**Optical Chemical Sensors: Design and Applications**

Aleksandra Lobnik1, Matejka Turel1 and Špela Korent Urek2 *1Universitiy of Maribor, Faculty of Mechanical Engineering, 2Institute for Environmental Protection and Sensors, Slovenia* 

## **1. Introduction**

Optical sensors, or opt(r)odes, represent a group of chemical sensors in which electromagnetic (EM) radiation is used to generate the analytical signal in a transduction element. The interaction of this radiation with the sample is evaluated from the change of a particular optical parameter and is related to the concentration of the analyte (Blum, 1997). Typically, an optical chemical sensor consists of a chemical recognition phase (sensing element or receptor) coupled with a transduction element (Fig. 1). The receptor identifies a parameter, e.g., the concentration of a given compound, pH, etc., and provides an optical signal proportional to the magnitude of this parameter. The function of the receptor is fulfilled in many cases by a thin layer that is able to interact with the analyte molecules, catalyse a reaction selectively, or participate in a chemical equilibrium together with the analyte. The transducer translates the optical signal produced by the receptor into a measurable signal that is suitable for processing by amplification, filtering, recording, display, etc. (Gründler, 2007; Nagl & Wolfbeis, 2008).

Sensors that have a receptor part based on a biochemical principle are usually called biosensors. The selectivity and sensitivity provided by Nature have been utilized in such sensors, frequently by immobilizing the biologically active compounds, such as enzymes and immunoglobulins, within a receptor part of the sensor (Patel et al., 2010). An effective way of obtaining the biological selectivity is a combination of cell cultures, tissue slices, organs and sometimes of whole living organisms with the transducer.

Optical sensors can be based on various optical principles (absorbance, reflectance, luminescence, fluorescence), covering different regions of the spectra (UV, Visible, IR, NIR) and allowing the measurement not only of the intensity of light, but also of other related properties, such as lifetime, refractive index, scattering, diffraction and polarization (Jerónimo et al., 2007). As an example, a luminescent sensor can be constructed by associating a sensing element, which emits light when in contact with a specific analyte, with a photodiode, which converts the energy of the incident light into a measurable signal.

Optical chemical sensors have numerous advantages over conventional electricity-based sensors, such as selectivity, immunity to electromagnetic interference, and safety while working with flammable and explosive compounds. They are also sensitive, inexpensive, non-destructive, and have many capabilities. Optrodes do not require a reference cell, as is

Optical Chemical Sensors: Design and Applications 5

For sensor applications only part of spectroscopic wavelength range is useful. From the practical point of view the following ways (Fig. 2) in which radiation can interact with an

**A** Sample

**<sup>D</sup>** λ-wave-

Fig. 2. General arrangement of spectroscopic measurements: A – light reflection, B – light

length selector

However, the most commonly applied methods in optical sensing are those based on light absorption or light emission. Compared to absorption-based methods, molecular emission (fluorescence, phosphorescence, and generally speaking, luminescence) is particularly important because of its extreme sensitivity and good specificity. The sensitivity of luminescence methods is about 1000 times greater than that of most spectrophotometric methods. In addition, lower limits of detection for the desired analytes can be achieved (Guilbault, 1990; Schulman, 1988; Wolfbeis, 2005). Measuring the emission intensity is also the most popular because the instrumentation needed is very simple and cheap. Nevertheless, measuring the light emission intensity has some disadvantages compared to emission lifetime measurements, in which the sample is excited only by a pulse of EM rather than via continuous illumination, which is the case with intensity-based methods. The precision and accuracy of luminescence intensity-based schemes are greatly affected by fluctuations in the light-source's intensity, detector sensitivity, inner filter effects, indicator concentration (bleaching and leaching), sample turbidity, and sensing layer thickness. However, some of these problems can be minimized or even overcome by measuring luminescence lifetimes instead of intensities. But again, lifetime measurements also have some drawbacks, which are the instrumentation complexity and high costs, along with a limited number of indicator dyes available that show significant analyte-dependent changes

Detector

**B**

**C**

Another way to reduce the problems associated with intensity as well as with lifetime detection principles is the use of ratiometric measurements. This technique employs dualemission or dual-excitation indicators or mixtures of two luminophores, exhibiting separated spectral areas with different behaviour. For example, the ratio of two fluorescent peaks is used instead of the absolute intensity of one peak. The sensors therefore typically contain a reference dye; the advantage of this approach is that factors such as excitation source fluctuations and sensor concentration will not affect the ratio between the

**1.1 Optical detection principles** 

• reflexion and refraction

• absorption

analytical sample are the most useful (Gründler, 2007):

• emission (fluorescence or phosphorescence)

Light

source

refraction, C – light absorption, D – light emission.

in the lifetime (Lippittsch & Draxler, 1993).

the case in potentiometry. Furthermore, they can easily be miniaturized and allow multiple analyses with a single control instrument at a central site (Lukowiak & Strek, 2009).

Fig. 1. Schematic representation of the composition and function of an optical chemical sensor

However, besides a number of advantages, optical sensors also exhibit disadvantages: ambient light can interfere with their operation, the long-term stability is limited due to indicator leaching or photobleaching, there may be a limited dynamic range, selectivity may be poor, and a mass transfer of the analyte from the sample into the indicator phase is necessary in order to obtain an analytical signal (Seitz, 1988).

Fiber-optic chemical sensors (FOCSs) represent a subclass of chemical sensors in which an optical fiber is commonly employed to transmit the electromagnetic radiation to and from a sensing region that is in direct contact with the sample. The spectroscopically detectable optical property can be measured through the fiber optic arrangement, which enables remote sensing. In addition to advantages in terms of cheapness, ease of miniaturization, obtaining safe, small, lightweight, compact and inexpensive sensing systems, a wide variety of sensor designs are possible (Jerónimo et al., 2007; Lukowiak & Strek, 2009; Seitz, 1988).

The most common classification of FOCs distinguishes between the intrinsic and extrinsic types of sensors (Seitz, 1988; Wolfbeis, 2008).


#### **1.1 Optical detection principles**

For sensor applications only part of spectroscopic wavelength range is useful. From the practical point of view the following ways (Fig. 2) in which radiation can interact with an analytical sample are the most useful (Gründler, 2007):

• absorption

4 Advances in Chemical Sensors

the case in potentiometry. Furthermore, they can easily be miniaturized and allow multiple

**MEASURABLE SIGNAL**

**signal processing**

Fig. 1. Schematic representation of the composition and function of an optical chemical sensor

**TRANSDUCER**

However, besides a number of advantages, optical sensors also exhibit disadvantages: ambient light can interfere with their operation, the long-term stability is limited due to indicator leaching or photobleaching, there may be a limited dynamic range, selectivity may be poor, and a mass transfer of the analyte from the sample into the indicator phase is

Fiber-optic chemical sensors (FOCSs) represent a subclass of chemical sensors in which an optical fiber is commonly employed to transmit the electromagnetic radiation to and from a sensing region that is in direct contact with the sample. The spectroscopically detectable optical property can be measured through the fiber optic arrangement, which enables remote sensing. In addition to advantages in terms of cheapness, ease of miniaturization, obtaining safe, small, lightweight, compact and inexpensive sensing systems, a wide variety of sensor designs are possible (Jerónimo et al., 2007; Lukowiak & Strek, 2009; Seitz, 1988). The most common classification of FOCs distinguishes between the intrinsic and extrinsic

• In the intrinsic type of FOCs, the sensing principle is based on the change in lighttransmission characteristics due to the change occurring in a fiber property (e.g., refractive index or length) upon the interaction with the analyte or the system being studied. The optical fibre itself has sensory characteristics. This type of sensor is mainly applied to measure physical or physicochemical parameters, such as the pressure,

• In the extrinsic type of FOCs, the optical fiber acts as a transporting media by means of guiding the radiation from the source to the sample or from the sample to the detection system. Extrinsic sensors can be subdivided into a) distal and b) lateral types. The most common are distal-type sensors, in which the indicator is immobilized at the distal end (tip) of the optical fibre. Alternatively, in a lateral sensor, the sensing chemistry can be immobilized along a section of the core of the optical fibre to make an evanescent field

necessary in order to obtain an analytical signal (Seitz, 1988).

