changes upon the binding of Ca2+ [Heredia & De Las Rivas, 2003, Kruk *et al.*, 2003], which is not the functional Ca2+ necessary for the water oxidation [Seidler & Rutherford, 1996]. In contrast, no significant Ca2+-induced stuctural change was found in cyanobacterial PsbO [Loll *et al.*, 2005], although it has been speculated that this protein serves as a lowaffinity binding site for functional Ca2+ [Murray & Barber, 2006, Rutherford & Faller,

PsbP is also indispensable for the regulation and stabilization of PSII in higher plants [Ifuku *et al.*, 2008]. Deletion of this protein disables the normal functions of the plant PSII [Ifuku *et al.*, 2005]. This protein is related to the stability of the Mn4Ca cluster as well as to the binding affinity of Ca2+ and Cl– ions, which are essential cofactors for water oxidation reactions [Seidler, 1996]. Although it is unclear whether PsbP proteins directly interact with the Mn4Ca cluster, the binding of this protein to the PSII core in the absence of Ca2+ is known to cause modification of its physicochemical properties, including redox potentials, magnetic structures, and ligation geometries of the Mn4Ca cluster. It has also been speculated that PsbP is a metal-binding protein which reserves Mn2+ or Ca2+ to keep or deliver it to the apo-

PsbV is also thought to be involved with the binding of inorganic factors. PsbV-lacking mutants were unable to grow photoautotrophically in the absence of Ca2+ or Cl- [Shen *et al.*, 1998]. In contrast to PsbP, PsbV was capable of supporting water oxidation even in the absence of PsbO [Shen *et al.*, 1995], suggesting that it serves to maintain a proper ion environment within the OEC for optimal oxygen-evolving activity [Nishiyama *et al.*, 1994, Shen *et al.*, 1995, Shen & Inoue, 1993, Shen *et al.*, 1998, Shen *et al.*, 1995]. Although none of residue from PsbV serves as a direct ligand for the Mn4Ca cluster, it has been suggested that this protein participates in stabilizing PsbA through electrostatic interactions [Sugiura *et al.*,

The roles of PsbQ protein in photosynthetic functions are not yet fully understood. The PsbQ protein is not necessary for normal growth in higher plants [Ifuku *et al.*, 2005, Yi *et al.*, 2006]. However, this protein is required for photoautotrophic growth under low-light conditions [Yi *et al.*, 2006] and it is involved in the binding of functional Cl ions [Balsera *et al.*, 2005]. In cyanobacteria, PsbU-lacking mutants were capable of photoautotrophic growth

ability was reduced by the removal of PsbU and restored in part by the addition of Cl- but

*al.*, 1997]. Another of its functions is the suppression of light-induced D1 degradation [Inoue-Kashino *et al.*, 2005]. Protection of the PSII core from reactive oxygen species (ROS)

, but at reduced rates [Shen *et al.*, 1998]. The oxygen-evolving

requirement [Inoue-Kashino *et al.*, 2005, Shen *et* 

2001].

2010].

**2.2.3 PsbQ and PsbU** 

in the absence of Ca2+ or Cl-

not Ca2+, indicating that PsbU regulates Cl-

[Balint *et al.*, 2006] has also been proposed.

**2.2.2 PsbP and PsbV** 

PSII [Bondarava *et al.*, 2005].

### **3.1.1 Depletion/reconstitution of extrinsic proteins and functional Ca2+**

In this study, we used two types of PSII membranes lacking functional Ca2+ with or without PsbP and PsbQ. The sample preparation method is shown in Fig. 3. Berthold-Babcock-Yocum (BBY)-type PS II membranes (untreated PSII, A) were prepared from spinach according to the method described previously [Ono *et al.*, 2001]. The O2-evolving activity was ~550 *μ*moles of O2/mgChl/h. For depletion of Ca2+, PsbP, and PsbQ, the membranes were suspended in medium A (2 M NaCl, 10 mM Mes/NaOH, and pH 6.5) at 0.5 mg of Chl per ml and gently stirred on ice under weak light(10 *μ*mol/s/m2)for 30 min. Next, the following procedures were carried out in complete darkness or dim green light unless otherwise noted: EDTA was added to the suspension to achieve a final concentration of 1 mM, followed by 10-min incubation in the dark. The suspension was centrifuged and extensively washed with Chelex-treated medium B (400 mM sucrose, 20 mM NaCl, 20 mM Mes/NaOH, and pH 6.5) to yield PSII membranes depleted of Ca2+, PsbP, and PsbQ (ExCa2+-depleted PSII, B). For depletion of PsbP and PsbQ proteins, PS II membranes were suspended in medium A at 0.5 mg of Chl per ml, and gently stirred on ice in darkness for 30 min. The extracted PsbP and PsbQ proteins were reconstituted into the NaCl/EDTA-treated PSII to obtain Ca2+-depleted PSII (C).

Alternatively, the PSII membranes were washed with medium C (400 mM sucrose, 20 mM NaCl, 0.1 mM Mes-NaOH, and pH 6.5) and then treated with medium D (400 mM sucrose, 20 mM NaCl, 40 mM citrate-NaOH , pH 3.0) at 2 mg of Chl per ml. After 5 min incubation on ice in darkness, the suspension was diluted with medium D (400 mM sucrose, 20 mM NaCl, 500 mM Mops-NaOH, pH 7.5), and incubated for 10 min to facilitate the rebinding of extrinsic proteins. Then, the sample was washed with medium E (400 mM sucrose, 20 mM NaCl, 40 mM Mes/NaOH, 0.5 mM EDTA, pH6.5) to obtain PSII membranes depleted of only Ca2+ (lowpH-treated PSII ,D). Finally, the resulting low-pH-treated PSII membranes were treated with medium A to produce PSII membranes depleted of both Ca2+ and extrinsic proteins (ExCa2+-depleted PSII, E).

Fig. 3. Schematic representation for each sample preparation.

Function of Extrinsic Proteins in Stabilization of the Photosynthetic Oxygen-Evolving Complex 115

The present findings are largely in agreement with previous findings, as shown in Table 1. FT-IR spectroscopy provides valuable information on the structure and interactions within the OEC. The ligation geometry around the Mn4Ca cluster is mostly similar between untreated and ExCa2+-depleted PSII, at least in the S1- and S2-states [Kimura & Ono, 2001]. However, Ca2+-depleted PSII exhibited marked deterioration in the carboxylate bands, which are thought to be from putative amino acid residues coordinating to the Mn4Ca cluster [Noguchi *et al.*, 1995]. Furthermore, the redox potential of the Mn4Ca cluster has been reported to be abnormal when the extrinsic proteins bound to the PSII core in the absence of Ca2+, as indicated by elevated peak temperatures of the thermoluminescence band for the

> 0 10 20 30 40 50 60 70 Incubation time / min

> > (a) PsbP & PsbQ (b) PsbP & PsbQ with Ca2+

difference specrum (b - a)

1566

1539

Fig. 4. Plots of relative absorbance at 680 nm as a function of incubation time at 50C for (a)

1800 1700 1600 1500 1400 Wavenumber / cm-1

Fig. 5. ATR-FTIR spectra of isolated PsbP and PsbQ proteins in the absence (a, dotted line) and presence of Ca2+ (b, solid line). The difference spectrum obtained by subtracting

1641

1693 1659

untreated, (b) ExCa2+-depleted, and (c) Ca2+-depleted PS II membranes.

Absorbance (a.u.)

spectrum a from spectrum b is shown in the lower panel.

(a)

(b)

(c)

1.0

0.9

0.8

0.7

Relative absorbace

0.6

0.5

0.4

### **3.1.2 Effects of extrinsic proteins on the properties of the OEC**

The PSII membranes prepared by the different methods were assessed by O2-evolving activity and Fv/Fm values of chlorophyll fluorescence and the resulting data are summarized in Table 1. The O2-evolving rate of the untreated PSII was decreased to 17% when PsbP, PsbQ and Ca2+ were depleted by the NaCl/EDTA treatments (B). The decreased activity was restored to 83% by reconstituting Ca2+, as reported previously [Kimura & Ono, 2001, Ono *et al.*, 2001]. However, the addition of PsbP and PsbQ to the ExCa2+-depleted PSII in the absence of Ca2+ lowered the O2-evolving rate to ~0% (C). Furthermore, the O2-evolving activity was almost completely lost upon Ca2+ depletion by the low-pH treatment (D) but restored to 79% by adding Ca2+. Notably, the lost activity was partially resotored by the further depletion of PsbP and PsbQ to 25% (E). These results indicate that PsbP and PsbQ proteins completely suppress O2 evolution in the absence of functional Ca2+. Similar effects are also evident in the chlorophyll fluorescence measurements: the Fv/Fm values were much lower in the Ca2+ depleted PSII (45%) than in the ExCa2+-depleted PSII (64%), and both values were recoverd to ~80% after the supplementation with Ca2+. In the Ca2+-depleted PSII, the partial recovery to 68% was induced by the following depletion of the extrinsic proteins. Since Fv/Fm values are related to O2-evolving activity, this strongly suggests that the functions of the OEC are disturbed by the extrinsic proteins in the absence of Ca2+.


aNo data, b[Kimura & Ono, 2001], c[Noguchi *et al.*, 1995], d[Ono *et al.*, 2001], e[Ono *et al.*, 1992], f [Ono & Inoue, 1989].

Table 1. Effects of Ca2+ and extrinsic proteins (PsbP and PsbQ) on the properties of the OEC.

Next, the effects of extrinsic proteins and Ca2+ on the thermal stability of the OEC were examined. Fig. 4 shows the relative absorbance at 680 nm of the untreated control PSII (circle), ExCa2+-depleted (triangle), and Ca2+-depleted (square) PSII membranes during incubation at 50C. The relative band intensity of the control PSII remained at ~85% after 64 min incubation, but was slightly decreased to ~75% in the ExCa2+-depleted PSII and was markedly decreased to 50% in the Ca2+-depleted PSII. This is consistent with the effects seen in the O2-evolving activity and Fv/Fm values. These results strongly support the idea that PsbP and PsbQ lower the structural stability and disturb the normal functioning of the OEC in the absence of Ca2+.

The PSII membranes prepared by the different methods were assessed by O2-evolving activity and Fv/Fm values of chlorophyll fluorescence and the resulting data are summarized in Table 1. The O2-evolving rate of the untreated PSII was decreased to 17% when PsbP, PsbQ and Ca2+ were depleted by the NaCl/EDTA treatments (B). The decreased activity was restored to 83% by reconstituting Ca2+, as reported previously [Kimura & Ono, 2001, Ono *et al.*, 2001]. However, the addition of PsbP and PsbQ to the ExCa2+-depleted PSII in the absence of Ca2+ lowered the O2-evolving rate to ~0% (C). Furthermore, the O2-evolving activity was almost completely lost upon Ca2+ depletion by the low-pH treatment (D) but restored to 79% by adding Ca2+. Notably, the lost activity was partially resotored by the further depletion of PsbP and PsbQ to 25% (E). These results indicate that PsbP and PsbQ proteins completely suppress O2 evolution in the absence of functional Ca2+. Similar effects are also evident in the chlorophyll fluorescence measurements: the Fv/Fm values were much lower in the Ca2+ depleted PSII (45%) than in the ExCa2+-depleted PSII (64%), and both values were recoverd to ~80% after the supplementation with Ca2+. In the Ca2+-depleted PSII, the partial recovery to 68% was induced by the following depletion of the extrinsic proteins. Since Fv/Fm values are related to O2-evolving activity, this strongly suggests that the functions of the OEC are

Fv/Fm FTIR S2/S1

No addition 100% 100% Normalb Normale,f Normalf

No addition 17% 64% Normalb Normalb,d,e Normald +Ca2+ 83% 79% Normalb Normalb,d,e Normald

No addition ~0% 45% Abnormalc Abnormale,f Modifiedf +Ca2+ 79% 81% Normalc Normale,f Normalf

Table 1. Effects of Ca2+ and extrinsic proteins (PsbP and PsbQ) on the properties of the OEC.

Next, the effects of extrinsic proteins and Ca2+ on the thermal stability of the OEC were examined. Fig. 4 shows the relative absorbance at 680 nm of the untreated control PSII (circle), ExCa2+-depleted (triangle), and Ca2+-depleted (square) PSII membranes during incubation at 50C. The relative band intensity of the control PSII remained at ~85% after 64 min incubation, but was slightly decreased to ~75% in the ExCa2+-depleted PSII and was markedly decreased to 50% in the Ca2+-depleted PSII. This is consistent with the effects seen in the O2-evolving activity and Fv/Fm values. These results strongly support the idea that PsbP and PsbQ lower the structural stability and disturb the normal functioning of the OEC

carboxylate bands

~0% –––a –––a Abnormale Modified

25% 68% ––– a Normale Normal

Thermoluminescen ce Q-band (C)

S2 EPR multiline signal

**3.1.2 Effects of extrinsic proteins on the properties of the OEC** 

disturbed by the extrinsic proteins in the absence of Ca2+.

evolving activity

aNo data, b[Kimura & Ono, 2001], c[Noguchi *et al.*, 1995], d[Ono *et al.*, 2001], e[Ono *et al.*, 1992], f

Additives O2-

+PsbP, +PsbQ


[Ono & Inoue, 1989].

(C)

(E)

PSII preparation

(A)

(B)

ExCa2+ depleted PSII

Untreated PSII

Ca2+-depleted PSII (D)

in the absence of Ca2+.

The present findings are largely in agreement with previous findings, as shown in Table 1. FT-IR spectroscopy provides valuable information on the structure and interactions within the OEC. The ligation geometry around the Mn4Ca cluster is mostly similar between untreated and ExCa2+-depleted PSII, at least in the S1- and S2-states [Kimura & Ono, 2001]. However, Ca2+-depleted PSII exhibited marked deterioration in the carboxylate bands, which are thought to be from putative amino acid residues coordinating to the Mn4Ca cluster [Noguchi *et al.*, 1995]. Furthermore, the redox potential of the Mn4Ca cluster has been reported to be abnormal when the extrinsic proteins bound to the PSII core in the absence of Ca2+, as indicated by elevated peak temperatures of the thermoluminescence band for the

Fig. 4. Plots of relative absorbance at 680 nm as a function of incubation time at 50C for (a) untreated, (b) ExCa2+-depleted, and (c) Ca2+-depleted PS II membranes.

Fig. 5. ATR-FTIR spectra of isolated PsbP and PsbQ proteins in the absence (a, dotted line) and presence of Ca2+ (b, solid line). The difference spectrum obtained by subtracting spectrum a from spectrum b is shown in the lower panel.

Function of Extrinsic Proteins in Stabilization of the Photosynthetic Oxygen-Evolving Complex 117

isolated PsbO [Loll *et al.*, 2005]. In contrast, the low-affinity Ca2+-binding site in PsbO located at the luminal exit of the proton channel has been suggested to be responsible for water oxidation [Murray & Barber, 2006, Rutherford & Faller, 2001]. These results strongly indicate that the structural-functional role of PsbO is not identical between higher plants and cyanobacteria. Interestingly, thermal stability was enhanced when plant PsbO proteins were replaced with thermally stable homologues from thermophilic *Phormidium laminosum* [Pueyo *et al.*, 2002]. Therefore, slight variation in the primary structure and/or the protein folding pattern is possibly responsible for the difference in thermal stability of PsbO

**4. Protective role of extrinsic proteins in regulation and stabilization of** 

geometry, redox potentials and magmetic structures of the Mn4Ca cluster.

The present study revealed that PsbP significantly affects the structure and function of the Mn4Ca cluster in the OEC only in the absence of sufficient Ca2+ in the OEC. This result is compatible with the previous analyses that involved FT-IR, thermoluminescence, and EPR spectoroscopies [Kimura & Ono, 2001, Noguchi *et al.*, 1995, Ono *et al.*, 2001, Ono & Inoue, 1989, Ono *et al.*, 1992]. In addition, it has been reported that PsbP has Ca2+-binding sites in the N-terminal region [Ifuku & Sato, 2002] and functions as a reserver of Mn2+ or Ca2+ ions to supply them as needed by the impaired OEC [Bondarava *et al.*, 2005]. Therefore, it is possible that the PsbP completely eliminates functional Ca2+ or interacts with the Mn4Ca cluster directly and/or indirectly to inhibit the O2-evolving activity and modify the ligation

It has been suggested that normal functioning of PSII requires 15 highly conserved residues in the N-terminal region of the PsbP protein as well as the PsbQ protein for retention of functional Ca2+ [Ifuku *et al.*, 2005]. A recent FTIR study indicated that the PsbP protein, but not the PsbQ protein, has an effect on S2/S1 conformational changes of the intrisic polypeptide backbone around the Mn4Ca cluster through the N-terminal region of the PsbP [Tomita *et al.*, 2009]. In addition, little change was found in characteristic carboxylate stretching modes from putative amino acid ligands for the Mn4Ca cluster in the presence of Ca2+ when PsbP and PsbQ were depleted by NaCl washing, or all the extrinsic proteins were eliminated by CaCl2 washing [Tomita *et al.*, 2009]. Based on these results, it is possible that the PsbP protein interacts with intrinsic proteins, which may be closely related to the Mn4Ca cluster, and preserves the OEC functions appropriately in the presence of Ca2+, but modifies the properties of the cluster directly and/or indirectly through intrinsic proteins in the

It is intriguing to note that PsbV in cyanobacteria exhibits functional similarity with PsbP in higher plants, although their primary and 3D crystallographic structures are largely different [Ifuku *et al.*, 2004, Kerfeld *et al.*, 2003]. The apparent inconsistency in the structuralfunctional consequence may reflect the fact that PsbP and PsbV in plant and cyanobacterial PSII are not involved in specific interactions between the protein and the Mn4Ca cluster, but serve to maintain indispensable inorganic cofactors in the proximity of the cluster and to protect it from invasion. Additionally, PsbQ and PsbU also play a key role for tuning O2-

between higher plant and thermophilic cyanobacteria.

**photosynthetic functions** 

**4.1 PsbP and PsbQ** 

absence of Ca2+.

S2QA recombination [Ono & Inoue, 1989, Ono *et al.*, 1992]. Additional support for this view was obtained from electron paramagnetic resonance (EPR) studies which demonstrated abnormal magnetic structures of PSII lacking Ca2+ but retaining the extrinsic proteins as revealed by modified S2-state multiline signals. These results are largely compatible with the present findings that the appropriate binding of extrinsic proteins in the presence of functional Ca2+ is required for the normal functioning of the OEC.

To understand function of these extrinsic proteins in the OEC, structural changes of the PsbP and PsbQ proteins induced by Ca2+ were observed by ATR-FTIR spectroscopy. Fig. 5 shows ATR-FTIR spectra of isolated PsbP and PsbQ (spectrum a) and those supplemented with Ca2+ (spectrum b). The control spectrum a exhibited characteristic bands for amide I (1700 – 1600 cm-1) and amide II (1600 – 1500 cm-1) vibrational modes from backbone polypeptides of the OEC. These bands were significantly modified when Ca2+ was added to the extrinsic proteins as can be clearly seen in the difference spectrum (lower part of Fig. 5). The IR bands at 1693, 1659 and 1539 cm-1 are decreased and new bands are visible at 1641 and 1566 cm-1, strongly indicating that PsbP and/or PsbQ are metal-binding proteins that alter their secondary structures upon the binding of Ca2+. Similar structural changes were evident in the spectrum of the purified PsbP protein (data not shown). Although high-resolution crystallographic studies have revealed the structure of the PsbP protein in *Nicotiana tabacum* [Ifuku *et al.*, 2004], this protein lacks the N-terminal region which are thought to contain the Ca2+-binding site, and therefore, the relationship between PsbP and Ca2+ remains unclear [Ifuku & Sato, 2002]. However, the authors of a previous study hypothesized that PsbP acts to reserve Mn2+ or Ca2+ ions [Bondarava *et al.*, 2005]. These results strongly support the idea that the PsbP protein is a metal-binding protein that directly and/or indirectly interacts with the catalytic center of the OEC in the absence of sufficient Ca2+.