**RECEPTOR** (chemical recognition phase)

types of sensors (Seitz, 1988; Wolfbeis, 2008).

**A**

**SAMPLE A** analyte interferents

**A**

**A**

**membrane**

**A**

**A**

**A**

temperature, or enthalpy of reactions.

sensor.

analyses with a single control instrument at a central site (Lukowiak & Strek, 2009).

**<sup>A</sup> LIGHT optical**


Fig. 2. General arrangement of spectroscopic measurements: A – light reflection, B – light refraction, C – light absorption, D – light emission.

However, the most commonly applied methods in optical sensing are those based on light absorption or light emission. Compared to absorption-based methods, molecular emission (fluorescence, phosphorescence, and generally speaking, luminescence) is particularly important because of its extreme sensitivity and good specificity. The sensitivity of luminescence methods is about 1000 times greater than that of most spectrophotometric methods. In addition, lower limits of detection for the desired analytes can be achieved (Guilbault, 1990; Schulman, 1988; Wolfbeis, 2005). Measuring the emission intensity is also the most popular because the instrumentation needed is very simple and cheap. Nevertheless, measuring the light emission intensity has some disadvantages compared to emission lifetime measurements, in which the sample is excited only by a pulse of EM rather than via continuous illumination, which is the case with intensity-based methods. The precision and accuracy of luminescence intensity-based schemes are greatly affected by fluctuations in the light-source's intensity, detector sensitivity, inner filter effects, indicator concentration (bleaching and leaching), sample turbidity, and sensing layer thickness. However, some of these problems can be minimized or even overcome by measuring luminescence lifetimes instead of intensities. But again, lifetime measurements also have some drawbacks, which are the instrumentation complexity and high costs, along with a limited number of indicator dyes available that show significant analyte-dependent changes in the lifetime (Lippittsch & Draxler, 1993).

Another way to reduce the problems associated with intensity as well as with lifetime detection principles is the use of ratiometric measurements. This technique employs dualemission or dual-excitation indicators or mixtures of two luminophores, exhibiting separated spectral areas with different behaviour. For example, the ratio of two fluorescent peaks is used instead of the absolute intensity of one peak. The sensors therefore typically contain a reference dye; the advantage of this approach is that factors such as excitation source fluctuations and sensor concentration will not affect the ratio between the

Optical Chemical Sensors: Design and Applications 7

optical chemical sensing, such as colorimetric - based on light absorption, and luminescent based on light emission (Demchenko, 2009; Guilbault, 1990; Lobnik, 2006; Wolfbeis, 1991). However, the latter are of primary importance due to their high interdisciplinarity, great sensitivity, and applicability to different detection principles. A large number of fluorescent synthetic organic products are available nowadays so that a researcher can easily select the proper dye (indicator) corresponding to a particular sensing application in terms of spectroscopic properties and chemical reactivity. On the other hand, the basic organic chemistry also offers great potential as it enables synthesizing tailor-made indicators for

Colorimetric sensing is accomplished using an indicator that changes its colour upon binding the analyte; this change is usually spectroscopically determined, but it is also visibly observed. Among the great variety of organic chromophores, such as azo dyes, nitrophenols, phtaleins, sulfophtaleins, aniline-sulfophtaleins, triphenylmethane dyes, the most popular applications using these materials is to measure the pH parameter. The pKa of these indicators indicates the center of the measurable pH range, for example, cresol red, bromophenol blue, and bromocresol purple respond to acidic pH (pH < 7), while cresol red, naphtolbenzene, and phenolphthalein respond at basic pH (pH > 7) (Lobnik, 2006; Wolfbeis,

Redox indicators are the next example of colorimetric reagents. These are all organic dyestuffs, exhibiting reversible redox reactions. Examples include materials such as anilinic acid, diphenylamine, eriogreen, m-cresol-indophenol, methylene blue, nile blue, etc. (Wolfbeis, 1991; Lobnik, 2006). For example, a redox indicator Meldola blue can be incorporated into a sol-gel layer for an optical sensor measuring hydrogen peroxide in the

Ion sensing is possible using metal indicators that form coloured complexes with metal ions (Kaur & Kumar, 2011; Kim et al., 2009). The so-called ionophores are ligands that selectively bind ions. Chromogenic ionophores are designed to bring about a specific colour change in the interaction with metal cations (Murković Steinberg, 2003). Typical representatives are

Among the new nano-based materials, nanoparticles (NPs), the colorimetric ones are less common in comparison with the luminescent NPs. However, some examples have recently been reported where gold NPs for sensing Cd2+ (Ying et al., 2011), Fe3+ (Shu-Pao et al., 2011), Pb2+ (Nan et al., 2010), nitrite, nitrate ions were utilized (Weston et al., 2009), and carbon nanotubes were reported for sensing nucleic acids (Ai Cheng, 2007), and nanowires were

The analyte concentration is determined by the change in the emission properties of a luminophore. Luminescence is intrinsically more sensitive than absorption as a sensing technique, so for many applications the literature more often reports on sensing with luminescent probes and sensors. A variety of fluorescent and luminescent materials in the form of molecules, complexes and NPs are available for implementation as the response

concentration range of 10-8 to 10-1 mol/L (Lobnik & Čajlaković, 2001).

macrocyclic molecules with an ion-binding cavity, crown ether dyes, etc.

specific applications.

1991).

**2.1.1 Colorimetric indicators** 

used for Hg2+ (Tsao-Yen, 2011).

**2.1.2 Luminescent indicators** 

fluorescence intensities of the indicator and the reference dye (Arduini et al., 2007; Buck et al., 2004; Cywinski et al., 2009; Doussineau et al., 2009; Frigoli et al., 2009; Sun et al., 2006).

Another important process that occurs in the excited state is the Förster or fluorescence resonance energy transfer (FRET). This process occurs whenever the emission spectrum of one fluorophore, which is the donor, overlaps with the absorption spectrum of another molecule, which is the acceptor. The acceptor must absorb the energy at the emission wavelength(s) of the donor, but does not necessarily have to remit the energy fluorescently itself. The transfer of energy leads to a reduction in the donor's fluorescence intensity and excited state lifetime, and an increase in the acceptor's emission intensity. The rate of energy transfer from donor to acceptor is highly dependent on many factors, such as the extent of the spectral overlap, the relative orientation on the transition dipoles, and, most importantly, the distance between the donor and the acceptor. Due to its sensitivity to distance, FRET has been used to investigate molecular interactions (Demchenko, 2009; Frigoli et al., 2009; Kikuchi et al., 2004; Lakowicz, 2006).

## **2. Design of an optical sensor**

The overall sensor quality is dependent on the total sensor system components, which are defined by the transduction, the sensitive layer, light source, data-acquisition electronics and evaluation software. In the next paragraphs the emphasis is devoted to the components when designing the sensitive sensor layer, namely, to the choice of suitable indicators, polymers, and immobilization techniques. Some selected, recently published applications using new nanomaterials are further presented.

An optical detection system may be based either on a) direct sensing or b) indicatormediated sensing. In a direct optical sensor, the analyte is detected directly via some intrinsic optical property such as, for example, absorption or luminescence. In an indicatormediated system, a change in the optical response of an intermediate agent, usually an analyte-sensitive dye molecule (indicator), is used to monitor the analyte concentration (McDonagh et al., 2008; Nagl & Wolfbeis, 2008). The principle of immobilized indicators relies on a large group of optical chemical sensors, because the measuring analytes mostly have no intrinsic optical property or this property is not convenient for their detection.

The reagent immobilised materials (sensitive layers) can be fabricated into several configurations, such as thin films, gels, to be interfaced with optical fibres (Gründler, 2007), in nanoparticles, etc. The most common are thin polymer films or membranes. This "smart" material responds to the species of interest by altering its optical properties (Seitz, 1988; Wolfbeis, 1991). For example, pH is measured optically by immobilizing a pH indicator on a solid support and observing the changes in the absorption or fluorescence of the indicator as the pH of the sample varies with time (Jerónimo et al., 2007; Lobnik, 1998,2006; Turel, 2008; Wolfbeis, 1991).

#### **2.1 Indicators**

The basic principle of the indicator chemistry (immobilized in or on the polymer matrix) in an optical chemical sensor is in transforming the measuring concentration of the analyte into a measurable analytical signal. The analyte concentration is measured indirectly, through the alteration of the indicator's optical properties. Various types of indicators are used in optical chemical sensing, such as colorimetric - based on light absorption, and luminescent based on light emission (Demchenko, 2009; Guilbault, 1990; Lobnik, 2006; Wolfbeis, 1991). However, the latter are of primary importance due to their high interdisciplinarity, great sensitivity, and applicability to different detection principles. A large number of fluorescent synthetic organic products are available nowadays so that a researcher can easily select the proper dye (indicator) corresponding to a particular sensing application in terms of spectroscopic properties and chemical reactivity. On the other hand, the basic organic chemistry also offers great potential as it enables synthesizing tailor-made indicators for specific applications.

#### **2.1.1 Colorimetric indicators**

6 Advances in Chemical Sensors

fluorescence intensities of the indicator and the reference dye (Arduini et al., 2007; Buck et al., 2004; Cywinski et al., 2009; Doussineau et al., 2009; Frigoli et al., 2009; Sun et al., 2006). Another important process that occurs in the excited state is the Förster or fluorescence resonance energy transfer (FRET). This process occurs whenever the emission spectrum of one fluorophore, which is the donor, overlaps with the absorption spectrum of another molecule, which is the acceptor. The acceptor must absorb the energy at the emission wavelength(s) of the donor, but does not necessarily have to remit the energy fluorescently itself. The transfer of energy leads to a reduction in the donor's fluorescence intensity and excited state lifetime, and an increase in the acceptor's emission intensity. The rate of energy transfer from donor to acceptor is highly dependent on many factors, such as the extent of the spectral overlap, the relative orientation on the transition dipoles, and, most importantly, the distance between the donor and the acceptor. Due to its sensitivity to distance, FRET has been used to investigate molecular interactions (Demchenko, 2009;

The overall sensor quality is dependent on the total sensor system components, which are defined by the transduction, the sensitive layer, light source, data-acquisition electronics and evaluation software. In the next paragraphs the emphasis is devoted to the components when designing the sensitive sensor layer, namely, to the choice of suitable indicators, polymers, and immobilization techniques. Some selected, recently published applications

An optical detection system may be based either on a) direct sensing or b) indicatormediated sensing. In a direct optical sensor, the analyte is detected directly via some intrinsic optical property such as, for example, absorption or luminescence. In an indicatormediated system, a change in the optical response of an intermediate agent, usually an analyte-sensitive dye molecule (indicator), is used to monitor the analyte concentration (McDonagh et al., 2008; Nagl & Wolfbeis, 2008). The principle of immobilized indicators relies on a large group of optical chemical sensors, because the measuring analytes mostly have no intrinsic optical property or this property is not convenient for their detection.

The reagent immobilised materials (sensitive layers) can be fabricated into several configurations, such as thin films, gels, to be interfaced with optical fibres (Gründler, 2007), in nanoparticles, etc. The most common are thin polymer films or membranes. This "smart" material responds to the species of interest by altering its optical properties (Seitz, 1988; Wolfbeis, 1991). For example, pH is measured optically by immobilizing a pH indicator on a solid support and observing the changes in the absorption or fluorescence of the indicator as the pH of the sample varies with time (Jerónimo et al., 2007; Lobnik, 1998,2006; Turel, 2008;

The basic principle of the indicator chemistry (immobilized in or on the polymer matrix) in an optical chemical sensor is in transforming the measuring concentration of the analyte into a measurable analytical signal. The analyte concentration is measured indirectly, through the alteration of the indicator's optical properties. Various types of indicators are used in

Frigoli et al., 2009; Kikuchi et al., 2004; Lakowicz, 2006).

using new nanomaterials are further presented.

**2. Design of an optical sensor** 

Wolfbeis, 1991).

**2.1 Indicators** 

Colorimetric sensing is accomplished using an indicator that changes its colour upon binding the analyte; this change is usually spectroscopically determined, but it is also visibly observed. Among the great variety of organic chromophores, such as azo dyes, nitrophenols, phtaleins, sulfophtaleins, aniline-sulfophtaleins, triphenylmethane dyes, the most popular applications using these materials is to measure the pH parameter. The pKa of these indicators indicates the center of the measurable pH range, for example, cresol red, bromophenol blue, and bromocresol purple respond to acidic pH (pH < 7), while cresol red, naphtolbenzene, and phenolphthalein respond at basic pH (pH > 7) (Lobnik, 2006; Wolfbeis, 1991).

Redox indicators are the next example of colorimetric reagents. These are all organic dyestuffs, exhibiting reversible redox reactions. Examples include materials such as anilinic acid, diphenylamine, eriogreen, m-cresol-indophenol, methylene blue, nile blue, etc. (Wolfbeis, 1991; Lobnik, 2006). For example, a redox indicator Meldola blue can be incorporated into a sol-gel layer for an optical sensor measuring hydrogen peroxide in the concentration range of 10-8 to 10-1 mol/L (Lobnik & Čajlaković, 2001).

Ion sensing is possible using metal indicators that form coloured complexes with metal ions (Kaur & Kumar, 2011; Kim et al., 2009). The so-called ionophores are ligands that selectively bind ions. Chromogenic ionophores are designed to bring about a specific colour change in the interaction with metal cations (Murković Steinberg, 2003). Typical representatives are macrocyclic molecules with an ion-binding cavity, crown ether dyes, etc.

Among the new nano-based materials, nanoparticles (NPs), the colorimetric ones are less common in comparison with the luminescent NPs. However, some examples have recently been reported where gold NPs for sensing Cd2+ (Ying et al., 2011), Fe3+ (Shu-Pao et al., 2011), Pb2+ (Nan et al., 2010), nitrite, nitrate ions were utilized (Weston et al., 2009), and carbon nanotubes were reported for sensing nucleic acids (Ai Cheng, 2007), and nanowires were used for Hg2+ (Tsao-Yen, 2011).