### **3.2 PsbO**

The most important physiological role of PsbO is to stabilize the binding of the Mn4Ca cluster, which is essential for oxygen-evolving activity [Debus, 2001]. The PsbO protein can be dissociated from the PSII by a variety of chemical treatments including washing with alkaline Tris buffer, a high concentration of CaCl2, and chaotropic agents [Enami *et al.*, 1994, Ghanotakis & Yocum, 1990]. In particular, Lys residue-modifying chemicals such as *N*succinimidyl propionate and 2,4,6-trinitrobenzene sulfonic acid caused release of PsbO from PSII and loss of oxygen-evolving activity [Miura *et al.*, 1997], suggesting that the positive charge of Lys is important for the electrostatic interaction between PsbO and PSII. Alternatively, the release of PsbO can be caused by thermal denaturation. However, PsbO itself is a thermostable protein [Lydakis-Simantiris *et al.*, 1999], and therefore, other factors might also be responsible for the release of PsbO as described later in this chapter.

Several spectroscopic studies using isolated PsbO reported different Ca2+-binding properties between higher plants and cyanobacteria. It has been suggested that plant PsbO can bind Ca2+, which induces slight changes in secondary structure from a -sheet to a loop or nonordered structure, and facilitated the association of PsbO with the PSII core [Heredia & De Las Rivas, 2003, Kruk *et al.*, 2003]. However, an EPR study indicated that the functional Ca2+ ion was not involved in the binding to PsbO [Seidler & Rutherford, 1996]. In cyanobacteria, PsbO does not bind Ca2+, at least before the protein associates with the PSII core, since no significant conformational change upon the Ca2+-binding was induced in isolated PsbO [Loll *et al.*, 2005]. In contrast, the low-affinity Ca2+-binding site in PsbO located at the luminal exit of the proton channel has been suggested to be responsible for water oxidation [Murray & Barber, 2006, Rutherford & Faller, 2001]. These results strongly indicate that the structural-functional role of PsbO is not identical between higher plants and cyanobacteria. Interestingly, thermal stability was enhanced when plant PsbO proteins were replaced with thermally stable homologues from thermophilic *Phormidium laminosum* [Pueyo *et al.*, 2002]. Therefore, slight variation in the primary structure and/or the protein folding pattern is possibly responsible for the difference in thermal stability of PsbO between higher plant and thermophilic cyanobacteria.

### **4. Protective role of extrinsic proteins in regulation and stabilization of photosynthetic functions**

### **4.1 PsbP and PsbQ**

116 Molecular Photochemistry – Various Aspects

To understand function of these extrinsic proteins in the OEC, structural changes of the PsbP and PsbQ proteins induced by Ca2+ were observed by ATR-FTIR spectroscopy. Fig. 5 shows ATR-FTIR spectra of isolated PsbP and PsbQ (spectrum a) and those supplemented with Ca2+ (spectrum b). The control spectrum a exhibited characteristic bands for amide I (1700 – 1600 cm-1) and amide II (1600 – 1500 cm-1) vibrational modes from backbone polypeptides of the OEC. These bands were significantly modified when Ca2+ was added to the extrinsic proteins as can be clearly seen in the difference spectrum (lower part of Fig. 5). The IR bands at 1693, 1659 and 1539 cm-1 are decreased and new bands are visible at 1641 and 1566 cm-1, strongly indicating that PsbP and/or PsbQ are metal-binding proteins that alter their secondary structures upon the binding of Ca2+. Similar structural changes were evident in the spectrum of the purified PsbP protein (data not shown). Although high-resolution crystallographic studies have revealed the structure of the PsbP protein in *Nicotiana tabacum* [Ifuku *et al.*, 2004], this protein lacks the N-terminal region which are thought to contain the Ca2+-binding site, and therefore, the relationship between PsbP and Ca2+ remains unclear [Ifuku & Sato, 2002]. However, the authors of a previous study hypothesized that PsbP acts to reserve Mn2+ or Ca2+ ions [Bondarava *et al.*, 2005]. These results strongly support the idea that the PsbP protein is a metal-binding protein that directly and/or indirectly interacts with the catalytic center of the

The most important physiological role of PsbO is to stabilize the binding of the Mn4Ca cluster, which is essential for oxygen-evolving activity [Debus, 2001]. The PsbO protein can be dissociated from the PSII by a variety of chemical treatments including washing with alkaline Tris buffer, a high concentration of CaCl2, and chaotropic agents [Enami *et al.*, 1994, Ghanotakis & Yocum, 1990]. In particular, Lys residue-modifying chemicals such as *N*succinimidyl propionate and 2,4,6-trinitrobenzene sulfonic acid caused release of PsbO from PSII and loss of oxygen-evolving activity [Miura *et al.*, 1997], suggesting that the positive charge of Lys is important for the electrostatic interaction between PsbO and PSII. Alternatively, the release of PsbO can be caused by thermal denaturation. However, PsbO itself is a thermostable protein [Lydakis-Simantiris *et al.*, 1999], and therefore, other factors

might also be responsible for the release of PsbO as described later in this chapter.

Ca2+, which induces slight changes in secondary structure from a

Several spectroscopic studies using isolated PsbO reported different Ca2+-binding properties between higher plants and cyanobacteria. It has been suggested that plant PsbO can bind

nonordered structure, and facilitated the association of PsbO with the PSII core [Heredia & De Las Rivas, 2003, Kruk *et al.*, 2003]. However, an EPR study indicated that the functional Ca2+ ion was not involved in the binding to PsbO [Seidler & Rutherford, 1996]. In cyanobacteria, PsbO does not bind Ca2+, at least before the protein associates with the PSII core, since no significant conformational change upon the Ca2+-binding was induced in


functional Ca2+ is required for the normal functioning of the OEC.

OEC in the absence of sufficient Ca2+.

**3.2 PsbO** 

 recombination [Ono & Inoue, 1989, Ono *et al.*, 1992]. Additional support for this view was obtained from electron paramagnetic resonance (EPR) studies which demonstrated abnormal magnetic structures of PSII lacking Ca2+ but retaining the extrinsic proteins as revealed by modified S2-state multiline signals. These results are largely compatible with the present findings that the appropriate binding of extrinsic proteins in the presence of

S2QA-

The present study revealed that PsbP significantly affects the structure and function of the Mn4Ca cluster in the OEC only in the absence of sufficient Ca2+ in the OEC. This result is compatible with the previous analyses that involved FT-IR, thermoluminescence, and EPR spectoroscopies [Kimura & Ono, 2001, Noguchi *et al.*, 1995, Ono *et al.*, 2001, Ono & Inoue, 1989, Ono *et al.*, 1992]. In addition, it has been reported that PsbP has Ca2+-binding sites in the N-terminal region [Ifuku & Sato, 2002] and functions as a reserver of Mn2+ or Ca2+ ions to supply them as needed by the impaired OEC [Bondarava *et al.*, 2005]. Therefore, it is possible that the PsbP completely eliminates functional Ca2+ or interacts with the Mn4Ca cluster directly and/or indirectly to inhibit the O2-evolving activity and modify the ligation geometry, redox potentials and magmetic structures of the Mn4Ca cluster.

It has been suggested that normal functioning of PSII requires 15 highly conserved residues in the N-terminal region of the PsbP protein as well as the PsbQ protein for retention of functional Ca2+ [Ifuku *et al.*, 2005]. A recent FTIR study indicated that the PsbP protein, but not the PsbQ protein, has an effect on S2/S1 conformational changes of the intrisic polypeptide backbone around the Mn4Ca cluster through the N-terminal region of the PsbP [Tomita *et al.*, 2009]. In addition, little change was found in characteristic carboxylate stretching modes from putative amino acid ligands for the Mn4Ca cluster in the presence of Ca2+ when PsbP and PsbQ were depleted by NaCl washing, or all the extrinsic proteins were eliminated by CaCl2 washing [Tomita *et al.*, 2009]. Based on these results, it is possible that the PsbP protein interacts with intrinsic proteins, which may be closely related to the Mn4Ca cluster, and preserves the OEC functions appropriately in the presence of Ca2+, but modifies the properties of the cluster directly and/or indirectly through intrinsic proteins in the absence of Ca2+.

It is intriguing to note that PsbV in cyanobacteria exhibits functional similarity with PsbP in higher plants, although their primary and 3D crystallographic structures are largely different [Ifuku *et al.*, 2004, Kerfeld *et al.*, 2003]. The apparent inconsistency in the structuralfunctional consequence may reflect the fact that PsbP and PsbV in plant and cyanobacterial PSII are not involved in specific interactions between the protein and the Mn4Ca cluster, but serve to maintain indispensable inorganic cofactors in the proximity of the cluster and to protect it from invasion. Additionally, PsbQ and PsbU also play a key role for tuning O2-

Function of Extrinsic Proteins in Stabilization of the Photosynthetic Oxygen-Evolving Complex 119

First, ROS attack trienoic fatty acids in thylakoid membranes, resulting in the generation of MDA. MDA attaches to critical Lys residues of PsbO and PsbB (CP47) for the interaction between PsbO and PSII in a temperature-dependent manner. When both sides of PsbO and PSII are modified by MDA, PsbO is released from PSII. Finally, the Mn4Ca cluster is

spontaneously released from PSII, causing loss of oxygen-evolving activity.

Fig. 6. A schematic model of MDA-induced loss of oxygen evolution in heat-stressed

In this article, we focused on the structural and functional roles of extrinsic proteins in the plant PSII. Since PSII is an integrated pigment-protein complex embedded in plant membranes, the structures and interactions of these extrinsic proteins in the membrane interface are of significance for protecting the RC. This involves protecting the Mn4Ca cluster from exogenous invasion and/or alteration of physiological conditions. However, based on the results presented here and reported previously, we consider it very likely that the extrinsic protein itself is also responsible for the deterioration of the normal functioning of the OEC under inappropriate conditions. Further studies on the plant PSII, including high-resolution crystallographic strudies, will be required for understanding the functions of extrinsic proteins in the structural stability and the water oxidation chemistry in PSII.

This research was supported by a grant from the Hyogo Science and Technology

Alfonso, M.; Collados, R.; Yruela, I. & Picorel, R. (2004). Photoinhibition and recovery in a

fatty acid unsaturation, *Planta*, Vol. 219, No.3, 428-439.

herbicide-resistant mutant from Glycine max (L.) Merr. cell cultures deficient in

spinach PSII complexes.

**6. Acknowledgment** 

Association of Japan.

**7. References** 

**5. Conclusion** 

evolving activity and enahncing structural stability through the interaction with PsbP and PsbV, respectively [Nishiyama *et al.*, 1997, Nishiyama *et al.*, 1999].

### **4.2 PsbO**

In higher plants, PSII is much more susceptible to high temperatures than PSI [Berry & Bjorkman, 1980]. The thermal stability of the PSII core is closely related to the acquisition of cellular thermal tolerance in oxyphototrophs. The thermosensitivity of oxygen evolution in higher plants has been studied through simple experiments using PSII particles or isolated thylakoid membranes. Previous in-vivo and in-vitro studies have estimated the heat-labile properties of the OEC [Berry & Bjorkman, 1980, Havaux & Tardy, 1996, Mamedov *et al.*, 1993]. These studies demonstrated that the release of PsbO occurs first, followed by liberation of two of the four Mn ions from the Mn4Ca cluster of the OEC [Enami *et al.*, 1998, Enami *et al.*, 1994, Nash *et al.*, 1985], and finally by the loss of oxygen evolution at high temperatures [Enami *et al.*, 1994, Yamane *et al.*, 1998].

Another form of damage to the physiological function of the PSII can be caused by reactive oxygen species (ROS) generated under high light conditions. The D1 proteins are degraded by the ROS species and inhibited in their ability to repair the photodamaged PSII by suppressing the synthesis of D1 proteins [Murata *et al.*, 2007]. The ROS species are thought to arise from heat-induced inactivation of a water-oxidizing manganese complex and through lipid peroxidation [Yamashita *et al.*, 2008]. On the other hand, saturation of polyunsaturated fatty acids (PUFAs) contributes to the acquisition of heat tolerance of photosynthesis by altering physicochemical properties [Alfonso *et al.*, 2001, Murakami *et al.*, 2000, Thomas *et al.*, 1986]. The increased saturation of PUFAs raises the temperature at which lipids phase-separate into non-bilayer structures, providing the proper assembly and dynamics of PSII tolerant to higher temperatures [Alfonso *et al.*, 2004].

Recently, we published biochemical evidence that the biological effect of reactive carbonyls such as malondialdehyde (MDA) and acrolein is greatly enhanced under heat-stressed conditions. [Yamauchi & Sugimoto, 2010]. PsbO is one of the proteins most frequently modified by MDA, which is an end-product of peroxidized polyunsaturated fatty acids. Detailed biochemical experiments indicated that the modification of PsbO by MDA affects its binding to the PSII complex and causes inactivation of the OEC (a schematic diagram is shown in Fig. 6). Purified PsbO and PSII membranes, from which extrinsic proteins had been eliminated, of the oxygen-evolving complex (PSII∆OEE) of spinach were separately treated with MDA. The binding was diminished when both PsbO and PSII∆OEE were modified, but when only PsbO or PSII∆OEE was treated, the binding was not impaired. In an experiment using thylakoid membranes, the release of PsbO from PSII and a corresponding loss of oxygen-evolving activity were observed when thylakoid membrances were treated with MDA at 40°C but not at 25°C. In spinach leaves treated at 40°C under light, the maximum efficiency of PSII photochemistry (Fv/Fm ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, the MDA content of the heat-stressed leaves increased, and PsbO and PSII core proteins (including 47 kDa and 43 kDa chlorophyll-binding proteins) were modified by MDA. In contrast, these changes were less profound when these experiments were performed at 40°C in the dark. Thus, MDA modification of PSII proteins likely causes the release of PsbO from PSII, an effect that is particuarly marked in heat and oxidative conditions.

First, ROS attack trienoic fatty acids in thylakoid membranes, resulting in the generation of MDA. MDA attaches to critical Lys residues of PsbO and PsbB (CP47) for the interaction between PsbO and PSII in a temperature-dependent manner. When both sides of PsbO and PSII are modified by MDA, PsbO is released from PSII. Finally, the Mn4Ca cluster is spontaneously released from PSII, causing loss of oxygen-evolving activity.

Fig. 6. A schematic model of MDA-induced loss of oxygen evolution in heat-stressed spinach PSII complexes.

### **5. Conclusion**

118 Molecular Photochemistry – Various Aspects

evolving activity and enahncing structural stability through the interaction with PsbP and

In higher plants, PSII is much more susceptible to high temperatures than PSI [Berry & Bjorkman, 1980]. The thermal stability of the PSII core is closely related to the acquisition of cellular thermal tolerance in oxyphototrophs. The thermosensitivity of oxygen evolution in higher plants has been studied through simple experiments using PSII particles or isolated thylakoid membranes. Previous in-vivo and in-vitro studies have estimated the heat-labile properties of the OEC [Berry & Bjorkman, 1980, Havaux & Tardy, 1996, Mamedov *et al.*, 1993]. These studies demonstrated that the release of PsbO occurs first, followed by liberation of two of the four Mn ions from the Mn4Ca cluster of the OEC [Enami *et al.*, 1998, Enami *et al.*, 1994, Nash *et al.*, 1985], and finally by the loss of oxygen evolution at high

Another form of damage to the physiological function of the PSII can be caused by reactive oxygen species (ROS) generated under high light conditions. The D1 proteins are degraded by the ROS species and inhibited in their ability to repair the photodamaged PSII by suppressing the synthesis of D1 proteins [Murata *et al.*, 2007]. The ROS species are thought to arise from heat-induced inactivation of a water-oxidizing manganese complex and through lipid peroxidation [Yamashita *et al.*, 2008]. On the other hand, saturation of polyunsaturated fatty acids (PUFAs) contributes to the acquisition of heat tolerance of photosynthesis by altering physicochemical properties [Alfonso *et al.*, 2001, Murakami *et al.*, 2000, Thomas *et al.*, 1986]. The increased saturation of PUFAs raises the temperature at which lipids phase-separate into non-bilayer structures, providing the proper assembly and

Recently, we published biochemical evidence that the biological effect of reactive carbonyls such as malondialdehyde (MDA) and acrolein is greatly enhanced under heat-stressed conditions. [Yamauchi & Sugimoto, 2010]. PsbO is one of the proteins most frequently modified by MDA, which is an end-product of peroxidized polyunsaturated fatty acids. Detailed biochemical experiments indicated that the modification of PsbO by MDA affects its binding to the PSII complex and causes inactivation of the OEC (a schematic diagram is shown in Fig. 6). Purified PsbO and PSII membranes, from which extrinsic proteins had been eliminated, of the oxygen-evolving complex (PSII∆OEE) of spinach were separately treated with MDA. The binding was diminished when both PsbO and PSII∆OEE were modified, but when only PsbO or PSII∆OEE was treated, the binding was not impaired. In an experiment using thylakoid membranes, the release of PsbO from PSII and a corresponding loss of oxygen-evolving activity were observed when thylakoid membrances were treated with MDA at 40°C but not at 25°C. In spinach leaves treated at 40°C under light, the maximum efficiency of PSII photochemistry (Fv/Fm ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, the MDA content of the heat-stressed leaves increased, and PsbO and PSII core proteins (including 47 kDa and 43 kDa chlorophyll-binding proteins) were modified by MDA. In contrast, these changes were less profound when these experiments were performed at 40°C in the dark. Thus, MDA modification of PSII proteins likely causes the release of PsbO from PSII, an effect that

PsbV, respectively [Nishiyama *et al.*, 1997, Nishiyama *et al.*, 1999].

temperatures [Enami *et al.*, 1994, Yamane *et al.*, 1998].

dynamics of PSII tolerant to higher temperatures [Alfonso *et al.*, 2004].

is particuarly marked in heat and oxidative conditions.

**4.2 PsbO** 

In this article, we focused on the structural and functional roles of extrinsic proteins in the plant PSII. Since PSII is an integrated pigment-protein complex embedded in plant membranes, the structures and interactions of these extrinsic proteins in the membrane interface are of significance for protecting the RC. This involves protecting the Mn4Ca cluster from exogenous invasion and/or alteration of physiological conditions. However, based on the results presented here and reported previously, we consider it very likely that the extrinsic protein itself is also responsible for the deterioration of the normal functioning of the OEC under inappropriate conditions. Further studies on the plant PSII, including high-resolution crystallographic strudies, will be required for understanding the functions of extrinsic proteins in the structural stability and the water oxidation chemistry in PSII.

### **6. Acknowledgment**

This research was supported by a grant from the Hyogo Science and Technology Association of Japan.