#### **2.1.2 Luminescent indicators**

The analyte concentration is determined by the change in the emission properties of a luminophore. Luminescence is intrinsically more sensitive than absorption as a sensing technique, so for many applications the literature more often reports on sensing with luminescent probes and sensors. A variety of fluorescent and luminescent materials in the form of molecules, complexes and NPs are available for implementation as the response

Optical Chemical Sensors: Design and Applications 9

prolongs the response time of the sensor (Lobnik et al., 1998). Although the impregnation technique is widely used and lowpriced, it is used first of all for test strips and in gas sensors due to its low stability (indicator leaching). Doping is one of the most used immobilizations as it is not restricted to certain indicators and polymers. The sensor stability (in terms of indicator leaching) is better compared to impregnation and worse compared to

Polymer chemistry is an extremely important part of optical sensor technology. Both the light guide (including its cladding and coating) and the sensing chemistry of indicator-mediated sensors are made from organic or inorganic polymers (Baldini et al., 2006; McDonagh et al., 2008; Orellana et al., 2005; Wolfbeis, 1991). The choice of polymer is governed by the permeability of the polymer for the analyte, its stability and availability, its suitability for dye immobilization, its compatibility with other materials used in the fabrication of optrodes, and its compatibility with the sample to be investigated. The polymer micro-environment has a strong effect on the spectral properties of the immobilized indicator, pKa value, luminescence lifetime, binding constant, etc. (Lobnik & Wolfbeis, 2001; Wolfbeis, 1991). Consequently, the choice of polymer material has a pronounced influence on the sensor performance and its characteristics, such as selectivity, sensitivity, working range, calibration, response time, (photo)stability (Orellana et al., 2005; Korent et al., 2007). The response time, for example, will be governed by the diffusion coefficients of the gases or liquids, and the quenching efficiency

However, although most authors have compiled a considerable amount of data on various polymers, numerous new materials are available for which no data exist. It is also known that copolymers and polymer mixtures do not necessarily display the properties that may be

On the other hand, nano-sized (polymer) materials pose new technological and analytical challenges in many different sensor designs to improve industrial process monitoring (air and water quality), food-quality surveillance, and medical diagnostics, and to provide the reliable, real-time detection of chemical, biological, radiological and nuclear hazards for military and anti-terrorism applications – all this by enabling improved sensor characteristics, such as sensitivity, selectivity and response time, along with dramatically reduced size, weight and power requirements of the resulting monitoring devices compared to the conventional, macroscaled alternatives (Basabe-Desmonts et al., 2007; Borisov &

Silicones have excellent optical and mechanical properties, and unique gas solubility. The main applications of silicone materials is in sensors for oxygen and other uncharged quenchers, such as sulfur dioxide and chlorine, and as gas-permeable covers in sensors for carbon dioxide or ammonia. Silicones cannot be easily plasticized by conventional plasticizers, but form copolymers, which may be used instead. Blackened silicone is a most useful material for optically isolating gas sensors in order to make them insensitive to the

optical properties of the sample (Baldini et al., 2006; Wolfbeis, 1991).

covalent bonding. The response time is better than in covalent immobilization.

**2.3 Polymers** 

by the solubility of the gas in the polymer.

Klimant, 2008; Demchenko, 2010).

**2.3.1 Hydrophobic polymers** 

expected from averaging the data of the pure components.

units into sensing technologies; among them, organic fluorescent dyes are of primary importance. However, there is an increasing application of other materials, such as luminescent metal-ion chelating complexes, fluorescent polymer molecules and especially, from different kinds of NPs (Demchenko, 2009; Basabe-Desmonts et al., 2007;).

As already mentioned, organic dyes are most commonly used in fluorescence sensing. Their advantages are easy availability, a low price, versatility. The best known are fluoresceins, rhodamines, cyanine dyes, Alexa dyes, and BODIPY dyes, which are frequently used for labelling. In addition to these, the environment-sensitive dyes (for example, Nile red), hydrogen-bond responsive dyes (ketocyanine dye), electric-field-sensitive dyes (styryl dyes), supersensitive multicolour ratiometric dyes (3-hydroxychromone dyes), phosphorescent dyes eosin and erythrosine derivatives, optimal FRET pairs (for example Pyrene/Coumarin) are known fluorescent reporters (Demchenko, 2009,2010; Lakowicz, 2006).

Luminescent metal complexes, with europium(III) (Eu3+) and terbium(III) (Tb3+) ions being the most used, represent advantages since they show longer lifetimes, large Stokes' shifts and, therefore, enable eliminating the light-scattering effects and short-lived background luminescence (Turel et al., 2009,2010), thus significantly increasing the sensitivity of the analysis. Transition-metal complexes that exhibit phosphorescence are also formed by ruthenium (Balzani et al., 2000), osmium, and rhenium ions and there are those based on porphyrin complexes (with Pt and Pd ions) (Papkovsky & O'Riordan, 2005).

Interesting alternatives to fluorescent indicators are represented by the dye-doped NPs, NPs made of organic polymer, silica-based NPs, dendrimeres, quantum dots, noble metal NPs, fluorescent conjugated polymers and visible fluorescent proteins (Borisov&Klimant, 2008; Demchenko, 2010; Wolfbeis, 2005).

#### **2.2 Immobilization techniques**

A method for indicator immobilization into a suitable polymer matrix also has an important influence on the sensing characteristics. The following possibilities are usually applied:


Covalent immobilization enables the sensor having good stability (no leaching, crystallization and evaporation of components) and a longer operational lifetime. The disadvantage is that the covalent bonding often lowers the sensitivity for the analyte and prolongs the response time of the sensor (Lobnik et al., 1998). Although the impregnation technique is widely used and lowpriced, it is used first of all for test strips and in gas sensors due to its low stability (indicator leaching). Doping is one of the most used immobilizations as it is not restricted to certain indicators and polymers. The sensor stability (in terms of indicator leaching) is better compared to impregnation and worse compared to covalent bonding. The response time is better than in covalent immobilization.

#### **2.3 Polymers**

8 Advances in Chemical Sensors

units into sensing technologies; among them, organic fluorescent dyes are of primary importance. However, there is an increasing application of other materials, such as luminescent metal-ion chelating complexes, fluorescent polymer molecules and especially,

As already mentioned, organic dyes are most commonly used in fluorescence sensing. Their advantages are easy availability, a low price, versatility. The best known are fluoresceins, rhodamines, cyanine dyes, Alexa dyes, and BODIPY dyes, which are frequently used for labelling. In addition to these, the environment-sensitive dyes (for example, Nile red), hydrogen-bond responsive dyes (ketocyanine dye), electric-field-sensitive dyes (styryl dyes), supersensitive multicolour ratiometric dyes (3-hydroxychromone dyes), phosphorescent dyes eosin and erythrosine derivatives, optimal FRET pairs (for example Pyrene/Coumarin) are known fluorescent reporters (Demchenko, 2009,2010; Lakowicz,

Luminescent metal complexes, with europium(III) (Eu3+) and terbium(III) (Tb3+) ions being the most used, represent advantages since they show longer lifetimes, large Stokes' shifts and, therefore, enable eliminating the light-scattering effects and short-lived background luminescence (Turel et al., 2009,2010), thus significantly increasing the sensitivity of the analysis. Transition-metal complexes that exhibit phosphorescence are also formed by ruthenium (Balzani et al., 2000), osmium, and rhenium ions and there are those based on

Interesting alternatives to fluorescent indicators are represented by the dye-doped NPs, NPs made of organic polymer, silica-based NPs, dendrimeres, quantum dots, noble metal NPs, fluorescent conjugated polymers and visible fluorescent proteins (Borisov&Klimant, 2008;

A method for indicator immobilization into a suitable polymer matrix also has an important influence on the sensing characteristics. The following possibilities are usually applied:

• Impregnation – the indicator is immobilized in the polymer matrix through physical adsorption, chemisorption or electrostatic bonding. The polymer thin film is dipped into a saturated indicator solution and the solvent is then left to evaporate (Wolfbeis,

• Covalent bonding – the indicator is covalently bonded to the polymer matrix. This may be achieved by a) choosing the indicator that contains a functional group for covalent bonding to the polymer, which is at the same time insensitive for the target analyte, or b) polymerizing the indicator to certain monomers to form a copolymer (Baldini et al.,

• Doping – the indicator is entrapped in the matrix during the polymerization process, where the indicator is simply added to the starting polymer solution (Lobnik et al.,

Covalent immobilization enables the sensor having good stability (no leaching, crystallization and evaporation of components) and a longer operational lifetime. The disadvantage is that the covalent bonding often lowers the sensitivity for the analyte and

from different kinds of NPs (Demchenko, 2009; Basabe-Desmonts et al., 2007;).

porphyrin complexes (with Pt and Pd ions) (Papkovsky & O'Riordan, 2005).

2006).

Demchenko, 2010; Wolfbeis, 2005).

**2.2 Immobilization techniques** 

2006; Lobnik et al., 1998).

1991).

1998).

Polymer chemistry is an extremely important part of optical sensor technology. Both the light guide (including its cladding and coating) and the sensing chemistry of indicator-mediated sensors are made from organic or inorganic polymers (Baldini et al., 2006; McDonagh et al., 2008; Orellana et al., 2005; Wolfbeis, 1991). The choice of polymer is governed by the permeability of the polymer for the analyte, its stability and availability, its suitability for dye immobilization, its compatibility with other materials used in the fabrication of optrodes, and its compatibility with the sample to be investigated. The polymer micro-environment has a strong effect on the spectral properties of the immobilized indicator, pKa value, luminescence lifetime, binding constant, etc. (Lobnik & Wolfbeis, 2001; Wolfbeis, 1991). Consequently, the choice of polymer material has a pronounced influence on the sensor performance and its characteristics, such as selectivity, sensitivity, working range, calibration, response time, (photo)stability (Orellana et al., 2005; Korent et al., 2007). The response time, for example, will be governed by the diffusion coefficients of the gases or liquids, and the quenching efficiency by the solubility of the gas in the polymer.

However, although most authors have compiled a considerable amount of data on various polymers, numerous new materials are available for which no data exist. It is also known that copolymers and polymer mixtures do not necessarily display the properties that may be expected from averaging the data of the pure components.

On the other hand, nano-sized (polymer) materials pose new technological and analytical challenges in many different sensor designs to improve industrial process monitoring (air and water quality), food-quality surveillance, and medical diagnostics, and to provide the reliable, real-time detection of chemical, biological, radiological and nuclear hazards for military and anti-terrorism applications – all this by enabling improved sensor characteristics, such as sensitivity, selectivity and response time, along with dramatically reduced size, weight and power requirements of the resulting monitoring devices compared to the conventional, macroscaled alternatives (Basabe-Desmonts et al., 2007; Borisov & Klimant, 2008; Demchenko, 2010).

#### **2.3.1 Hydrophobic polymers**

Silicones have excellent optical and mechanical properties, and unique gas solubility. The main applications of silicone materials is in sensors for oxygen and other uncharged quenchers, such as sulfur dioxide and chlorine, and as gas-permeable covers in sensors for carbon dioxide or ammonia. Silicones cannot be easily plasticized by conventional plasticizers, but form copolymers, which may be used instead. Blackened silicone is a most useful material for optically isolating gas sensors in order to make them insensitive to the optical properties of the sample (Baldini et al., 2006; Wolfbeis, 1991).

Optical Chemical Sensors: Design and Applications 11

smaller than the characteristic lengths associated with the specific phenomena often display new chemistry and new physics that lead to new properties that depend on size. Perhaps one of the most intuitive effects is due to the change in the surface-to-volume ratio. When the size of the structure is decreased, this ratio increases considerably and the surface phenomena predominate over the chemistry and physics in the bulk. Therefore, the sensor characteristics, such as sensitivity (Chu & Lo, 2009) and response time (Waich et al., 2008),

Nanoparticles containing indicator dyes can either be used directly as nanosensors (NSs) or as the components of optical sensor materials. In the sensor matrix, more than one component can be encapsulated, thus allowing a synergistic approach to be employed (Aylott, 2003). Most of the NSs reported so far have used fluorophores as the sensitive and selective indicators. For making quantitative measurements in the intracellular environment, the so-called PEBBLE (Probes encapsulated by biologically localized embedding) NSs have been designed as they are small enough to be inserted into living cells with a minimum of physical perturbation (Buck et al., 2004). PEBBLEs have many advantages over widely used fluorescence-dye-based methods, such as: a) the increased number of analytes that can be measured because NSs are not limited to using a single fluorophore and can utilize cooperative interactions between ionophores, enzymes, reporter dyes, etc. (e.g., pH-sensitive and oxygen-sensitive beads can be incorporated into one polymer), b) the matrix protects the intracellular environment from any potentially toxic effects of the sensing dye, c) the matrix protects the sensing dye from potential interferences in the cellular environment, e.g., non-specific binding proteins and organelles, d) no selective sequestration of the NSs into cellular compartments or leaking from, or being pumped out of, cells, e) enhanced ability to carry out ratiometric measurements, and f) the

in-vitro calibration of NSs is valid for in-vivo measurements (Aylott, 2003).

(Arduini et al., 2007; Hun & Zhang, 2007; Sun et al., 2006).

**3. Selected applications** 

NSs provide advantages, such as an improved sensitivity, response time and ability to perform in-vivo measurements. However, the down side of using "free" NSs for in-vivo measurements needs to be considered. The prime concern is the retention of these particles in the body and the harmful effect in the long run (Sounderya & Zhang, 2008) since NPs can be responsible for a number of material interactions that could lead to toxicological effects (Nel et al., 2006). In any case, the optical properties that can be controlled at the nanoscale are of great interest in the field of optical sensor designing (Borisov & Klimant, 2008). Some optical chemical NSs rely on quantum dots (Asefa et al. 2009), metal beads (Shtykov & Rusanova, 2008) and other materials; however, most of them make use of indicators embedded in polymer beads (Lapresta et al., 2009; Zenkl & Klimant, 2009) and sol–gels

Optical chemical sensors provide the opportunity to continuously monitor chemical species and have thus found numerous applications in areas such as the chemical industry, biotechnology, medicine, environmental sciences, personal protection, etc. Books and reviews presenting various optical sensing schemes (fiber optics, capillary waveguides, microsystems and microstructures, refractive index-based, surface plasmon resonance-based, biosensing, etc.) and various applications (sensing gases, vapours, humidity, pH, ions, organic chemicals, certain bacteria, DNA, etc. in medical and chemical analyses, molecular biotechnology, marine

can be dramatically improved.