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**7** 

**UV Light Effects on Proteins:** 

and Steffen B. Petersen1,2

*2Aalborg University, Aalborg,* 

*Kalpakkam, 1Portugal 2Denmark 3India* 

**From Photochemistry to Nanomedicine** 

*3Materials and Metallurgy Group, Indira Gandhi Centre for Atomic Research,* 

Throughout 4.5 billion year of molecular evolution, proteins have evolved in order to maintain the spatial proximity between aromatic residues (Trp, Tyr and Phe) and disulphide bridges (SS) (Petersen et al, 1999). Aromatic residues are the nanosized antennas in the protein world that can capture UV light (from ~250-298nm). Once excited by UV light they can enter photochemical pathways likely to have harmful effects on protein structures. However, disulphide bridges in proteins are excellent quenchers of the excited state of aromatic residues, contributing this way to protein stability and activity. UV light excitation of the aromatic residues is known to trigger electron ejection from their side chains (Bent & Hayon, 1975a; Bent & Hayon, 1975b; Bent & Hayon, 1975c; Creed, 1984a; Creed, 1984b; Kerwin & Rammele, 2007, Neves-Petersen et al., 2009a). These electrons can be captured by disulphide bridges, leading to the formation of a transient disulphide electron adduct radical, which will dissociate leading to the formation of free thiol groups in the protein. This observation lead to the development in our lab of a new photonic technology, Light Assisted Molecular Immobilization (LAMI), used to functionalize surfaces with biomolecules. This technology is being used in order to create a new generation of biosensors with unsurpassed density (number of spots per mm2). This technology is also being used in order to create nanoparticles based drug delivery systems relevant to

In this chapter we will describe the effects of UV excitation of proteins. Furthermore, we will also describe the specific, conserved structural motif in protein molecules that can be activated by UV light, leading to the formation of reactive free thiol groups. The dynamics of formation and the lifetimes of transient species will be described. Afterwards, the different applications of LAMI will be shown. LAMI has been successfully used for the creation of protein microarrays and in order to immobilize proteins on a surface according

**1. Introduction** 

nanomedical applications (Parracino et al., 2011).

Maria Teresa Neves-Petersen1,2, Gnana Prakash Gajula3

*1International Iberian Nanotechnology Laboratory (INL), Braga* 


## **UV Light Effects on Proteins: From Photochemistry to Nanomedicine**

Maria Teresa Neves-Petersen1,2, Gnana Prakash Gajula3 and Steffen B. Petersen1,2 *1International Iberian Nanotechnology Laboratory (INL), Braga 2Aalborg University, Aalborg, 3Materials and Metallurgy Group, Indira Gandhi Centre for Atomic Research, Kalpakkam, 1Portugal 2Denmark 3India* 

### **1. Introduction**

124 Molecular Photochemistry – Various Aspects

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the interaction between the oxygen-evolving complex 33 kDa protein and

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Crystal structure of photosystem II from Synechococcus elongatus at 3.8 angstrom

Throughout 4.5 billion year of molecular evolution, proteins have evolved in order to maintain the spatial proximity between aromatic residues (Trp, Tyr and Phe) and disulphide bridges (SS) (Petersen et al, 1999). Aromatic residues are the nanosized antennas in the protein world that can capture UV light (from ~250-298nm). Once excited by UV light they can enter photochemical pathways likely to have harmful effects on protein structures. However, disulphide bridges in proteins are excellent quenchers of the excited state of aromatic residues, contributing this way to protein stability and activity. UV light excitation of the aromatic residues is known to trigger electron ejection from their side chains (Bent & Hayon, 1975a; Bent & Hayon, 1975b; Bent & Hayon, 1975c; Creed, 1984a; Creed, 1984b; Kerwin & Rammele, 2007, Neves-Petersen et al., 2009a). These electrons can be captured by disulphide bridges, leading to the formation of a transient disulphide electron adduct radical, which will dissociate leading to the formation of free thiol groups in the protein. This observation lead to the development in our lab of a new photonic technology, Light Assisted Molecular Immobilization (LAMI), used to functionalize surfaces with biomolecules. This technology is being used in order to create a new generation of biosensors with unsurpassed density (number of spots per mm2). This technology is also being used in order to create nanoparticles based drug delivery systems relevant to nanomedical applications (Parracino et al., 2011).

In this chapter we will describe the effects of UV excitation of proteins. Furthermore, we will also describe the specific, conserved structural motif in protein molecules that can be activated by UV light, leading to the formation of reactive free thiol groups. The dynamics of formation and the lifetimes of transient species will be described. Afterwards, the different applications of LAMI will be shown. LAMI has been successfully used for the creation of protein microarrays and in order to immobilize proteins on a surface according

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 127

Excitation to higher energy states is followed by relaxation to ground state (*e.g.* fluorescence, phosphorescence) or to excited state photochemical or photophysical processes, such as

Flash photolysis studies have revealed two non-radiative relaxation channels from the

*Trp h Trp e* 

2. Intersystem crossing, yielding the triplet-state 3Trp which has its maximum absorption at ~450 nm. The triplet state tryptophan can transfer an electron to a nearby disulphide

> 1 1 *Trp h Tr*

Another aromatic residue with non-negligible absorption in the near-UV region is tyrosine (Tyr-OH). At neutral pH tyrosine has absorption maxima at 220nm (є~9000 M-1cm-1) and 275nm (є~1400 M-1cm-1) (Creed, 1984a). At alkaline pH the OH group of tyrosine side chain deprotonates. The resulting tyrosinate (Tyr-O●−) has a slightly red-shifted absorption compared to tyrosine, with maxima at 240nm (є~11000 M-1cm-1) and 290nm (є~2300 M-1cm-1) (Creed, 1984a). Photoexcited tyrosine can fluoresce, decay non-radiatively, or undergo intersystem crossing to the triplet state, from which most of the photochemistry proceeds. Alternatively, at neutral pH, tyrosine can be photoionized through a biphotonic process that involves absorption of a second photon from the triplet state. This results in a solvated

neutral radical (Tyr-OH●). Photoionization of tyrosinate at high pH is monophotonic and

*T aq yr OH h T* 

aq) and a radical cation (Tyr-OH●+) that will rapidly deprotonate to create the

bridge to give Trp●+ and the disulphide bridge electron adduct RSSR●-

has its maximum absorption at ~420 nm (Bent & Hayon, 1975a).

3

results in a neutral radical (Tyr-O●) and a solvated electron (e-

absorption peak centred at ~720 nm and the tryptophan radical cation Trp●+ which has its maximum absorption at ~560 nm. Trp●+ deprotonates rapidly, yielding the neutral

aq, which have a broad

, where the latter

*aq* (1)

*Trp Trp H* (2)

*p* (3)

1 3 *Trp Trp* (4)

*Trp RSSR Trp RSSR* (5)

aq).

*yr OH e* (6)

*Tyr OH Tyr OH H* (7)

photoionization (Creed, 1984b).

singlet excited state of Tryptophan (Bent & Hayon, 1975a):

1. Electron ejection to the solvent, yielding solvated electrons, e-

radical Trp● that has its maximum absorption at ~510 nm.

**Tryptophan** 

**Tyrosine** 

electron (e-

<sup>3</sup>

to any desired pattern, with submicrometer and nanometer resolution. LAMI has also been used for the creation of nanoparticle based drug delivery systems. An overview on different protein immobilization technologies will be given and the advantages of the new photonic technology will be highlighted. An overview of the use of nanoparticles in nanomedicine will also be given. Furthermore, a new light based cancer therapy which makes use of the knowledge derived from the effects of UV light on proteins will be described.

Light can change the properties of biomolecules and the number of drugs found to be photochemically unstable is steadily increasing. The effects of light on drugs include not only degradation reactions but also other processes, such as the formation of radicals, energy transfer, and luminescence. Adequate protection for most drug products during storage and distribution is needed. Indeed, proper storage conditions that secure protection from UV and visible radiation are essential for the efficacy of many common dermatologic drugs. If a drug is exposed to fluorescent tubes and/or filtered daylight for several weeks or months before it is finally administered to the patient, the drug may be altered. The most common consequence of drug photodecomposition is loss of potency with concomitant loss of therapeutic activity. It is therefore of interest to be aware of light induced reaction in biomolecules.

### **2. UV light induced photochemical reactions**

Cells, their proteins and genes are sensitive to light. The vision process itself is initiated when photoreceptor cells are activated by light (photo-isomerization). Several papers report effects of UV light in cells and their proteins/genes. For example, UV-light is known to inhibit photosystem II activity in cyanobacterium and to enhance the transcription of particular genes (310nm light) (Vass et al., 2000). It is also known that near UV (290nm) exposed prion protein fails to form amyloid fibrils (Thakur & Rao, 2008). Nucleic acids in living cells are associated with a large variety of proteins. Therefore, it is logical to assume that the ultraviolet (UV) irradiation of cells could lead to reactive interactions between DNA and the proteins that are in contact with it. One reaction that does occur is the cross-linking between the amino acids in these associated proteins and the bases in DNA. Such reaction appears to be an important process that photoexcited DNA and proteins undergo *in vivo*, as well as in DNA-protein complexes *in vitro*. Since the crosslinking of DNA and protein by UV radiation is many times more sensitive than is thymine dimer formation, it was suggested that DNA-protein crosslinks may play a significant role in the inactivation of bacteria by UV radiation (Smith, 1962). The first amino acid shown to photochemically add to uracil was cysteine, to form 5-S-cysteinyl-6 hydrouracil (Smith and Aplin, 1966). The structure of the mixed photoproduct of thymine and cysteine was also determined (Smith, 1970). The first survey performed determined the ability of the 22 common amino acids to bind photochemically (upon 254nm excitation) to uracil. The 11 reactive amino acids were glycine, serine, phenylalanine, tyrosine, tryptophan, cystine, cysteine, methionine, histidine, arginine and lysine. The most reactive amino acids were phenylalanine, tyrosine and cysteine.

The three amino acid residues which side chains absorb in the UV range are the aromatic residues tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). Several reviews have been published on the photochemistry and photophysics of Trp (Bent & Hayon, 1975a; Creed, 1984b), Tyr (Bent & Hayon, 1975b; Creed, 1984a), Phe (Bent & Hayon, 1975c), and Cystine (name given to each bridged cysteine in a disulphide bridge) (Creed, 1984a).

Excitation to higher energy states is followed by relaxation to ground state (*e.g.* fluorescence, phosphorescence) or to excited state photochemical or photophysical processes, such as photoionization (Creed, 1984b).

### **Tryptophan**

126 Molecular Photochemistry – Various Aspects

to any desired pattern, with submicrometer and nanometer resolution. LAMI has also been used for the creation of nanoparticle based drug delivery systems. An overview on different protein immobilization technologies will be given and the advantages of the new photonic technology will be highlighted. An overview of the use of nanoparticles in nanomedicine will also be given. Furthermore, a new light based cancer therapy which makes use of the

Light can change the properties of biomolecules and the number of drugs found to be photochemically unstable is steadily increasing. The effects of light on drugs include not only degradation reactions but also other processes, such as the formation of radicals, energy transfer, and luminescence. Adequate protection for most drug products during storage and distribution is needed. Indeed, proper storage conditions that secure protection from UV and visible radiation are essential for the efficacy of many common dermatologic drugs. If a drug is exposed to fluorescent tubes and/or filtered daylight for several weeks or months before it is finally administered to the patient, the drug may be altered. The most common consequence of drug photodecomposition is loss of potency with concomitant loss of therapeutic activity. It is therefore of interest to be aware of light induced reaction in

Cells, their proteins and genes are sensitive to light. The vision process itself is initiated when photoreceptor cells are activated by light (photo-isomerization). Several papers report effects of UV light in cells and their proteins/genes. For example, UV-light is known to inhibit photosystem II activity in cyanobacterium and to enhance the transcription of particular genes (310nm light) (Vass et al., 2000). It is also known that near UV (290nm) exposed prion protein fails to form amyloid fibrils (Thakur & Rao, 2008). Nucleic acids in living cells are associated with a large variety of proteins. Therefore, it is logical to assume that the ultraviolet (UV) irradiation of cells could lead to reactive interactions between DNA and the proteins that are in contact with it. One reaction that does occur is the cross-linking between the amino acids in these associated proteins and the bases in DNA. Such reaction appears to be an important process that photoexcited DNA and proteins undergo *in vivo*, as well as in DNA-protein complexes *in vitro*. Since the crosslinking of DNA and protein by UV radiation is many times more sensitive than is thymine dimer formation, it was suggested that DNA-protein crosslinks may play a significant role in the inactivation of bacteria by UV radiation (Smith, 1962). The first amino acid shown to photochemically add to uracil was cysteine, to form 5-S-cysteinyl-6 hydrouracil (Smith and Aplin, 1966). The structure of the mixed photoproduct of thymine and cysteine was also determined (Smith, 1970). The first survey performed determined the ability of the 22 common amino acids to bind photochemically (upon 254nm excitation) to uracil. The 11 reactive amino acids were glycine, serine, phenylalanine, tyrosine, tryptophan, cystine, cysteine, methionine, histidine, arginine and lysine. The most reactive amino acids were

The three amino acid residues which side chains absorb in the UV range are the aromatic residues tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). Several reviews have been published on the photochemistry and photophysics of Trp (Bent & Hayon, 1975a; Creed, 1984b), Tyr (Bent & Hayon, 1975b; Creed, 1984a), Phe (Bent & Hayon, 1975c), and Cystine (name given to each bridged cysteine in a disulphide bridge) (Creed, 1984a).

knowledge derived from the effects of UV light on proteins will be described.

**2. UV light induced photochemical reactions** 

phenylalanine, tyrosine and cysteine.

biomolecules.

Flash photolysis studies have revealed two non-radiative relaxation channels from the singlet excited state of Tryptophan (Bent & Hayon, 1975a):

1. Electron ejection to the solvent, yielding solvated electrons, eaq, which have a broad absorption peak centred at ~720 nm and the tryptophan radical cation Trp●+ which has its maximum absorption at ~560 nm. Trp●+ deprotonates rapidly, yielding the neutral radical Trp● that has its maximum absorption at ~510 nm.

$$Trp + h\nu \to Trp^{\bullet \text{+}} + e^{-}\_{aq} \tag{1}$$

$$Tr\mathfrak{p}^{\bullet+} \to Tr\mathfrak{p}^{\bullet} + H^{+} \tag{2}$$

2. Intersystem crossing, yielding the triplet-state 3Trp which has its maximum absorption at ~450 nm. The triplet state tryptophan can transfer an electron to a nearby disulphide bridge to give Trp●+ and the disulphide bridge electron adduct RSSR●-, where the latter has its maximum absorption at ~420 nm (Bent & Hayon, 1975a).

$$\mathop{\rm tr}^{1}Trp + h\nu \to \mathop{\rm tr}^{1}Trp^\* \tag{3}$$

$$^{1}Tr p^{\*} \to \,^{3}Tr p \tag{4}$$

$$^3Trp + RSSR \to Trp \stackrel{\bullet \text{ } +}{\rightleftharpoons} RSSR \stackrel{\bullet -}{\tag{5}} \tag{5}$$

### **Tyrosine**

Another aromatic residue with non-negligible absorption in the near-UV region is tyrosine (Tyr-OH). At neutral pH tyrosine has absorption maxima at 220nm (є~9000 M-1cm-1) and 275nm (є~1400 M-1cm-1) (Creed, 1984a). At alkaline pH the OH group of tyrosine side chain deprotonates. The resulting tyrosinate (Tyr-O●−) has a slightly red-shifted absorption compared to tyrosine, with maxima at 240nm (є~11000 M-1cm-1) and 290nm (є~2300 M-1cm-1) (Creed, 1984a). Photoexcited tyrosine can fluoresce, decay non-radiatively, or undergo intersystem crossing to the triplet state, from which most of the photochemistry proceeds. Alternatively, at neutral pH, tyrosine can be photoionized through a biphotonic process that involves absorption of a second photon from the triplet state. This results in a solvated electron (eaq) and a radical cation (Tyr-OH●+) that will rapidly deprotonate to create the neutral radical (Tyr-OH●). Photoionization of tyrosinate at high pH is monophotonic and results in a neutral radical (Tyr-O●) and a solvated electron (eaq).

$$\mathrm{^3Tyr-OH} + \mathrm{h}\nu \rightarrow \mathrm{Tyr-OH}^{\bullet+} + \mathrm{e}\_{\mathrm{aq}}^{-} \tag{6}$$

$$\text{Tyr}-\text{OH}^{\bullet+} \rightarrow \text{Tyr}-\text{OH}^{\bullet} + \text{H}^{+} \tag{7}$$

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 129

Fig. 1. The spatial proximity between aromatic residues and disulphide bridges (SS) has been conserved throughout molecular evolution. Trp is the preferred spatial neighbor of SS (Petersen et al., 1999). UV excitation of the side chain of aromatic residues leads to electron

**2.2 Observing the solvated electron and other transient species formed upon UV** 

between the donor and acceptor increases). This process will result in a RSSR●-

der Waals contact of a disulphide bridge (closest distance ~3.8 Å).

which again can result in breakage of the disulphide bridge, as shown above (scheme 12).

It is clear from the above that there are many possible pathways for the breakage of intramolecular disulphide bridges in proteins upon UV excitation of aromatic residues, even in the absence of molecular oxygen. Breakage of the disulphide bridge can lead to conformational changes in the protein, not necessarily resulting in inactivation of the protein. Transient absorption data of *Fusarium solani pisi* cutinase has also been acquired, with supplemental experimental data on tryptophan and lysozyme as a reference (Neves-Petersen et al., 2009a). Cutinase is a good model protein for studying the UV induced breakage of disulphide bridges is since it contains only one tryptophan that is within van

Data showed that UV excitation of cutinase lead to the formation of the solvated electron (transient species with absorption maximum around 710-720nm, see Fig. 2) and of the disulphide bridge electron adduct radical, RSSR●- (transient species with absorption maximum around 420nm, see Fig. 2) (Neves-Petersen et al., 2009a). Figs. 2 and 3 show the kinetics of formation of the solvated electron. The increase in absorption of light at 710nm can clearly be seen following excitation at 266nm, which coincides with time zero in Figure 2. The data displayed in Fig. 3 is the intensity of absorbed light at 710nm (the intense peak displayed in Fig. 2) during the initial 43ns after excitation of cutinase with

In the flash photolysis experiments on lysozyme by Grossweiner and Usui (Grossweiner & Usui, 1971) it was shown that the initial photoproducts upon UV excitation of lysozyme are the photo-oxidized tryptophan residue, solvated electrons, and the cystine residue (disulphide bridge) electron adduct. In a more recent paper by Zhi Li *et al.* (Z. Li et al., 1989), experiments on a model system demonstrated that the fast electron transfer is consistent with direct electron transfer between the tryptophan triplet state and a nearby disulphide bridge (this is a very short range interaction that decays exponentially as the distance

radical,

ejection. The electron can then be captured by disulphide bridges, leading to their

dissociation.