Poly(vinil chloride) (PVC), poly(methyl methacrylate) (PMMA), polyethylene, poly(tetrafluoroethylene) (PTFE), polystyrene (PS), and ethylcellulose comprise another group of hydrophobic materials that efficiently reject ionic species (Amao, 2003). Except for polystyrene, they are difficult to chemically modify so that their function is confined to that of a "solvent" for indicators, or as a gas-permeable cover. For example, PMMA and PDMS have been selected as the optimum matrix for oxygen sensing.

## **2.3.2 Hydrophilic polymers**

Hydrophilic polymers provide a matrix that corresponds to an aqueous environment. Hydrophilic supports are characterized by a large number of hydrogen-bridging functions, such as hydroxyl, amino, or carboxamide groups, or by anionic groups (mainly carboxyl and sulfo) linked to the polymer backbone. Typical examples are the polysaccharides (cellulose), polyacrylates, polyacrylamides, polyimines, polyglycols, and variety of so-called hydrogels. Depending on the degree of polymerization and cross-linking, they are watersoluble or water-insoluble. The ions can diffuse quite freely, but the possible water uptake (10-1000%) can cause significant swelling of the polymer. Swelling of the matrix affects the optical properties of the sensors and, consequently, the signal changes. They display poor compatibility with hydrophobic polymers, such as silicone and polystyrene. Most hydrophilic polymer membranes are easily penetrated by both charged and uncharged lowmolecular-weight analytes, but not by large proteins, and have found widespread application as support for indicators (Baldini et al. 2006; Wolfbeis, 1991). Hydrophilic matrices have been widely used for pH sensing.

#### **2.3.3 Hydrophobic/hydrophilic polymers**

Glass is widely used for manufacturing optical fibres. Its surface may be made either hydrophilic or hydrophobic by treatment with a proper surface-modification reagent. Surface derivatization is usually performed with reagents, such as amino-propyltriethoxysilane, which introduces a free amino group onto the surface of the glass to which dyes or proteins may be covalently attached. Glass does not measurably swell, but is difficult to handle because of its brittleness.

Sol-gel forms an attractive alternative to conventional glass (Baldini et al. 2006; Lobnik & Wolfbeis, 2001). By changing parameters, such as the sol pH, precursor type and concentration, water content, and curing temperature, materials of the desired porosity and polarity can be produced. The versatility of the process facilitates tailoring of the physicochemical properties of the material in order to optimize sensor performance. The basic process involves the hydrolysis and polycondensation of the appropriate metal alkoxide solution to produce a porous glass matrix. The reagent is entrapped in such a matrix and the analyte can diffuse to it. By altering its polarity, the sol-gel matrix makes it possible to sense either ions or gas molecules (Lobnik & Wolfbeis, 1998; Lobnik & Čajlaković, 2001; Murković, 2003; Pagliaro, 2009; Turel et al., 2008).

#### **2.4 Effect of nanodimensions on sensor characteristics**

The sensor characteristics can be tuned not only by the choice of the indicator and polymeric support but also by merely reducing the size (< 100 nm). This is because materials that are

Poly(vinil chloride) (PVC), poly(methyl methacrylate) (PMMA), polyethylene, poly(tetrafluoroethylene) (PTFE), polystyrene (PS), and ethylcellulose comprise another group of hydrophobic materials that efficiently reject ionic species (Amao, 2003). Except for polystyrene, they are difficult to chemically modify so that their function is confined to that of a "solvent" for indicators, or as a gas-permeable cover. For example, PMMA and PDMS

Hydrophilic polymers provide a matrix that corresponds to an aqueous environment. Hydrophilic supports are characterized by a large number of hydrogen-bridging functions, such as hydroxyl, amino, or carboxamide groups, or by anionic groups (mainly carboxyl and sulfo) linked to the polymer backbone. Typical examples are the polysaccharides (cellulose), polyacrylates, polyacrylamides, polyimines, polyglycols, and variety of so-called hydrogels. Depending on the degree of polymerization and cross-linking, they are watersoluble or water-insoluble. The ions can diffuse quite freely, but the possible water uptake (10-1000%) can cause significant swelling of the polymer. Swelling of the matrix affects the optical properties of the sensors and, consequently, the signal changes. They display poor compatibility with hydrophobic polymers, such as silicone and polystyrene. Most hydrophilic polymer membranes are easily penetrated by both charged and uncharged lowmolecular-weight analytes, but not by large proteins, and have found widespread application as support for indicators (Baldini et al. 2006; Wolfbeis, 1991). Hydrophilic

Glass is widely used for manufacturing optical fibres. Its surface may be made either hydrophilic or hydrophobic by treatment with a proper surface-modification reagent. Surface derivatization is usually performed with reagents, such as amino-propyltriethoxysilane, which introduces a free amino group onto the surface of the glass to which dyes or proteins may be covalently attached. Glass does not measurably swell, but is

Sol-gel forms an attractive alternative to conventional glass (Baldini et al. 2006; Lobnik & Wolfbeis, 2001). By changing parameters, such as the sol pH, precursor type and concentration, water content, and curing temperature, materials of the desired porosity and polarity can be produced. The versatility of the process facilitates tailoring of the physicochemical properties of the material in order to optimize sensor performance. The basic process involves the hydrolysis and polycondensation of the appropriate metal alkoxide solution to produce a porous glass matrix. The reagent is entrapped in such a matrix and the analyte can diffuse to it. By altering its polarity, the sol-gel matrix makes it possible to sense either ions or gas molecules (Lobnik & Wolfbeis, 1998; Lobnik &

The sensor characteristics can be tuned not only by the choice of the indicator and polymeric support but also by merely reducing the size (< 100 nm). This is because materials that are

have been selected as the optimum matrix for oxygen sensing.

matrices have been widely used for pH sensing.

**2.3.3 Hydrophobic/hydrophilic polymers** 

difficult to handle because of its brittleness.

Čajlaković, 2001; Murković, 2003; Pagliaro, 2009; Turel et al., 2008).

**2.4 Effect of nanodimensions on sensor characteristics** 

**2.3.2 Hydrophilic polymers** 

smaller than the characteristic lengths associated with the specific phenomena often display new chemistry and new physics that lead to new properties that depend on size. Perhaps one of the most intuitive effects is due to the change in the surface-to-volume ratio. When the size of the structure is decreased, this ratio increases considerably and the surface phenomena predominate over the chemistry and physics in the bulk. Therefore, the sensor characteristics, such as sensitivity (Chu & Lo, 2009) and response time (Waich et al., 2008), can be dramatically improved.

Nanoparticles containing indicator dyes can either be used directly as nanosensors (NSs) or as the components of optical sensor materials. In the sensor matrix, more than one component can be encapsulated, thus allowing a synergistic approach to be employed (Aylott, 2003). Most of the NSs reported so far have used fluorophores as the sensitive and selective indicators. For making quantitative measurements in the intracellular environment, the so-called PEBBLE (Probes encapsulated by biologically localized embedding) NSs have been designed as they are small enough to be inserted into living cells with a minimum of physical perturbation (Buck et al., 2004). PEBBLEs have many advantages over widely used fluorescence-dye-based methods, such as: a) the increased number of analytes that can be measured because NSs are not limited to using a single fluorophore and can utilize cooperative interactions between ionophores, enzymes, reporter dyes, etc. (e.g., pH-sensitive and oxygen-sensitive beads can be incorporated into one polymer), b) the matrix protects the intracellular environment from any potentially toxic effects of the sensing dye, c) the matrix protects the sensing dye from potential interferences in the cellular environment, e.g., non-specific binding proteins and organelles, d) no selective sequestration of the NSs into cellular compartments or leaking from, or being pumped out of, cells, e) enhanced ability to carry out ratiometric measurements, and f) the in-vitro calibration of NSs is valid for in-vivo measurements (Aylott, 2003).

NSs provide advantages, such as an improved sensitivity, response time and ability to perform in-vivo measurements. However, the down side of using "free" NSs for in-vivo measurements needs to be considered. The prime concern is the retention of these particles in the body and the harmful effect in the long run (Sounderya & Zhang, 2008) since NPs can be responsible for a number of material interactions that could lead to toxicological effects (Nel et al., 2006). In any case, the optical properties that can be controlled at the nanoscale are of great interest in the field of optical sensor designing (Borisov & Klimant, 2008). Some optical chemical NSs rely on quantum dots (Asefa et al. 2009), metal beads (Shtykov & Rusanova, 2008) and other materials; however, most of them make use of indicators embedded in polymer beads (Lapresta et al., 2009; Zenkl & Klimant, 2009) and sol–gels (Arduini et al., 2007; Hun & Zhang, 2007; Sun et al., 2006).

## **3. Selected applications**

Optical chemical sensors provide the opportunity to continuously monitor chemical species and have thus found numerous applications in areas such as the chemical industry, biotechnology, medicine, environmental sciences, personal protection, etc. Books and reviews presenting various optical sensing schemes (fiber optics, capillary waveguides, microsystems and microstructures, refractive index-based, surface plasmon resonance-based, biosensing, etc.) and various applications (sensing gases, vapours, humidity, pH, ions, organic chemicals, certain bacteria, DNA, etc. in medical and chemical analyses, molecular biotechnology, marine

Optical Chemical Sensors: Design and Applications 13

2008b). Swelling is another widespread method for the encapsulation of indicators in NPs (Méallet-Renault, 2004; Frigoli et al., 2009). This method is only useful for hydrophobic materials that are not swellable in water. It is essential to use water-insoluble indicators for

Nanosensors based on hydrophobic materials even allow the monitoring of hydrophilic species with acceptable response times (in contrast to monolithic films based on the identical composition), due to small diffusion distances. Polymers with polar properties (such as polyacrylonitrile) have a large surface-to-volume ratio and are therefore especially useful for ion sensing. Indicators of an amphiphilic nature are often located on the surface of the bead,

Sol-gels (inorganic silica beads and organically modified silica – Ormosils) are very popular materials for designing optical nanosensors (Jain et al., 1998; Rossi et al., 2005; Shibata et al. 1997). This is due to the fact that the beads can easily be manufactured, are porous to allow an analyte to diffuse freely inside, are robust, and are biocompatible, making them suitable for intracellular measurements. Compared with polymer NPs, silica NPs possess several advantages. Silica NPs are easy to separate via centrifugation during particle preparation, surface modification, and other solution-treatment processes because of the higher density of silica (e.g., 1.96 g/cm3 for silica vs. 1.05 g/cm3 for polystyrene). Silica NPs are more hydrophilic and biocompatible, they are not subject to microbial attack, and no swelling or

Nanoparticles based on sol-gel materials can be prepared by two general synthetic routes: the Stöber (Rossi et al., 2005; Shibata et al., 1997) and reverse microemulsion processes (Bagwe et al., 2004; Santra et al., 2005; Wang et al., 2005). In a typical Stöber-based protocol, a silica alkoxide precursor (such as tetraethyl orthosilicate, TEOS) is hydrolyzed in an ethanol and ammonium hydroxide mixture. The hydrolysis of TEOS produces silicic acid, which then undergoes a condensation process to form amorphous silica particles. In general, the lower the concentration of water and ammonia, the smaller are the particles. Indicators are typically entrapped inside the pores of the beads. However, that does not always prevent them from leaching into solution. Therefore, similar to the polymer beads, covalent

Dye-doped silica NPs can also be synthesized by hydrolyzing TEOS in a reverse-micelle or water-in-oil (w/o) microemulsion system. In a typical w/o microemulsion system, water droplets are stabilized by surfactant molecules and remain dispersed in the bulk oil. The nucleation and growth kinetics of the silica are highly regulated in the water droplets of the microemulsion system, and the dye molecules are physically encapsulated in the silica network, resulting in the formation of highly monodispersed dye-doped silica NPs (Schmidt et al., 1999). Polar dye molecules are used in the w/o microemulsion system to increase the electrostatic attraction of the dye molecules to the negatively charged silica matrix, so that dye molecules are successfully entrapped inside the silica matrix. Water-soluble inorganic dyes, such as ruthenium complexes, can be readily encapsulated into nano-particles using this method (Brasola et al., 2003; Frasco & Chaniotakis, 2009; Méallet-Renault et al., 2004; Ramazzo et al., 2005). Various trapping methods can be used, such as introducing a

this method otherwise leaching can occur over time.

allowing a response even to hydrophilic analytes.

porosity change occurs with changes in the pH (Jain et al., 1998).

**3.2 Sol-gel based nanoparticles** 

coupling is often preferred.

and environmental analysis, industrial production monitoring, bioprocess control, automotive industry) have been published in recent years. The most comprehensive studies include work by Baldini et al., 2006; Gauglitz, 2005; Gründler, 2007; McDonagh et al., 2008; Nagl & Wolfbeis, 2008; Wolfbeis, 2005; Wolfbeis, 2008.

The broad variety of applications of optical chemical sensors would deserve a special chapter devoted only to the applications. However, due to the limited space, we had to restrict our contribution to selected materials. Since the nano-world continues to rapidly enter our lives in many different ways, the following pages will survey the recently developed optical chemical nanosensors. The applications are selected for sensors or probes based on sol-gel and polymer NPs that have their dimensions ≤ 100 nm. Among the many optical methods employed in nanosensing, fluorescence has attracted particular attention because it is sensitive, offers several techniques to be used to explore various parameters that can serve as an analytical information; therefore, the luminescent approach is selected for the two types of nanoparticles mentioned.

#### **3.1 Polymer-based nanoparticles**

Polymer NPs are usually obtained by microemulsion polymerization. Microemulsions are clear, stable, isotropic liquid mixtures of oil, water and surfactant, sometimes in combination with a cosurfactant. The microemulsion polymerization of monomers may be achieved by incorporating a monomer in any of the water and oil phases of the system (Pavel, 2004). The two basic types of microemulsions, direct (oil dispersed in water, o/w) and reversed (water dispersed in oil, w/o) are frequently used. In w/o microemulsion nanodroplets of oil surrounded by the surfactant are dispersed in the continuous bulk water phase. The size of the synthesized particles is determined by the size of those droplets (Košak et al., 2004,2005).

NPs can also be prepared by the precipitation method, which is based on the use of two miscible solvents (Borisov et al., 2009; Higuchi et al., 2006). The nanobeads are formed by diluting the polymer solution with a poor solvent. Gradually, evaporation of the good solvent at room temperature causes precipitation of the polymer solute as fine particles. Using this method, NPs can be prepared from a variety of polymers (e.g., engineering plastics, biodegradable polymers and electro-conductive polymers, etc.). The diameter of the particles can be controlled by changing the concentration of the solution, and the mixing ratio of the good solvent and the poor solvent, respectively. It should be emphasized that this process does not require the addition of surfactants (and their subsequent removal) as in the case of NSs prepared via polymerization (Borisov et al., 2009).