**excitation of proteins** 

$$Tyr - O^{\bullet-} + h\nu \to Tyr - O^{\bullet} + e\_{aq}^{-} \tag{8}$$

The triplet state tyrosine is rapidly quenched by molecular oxygen or nearby residues like tryptophan or disulphide bridges (Bent & Hayon, 1975b):

$$\text{T}^{\text{A}}\text{Tyr}-\text{OH} + \text{RSSR} \rightarrow \text{Tyr}-\text{O}^{\bullet}\text{ } + \text{H}^{+} + \text{RSSR}^{\bullet -} \tag{9}$$

### **2.1 Important photochemical mechanism in disulphide bridge containing proteins**

An important photochemical mechanism in proteins involves reduction of disulphide bridges (SS) upon UV excitation of Trp and Tyr side chains (Kerwin & Rammele, 2007, Neves-Petersen et al., 2002 & 2009a). As shown above, UV-excitation of tryptophan or tyrosine can result in their photoionization and to the generation of solvated electrons (Bent & Hayon, 1975a & 1975b; Creed, 1984b, Kerwin & Rammele, 2007, Neves-Petersen et al., 2009a). The generated solvated electrons can subsequently undergo fast geminate recombination with their parent molecule, or they can be captured by electrophillic species like molecular oxygen, H3O+ (at low pH), and cystines as summarized below:

$$\text{Co}\_{aq}^{-} + \text{O}\_{2} \rightarrow \text{O}\_{2}^{\bullet -} \tag{10}$$

$$e\_{aq}^{-} + H\_{3}O^{+} \rightarrow H^{\bullet} + H\_{2}O \tag{11}$$

$$
\bar{e\_{aq}} + RSSR \to RSSR^{\bullet -} \tag{12}
$$

In the case where the electron is captured by the cystine, the result can also be the breakage of the disulphide bridge (Hoffman & Hayon, 1972):

$$
\star e\_{aq}^- + RSSR \to RSSR \, ^{\bullet-}\tag{10}
$$

$$\text{RSSR}^{\bullet-} \Leftrightarrow \text{RS}^{\bullet} + \text{RS}^{-} \tag{11}$$

$$\text{RSSR}^{\bullet-} + H^{+} \Leftrightarrow \text{RS}^{\bullet} + \text{RSH} \tag{12}$$

The resultant free thiol radicals/groups can then subsequently react with other free thiol groups to create a new disulphide bridge. Reduction of SS upon UV excitation of aromatic residues has been shown for proteins such as cutinase and lysozyme (Neves-Petersen et al., 2009a, 2006 & 2002), bovine serum albumin (Skovsen et al., 2009a; Parracino et al., 2011) prostate specific antigen (Parracino et al., 2010), and antibody Fab fragments (Duroux et al., 2007). As mentioned in the introduction, this phenomenon has led to a new technology for protein immobilization (LAMI, light assisted molecular immobilization) since the created thiol groups can bind thiol reactive surfaces leading to oriented covalent protein immobilization (Neves-Petersen et al., 2006;Snabe et al., 2006; Duroux et al., 2007a, 2007b & 2007c; Skovsen et al., 2007, 2009a & 2009b; Neves-Petersen et al., 2009b; Parracino et al., 2010 & 2011).

*T aq yrO h T* 

The triplet state tyrosine is rapidly quenched by molecular oxygen or nearby residues like

**2.1 Important photochemical mechanism in disulphide bridge containing proteins** 

like molecular oxygen, H3O+ (at low pH), and cystines as summarized below:

An important photochemical mechanism in proteins involves reduction of disulphide bridges (SS) upon UV excitation of Trp and Tyr side chains (Kerwin & Rammele, 2007, Neves-Petersen et al., 2002 & 2009a). As shown above, UV-excitation of tryptophan or tyrosine can result in their photoionization and to the generation of solvated electrons (Bent & Hayon, 1975a & 1975b; Creed, 1984b, Kerwin & Rammele, 2007, Neves-Petersen et al., 2009a). The generated solvated electrons can subsequently undergo fast geminate recombination with their parent molecule, or they can be captured by electrophillic species

*aq* 2 2 *eO O*

*aq* 3 2 *e HO H HO*

*aq e RSSR RSSR*

*aq e RSSR RSSR*

The resultant free thiol radicals/groups can then subsequently react with other free thiol groups to create a new disulphide bridge. Reduction of SS upon UV excitation of aromatic residues has been shown for proteins such as cutinase and lysozyme (Neves-Petersen et al., 2009a, 2006 & 2002), bovine serum albumin (Skovsen et al., 2009a; Parracino et al., 2011) prostate specific antigen (Parracino et al., 2010), and antibody Fab fragments (Duroux et al., 2007). As mentioned in the introduction, this phenomenon has led to a new technology for protein immobilization (LAMI, light assisted molecular immobilization) since the created thiol groups can bind thiol reactive surfaces leading to oriented covalent protein immobilization (Neves-Petersen et al., 2006;Snabe et al., 2006; Duroux et al., 2007a, 2007b & 2007c; Skovsen et al., 2007, 2009a & 2009b; Neves-Petersen et al., 2009b; Parracino

In the case where the electron is captured by the cystine, the result can also be the breakage

tryptophan or disulphide bridges (Bent & Hayon, 1975b):

3

of the disulphide bridge (Hoffman & Hayon, 1972):

et al., 2010 & 2011).

*yrO e* (8)

(10)

(11)

(12)

(10)

*RSSR RS RS* (11)

*RSSR H RS RSH* (12)

*Tyr OH RSSR Tyr O H RSSR* (9)

Fig. 1. The spatial proximity between aromatic residues and disulphide bridges (SS) has been conserved throughout molecular evolution. Trp is the preferred spatial neighbor of SS (Petersen et al., 1999). UV excitation of the side chain of aromatic residues leads to electron ejection. The electron can then be captured by disulphide bridges, leading to their dissociation.

### **2.2 Observing the solvated electron and other transient species formed upon UV excitation of proteins**

In the flash photolysis experiments on lysozyme by Grossweiner and Usui (Grossweiner & Usui, 1971) it was shown that the initial photoproducts upon UV excitation of lysozyme are the photo-oxidized tryptophan residue, solvated electrons, and the cystine residue (disulphide bridge) electron adduct. In a more recent paper by Zhi Li *et al.* (Z. Li et al., 1989), experiments on a model system demonstrated that the fast electron transfer is consistent with direct electron transfer between the tryptophan triplet state and a nearby disulphide bridge (this is a very short range interaction that decays exponentially as the distance between the donor and acceptor increases). This process will result in a RSSR● radical, which again can result in breakage of the disulphide bridge, as shown above (scheme 12).

It is clear from the above that there are many possible pathways for the breakage of intramolecular disulphide bridges in proteins upon UV excitation of aromatic residues, even in the absence of molecular oxygen. Breakage of the disulphide bridge can lead to conformational changes in the protein, not necessarily resulting in inactivation of the protein. Transient absorption data of *Fusarium solani pisi* cutinase has also been acquired, with supplemental experimental data on tryptophan and lysozyme as a reference (Neves-Petersen et al., 2009a). Cutinase is a good model protein for studying the UV induced breakage of disulphide bridges is since it contains only one tryptophan that is within van der Waals contact of a disulphide bridge (closest distance ~3.8 Å).

Data showed that UV excitation of cutinase lead to the formation of the solvated electron (transient species with absorption maximum around 710-720nm, see Fig. 2) and of the disulphide bridge electron adduct radical, RSSR●- (transient species with absorption maximum around 420nm, see Fig. 2) (Neves-Petersen et al., 2009a). Figs. 2 and 3 show the kinetics of formation of the solvated electron. The increase in absorption of light at 710nm can clearly be seen following excitation at 266nm, which coincides with time zero in Figure 2. The data displayed in Fig. 3 is the intensity of absorbed light at 710nm (the intense peak displayed in Fig. 2) during the initial 43ns after excitation of cutinase with

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 131

Fig. 3. Normalized transient absorption data at 710nm for 0-43ns probe times displaying the kinetics of formation of the solvated electron for tryptophan (Trp), lysozyme (Lys) and

Time (s)

Likewise, fitting the decay in the absorption peaks at 710nm and 420nm will allow us to recover the lifetime of the solvated electrons and of the disulphide bridge electron adduct radical, respectively. The lifetimes of the solvated electron in lysozyme and cutinase samples at different pH values can be found in Table I. Below is shown the decay kinetics of the solvated electron within 10 s after 266nm excitation of cutinase (Neves-Petersen et al.,

+pH 4.0 ●pH8.5 ○pH 10.0

Fig. 4. Decay kinetics of the solvated electron absorption peak at 710nm within 10s after

Time after 266nm pump beam (s)

cutinase (Cut) samples at pH 8.5

2009a).

Transient absorption at 710nm

excitation.

266nm laser light. Light is absorbed at 710nm due to the presence of a new transient species created upon 266nm excitation of cutinase: the solvated electron. Solvated electrons are transient, i.e., short lived. One group that will capture them are disulphide bridges (RSSR). The capture of the solvated electrons by disulphide bridges leads to a decrease of the concentration of the solvated electron, and therefore, to a decrease in the intensity of absorbed light at 710nm. Such decay is displayed in Fig. 4. Furthermore, the combination of the solvated electron with the disulphide bridge leads to the formation of the disulphide-electron adduct radical, RSSR●-, a group which has its maximum absorption around 420nm. The presence of such group can be seen in Fig. 2, since absorption around 420nm can be observed.

Fig. 2. Cutinase transient absorption data. Transient absorption data collected at probe times from 0 to 50s. The transient solvated electron absorbs maximally light at ~710-720nm (intense peak) and the disulphide bridge electron adduct radical has its maximum absorption at ~420nm. The intensity of the peaks displayed in the 2D image to the right can be seen in the 3D image to the left.

Determination of the lifetimes of the different transient species formed upon UV excitation of proteins can be carried out by fitting the kinetic data displayed in Figs. 3 and 4. Upon fitting the initial increase of absorption at a 710nm and 420nm one recovers the rate of formation of solvated electrons and of the disulphide bridge electron adduct radical, respectively.

266nm laser light. Light is absorbed at 710nm due to the presence of a new transient species created upon 266nm excitation of cutinase: the solvated electron. Solvated electrons are transient, i.e., short lived. One group that will capture them are disulphide bridges (RSSR). The capture of the solvated electrons by disulphide bridges leads to a decrease of the concentration of the solvated electron, and therefore, to a decrease in the intensity of absorbed light at 710nm. Such decay is displayed in Fig. 4. Furthermore, the combination of the solvated electron with the disulphide bridge leads to the formation of the disulphide-electron adduct radical, RSSR●-, a group which has its maximum absorption around 420nm. The presence of such group can be seen in Fig. 2, since

Fig. 2. Cutinase transient absorption data. Transient absorption data collected at probe times from 0 to 50s. The transient solvated electron absorbs maximally light at ~710-720nm (intense peak) and the disulphide bridge electron adduct radical has its maximum

absorption at ~420nm. The intensity of the peaks displayed in the 2D image to the right can

Determination of the lifetimes of the different transient species formed upon UV excitation of proteins can be carried out by fitting the kinetic data displayed in Figs. 3 and 4. Upon fitting the initial increase of absorption at a 710nm and 420nm one recovers the rate of formation of solvated electrons and of the disulphide bridge electron adduct radical,

absorption around 420nm can be observed.

be seen in the 3D image to the left.

respectively.

Fig. 3. Normalized transient absorption data at 710nm for 0-43ns probe times displaying the kinetics of formation of the solvated electron for tryptophan (Trp), lysozyme (Lys) and cutinase (Cut) samples at pH 8.5

Likewise, fitting the decay in the absorption peaks at 710nm and 420nm will allow us to recover the lifetime of the solvated electrons and of the disulphide bridge electron adduct radical, respectively. The lifetimes of the solvated electron in lysozyme and cutinase samples at different pH values can be found in Table I. Below is shown the decay kinetics of the solvated electron within 10 s after 266nm excitation of cutinase (Neves-Petersen et al., 2009a).

Time after 266nm pump beam (s)

Fig. 4. Decay kinetics of the solvated electron absorption peak at 710nm within 10s after excitation.

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 133

Therefore, the lower the pH the faster the rate of decay of solvated electrons formed upon UV excitation of Trp molecules. In a protein, besides H3O+, different groups can act as electron scavengers, e.g. positively charged residues, the carbonyl group of the peptide chain (Faraggi & Bettelheim, 1977) as well as disulphide bridges, according to the reaction

Data shows that the higher the pH the longer time it takes for the solvated electron to recombine with the parent molecule (geminate recombination) or another electron scavenger molecule, such as H3O+. The observed lifetime increase with pH can be explained since the lower the pH, the higher the concentration of H3O+ and therefore the larger the probability of recombination of the solvated electron with the hydronium ion. Furthermore, for proteins, the higher the pH of the solution, the larger the number of basic titratable residues that have lost their positive charge and became neutral (His, Lys, Arg) and the larger the number of acidic titratable residues that have acquired a negative charge (Asp, Gly, Tyr, Cys not bridged). This means that an increase of pH leads to a loss of positive charge in the protein and a gain of neutral and negative charged residues in the protein. This will lead to an increase of the areas in the protein that carry a negative electrostatic potential. Therefore, an increase in pH will decrease the efficiency of electron recombination with the molecule due to electrostatic repulsion. This will lead to an increase of the solvated

The importance of immobilisation technology is demonstrated by the recent development of DNA microarrays, where multiple oligonucleotide or cDNA samples are immobilised on a solid surface in a spatially addressable manner. These arrays have revolutionised genetic studies by facilitating the global analysis of gene expression in living organisms. Similar approaches have been developed for protein analysis, where as little as one picogram of protein need be bound to each point on a microarray for subsequent analysis. The proteins bound to the microarrays, can then be assayed for functional or structural properties, facilitating screening on a scale and with a speed previously unknown. The biomolecules bound to the solid surface may additionally be used to capture other unbound molecules present in the mixture. Development of this technology, with the goal of immobilising a biomolecule on a solid surface in a controlled manner, with minimal surface migration of the bound moiety and with full retention of its native structure and function, has been the subject of intensive investigation in recent years (Veilleux & Duran, 1996). The simplest type of protein immobilisation exploits the high inherent binding affinity of surfaces to proteins in general. For example, proteins will physically adsorb to hydrophobic substrates via numerous weak contacts, comprising van der Waals and hydrogen bonding interactions. The advantage of this method is that it avoids modification of the protein to be bound. On the other hand, adsorbed proteins may be distributed unevenly over the solid support and/or inactivated since, e.g., their clustering may lead to steric hindrance of the active

*aq e RSSR RSSR* (17)

below:

electron lifetime, as observed in Fig. 4.

**3. Protein Immobilization onto surfaces: An overview** 

site/binding region in any subsequent functional assay.

The governing equations for the time-resolved intensity decay data were assumed to be a sum of exponentials as in

$$Abs(t) = \sum \alpha\_i \cdot \exp\left(\frac{-t}{\tau\_i}\right) \tag{13}$$

where *Abs*(t) is the intensity decay, i is the amplitude (pre-exponential factor), i the lifetime of the i-th component and i = 1.0. Data was analysed using a global analysis approach.

The fractional intensity *f*i of each decay time is given by

$$f\_i = \frac{\alpha\_i \tau\_i}{\sum\_j \alpha\_j \tau\_j} \tag{14}$$

and the mean lifetime is

$$\{\pi\} = \sum\_{i} f\_i \pi\_i \tag{15}$$

It was observed that the solvated electron average lifetime is shorter at acidic pH values, which is correlated with the fact that H3O+ captures the solvated electron. Furthermore, the solvated electron lifetime is significantly shorter in protein systems as compared to from Trp alone in solution, thus indicating that a protein offers other pathways involving capture of the solvated electron.


Table 1. Mean lifetime of the solvated electron in all samples at different pH values.

Data analysis shows that the solvated electron has ns and sub s decay lifetimes (Neves-Petersen et al., 2009a). These different lifetimes can be explained due to different recombination pathways of the solvated electron: recombination with the parent molecule (geminate recombination), with the hydronium ion present in the solvent or with other electron acceptor, such as disulphide bridges and positively charged groups. The intensity of the solvated electron peak is clearly pH dependent (Neves-Petersen et al., 2009a). This is correlated with the fact that the hydronium ion H3O+ is an electron scavenger. Recombination happens according to the reaction (Spanel & Smith, 1995):

$$e\_{aq}^{-} + H\_{3}O^{+} \rightarrow H^{\bullet} + H\_{2}O \tag{16}$$

The governing equations for the time-resolved intensity decay data were assumed to be a

*i*

where *Abs*(t) is the intensity decay, i is the amplitude (pre-exponential factor), i the lifetime of the i-th component and i = 1.0. Data was analysed using a global analysis

*i i*

 

 

*j j j*

> *i i i*

It was observed that the solvated electron average lifetime is shorter at acidic pH values, which is correlated with the fact that H3O+ captures the solvated electron. Furthermore, the solvated electron lifetime is significantly shorter in protein systems as compared to from Trp alone in solution, thus indicating that a protein offers other pathways involving capture of

> Tryptophan Lysozyme Cutinase pH <t> (µs) <t> (µs) <t> (µs) 4.0 1.0 0.3 0.3

Data analysis shows that the solvated electron has ns and sub s decay lifetimes (Neves-Petersen et al., 2009a). These different lifetimes can be explained due to different recombination pathways of the solvated electron: recombination with the parent molecule (geminate recombination), with the hydronium ion present in the solvent or with other electron acceptor, such as disulphide bridges and positively charged groups. The intensity of the solvated electron peak is clearly pH dependent (Neves-Petersen et al., 2009a). This is correlated with the fact that the hydronium ion H3O+ is an electron scavenger.

*aq* 3 2 *e HO H HO*

(16)

8.5 2.3 0.8 1.0 10.0 2.8 1.6 2.1 Table 1. Mean lifetime of the solvated electron in all samples at different pH values.

7.5 1.1 0.7

Recombination happens according to the reaction (Spanel & Smith, 1995):

*i*

*<sup>f</sup>*

*f*

*<sup>t</sup> Abs(t) exp* 

*i*

(13)

(14)

(15)

sum of exponentials as in

and the mean lifetime is

the solvated electron.

The fractional intensity *f*i of each decay time is given by

approach.

Therefore, the lower the pH the faster the rate of decay of solvated electrons formed upon UV excitation of Trp molecules. In a protein, besides H3O+, different groups can act as electron scavengers, e.g. positively charged residues, the carbonyl group of the peptide chain (Faraggi & Bettelheim, 1977) as well as disulphide bridges, according to the reaction below:

$$\text{R}\,e\_{aq}^{-} + \text{RSSR} \rightarrow \text{RSSR}\,\text{\textbullet}^{\bullet-} \tag{17}$$

Data shows that the higher the pH the longer time it takes for the solvated electron to recombine with the parent molecule (geminate recombination) or another electron scavenger molecule, such as H3O+. The observed lifetime increase with pH can be explained since the lower the pH, the higher the concentration of H3O+ and therefore the larger the probability of recombination of the solvated electron with the hydronium ion. Furthermore, for proteins, the higher the pH of the solution, the larger the number of basic titratable residues that have lost their positive charge and became neutral (His, Lys, Arg) and the larger the number of acidic titratable residues that have acquired a negative charge (Asp, Gly, Tyr, Cys not bridged). This means that an increase of pH leads to a loss of positive charge in the protein and a gain of neutral and negative charged residues in the protein. This will lead to an increase of the areas in the protein that carry a negative electrostatic potential. Therefore, an increase in pH will decrease the efficiency of electron recombination with the molecule due to electrostatic repulsion. This will lead to an increase of the solvated electron lifetime, as observed in Fig. 4.

### **3. Protein Immobilization onto surfaces: An overview**

The importance of immobilisation technology is demonstrated by the recent development of DNA microarrays, where multiple oligonucleotide or cDNA samples are immobilised on a solid surface in a spatially addressable manner. These arrays have revolutionised genetic studies by facilitating the global analysis of gene expression in living organisms. Similar approaches have been developed for protein analysis, where as little as one picogram of protein need be bound to each point on a microarray for subsequent analysis. The proteins bound to the microarrays, can then be assayed for functional or structural properties, facilitating screening on a scale and with a speed previously unknown. The biomolecules bound to the solid surface may additionally be used to capture other unbound molecules present in the mixture. Development of this technology, with the goal of immobilising a biomolecule on a solid surface in a controlled manner, with minimal surface migration of the bound moiety and with full retention of its native structure and function, has been the subject of intensive investigation in recent years (Veilleux & Duran, 1996). The simplest type of protein immobilisation exploits the high inherent binding affinity of surfaces to proteins in general. For example, proteins will physically adsorb to hydrophobic substrates via numerous weak contacts, comprising van der Waals and hydrogen bonding interactions. The advantage of this method is that it avoids modification of the protein to be bound. On the other hand, adsorbed proteins may be distributed unevenly over the solid support and/or inactivated since, e.g., their clustering may lead to steric hindrance of the active site/binding region in any subsequent functional assay.

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 135

biomolecules. Following illumination, the photochemically active compound anthraquinone will react as a free radical and form a stable ether bond with a polymer surface. Since anthraquinone is not found in native biomolecules, appropriate ligands have to be introduced into the biomolecule. In the case of proteins, this additional sample preparation step may require thermochemical coupling to the quinone and may not be site specific. A further development of light-induced immobilization technology is disclosed in US patents US 5,412,087 and US 6406844, which describe a method for preparing a linker bound to a substrate. The terminal end of the linker molecule is provided with a reactive functional group protected with a photo-removable protective group, e.g. a nitro-aromatic compound. Following exposure to light, the protective group is lost and the linker can react with a monomer such as an amino acid at its amino or carboxy-terminus. The monomer, furthermore, may itself carry a similar photo-removable protective group which can also be displaced by light during a subsequent reaction cycle. The method has particular application to solid phase synthesis, but does not facilitate orientated binding of proteins to a support. Bifunctional agents possessing thermochemical and photochemical functional substituents for immobilizing an enzyme are disclosed in US patent US 3,959,078. Derivatives of arylazides are described which allow light mediated activation and covalent coupling of the azide group to an enzyme, and substituents which react thermochemically with a solid support. The orientation of the enzyme molecules is not controlled. A method for oriented, light-dependent, covalent immobilization of proteins on a solid support, using the heterobifunctional wetting-agent N-(m-(3-(trifluoromethyl)diazirin-3 yl)phenyl)-4-maleimidobutyramine, is described by Collioud et al. (Collioud et al., 1993). The aryldiazirine function of this cross-linking reagent facilitates light-dependent, carbenemediated, covalent binding to either inert supports or to biomolecules such as proteins, carbohydrates and nucleic acids. The maleimide function of the cross-linker allows binding to a thiolated surface by thermochemical modification of cysteine thiols. However this treatment may modify the structure and activity of the target protein. Light-induced covalent coupling of the cross-linking reagent to a protein via the carbene function, however, has the disadvantage

that it does not provide controlled orientation of the target protein.

**3.1 Light Assisted Molecular Immobilization technology (LAMI) Photochemistry, biosensor microarrays and drug delivery systems** 

Common for most of the described immobilization methods is their use of one or more thermochemical/chemical steps, sometimes with hazardous chemicals, some of which are likely to have a deleterious effect on the structure and/or function of the bound protein. The available methods are often invasive, whereby foreign groups are introduced into a protein to act as functional groups, which cause protein denaturation, as well as lower its biological activity and substrate specificity. There is a need in the art of protein coupling and immobilization to improve the method of coupling, where the structural and functional properties of the coupled or immobilized component are preserved and the orientation of coupling can be controlled. We believe that LAMI represents a significant step in this direction.

Light assisted molecular immobilization technology provides a photonic method for coupling a protein or a peptide on a carrier via stable bonds (covalent bond or thiol-Au bond) while preserving the native structural and functional properties of the coupled protein or peptide. This technology avoids the use of one or more chemical steps, in contrast with traditional coupling methods for protein immobilisation, which typically involve several chemical

Molecules can be immobilised on a carrier or solid surface either passively through hydrophobic or ionic interactions, or covalently by attachment to surface groups. In response to the enormous importance of immobilisation for solid phase chemistry and biological screening, the analytical uses of the technology have been widely explored. The technology has found broad application in different areas of biotechnology, e.g. diagnostics, biosensors, affinity chromatography and immobilisation of molecules in ELISA assays. Alternative methods of immobilisation rely on the use of a few strong covalent bonds to bind the protein to the solid surface (Wilson & Nock , 2001). Examples include immobilisation of biotinylated proteins onto streptavidin-coated supports, and immobilisation of His-tagged proteins, containing a poly-histidine sequence, to Ni2+-chelating supports. Other functional groups on the surface of proteins which can be used for attachment to an appropriate surface include reacting an amine with an aldehyde via a Schiff-base, cross-linking amine groups to an amine surface with gluteraldehyde to form peptide bonds, cross-linking carboxylic acid groups present on the protein and support surface with carbodiimide, cross-linking based on disulphide bridge formation between two thiol groups and the formation of a thiol-Au bond between a thiol group and a gold surface. Amine groups in proteins are widely used for protein covalent immobilization via NHS (N-hydroxysuccinimide)-EDC (N-ethyl-N'- (dimethylaminopropyl) carbodiimide hydrochloride) chemistry (Johnsson et. al., 1991). Following immobilisation, un-reacted N-hydroxysuccinimide esters on the support are deactivated with ethanolamine hydrochloride to block areas devoid of bound proteins. The method is laborious since the reagents, used at each step of a chemical immobilization method, usually need to be removed prior to initiating the next step.

Methods for the immobilization of biomolecules via disulphide bridges are described by Veilleux J (1996). Protein samples are treated with a mild reducing agent, such as dithiothreitol, 2-mercaptoethanol or tris(2-carboxyethyl)phosphine hydrochloride to reduce disulphide bonds between cysteine residues, which are then bound to a support surface coated with maleimide. Alternatively primary amine groups on the protein can be modified with 2-iminothiolane hydrochloride (Traut's reagent) to introduce novel sulfhydryl groups, which are thereafter immobilized to the maleimide surface. Immobilization of proteins on a gold substrate via SH groups formed upon intracellular reduction of surface engineered disulphide bridges is shown for cupredoxin protein plastocyanin (Andolfi et al., 2002). The disulphide bridge has been engineered upon mutating the solvent accessible residues Ile21 and Glu25. An alternative approach to engineering thiol-groups into a protein has been described for ribonuclease (RNaseA), which has four essential cystines (Sweeney et al., 2000). In this case a single cysteine residue was substituted for Ala19, located in a surface loop near the N-terminus of RNase A. The cysteine in the expressed RNase was protected as a mixed disulphide with 2-nitro-5-thiobenzoic acid. Following subsequent de-protection with an excess of dithiothreitol, the RNase was coupled to the iodoacetyl groups attached to a cross-linked agarose resin, without loss of enzymatic activity. Again, preparation of the protein for immobilization requires its exposure to both protecting and de-protecting agents, which may negatively impact its native structure and/or function.

Light-induced immobilization techniques have also been explored, leading to the use of quinone compounds for photochemical linking to a carbon-containing support (European patent EP0820483). Activation occurs following irradiation with non-ionizing UV and visible light. Masks can be used to activate certain areas of the support for subsequent attachment of

Molecules can be immobilised on a carrier or solid surface either passively through hydrophobic or ionic interactions, or covalently by attachment to surface groups. In response to the enormous importance of immobilisation for solid phase chemistry and biological screening, the analytical uses of the technology have been widely explored. The technology has found broad application in different areas of biotechnology, e.g. diagnostics, biosensors, affinity chromatography and immobilisation of molecules in ELISA assays. Alternative methods of immobilisation rely on the use of a few strong covalent bonds to bind the protein to the solid surface (Wilson & Nock , 2001). Examples include immobilisation of biotinylated proteins onto streptavidin-coated supports, and immobilisation of His-tagged proteins, containing a poly-histidine sequence, to Ni2+-chelating supports. Other functional groups on the surface of proteins which can be used for attachment to an appropriate surface include reacting an amine with an aldehyde via a Schiff-base, cross-linking amine groups to an amine surface with gluteraldehyde to form peptide bonds, cross-linking carboxylic acid groups present on the protein and support surface with carbodiimide, cross-linking based on disulphide bridge formation between two thiol groups and the formation of a thiol-Au bond between a thiol group and a gold surface. Amine groups in proteins are widely used for protein covalent immobilization via NHS (N-hydroxysuccinimide)-EDC (N-ethyl-N'- (dimethylaminopropyl) carbodiimide hydrochloride) chemistry (Johnsson et. al., 1991). Following immobilisation, un-reacted N-hydroxysuccinimide esters on the support are deactivated with ethanolamine hydrochloride to block areas devoid of bound proteins. The method is laborious since the reagents, used at each step of a chemical immobilization method,

Methods for the immobilization of biomolecules via disulphide bridges are described by Veilleux J (1996). Protein samples are treated with a mild reducing agent, such as dithiothreitol, 2-mercaptoethanol or tris(2-carboxyethyl)phosphine hydrochloride to reduce disulphide bonds between cysteine residues, which are then bound to a support surface coated with maleimide. Alternatively primary amine groups on the protein can be modified with 2-iminothiolane hydrochloride (Traut's reagent) to introduce novel sulfhydryl groups, which are thereafter immobilized to the maleimide surface. Immobilization of proteins on a gold substrate via SH groups formed upon intracellular reduction of surface engineered disulphide bridges is shown for cupredoxin protein plastocyanin (Andolfi et al., 2002). The disulphide bridge has been engineered upon mutating the solvent accessible residues Ile21 and Glu25. An alternative approach to engineering thiol-groups into a protein has been described for ribonuclease (RNaseA), which has four essential cystines (Sweeney et al., 2000). In this case a single cysteine residue was substituted for Ala19, located in a surface loop near the N-terminus of RNase A. The cysteine in the expressed RNase was protected as a mixed disulphide with 2-nitro-5-thiobenzoic acid. Following subsequent de-protection with an excess of dithiothreitol, the RNase was coupled to the iodoacetyl groups attached to a cross-linked agarose resin, without loss of enzymatic activity. Again, preparation of the protein for immobilization requires its exposure to both protecting and de-protecting agents,

Light-induced immobilization techniques have also been explored, leading to the use of quinone compounds for photochemical linking to a carbon-containing support (European patent EP0820483). Activation occurs following irradiation with non-ionizing UV and visible light. Masks can be used to activate certain areas of the support for subsequent attachment of

usually need to be removed prior to initiating the next step.

which may negatively impact its native structure and/or function.

biomolecules. Following illumination, the photochemically active compound anthraquinone will react as a free radical and form a stable ether bond with a polymer surface. Since anthraquinone is not found in native biomolecules, appropriate ligands have to be introduced into the biomolecule. In the case of proteins, this additional sample preparation step may require thermochemical coupling to the quinone and may not be site specific. A further development of light-induced immobilization technology is disclosed in US patents US 5,412,087 and US 6406844, which describe a method for preparing a linker bound to a substrate. The terminal end of the linker molecule is provided with a reactive functional group protected with a photo-removable protective group, e.g. a nitro-aromatic compound. Following exposure to light, the protective group is lost and the linker can react with a monomer such as an amino acid at its amino or carboxy-terminus. The monomer, furthermore, may itself carry a similar photo-removable protective group which can also be displaced by light during a subsequent reaction cycle. The method has particular application to solid phase synthesis, but does not facilitate orientated binding of proteins to a support. Bifunctional agents possessing thermochemical and photochemical functional substituents for immobilizing an enzyme are disclosed in US patent US 3,959,078. Derivatives of arylazides are described which allow light mediated activation and covalent coupling of the azide group to an enzyme, and substituents which react thermochemically with a solid support. The orientation of the enzyme molecules is not controlled. A method for oriented, light-dependent, covalent immobilization of proteins on a solid support, using the heterobifunctional wetting-agent N-(m-(3-(trifluoromethyl)diazirin-3 yl)phenyl)-4-maleimidobutyramine, is described by Collioud et al. (Collioud et al., 1993). The aryldiazirine function of this cross-linking reagent facilitates light-dependent, carbenemediated, covalent binding to either inert supports or to biomolecules such as proteins, carbohydrates and nucleic acids. The maleimide function of the cross-linker allows binding to a thiolated surface by thermochemical modification of cysteine thiols. However this treatment may modify the structure and activity of the target protein. Light-induced covalent coupling of the cross-linking reagent to a protein via the carbene function, however, has the disadvantage that it does not provide controlled orientation of the target protein.

Common for most of the described immobilization methods is their use of one or more thermochemical/chemical steps, sometimes with hazardous chemicals, some of which are likely to have a deleterious effect on the structure and/or function of the bound protein. The available methods are often invasive, whereby foreign groups are introduced into a protein to act as functional groups, which cause protein denaturation, as well as lower its biological activity and substrate specificity. There is a need in the art of protein coupling and immobilization to improve the method of coupling, where the structural and functional properties of the coupled or immobilized component are preserved and the orientation of coupling can be controlled. We believe that LAMI represents a significant step in this direction.

### **3.1 Light Assisted Molecular Immobilization technology (LAMI)**

### **Photochemistry, biosensor microarrays and drug delivery systems**

Light assisted molecular immobilization technology provides a photonic method for coupling a protein or a peptide on a carrier via stable bonds (covalent bond or thiol-Au bond) while preserving the native structural and functional properties of the coupled protein or peptide. This technology avoids the use of one or more chemical steps, in contrast with traditional coupling methods for protein immobilisation, which typically involve several chemical

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 137

quartz or a gold surface, the protein is immobilized onto the surface. Since this happens where the UV photons are present, the size of the focal spot e.g. in a simple focusing setup determines where immobilization takes place. We are able to control this process such that spot size is ~3-5 micron. The process is relatively fast, being determined by physical chemical parameters as well as the light fluency (power per unit area). Currently we are operating with 100 ms illumination per spot with ~1mW 280nm 8MHz femtosecond pulses (Duroux et al, 2007). With a pitch of 10 micron and spot size of 5 micron, this allows for about 40.000 spots per mm2. We have verified that Fab anti prostate specific antigen can be immobilized with our

Fig. 6. (Left) Protein microarray engineered with LAMI using 280nm laser light focused to a spot size of 5 micron. The displayed array has 5 micron spots, 10 micron pitch leading to an array density equivalent to 104 spots per 1mm2 of sensor surface. (Right) Atomic Force Microscopy visualization of protein immobilized using LAMI, of the central area of 1 spot of

Although light assisted immobilization technology has obvious applications in the area of biosensor microarraying, any pattern of immobilized proteins can be generated. As such, we have successfully transferred several different bitmaps to selected surfaces. As seen in the figure a bitmap of a fullerene was printed into a protein pattern that retained almost all graphical details of the original bitmap (Neves-Petersen et al., 2009b). The size of the protein

Fig. 7. Fluorescence is emitted from proteins that have been immobilized with LAMI technology. A film of protein has been illuminated according to a fullerene pattern. The illuminated proteins (280nm excitation) were immobilized onto the thiol reactive surface.

The image has 10m resolution and dimensions ~1260m x 1220m

technology and still remains biologically active (Parracino et al., 2010).

the displayed array.

printed surface is 1mm2.

reactions. That can be costly, time-consuming as well as deleterious to the structure/function of the bound protein. Furthermore the orientation of the protein or peptide, coupled according to the method of the present invention, can be controlled, such that their functional properties, e.g. enzymatic, may be preserved. In comparison, the majority of known protein coupling methods lead to a random orientation of the proteins immobilised on a carrier, with the significant risk of lower biological activity and raised detection limits.

LAMI technology exploits an inherent natural property of proteins and peptides, whereby a disulphide bridge in a protein or peptide, located in close proximity to an aromatic amino acid residue, is disrupted following excitation of aromatic amino acids. The aromatic residues are actually the preferred spatial neighbours of disulphide bridges (Petersen et al., 1999). The thiol groups created by light induced disulphide bridge disruption in a protein or peptide are then used to immobilise the protein or peptide to a carrier. The formed free thiol groups in the protein can afterwards attach the protein onto a thiol reactive surface, such as gold, thiol derivatized glass and quartz, or even plastics (see Fig. 5). The new protein immobilization technology has led to the development of (Neves-Petersen et al., 2006;Snabe et al., 2006; Duroux et al., 2007a, 2007b & 2007c; Skovsen et al., 2007, 2009a & 2009b; Neves-Petersen et al., 2009b; Parracino et al., 2010 & 2011):


Fig. 5. The principle of light assisted molecular immobilization (LAMI) sketched with tryptophan near a disulphide bridge on a protein molecule. UV illumination of aromatic residues leads to disulphide bridge (SS) opening and to the formation of free SH groups, which will react to thiol reactive surfaces.

### **3.2 Bio-functionalization of surfaces with micrometer-resolution**

With a beam of UV laser light we are able to open disulphide bonds in most SS-containing proteins. If this happens at or close to a thiol reactive surface, such as thiol derivatized glass,

reactions. That can be costly, time-consuming as well as deleterious to the structure/function of the bound protein. Furthermore the orientation of the protein or peptide, coupled according to the method of the present invention, can be controlled, such that their functional properties, e.g. enzymatic, may be preserved. In comparison, the majority of known protein coupling methods lead to a random orientation of the proteins immobilised on a carrier, with the

LAMI technology exploits an inherent natural property of proteins and peptides, whereby a disulphide bridge in a protein or peptide, located in close proximity to an aromatic amino acid residue, is disrupted following excitation of aromatic amino acids. The aromatic residues are actually the preferred spatial neighbours of disulphide bridges (Petersen et al., 1999). The thiol groups created by light induced disulphide bridge disruption in a protein or peptide are then used to immobilise the protein or peptide to a carrier. The formed free thiol groups in the protein can afterwards attach the protein onto a thiol reactive surface, such as gold, thiol derivatized glass and quartz, or even plastics (see Fig. 5). The new protein immobilization technology has led to the development of (Neves-Petersen et al., 2006;Snabe et al., 2006; Duroux et al., 2007a, 2007b & 2007c; Skovsen et al., 2007, 2009a & 2009b; Neves-

biofunctionalization of thiol reactive nanoparticles, aiming at engineering drug delivery

Fig. 5. The principle of light assisted molecular immobilization (LAMI) sketched with tryptophan near a disulphide bridge on a protein molecule. UV illumination of aromatic residues leads to disulphide bridge (SS) opening and to the formation of free SH groups,

With a beam of UV laser light we are able to open disulphide bonds in most SS-containing proteins. If this happens at or close to a thiol reactive surface, such as thiol derivatized glass,

**3.2 Bio-functionalization of surfaces with micrometer-resolution** 

significant risk of lower biological activity and raised detection limits.

Petersen et al., 2009b; Parracino et al., 2010 & 2011):

microarrays of active biosensors and

which will react to thiol reactive surfaces.

systems

quartz or a gold surface, the protein is immobilized onto the surface. Since this happens where the UV photons are present, the size of the focal spot e.g. in a simple focusing setup determines where immobilization takes place. We are able to control this process such that spot size is ~3-5 micron. The process is relatively fast, being determined by physical chemical parameters as well as the light fluency (power per unit area). Currently we are operating with 100 ms illumination per spot with ~1mW 280nm 8MHz femtosecond pulses (Duroux et al, 2007). With a pitch of 10 micron and spot size of 5 micron, this allows for about 40.000 spots per mm2. We have verified that Fab anti prostate specific antigen can be immobilized with our technology and still remains biologically active (Parracino et al., 2010).

Fig. 6. (Left) Protein microarray engineered with LAMI using 280nm laser light focused to a spot size of 5 micron. The displayed array has 5 micron spots, 10 micron pitch leading to an array density equivalent to 104 spots per 1mm2 of sensor surface. (Right) Atomic Force Microscopy visualization of protein immobilized using LAMI, of the central area of 1 spot of the displayed array.

Although light assisted immobilization technology has obvious applications in the area of biosensor microarraying, any pattern of immobilized proteins can be generated. As such, we have successfully transferred several different bitmaps to selected surfaces. As seen in the figure a bitmap of a fullerene was printed into a protein pattern that retained almost all graphical details of the original bitmap (Neves-Petersen et al., 2009b). The size of the protein printed surface is 1mm2.

Fig. 7. Fluorescence is emitted from proteins that have been immobilized with LAMI technology. A film of protein has been illuminated according to a fullerene pattern. The illuminated proteins (280nm excitation) were immobilized onto the thiol reactive surface. The image has 10m resolution and dimensions ~1260m x 1220m

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 139

Fig. 9. Consider a thin lens illuminated by a monochromatic plan wave. According to the theory of Fourier optics, an aperture (transmission mask) placed in the back focal plane of a lens will generate a diffraction pattern in the front focal plane that will be identical to the FT

Fig. 10. A spatial mask of eight holes arranged in a square (total dimension of mask is 1cmx1cm) placed in the back focal plane of a lens, will give rise to a diffraction pattern

Fig. 11. Fluorescence image of the fluorescently labeled proteins immobilized using the simple eight-hole mask, depicting the fluorescence emission from FITC. The peaks in the array are interspaced by ~1.5µm and have a FWHM of 750nm. The pitch of 1.5µm

Using the light pattern produced by this simple mask with 8 x 1mm sized holes, in a single shot we produce multiple spots with immobilized protein with a spot size of ~700 nm (Fig. 11 and Skovsen et al., 2009b). With this spot density, we can populate 1 mm2 with close to 1 million sensor spots, which represents an improvement of 10 fold over existing commercially available high density protein arraying methods. Our approach bypasses the use of micro dispenser techniques – and the technical difficulties associated with the use of

displayed to the right (central part of the diffraction pattern).

corresponds to a spot density of ~4.5x105 spots per mm2.

of the aperture.

In Fig. 8 is displayed the optical setup used in order to immobilize the proteins according to a particular bitmap:


Fig. 8. Optical setup used in order to immobilize the proteins according to a particular bitmap. The slide were the protein film is placed on is optically flat and derivatised with thiol (SH) groups using silane chemistry.

### **3.3 Biofunctionalization of surfaces with nm/submicrometer-resolution**

The technique of UV-light assisted immobilization of disulphide containing proteins has been combined with the Fourier transforming properties of lenses as well as with a simple mm scale feature size spatial mask. Theory predicts that when light passes through a spatial mask placed in the back focal plane of a focusing lens, we should obtain an intensity pattern in the front focal plane corresponding to the Fourier transform of the spatial mask (see Fig. 9).

A spatial mask consisting of eight holes arranged in a square with a hole in each corner and in the middle of each side is displayed below (Fig. 10). This pattern was then Fourier transformed in order to evaluate the diffraction pattern that the simplified eight-hole mask would generate after being Fourier transformed by a lens. The diffraction patter is displayed in Fig. 10

In Fig. 8 is displayed the optical setup used in order to immobilize the proteins according to

The surface is illuminated according to the bitmap, i.e., light will hit the surface

The slide is washed, removing the non-illuminated and therefore non-immobilized

Molecules will only be immobilized on the surface if they have been illuminated

Fig. 8. Optical setup used in order to immobilize the proteins according to a particular bitmap. The slide were the protein film is placed on is optically flat and derivatised with

The technique of UV-light assisted immobilization of disulphide containing proteins has been combined with the Fourier transforming properties of lenses as well as with a simple mm scale feature size spatial mask. Theory predicts that when light passes through a spatial mask placed in the back focal plane of a focusing lens, we should obtain an intensity pattern in the front focal plane corresponding to the Fourier transform of the spatial mask (see Fig. 9).

A spatial mask consisting of eight holes arranged in a square with a hole in each corner and in the middle of each side is displayed below (Fig. 10). This pattern was then Fourier transformed in order to evaluate the diffraction pattern that the simplified eight-hole mask would generate

after being Fourier transformed by a lens. The diffraction patter is displayed in Fig. 10

**3.3 Biofunctionalization of surfaces with nm/submicrometer-resolution** 

The fluorescence of the immobilized molecules can be observed

a particular bitmap:

proteins

The slide surface is covered with a protein film

A bit map is loaded into the computer

thiol (SH) groups using silane chemistry.

reproducing the image in the bitmap

Fig. 9. Consider a thin lens illuminated by a monochromatic plan wave. According to the theory of Fourier optics, an aperture (transmission mask) placed in the back focal plane of a lens will generate a diffraction pattern in the front focal plane that will be identical to the FT of the aperture.

Fig. 10. A spatial mask of eight holes arranged in a square (total dimension of mask is 1cmx1cm) placed in the back focal plane of a lens, will give rise to a diffraction pattern displayed to the right (central part of the diffraction pattern).

Fig. 11. Fluorescence image of the fluorescently labeled proteins immobilized using the simple eight-hole mask, depicting the fluorescence emission from FITC. The peaks in the array are interspaced by ~1.5µm and have a FWHM of 750nm. The pitch of 1.5µm corresponds to a spot density of ~4.5x105 spots per mm2.

Using the light pattern produced by this simple mask with 8 x 1mm sized holes, in a single shot we produce multiple spots with immobilized protein with a spot size of ~700 nm (Fig. 11 and Skovsen et al., 2009b). With this spot density, we can populate 1 mm2 with close to 1 million sensor spots, which represents an improvement of 10 fold over existing commercially available high density protein arraying methods. Our approach bypasses the use of micro dispenser techniques – and the technical difficulties associated with the use of

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 141

digestion time than free enzyme molecules and easy separation from the solution (Y.Li et al. 2007). Earlier studies have shown that immobilization of biomolecules not only makes separation easier but also increases the stability of enzyme towards pH, temperature, chemical denaturants and organic solvents (Z. Yang et al. 2008, H.Yang et al., 2004). Water soluble carbodiimide was used to activate the direct adsorption of glucose oxidase, streptokinase, chymotrypsin, dispase, BSA and alkaline phosphatase on magnetic particles (Koneracka et al. 2002). Recently, decanthiol capped gold nanoparticles were modified with dithiobis (succinimidyl propionate) for BSA coupling by ligand exchange (H.-Y.Park et al. 2007). Ma and colleagues used condensation product of 3-glycidoxypropyltrimethoxysilane and iminodiacetic acid charged with Cu2+ to immobilize BSA onto the silica coated magnetic

We are tagging biomolecules directly onto magnetic nanoparticles using our new photonic technology, light assisted molecular immobilization. The surface of these nanoparticles is thiol reactive, being gold or thiol derivatised silica. These particles provide very high surface area to tag protein efficiently and also there is no need to use other reagents to enhance the binding. The surface affinity towards the thiol groups present in the protein will be used to immobilize the protein molecule onto the nanoparticle. Bovine serum albumin (BSA, a carrier protein) and insulin have been successfully immobilized with our new photonic technology. Recently, LAMI technology has been used to create free and active thiol functional groups in BSA to be linked to

Fig. 12. Covalent immobilization of proteins onto the thiol reactive surface of nanoparticles using light assisted molecular immobilization (LAMI) technology, in order to engineer drug

We have developed the necessary technology that allows us to produce a variety of nanoparticles, from gold and silica nanoparticles to core-shell superparamagnetic nanoparticles. Furthermore, we can further derivatise the silica outer layer of those nanoparticles with chemical functional groups, such as thiol, amino and carboxylic groups. The combination of such knowledge with our new photonic immobilization technology allows us to build protein bioconjugates in a new way. Our new photonic immobilization technology is ideal to couple drugs, proteins, peptides, DNA and other molecules to nanoparticles such as gold or biopolymer nanospheres, which can subsequently be used as

nanoparticles through metal ion affinity towards protein (Ma et al., 2006).

Fe3O4@Au core-shell nanoparticle (Parracino et al., 2011).

molecular carriers into cells for therapeutic purposes.

delivery systems.

such. It is simple, and fast. With our current ability to generate immobilized patterns with a spot size down to 700 nanometer, we believe that we can design spatial patterns of binding proteins which could identify and bind, e.g., specific cells such as stem cells.

Our previous works report that we do see patterns similar to what theory predicts (Skovsen et al., 2009b; Petersen et al., 2010) but not always identical (Petersen et al., 2010). We have also shown that the presence of biomolecules on the slide surface can break the diffraction pattern of light (Petersen et al., 2010).

### **4. Biofunctionalization of thiol reactive nanoparticles, aiming at the development of drug delivery systems**

For the last few decades, research efforts have been focused on the development of new materials at the atomic, molecular and macromolecular levels, on the length scale of approximately 1-100nm in order to build materials at the nanoscale, and to explore structures, devices and systems that have novel properties. It has been shown that it is possible to tune precisely the physico-chemical properties of nano-materials by modifying the crystal size, shape and composition. Among various nano materials, magnetic core-shell particles have been broadly used in many technological applications, especially for biological applications such as drug targeting and delivery, cell labeling and separation, cancer therapy, magnetic resonance image (MRI) contrast agents, bio-sensors and bioimaging. Core-shell particles result from the combination of different metals that together display new properties compared to their monometallic counterparts. Therefore, the combination of both metal's properties such as optical, electrical, magnetic and catalytic can be used for technological applications. Among the core-shell nanoparticles, Fe3O4@Au and Fe3O4@SiO2 particles are widely used not only for its magnetic, optical and chemical properties but also for chemical stability, good biocompatibility, low toxicity, easy dispersibility, affinity towards biomolecules with amine/thiol/carboxylic terminal groups and convenient preparation techniques. Magnetite nanoparticles of size below 26.1 nm are super-paramagnetic in nature which means that these particles can be controlled by external magnetic field but retain no coercivity value (no residual magnetism) once the field is removed (Gnanaprakash et al., 2007). This magnetic property is being used extensively for biosciences in various applications, including bioseparation and imaging. Since biomolecules are highly sensitive to pH, temperature and chemical environment, immobilization protocols should be developed in order to secure high molecular activity and stability.

### **4.1 New photonic methodology used to create functional nanoparticles**

Recently, many protocols have been proposed for the immobilization of various biomolecules on to the particle surfaces for novel properties and various applications. The review paper on "Chemical Strategies for Generating Protein Biochips" by Jonkheijm et al. (Jonkheijm et al., 2008) describes different approaches using covalent and non-covalent immobilization chemistry are reviewed. Recent studies demonstrated that the incorporation of chiral molecules onto nanoparticles provides new opportunities for achieving specificity in the recognition of protein surfaces (You et al. 2008). Immobilization of trypsin on superparamagnetic nanoparticles allows using higher enzyme concentrations, leading to shorter

such. It is simple, and fast. With our current ability to generate immobilized patterns with a spot size down to 700 nanometer, we believe that we can design spatial patterns of binding

Our previous works report that we do see patterns similar to what theory predicts (Skovsen et al., 2009b; Petersen et al., 2010) but not always identical (Petersen et al., 2010). We have also shown that the presence of biomolecules on the slide surface can break the diffraction

For the last few decades, research efforts have been focused on the development of new materials at the atomic, molecular and macromolecular levels, on the length scale of approximately 1-100nm in order to build materials at the nanoscale, and to explore structures, devices and systems that have novel properties. It has been shown that it is possible to tune precisely the physico-chemical properties of nano-materials by modifying the crystal size, shape and composition. Among various nano materials, magnetic core-shell particles have been broadly used in many technological applications, especially for biological applications such as drug targeting and delivery, cell labeling and separation, cancer therapy, magnetic resonance image (MRI) contrast agents, bio-sensors and bioimaging. Core-shell particles result from the combination of different metals that together display new properties compared to their monometallic counterparts. Therefore, the combination of both metal's properties such as optical, electrical, magnetic and catalytic can be used for technological applications. Among the core-shell nanoparticles, Fe3O4@Au and Fe3O4@SiO2 particles are widely used not only for its magnetic, optical and chemical properties but also for chemical stability, good biocompatibility, low toxicity, easy dispersibility, affinity towards biomolecules with amine/thiol/carboxylic terminal groups and convenient preparation techniques. Magnetite nanoparticles of size below 26.1 nm are super-paramagnetic in nature which means that these particles can be controlled by external magnetic field but retain no coercivity value (no residual magnetism) once the field is removed (Gnanaprakash et al., 2007). This magnetic property is being used extensively for biosciences in various applications, including bioseparation and imaging. Since biomolecules are highly sensitive to pH, temperature and chemical environment, immobilization protocols should be developed in order to secure high molecular activity

proteins which could identify and bind, e.g., specific cells such as stem cells.

**4. Biofunctionalization of thiol reactive nanoparticles, aiming at the** 

**4.1 New photonic methodology used to create functional nanoparticles** 

Recently, many protocols have been proposed for the immobilization of various biomolecules on to the particle surfaces for novel properties and various applications. The review paper on "Chemical Strategies for Generating Protein Biochips" by Jonkheijm et al. (Jonkheijm et al., 2008) describes different approaches using covalent and non-covalent immobilization chemistry are reviewed. Recent studies demonstrated that the incorporation of chiral molecules onto nanoparticles provides new opportunities for achieving specificity in the recognition of protein surfaces (You et al. 2008). Immobilization of trypsin on superparamagnetic nanoparticles allows using higher enzyme concentrations, leading to shorter

pattern of light (Petersen et al., 2010).

and stability.

**development of drug delivery systems** 

digestion time than free enzyme molecules and easy separation from the solution (Y.Li et al. 2007). Earlier studies have shown that immobilization of biomolecules not only makes separation easier but also increases the stability of enzyme towards pH, temperature, chemical denaturants and organic solvents (Z. Yang et al. 2008, H.Yang et al., 2004). Water soluble carbodiimide was used to activate the direct adsorption of glucose oxidase, streptokinase, chymotrypsin, dispase, BSA and alkaline phosphatase on magnetic particles (Koneracka et al. 2002). Recently, decanthiol capped gold nanoparticles were modified with dithiobis (succinimidyl propionate) for BSA coupling by ligand exchange (H.-Y.Park et al. 2007). Ma and colleagues used condensation product of 3-glycidoxypropyltrimethoxysilane and iminodiacetic acid charged with Cu2+ to immobilize BSA onto the silica coated magnetic nanoparticles through metal ion affinity towards protein (Ma et al., 2006).

We are tagging biomolecules directly onto magnetic nanoparticles using our new photonic technology, light assisted molecular immobilization. The surface of these nanoparticles is thiol reactive, being gold or thiol derivatised silica. These particles provide very high surface area to tag protein efficiently and also there is no need to use other reagents to enhance the binding. The surface affinity towards the thiol groups present in the protein will be used to immobilize the protein molecule onto the nanoparticle. Bovine serum albumin (BSA, a carrier protein) and insulin have been successfully immobilized with our new photonic technology. Recently, LAMI technology has been used to create free and active thiol functional groups in BSA to be linked to Fe3O4@Au core-shell nanoparticle (Parracino et al., 2011).

Fig. 12. Covalent immobilization of proteins onto the thiol reactive surface of nanoparticles using light assisted molecular immobilization (LAMI) technology, in order to engineer drug delivery systems.

We have developed the necessary technology that allows us to produce a variety of nanoparticles, from gold and silica nanoparticles to core-shell superparamagnetic nanoparticles. Furthermore, we can further derivatise the silica outer layer of those nanoparticles with chemical functional groups, such as thiol, amino and carboxylic groups. The combination of such knowledge with our new photonic immobilization technology allows us to build protein bioconjugates in a new way. Our new photonic immobilization technology is ideal to couple drugs, proteins, peptides, DNA and other molecules to nanoparticles such as gold or biopolymer nanospheres, which can subsequently be used as molecular carriers into cells for therapeutic purposes.

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 143

molecules and therapeutic molecules to track particles inside the human body and targeted drug delivery. However, intense research studies are needed in order to overcome the barriers in the human body that challenge the efficiency of nanoparticle delivery such as walls of blood vessels, physical entrapment of particles in organs and removal of particles by phagocytic cells. Therefore, the ideal nanoparticle system to be used in nanomedicine should not only overcome such barriers but also allow for real time visualisation of particles, detection of the damaged tissues, selective and rapid accumulation at diseased tissue, effective drug delivery or effective therapy (in the case of hyperthermia). Hence, pioneering works are being carried out at related interdisciplinary fields such as chemistry, biology,

Fig. 13. The surface of silica nanoparticles can be functionalized with different chemical groups which can be used to bind fluorophores (e.g. in this example the fluorophore AF 488 has been coupled to the SH groups introduced on the surface of bare silica nanoparticles),

Nanoparticles based drug delivery is particularly advantageous when planning cancer therapy because the leaky blood cells in tumours mean that particles of certain size tend to accumulate more in cancer tissues than in normal tissues (Peer D et al, 2007). By coating nanoparticles with biomolecules that recognize receptors on cell membranes, a wide range of drugs and imaging agents can enter cells (endocytosis). Drug loaded magnetic particles can be localized to the specific site by an external magnetic field (Hu et al., 2008). This allows more concentrated doses of the anticancer drugs to be delivered to the cancer cells and keep them on site for longer periods of time. In order to prevent dangerous agglomeration of the particles in the blood stream, the particles must be of a small size relative to the dimensions of the capillaries, monodisperse and spherical in shape. In addition, the particles must have a high magnetic moment and switch their magnetisation quickly at low elds (Ankamwar et al., 2010; Shah et al., 2011). Nanoparticles surface modified with amphiphilic polymeric surfactants such as poloxamers, poloxamines or polyethylene glycol derivatives (S.-M. Lee et al., 2010), folic acid derivatives (Das et al, 2008; Landmark et al., 2008), silica derivatized with different functional groups or with porous structure (Wang et al., 2008; Slowing et al., 2007) are used for drug targeting and delivery. Moreover, triggerable drug delivery systems enable on-demand controlled release of drugs that may enhance therapeutic effectiveness and reduce systemic toxicity. Recently, a number of new materials have been developed that exhibit, e.g., sensitivity to UV and visible light, such that irradiation can release covalently bound drugs from dendrimers or dendrons with photocleavable cores or photoactivated surfaces (C. Park et al., 2008), temperature (Bikram et al., 2007; Peng et al., 2011), nearinfrared light (S.-M.Lee et al., 2010), pH (Benarjee & Chen, 2008), ultrasound (Bawa et al.,

pharmacy, nanotechnology, medicine and imaging.

proteins or other molecules.

### **4.2 Medical applications of bio-functionalized nanoparticles**

Nanomedicine is the medical application of nanotechnology and related disciplines, which mostly include biocompatible nanoparticle platforms that contain both therapeutic and/or imaging components. Since biomolecules such as individual cells, mRNA, DNA and proteins are nanoscale sized, probes of equivalent dimensions can provide very effective detection of individual chemical interactions of biomolecules, understanding of the chemical reactions and manipulation of the same. Therefore, the integration of nanotechnology and medical sciences has led to fundamental understanding in molecular biology, as well as new advanced technological applications such as drug targeting and delivery, cell labeling and separation, cancer therapy, magnetic resonance image (MRI) contrast agents, bio-sensors and bio-imaging. Nanoparticles may carry chemo-, radio-, and gene therapeutics or combination of these. They can be inorganic nanoparticles (noble metal, metal oxide, silica, mesoporous silica and combination of these components), lipid aggregates, and synthetic surfactant-polymer systems (such as vesicles, micelles). Inorganic nanoparticles that have unique physicochemical properties allow applications in nanomedicine after proper synthesis, coating, surface functionalization and bioconjugation. These nanosized materials provide a robust framework in which two or more components can be incorporated to give multifunctional capabilities (Wang et al., 2008; Salgueiriño-Maceira & Correa-Duarte, 2007). The combination of metals or polymer molecules should provide suitable and tunable magnetic, optical and chemical properties, chemical stability, low toxicity, easy dispersibility, affinity towards biomolecules with amine/thiol/carboxylic terminal groups and convenient preparation techniques.

Among the various compositions, gold composites are used for the development of various clinical diagnosis methods (Baptista et al., 2008; Raj et al., 2011) because of its size dependent optical properties. Time-resolved single-photon counting fluorescence studies on porphyrin monolayer-modified gold clusters revealed resonant energy transfer between the porphyrin and the gold surface, which is a phenomenon of considerable interest in biophotonics (Imahori & Fukuzumi, 2001). The ability to control the size and shape of gold nanoparticles and their surface conjugation with antibodies allow for both selective imaging and photothermal killing of cancer cells by using light with longer wavelengths for tissue penetration (Gobin et al., 2007). Similar success was also demonstrated with polymer-coated superparamagnetic iron oxide nanoparticles conjugated with, e.g., uorescent molecules, tumor-targeting moieties and anticancer drugs which aim targeting human cancers. Imaging inside the body can either be done using magnetic resonance or uorescence imaging (Kohler et al., 2006). Quantum dots optical properties including bright emission, photostability, size dependant luminescence and long fluorescence lifetimes make them also suitable for bioimaging applications. In combination with superparamagnetic nanoparticles and surface modification with peptides or other functional groups, these multifunctional particles are being used in bioimaging, bioseparation and in order to understand the behaviour of nanoparticles in cells such as tracking the particles, cell uptake of particles, drug dose evolution at targeted site (Janczewski et al., 2011; Summers H.D et al., 2011). Many biosensors and bioseparation protocols were also demonstrated using such multifunctional nanoparticles (Rossi et al., 2006; Liu and Xu, 1995; Fan et al., 2003). Figure 13 shows the example of functional nanoparticles. Silica nanoparticles can be linked to functional groups like carboxylic, thiol, amine or hydroxide, in order to attach dye

Nanomedicine is the medical application of nanotechnology and related disciplines, which mostly include biocompatible nanoparticle platforms that contain both therapeutic and/or imaging components. Since biomolecules such as individual cells, mRNA, DNA and proteins are nanoscale sized, probes of equivalent dimensions can provide very effective detection of individual chemical interactions of biomolecules, understanding of the chemical reactions and manipulation of the same. Therefore, the integration of nanotechnology and medical sciences has led to fundamental understanding in molecular biology, as well as new advanced technological applications such as drug targeting and delivery, cell labeling and separation, cancer therapy, magnetic resonance image (MRI) contrast agents, bio-sensors and bio-imaging. Nanoparticles may carry chemo-, radio-, and gene therapeutics or combination of these. They can be inorganic nanoparticles (noble metal, metal oxide, silica, mesoporous silica and combination of these components), lipid aggregates, and synthetic surfactant-polymer systems (such as vesicles, micelles). Inorganic nanoparticles that have unique physicochemical properties allow applications in nanomedicine after proper synthesis, coating, surface functionalization and bioconjugation. These nanosized materials provide a robust framework in which two or more components can be incorporated to give multifunctional capabilities (Wang et al., 2008; Salgueiriño-Maceira & Correa-Duarte, 2007). The combination of metals or polymer molecules should provide suitable and tunable magnetic, optical and chemical properties, chemical stability, low toxicity, easy dispersibility, affinity towards biomolecules with amine/thiol/carboxylic terminal groups

Among the various compositions, gold composites are used for the development of various clinical diagnosis methods (Baptista et al., 2008; Raj et al., 2011) because of its size dependent optical properties. Time-resolved single-photon counting fluorescence studies on porphyrin monolayer-modified gold clusters revealed resonant energy transfer between the porphyrin and the gold surface, which is a phenomenon of considerable interest in biophotonics (Imahori & Fukuzumi, 2001). The ability to control the size and shape of gold nanoparticles and their surface conjugation with antibodies allow for both selective imaging and photothermal killing of cancer cells by using light with longer wavelengths for tissue penetration (Gobin et al., 2007). Similar success was also demonstrated with polymer-coated superparamagnetic iron oxide nanoparticles conjugated with, e.g., uorescent molecules, tumor-targeting moieties and anticancer drugs which aim targeting human cancers. Imaging inside the body can either be done using magnetic resonance or uorescence imaging (Kohler et al., 2006). Quantum dots optical properties including bright emission, photostability, size dependant luminescence and long fluorescence lifetimes make them also suitable for bioimaging applications. In combination with superparamagnetic nanoparticles and surface modification with peptides or other functional groups, these multifunctional particles are being used in bioimaging, bioseparation and in order to understand the behaviour of nanoparticles in cells such as tracking the particles, cell uptake of particles, drug dose evolution at targeted site (Janczewski et al., 2011; Summers H.D et al., 2011). Many biosensors and bioseparation protocols were also demonstrated using such multifunctional nanoparticles (Rossi et al., 2006; Liu and Xu, 1995; Fan et al., 2003). Figure 13 shows the example of functional nanoparticles. Silica nanoparticles can be linked to functional groups like carboxylic, thiol, amine or hydroxide, in order to attach dye

**4.2 Medical applications of bio-functionalized nanoparticles** 

and convenient preparation techniques.

molecules and therapeutic molecules to track particles inside the human body and targeted drug delivery. However, intense research studies are needed in order to overcome the barriers in the human body that challenge the efficiency of nanoparticle delivery such as walls of blood vessels, physical entrapment of particles in organs and removal of particles by phagocytic cells. Therefore, the ideal nanoparticle system to be used in nanomedicine should not only overcome such barriers but also allow for real time visualisation of particles, detection of the damaged tissues, selective and rapid accumulation at diseased tissue, effective drug delivery or effective therapy (in the case of hyperthermia). Hence, pioneering works are being carried out at related interdisciplinary fields such as chemistry, biology, pharmacy, nanotechnology, medicine and imaging.

Fig. 13. The surface of silica nanoparticles can be functionalized with different chemical groups which can be used to bind fluorophores (e.g. in this example the fluorophore AF 488 has been coupled to the SH groups introduced on the surface of bare silica nanoparticles), proteins or other molecules.

Nanoparticles based drug delivery is particularly advantageous when planning cancer therapy because the leaky blood cells in tumours mean that particles of certain size tend to accumulate more in cancer tissues than in normal tissues (Peer D et al, 2007). By coating nanoparticles with biomolecules that recognize receptors on cell membranes, a wide range of drugs and imaging agents can enter cells (endocytosis). Drug loaded magnetic particles can be localized to the specific site by an external magnetic field (Hu et al., 2008). This allows more concentrated doses of the anticancer drugs to be delivered to the cancer cells and keep them on site for longer periods of time. In order to prevent dangerous agglomeration of the particles in the blood stream, the particles must be of a small size relative to the dimensions of the capillaries, monodisperse and spherical in shape. In addition, the particles must have a high magnetic moment and switch their magnetisation quickly at low elds (Ankamwar et al., 2010; Shah et al., 2011). Nanoparticles surface modified with amphiphilic polymeric surfactants such as poloxamers, poloxamines or polyethylene glycol derivatives (S.-M. Lee et al., 2010), folic acid derivatives (Das et al, 2008; Landmark et al., 2008), silica derivatized with different functional groups or with porous structure (Wang et al., 2008; Slowing et al., 2007) are used for drug targeting and delivery. Moreover, triggerable drug delivery systems enable on-demand controlled release of drugs that may enhance therapeutic effectiveness and reduce systemic toxicity. Recently, a number of new materials have been developed that exhibit, e.g., sensitivity to UV and visible light, such that irradiation can release covalently bound drugs from dendrimers or dendrons with photocleavable cores or photoactivated surfaces (C. Park et al., 2008), temperature (Bikram et al., 2007; Peng et al., 2011), nearinfrared light (S.-M.Lee et al., 2010), pH (Benarjee & Chen, 2008), ultrasound (Bawa et al.,

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 145

The magnetic properties and self-organization of magnetic nanoparticles has to be understood in order to use these particles for biological applications. Extensive studies have been carried out in this direction. Superparamagnetic nanoparticles consisting of singledomain magnetite nanoparticles are randomly dispersed in liquid in the absence of external magnetic field. Under an external magnetic eld, these particles are assembled to form chain-like structures along the magnetic eld lines. An attractive magnetic force due to the magnetic dipole is balanced by repulsive electrostatic and solvation forces. Upon removal of the external magnetic field, thermal energy dominates and the particles disintegrate (Fig. 14). A wide range of studies were carried out for quantitative investigation of the temporal self-organization of superparamagnetic composite particles in the presence of an external magnetic eld. The kinetics of eld-induced self-organization into linear chains, timedependent chain-size distribution, resolved growth steps condensation, polarization, colinearity, and concatenation, the average chain growth rate, and inter-particle interaction length were calculated in the presence of external magnetic elds (Gajula et al., 2010). These studies give us valuable information relevant to hyperthermia treatment, MRI contrast agents, bioseparation, drug targeting studies and is relevant for making 2D and 3D biocompatible structures for tissue engineering. Moreover, the LAMI technique will enhance the nanomedical applications by creating strong, covalent bond interactions between the

Fig. 14. Bright field microscope images of superparamagnetic nanoparticles in the (left) presence and (right) absence of an external magnetic field (2s after field removal).

Most tissues in organisms are composed of repeating basic cellular structures that are embedded in an extracellular 3D matrix. Tissue functionality arises from these components and the relative spatial locations of these components (Nichol & Khademhosseini, 2009). Tissue engineering enables to recreate the native 3D architecture *in vitro*. Artificial biocompatible 3D structures enable researchers to create organs for transplantation and to understand the structure/function relationship, to characterize cell-membrane mechanical properties, enables the theoretical analyses and to model cellular events and diseases. Xu and co-workers fabricated magnetic nanoparticles loaded cell-encapsulating microscale hydrogels and assembled these gels into 3D multilayer constructs using magnetic fields (Xu et al., 2011). A three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage has been exploited for direct cell manipulation and cell sorting. By spatially controlling the magnetic field, the geometry of the cell mass is manipulated (Souza et al., 2010). Ito and co-workers have demonstrated the successful construction and

2s

**4.3 Self-organization of magnetic nanoparticles** 

nanoparticles and therapeutical biomolecules.

2009), or external magnetic elds (Hu et al., 2008). Long-wavelength light such as radio frequency radiation and microwave radiation have also been used to trigger drug release. This responsiveness can be triggered remotely to provide exible control of dose magnitude and timing. Mann et al. (Mann et al., 2011) demonstrated enhanced delivery of therapeutic liposomes carried in E-selectin thioaptamer conjugated porous silica particles to the bone marrow tissue. These particles can also be utilised to deliver imaging agents and growth factors (eg. colony stimulating factor) for the protection of bone marrow against chemotherapy and radiation.

The use of biocompatible iron oxide particles for hyperthermia is increasing in cancer therapy. Iron oxide magnetic nanoparticles exposed to an alternating magnetic eld act as localized heat sources at certain target regions inside the human body. The heating of magnetic oxide particles with low electrical conductivity in an external alternating magnetic eld is mainly due to either loss processes during the reversal of coupled spins within the particles or due to frictional losses if the particles rotate in an environment of appropriate viscosity. Magnetic nanoparticles coated with amphipathic polymer pullulan acetate (food additive) were examined for their cytotoxicity and cellular uptake. Moreover, in vitro hyperthermia treatment of KB cells produced therapeutic efficacies of 56% and 78% at 45º C and 47ºC, respectively, indicating the great potential of surface modified magnetic nanoparticles as magnetic hyperthermia mediators (Gao et al., 2010). Gonzalez-Fernandez and co-workers have presented a study on the magnetic properties of bare and silica-coated ferrite nanoparticles with sizes between 5 and 110 nm (Gonzalez-Fernandez et al., 2009). Their results show a strong dependence of the power absorption with particle size, with a maximum around 30 nm, as expected for a Neel relaxation mechanism in single-domain particles. Recently, in order to enhance the heat conversion capacity of nanoparticles from electromagnetic energy into heat, core-shell nanoparticles were designed to exploit the advantage of exchange coupling between a magnetically hard core and magnetically soft shell to maximize the specific loss power (J.H. Lee et al., 2011). However, in order to avoid the risk of overheating during the hyperthermia effect, curie temperature tuning is done by designing the composition of the core magnetic nanoparticles (Kaman et al., 2009).

MRI is one of the most useful diagnostic tools for medical sciences. MRI contrast agents are chemical substances introduced to the region being imaged to increase the contrast between different tissues or between normal and abnormal tissue, by altering the relaxation times. Generally, gadolinium or manganese salts as well as superparamagnetic iron-oxide based particles are by far the most commonly used materials as MRI contrast agents. Superparamagnetic iron oxide based contrast agents have the advantage of producing an enhanced proton relaxation in MRI better than those produced by paramagnetic ions. Consequently, lower doses are needed which reduce to a great extent the secondary effects in the human body. Particle's negligible remanence after removing the magnetic field (minimizes the particles aggregation) and low toxicity makes them beneficial for in vivo applications (Kinsella et al., 2011; Y.Park et al., 2009).

Various biosensors were developed exploiting novel properties of nanomaterials. Rossi and co-workers have shown the utility of uorescent nanospheres to detect the breast cancer marker HER2/neu in a glass slide based assay (Rossi et al., 2006). For the detection of cholesterol, plasmon resonance properties of gold nanoparticles were used after conjugating digitonin onto the surface of gold nanoparticles (Raj et al., 2011).

### **4.3 Self-organization of magnetic nanoparticles**

144 Molecular Photochemistry – Various Aspects

2009), or external magnetic elds (Hu et al., 2008). Long-wavelength light such as radio frequency radiation and microwave radiation have also been used to trigger drug release. This responsiveness can be triggered remotely to provide exible control of dose magnitude and timing. Mann et al. (Mann et al., 2011) demonstrated enhanced delivery of therapeutic liposomes carried in E-selectin thioaptamer conjugated porous silica particles to the bone marrow tissue. These particles can also be utilised to deliver imaging agents and growth factors (eg. colony stimulating factor) for the protection of bone marrow against

The use of biocompatible iron oxide particles for hyperthermia is increasing in cancer therapy. Iron oxide magnetic nanoparticles exposed to an alternating magnetic eld act as localized heat sources at certain target regions inside the human body. The heating of magnetic oxide particles with low electrical conductivity in an external alternating magnetic eld is mainly due to either loss processes during the reversal of coupled spins within the particles or due to frictional losses if the particles rotate in an environment of appropriate viscosity. Magnetic nanoparticles coated with amphipathic polymer pullulan acetate (food additive) were examined for their cytotoxicity and cellular uptake. Moreover, in vitro hyperthermia treatment of KB cells produced therapeutic efficacies of 56% and 78% at 45º

and 47ºC, respectively, indicating the great potential of surface modified magnetic nanoparticles as magnetic hyperthermia mediators (Gao et al., 2010). Gonzalez-Fernandez and co-workers have presented a study on the magnetic properties of bare and silica-coated ferrite nanoparticles with sizes between 5 and 110 nm (Gonzalez-Fernandez et al., 2009). Their results show a strong dependence of the power absorption with particle size, with a maximum around 30 nm, as expected for a Neel relaxation mechanism in single-domain particles. Recently, in order to enhance the heat conversion capacity of nanoparticles from electromagnetic energy into heat, core-shell nanoparticles were designed to exploit the advantage of exchange coupling between a magnetically hard core and magnetically soft shell to maximize the specific loss power (J.H. Lee et al., 2011). However, in order to avoid the risk of overheating during the hyperthermia effect, curie temperature tuning is done by

designing the composition of the core magnetic nanoparticles (Kaman et al., 2009).

applications (Kinsella et al., 2011; Y.Park et al., 2009).

digitonin onto the surface of gold nanoparticles (Raj et al., 2011).

MRI is one of the most useful diagnostic tools for medical sciences. MRI contrast agents are chemical substances introduced to the region being imaged to increase the contrast between different tissues or between normal and abnormal tissue, by altering the relaxation times. Generally, gadolinium or manganese salts as well as superparamagnetic iron-oxide based particles are by far the most commonly used materials as MRI contrast agents. Superparamagnetic iron oxide based contrast agents have the advantage of producing an enhanced proton relaxation in MRI better than those produced by paramagnetic ions. Consequently, lower doses are needed which reduce to a great extent the secondary effects in the human body. Particle's negligible remanence after removing the magnetic field (minimizes the particles aggregation) and low toxicity makes them beneficial for in vivo

Various biosensors were developed exploiting novel properties of nanomaterials. Rossi and co-workers have shown the utility of uorescent nanospheres to detect the breast cancer marker HER2/neu in a glass slide based assay (Rossi et al., 2006). For the detection of cholesterol, plasmon resonance properties of gold nanoparticles were used after conjugating

C

chemotherapy and radiation.

The magnetic properties and self-organization of magnetic nanoparticles has to be understood in order to use these particles for biological applications. Extensive studies have been carried out in this direction. Superparamagnetic nanoparticles consisting of singledomain magnetite nanoparticles are randomly dispersed in liquid in the absence of external magnetic field. Under an external magnetic eld, these particles are assembled to form chain-like structures along the magnetic eld lines. An attractive magnetic force due to the magnetic dipole is balanced by repulsive electrostatic and solvation forces. Upon removal of the external magnetic field, thermal energy dominates and the particles disintegrate (Fig. 14). A wide range of studies were carried out for quantitative investigation of the temporal self-organization of superparamagnetic composite particles in the presence of an external magnetic eld. The kinetics of eld-induced self-organization into linear chains, timedependent chain-size distribution, resolved growth steps condensation, polarization, colinearity, and concatenation, the average chain growth rate, and inter-particle interaction length were calculated in the presence of external magnetic elds (Gajula et al., 2010). These studies give us valuable information relevant to hyperthermia treatment, MRI contrast agents, bioseparation, drug targeting studies and is relevant for making 2D and 3D biocompatible structures for tissue engineering. Moreover, the LAMI technique will enhance the nanomedical applications by creating strong, covalent bond interactions between the nanoparticles and therapeutical biomolecules.

Fig. 14. Bright field microscope images of superparamagnetic nanoparticles in the (left) presence and (right) absence of an external magnetic field (2s after field removal).

Most tissues in organisms are composed of repeating basic cellular structures that are embedded in an extracellular 3D matrix. Tissue functionality arises from these components and the relative spatial locations of these components (Nichol & Khademhosseini, 2009). Tissue engineering enables to recreate the native 3D architecture *in vitro*. Artificial biocompatible 3D structures enable researchers to create organs for transplantation and to understand the structure/function relationship, to characterize cell-membrane mechanical properties, enables the theoretical analyses and to model cellular events and diseases. Xu and co-workers fabricated magnetic nanoparticles loaded cell-encapsulating microscale hydrogels and assembled these gels into 3D multilayer constructs using magnetic fields (Xu et al., 2011). A three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage has been exploited for direct cell manipulation and cell sorting. By spatially controlling the magnetic field, the geometry of the cell mass is manipulated (Souza et al., 2010). Ito and co-workers have demonstrated the successful construction and

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 147

therapy resistance and poor outcome. Therefore, inhibition of EGFR function is a rational

Fig. 15. Overview of the cellular pathways affected by the laser-pulsed UV illumination of the EGF (epidermal growth factor) receptor leading to attenuation of the EGFR signalling cascade. Photoactivation of aromatic residues within the extracellular domain of the EGF receptor, leads to disruption of nearby disulphide bridges. This prevents the ligand, e.g. EGF, from binding to the receptor and activating the EGFR pathways. In addition, it is possible that laser-pulsed UV illumination targets the intracellular domain of the EGF receptor causing photodegradation of phosphorylation-targeted tyrosine residues again

Bioinformatics studies show that the extracellular domain of human HER3 (ErbB3), a member of the epidermal growth factor receptor (EGFR) family, is exceedingly rich in SS bridges and aromatic residues (Petersen et al, 2008, Fig. 15). The structure of the extracellular domain consists of four domains. Tethered domains II and IV are displayed. A total of 22 disulphide bridges can be seen in one domain and 25 in the other domain. On the other hand, biophysical studies show that UV illumination of aromatic residues nearby disulphide bridges leads to the SS disruption (Neves-Petersen et al, 2002, Fig. 1). Therefore, it is likely that we induce structural changes in the 3D structure of EGFR upon prolonged UV excitation. If the UV light fluency (power per unit area) is above a certain threshold it is likely that 3D structural changes occur that will prevent the correct binding to, e.g., EGF. We have shown that relatively low intensity UV light (0.273 mW 200fs femtosecond pulses at 280nm diffused onto a petri dish area) can be used to induce cancer cell death (apoptosis) in skin cancer cells in culture (Olsen B.B. et al., 2007, Petersen S.B. et al., 2008). We have also shown that most likely the epidermal growth factor receptor in cancer cell membranes upon

preventing the proteins from binding to the phosphorylated tyrosine residues.

treatment approach.

delivery of human retinal pigment epithelial cell sheets using magnetic nanoparticles (Ito et al., 2005). Biomimetic nanopatterns can also be explored for analysis and control of live cells growth (Kim et al., 2010). Richert and co-workers observed the control over the growth of the osteogenic cells, fibroblastic cells and smooth muscle cells using nano-porous titanium alloy explaining that nanotopography may modulate cytoskeletal organization and membrane receptor organization (Richert et al., 2008). This work indicates the impact of nanoscale engineering in controlling cell-material interfaces, which can have profound implications for the development of tissue engineering and regeneration medicine.

Structural colour originates from physical configurations of materials e.g. upon light interaction the lattice spacing of the melanin rods generate various colours in the feathers of a peacock. Structural colour is free from photobleaching, unlike traditional pigments or dyes. Owing to its unique characteristics, there have been many attempts to make articial structural colour through various technological approaches such as colloidal crystallization, dielectric layer stacking and direct lithographic patterning. However, these techniques are time consuming, cost, and great effort is needed to produce multicoloured patterns on a substrate. Using the self-organization of nanoparticles, external magnetic field tunable bandgaps can be created on various substrates within a few seconds (Philip et al., 2003; H. Kim et al., 2009). During the chaining process under external magnetic field, the combination of attractive and repulsive forces determines the interparticle distance, and the interparticle distance in a chain determines the colour of the light diffracted from the chain, which can be explained by Bragg diffraction theory. Thus, the colour can be tuned by simply varying the interparticle distance using external magnetic elds. The spacing between the particles is sensitive towards charges over the particle's surface. This property can be used to develop biosensors. For example, the specific molecular recognition of cholesterol by digitonin on nanoparticles may result in a reduction of the inter-particle spacing due to the enhanced hydrophobicity of the surface after cholesterol binding. Therefore, the light diffracted from the chain varies indicating the adsorption of cholesterol onto the nanoparticles surface. Cassagneau et al., have shown that the reversible colour tuning of a colloidal crystal could be potentially adopted for biosensing. Biospecific binding of avidin with biotin is demonstrated by monitoring changes in the bandgap spectral peak position caused by Bragg-diffraction of electromagnetic waves within the structure **(**Cassagneau & Caruso, 2002). The same chaining technique is explored for the DNA separation (Doyle et al., 2002).

### **5. Photonic cancer therapy**

The epidermal growth factor receptor (EGFR), also known as HER1/Erb-B1, belongs to the ErbB family of receptor tyrosine kinases (RTKs) (Riese & Stern, 1998; Yarden & Sliwkowski, 2001; Olayioye et al., 2000). Binding of ligands such as EGF and TGF, leads to homo- and heterodimerization of the receptors (Olayioye et al., 20003). Dimerization in the case of EGFR leads to autophosphorylation of specific tyrosine residues in the intracellular tyrosine kinase domain. EGFR activation results in cell signaling cascades that promote tumor cell proliferation, survival and inhibits apoptosis (Fig. 14). EGFR is expressed or highly expressed in non-small-cell lung cancer (NSCLC) and in a variety of common solid tumors, and has also been associated with poor prognosis. High EGFR expression is generally associated with invasion, metastasis, late-stage disease, chemotherapy resistance, hormonal

delivery of human retinal pigment epithelial cell sheets using magnetic nanoparticles (Ito et al., 2005). Biomimetic nanopatterns can also be explored for analysis and control of live cells growth (Kim et al., 2010). Richert and co-workers observed the control over the growth of the osteogenic cells, fibroblastic cells and smooth muscle cells using nano-porous titanium alloy explaining that nanotopography may modulate cytoskeletal organization and membrane receptor organization (Richert et al., 2008). This work indicates the impact of nanoscale engineering in controlling cell-material interfaces, which can have profound

Structural colour originates from physical configurations of materials e.g. upon light interaction the lattice spacing of the melanin rods generate various colours in the feathers of a peacock. Structural colour is free from photobleaching, unlike traditional pigments or dyes. Owing to its unique characteristics, there have been many attempts to make articial structural colour through various technological approaches such as colloidal crystallization, dielectric layer stacking and direct lithographic patterning. However, these techniques are time consuming, cost, and great effort is needed to produce multicoloured patterns on a substrate. Using the self-organization of nanoparticles, external magnetic field tunable bandgaps can be created on various substrates within a few seconds (Philip et al., 2003; H. Kim et al., 2009). During the chaining process under external magnetic field, the combination of attractive and repulsive forces determines the interparticle distance, and the interparticle distance in a chain determines the colour of the light diffracted from the chain, which can be explained by Bragg diffraction theory. Thus, the colour can be tuned by simply varying the interparticle distance using external magnetic elds. The spacing between the particles is sensitive towards charges over the particle's surface. This property can be used to develop biosensors. For example, the specific molecular recognition of cholesterol by digitonin on nanoparticles may result in a reduction of the inter-particle spacing due to the enhanced hydrophobicity of the surface after cholesterol binding. Therefore, the light diffracted from the chain varies indicating the adsorption of cholesterol onto the nanoparticles surface. Cassagneau et al., have shown that the reversible colour tuning of a colloidal crystal could be potentially adopted for biosensing. Biospecific binding of avidin with biotin is demonstrated by monitoring changes in the bandgap spectral peak position caused by Bragg-diffraction of electromagnetic waves within the structure **(**Cassagneau & Caruso, 2002). The same

implications for the development of tissue engineering and regeneration medicine.

chaining technique is explored for the DNA separation (Doyle et al., 2002).

The epidermal growth factor receptor (EGFR), also known as HER1/Erb-B1, belongs to the ErbB family of receptor tyrosine kinases (RTKs) (Riese & Stern, 1998; Yarden & Sliwkowski, 2001; Olayioye et al., 2000). Binding of ligands such as EGF and TGF, leads to homo- and heterodimerization of the receptors (Olayioye et al., 20003). Dimerization in the case of EGFR leads to autophosphorylation of specific tyrosine residues in the intracellular tyrosine kinase domain. EGFR activation results in cell signaling cascades that promote tumor cell proliferation, survival and inhibits apoptosis (Fig. 14). EGFR is expressed or highly expressed in non-small-cell lung cancer (NSCLC) and in a variety of common solid tumors, and has also been associated with poor prognosis. High EGFR expression is generally associated with invasion, metastasis, late-stage disease, chemotherapy resistance, hormonal

**5. Photonic cancer therapy** 

therapy resistance and poor outcome. Therefore, inhibition of EGFR function is a rational treatment approach.

Fig. 15. Overview of the cellular pathways affected by the laser-pulsed UV illumination of the EGF (epidermal growth factor) receptor leading to attenuation of the EGFR signalling cascade. Photoactivation of aromatic residues within the extracellular domain of the EGF receptor, leads to disruption of nearby disulphide bridges. This prevents the ligand, e.g. EGF, from binding to the receptor and activating the EGFR pathways. In addition, it is possible that laser-pulsed UV illumination targets the intracellular domain of the EGF receptor causing photodegradation of phosphorylation-targeted tyrosine residues again preventing the proteins from binding to the phosphorylated tyrosine residues.

Bioinformatics studies show that the extracellular domain of human HER3 (ErbB3), a member of the epidermal growth factor receptor (EGFR) family, is exceedingly rich in SS bridges and aromatic residues (Petersen et al, 2008, Fig. 15). The structure of the extracellular domain consists of four domains. Tethered domains II and IV are displayed. A total of 22 disulphide bridges can be seen in one domain and 25 in the other domain. On the other hand, biophysical studies show that UV illumination of aromatic residues nearby disulphide bridges leads to the SS disruption (Neves-Petersen et al, 2002, Fig. 1). Therefore, it is likely that we induce structural changes in the 3D structure of EGFR upon prolonged UV excitation. If the UV light fluency (power per unit area) is above a certain threshold it is likely that 3D structural changes occur that will prevent the correct binding to, e.g., EGF. We have shown that relatively low intensity UV light (0.273 mW 200fs femtosecond pulses at 280nm diffused onto a petri dish area) can be used to induce cancer cell death (apoptosis) in skin cancer cells in culture (Olsen B.B. et al., 2007, Petersen S.B. et al., 2008). We have also shown that most likely the epidermal growth factor receptor in cancer cell membranes upon

UV Light Effects on Proteins: From Photochemistry to Nanomedicine 149

(PDT) which requires the use of a photosensitizer molecule that upon excitation and interaction to molecular oxygen leads to the formation of singlet oxygen, which kills the cells. The new photonic cancer therapy does not require the use of photosensitizer molecules. UV light induced 3D structural changes in the EGFR protein prevents binding/activation by EGF, halting this way the phosphorylation of the intracellular domain of EGFR and stopping metabolic pathways that lead to cancer proliferation. The new photonic cancer therapy can be used in combination to PDT and other cancer therapies.

Several reports describe how UV light can activate the EGF receptor hence activating the AKT and MAPK pathway (Warmuth et al., 1994; Coffer PJ at al., 1995; Huang et al., 1996; Katiyar, 2001; Wan et al., 2001; Iordanov et al., 2002; Matsumura & Ananthaswamy, 2004; El-Abaseri at al., 2006). Our observations seem to point at another effect of UV light, in apparent contrast with those results. The reason for this discrepancy could be found in the illumination power per unit of illuminated area (fluency). In our experiments the total integrated power over a second is significantly less than the average solar UV output but comparing the actual output during a pulse event we have 1000-fold higher intensity during

Our work on the interplay between the protein molecule and UV light has resulted in new basic science insights. It has also led to the development of a new protein covalent immobilization technique. The new photonic technology has been used successfully to design and engineer drug delivery systems and biosensors at the micro and nanoscale relevant to nanomedicine. The new engineering principle is made possible due to the presence of a conserved structural motif in proteins conserved by nature throughout

As a surprising spin-off, our work has resulted in new knowledge concerning how UV light can stop skin cancer. Pulsed UV illumination can halt activation of cancer cell membrane receptors and thereby stop all downstream reactions that would lead to cancer, shutting down the cells' biological functions. Moreover, this new treatment activated the cell's own cell death program (apoptosis). In particular we have realized that UV light chemically modifies the same receptor protein that many cancer therapeutic treatments are trying to target chemically. We believe that this holds promise for a totally new approach to treat some types of localized cancer. We will strive to develop an in-depth understanding of how and why nanometer-sized protein structures respond to light

Maria Teresa Neves-Petersen acknowledges the leave of absence granted by AAU.

Andolfi, L.; Cannistraro, S.; Canters, G.W.; Facci, P.; Ficca, A.G.; Amsterdam, I.M.C.V. &

Verbeet, M.Ph. (2002). A poplar plastocyanin mutant suitable for adsorption onto

the 200 femtosecond long pulse event (Olsen et al., 2007, Petersen et al., 2008).

**6. Conclusion** 

evolution.

exposure.

**7. Acknowledgment** 

**8. References** 

absorption of the UV photons changes its molecular structure as a consequence. The net result is that phosphorylation and cell signaling is abolished, which in turn leads to apoptosis. The technology may lead to an important new modality in the treatment of various cancers. Pulsed UV illumination can halt activation of cancer cell membrane receptors and thereby all downstream reactions that would lead to cancer, shutting down the cells' biological functions. Moreover, this new treatment activated the cell's own cell death program. This has been documented on two human epidermal cancer cell lines (Olsen B.B. et al., 2007). The photonic dosage necessary for therapeutical results has additionally been determined.

Fig. 16. Molecular structure of the intracellular and extracellular domains of human HER3 (ErbB3), a member of the epidermal growth factor receptor (EGFR) family. Tethered extracellular domains II and IV are displayed. The pdb code of the extracellular domain is 1m6b.pdb. The extracellular domain is extremely rich in disulphide bridges. Tethered extracellular domains II and IV are displayed. A total of 22 disulphide bridges can be seen in each of the displayed domains. The intracellular domain is a protein tyrosine kinase. In the extracellular domain, disulphide bridges are depicted as black sticks, the tryptophan amino acids as rendered grey CPK models, and the tyrosine and phenylalanine residues are depicted as white CPK models. As expected, no disulphide bridges are observed in the intracellular domain due to the reduction environment of cells. However, this domain is also rich in aromatic residues.

This technology is applicable to the treatment of various forms of cancer. Using optical fibers it is possible to illuminate localized areas in any region of the human body. Therefore, both external and internal tumors can be treated. This method may also offer a better treatment of a surgical wound cavity prior to its closure in order to prevent cancer reappearance. This new photonic method differs from the classical photodynamic therapy (PDT) which requires the use of a photosensitizer molecule that upon excitation and interaction to molecular oxygen leads to the formation of singlet oxygen, which kills the cells. The new photonic cancer therapy does not require the use of photosensitizer molecules. UV light induced 3D structural changes in the EGFR protein prevents binding/activation by EGF, halting this way the phosphorylation of the intracellular domain of EGFR and stopping metabolic pathways that lead to cancer proliferation. The new photonic cancer therapy can be used in combination to PDT and other cancer therapies.

Several reports describe how UV light can activate the EGF receptor hence activating the AKT and MAPK pathway (Warmuth et al., 1994; Coffer PJ at al., 1995; Huang et al., 1996; Katiyar, 2001; Wan et al., 2001; Iordanov et al., 2002; Matsumura & Ananthaswamy, 2004; El-Abaseri at al., 2006). Our observations seem to point at another effect of UV light, in apparent contrast with those results. The reason for this discrepancy could be found in the illumination power per unit of illuminated area (fluency). In our experiments the total integrated power over a second is significantly less than the average solar UV output but comparing the actual output during a pulse event we have 1000-fold higher intensity during the 200 femtosecond long pulse event (Olsen et al., 2007, Petersen et al., 2008).

### **6. Conclusion**

148 Molecular Photochemistry – Various Aspects

absorption of the UV photons changes its molecular structure as a consequence. The net result is that phosphorylation and cell signaling is abolished, which in turn leads to apoptosis. The technology may lead to an important new modality in the treatment of various cancers. Pulsed UV illumination can halt activation of cancer cell membrane receptors and thereby all downstream reactions that would lead to cancer, shutting down the cells' biological functions. Moreover, this new treatment activated the cell's own cell death program. This has been documented on two human epidermal cancer cell lines (Olsen B.B. et al., 2007). The photonic dosage necessary for therapeutical results has additionally

**Extracellular domain** 

Fig. 16. Molecular structure of the intracellular and extracellular domains of human HER3 (ErbB3), a member of the epidermal growth factor receptor (EGFR) family. Tethered extracellular domains II and IV are displayed. The pdb code of the extracellular domain is 1m6b.pdb. The extracellular domain is extremely rich in disulphide bridges. Tethered extracellular domains II and IV are displayed. A total of 22 disulphide bridges can be seen in each of the displayed domains. The intracellular domain is a protein tyrosine kinase. In the extracellular domain, disulphide bridges are depicted as black sticks, the tryptophan amino acids as rendered grey CPK models, and the tyrosine and phenylalanine residues are depicted as white CPK models. As expected, no disulphide bridges are observed in the intracellular domain due to the reduction environment of cells. However, this domain is also

This technology is applicable to the treatment of various forms of cancer. Using optical fibers it is possible to illuminate localized areas in any region of the human body. Therefore, both external and internal tumors can be treated. This method may also offer a better treatment of a surgical wound cavity prior to its closure in order to prevent cancer reappearance. This new photonic method differs from the classical photodynamic therapy

been determined.

**Intracellular domain** 

rich in aromatic residues.

Our work on the interplay between the protein molecule and UV light has resulted in new basic science insights. It has also led to the development of a new protein covalent immobilization technique. The new photonic technology has been used successfully to design and engineer drug delivery systems and biosensors at the micro and nanoscale relevant to nanomedicine. The new engineering principle is made possible due to the presence of a conserved structural motif in proteins conserved by nature throughout evolution.

As a surprising spin-off, our work has resulted in new knowledge concerning how UV light can stop skin cancer. Pulsed UV illumination can halt activation of cancer cell membrane receptors and thereby stop all downstream reactions that would lead to cancer, shutting down the cells' biological functions. Moreover, this new treatment activated the cell's own cell death program (apoptosis). In particular we have realized that UV light chemically modifies the same receptor protein that many cancer therapeutic treatments are trying to target chemically. We believe that this holds promise for a totally new approach to treat some types of localized cancer. We will strive to develop an in-depth understanding of how and why nanometer-sized protein structures respond to light exposure.

### **7. Acknowledgment**

Maria Teresa Neves-Petersen acknowledges the leave of absence granted by AAU.

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**Part 4** 

**Computational Aspects of Photochemistry** 