An indicator can be added to the mixture of monomers to be entrapped in the bead during polymerization. Both physical (Borisov et al., 2009) entrapment and covalent (Sun et al., 2006) coupling are used. Physical entrapment of an indicator in NPs is preferred because of its simplicity and reproducibility. In contrast to bulk sensor films (typically several microns thick), in nanosensors many indicator molecules are located close to the surface so that leaching can become a serious problem. To avoid leaching, covalent binding can be used. However, in this case, both the dye and the beads require having a reactive group through which a covalent bond can be formed between the polymer and the indicator. This situation is often undesirable because excess reactive groups on the surface of the beads may compromise their properties and often make them more prone to aggregation (Borisov et al.,

and environmental analysis, industrial production monitoring, bioprocess control, automotive industry) have been published in recent years. The most comprehensive studies include work by Baldini et al., 2006; Gauglitz, 2005; Gründler, 2007; McDonagh et al., 2008; Nagl & Wolfbeis,

The broad variety of applications of optical chemical sensors would deserve a special chapter devoted only to the applications. However, due to the limited space, we had to restrict our contribution to selected materials. Since the nano-world continues to rapidly enter our lives in many different ways, the following pages will survey the recently developed optical chemical nanosensors. The applications are selected for sensors or probes based on sol-gel and polymer NPs that have their dimensions ≤ 100 nm. Among the many optical methods employed in nanosensing, fluorescence has attracted particular attention because it is sensitive, offers several techniques to be used to explore various parameters that can serve as an analytical information; therefore, the luminescent approach is selected

Polymer NPs are usually obtained by microemulsion polymerization. Microemulsions are clear, stable, isotropic liquid mixtures of oil, water and surfactant, sometimes in combination with a cosurfactant. The microemulsion polymerization of monomers may be achieved by incorporating a monomer in any of the water and oil phases of the system (Pavel, 2004). The two basic types of microemulsions, direct (oil dispersed in water, o/w) and reversed (water dispersed in oil, w/o) are frequently used. In w/o microemulsion nanodroplets of oil surrounded by the surfactant are dispersed in the continuous bulk water phase. The size of the synthesized particles is determined by the size of those droplets (Košak et al., 2004,2005). NPs can also be prepared by the precipitation method, which is based on the use of two miscible solvents (Borisov et al., 2009; Higuchi et al., 2006). The nanobeads are formed by diluting the polymer solution with a poor solvent. Gradually, evaporation of the good solvent at room temperature causes precipitation of the polymer solute as fine particles. Using this method, NPs can be prepared from a variety of polymers (e.g., engineering plastics, biodegradable polymers and electro-conductive polymers, etc.). The diameter of the particles can be controlled by changing the concentration of the solution, and the mixing ratio of the good solvent and the poor solvent, respectively. It should be emphasized that this process does not require the addition of surfactants (and their subsequent removal) as

An indicator can be added to the mixture of monomers to be entrapped in the bead during polymerization. Both physical (Borisov et al., 2009) entrapment and covalent (Sun et al., 2006) coupling are used. Physical entrapment of an indicator in NPs is preferred because of its simplicity and reproducibility. In contrast to bulk sensor films (typically several microns thick), in nanosensors many indicator molecules are located close to the surface so that leaching can become a serious problem. To avoid leaching, covalent binding can be used. However, in this case, both the dye and the beads require having a reactive group through which a covalent bond can be formed between the polymer and the indicator. This situation is often undesirable because excess reactive groups on the surface of the beads may compromise their properties and often make them more prone to aggregation (Borisov et al.,

in the case of NSs prepared via polymerization (Borisov et al., 2009).

2008; Wolfbeis, 2005; Wolfbeis, 2008.

for the two types of nanoparticles mentioned.

**3.1 Polymer-based nanoparticles** 

2008b). Swelling is another widespread method for the encapsulation of indicators in NPs (Méallet-Renault, 2004; Frigoli et al., 2009). This method is only useful for hydrophobic materials that are not swellable in water. It is essential to use water-insoluble indicators for this method otherwise leaching can occur over time.

Nanosensors based on hydrophobic materials even allow the monitoring of hydrophilic species with acceptable response times (in contrast to monolithic films based on the identical composition), due to small diffusion distances. Polymers with polar properties (such as polyacrylonitrile) have a large surface-to-volume ratio and are therefore especially useful for ion sensing. Indicators of an amphiphilic nature are often located on the surface of the bead, allowing a response even to hydrophilic analytes.

## **3.2 Sol-gel based nanoparticles**

Sol-gels (inorganic silica beads and organically modified silica – Ormosils) are very popular materials for designing optical nanosensors (Jain et al., 1998; Rossi et al., 2005; Shibata et al. 1997). This is due to the fact that the beads can easily be manufactured, are porous to allow an analyte to diffuse freely inside, are robust, and are biocompatible, making them suitable for intracellular measurements. Compared with polymer NPs, silica NPs possess several advantages. Silica NPs are easy to separate via centrifugation during particle preparation, surface modification, and other solution-treatment processes because of the higher density of silica (e.g., 1.96 g/cm3 for silica vs. 1.05 g/cm3 for polystyrene). Silica NPs are more hydrophilic and biocompatible, they are not subject to microbial attack, and no swelling or porosity change occurs with changes in the pH (Jain et al., 1998).

Nanoparticles based on sol-gel materials can be prepared by two general synthetic routes: the Stöber (Rossi et al., 2005; Shibata et al., 1997) and reverse microemulsion processes (Bagwe et al., 2004; Santra et al., 2005; Wang et al., 2005). In a typical Stöber-based protocol, a silica alkoxide precursor (such as tetraethyl orthosilicate, TEOS) is hydrolyzed in an ethanol and ammonium hydroxide mixture. The hydrolysis of TEOS produces silicic acid, which then undergoes a condensation process to form amorphous silica particles. In general, the lower the concentration of water and ammonia, the smaller are the particles. Indicators are typically entrapped inside the pores of the beads. However, that does not always prevent them from leaching into solution. Therefore, similar to the polymer beads, covalent coupling is often preferred.

Dye-doped silica NPs can also be synthesized by hydrolyzing TEOS in a reverse-micelle or water-in-oil (w/o) microemulsion system. In a typical w/o microemulsion system, water droplets are stabilized by surfactant molecules and remain dispersed in the bulk oil. The nucleation and growth kinetics of the silica are highly regulated in the water droplets of the microemulsion system, and the dye molecules are physically encapsulated in the silica network, resulting in the formation of highly monodispersed dye-doped silica NPs (Schmidt et al., 1999). Polar dye molecules are used in the w/o microemulsion system to increase the electrostatic attraction of the dye molecules to the negatively charged silica matrix, so that dye molecules are successfully entrapped inside the silica matrix. Water-soluble inorganic dyes, such as ruthenium complexes, can be readily encapsulated into nano-particles using this method (Brasola et al., 2003; Frasco & Chaniotakis, 2009; Méallet-Renault et al., 2004; Ramazzo et al., 2005). Various trapping methods can be used, such as introducing a

Optical Chemical Sensors: Design and Applications 15

indicator and matrix used, dynamic range, response time, limit of detection (LOD), indicator

The determination of pH has great importance for a wide range of applications, e.g., in the medical, environmental and biotechnological fields. The most common pH sensors are electrochemical devices (glass electrodes). Although they can be reliable analytical tools in many situations, their use for intracellular measurements is limited. In the field of biology and medicine, sensors able to monitor pH in real time are in great demand, as they can be used for the general understanding of biological processes and for biomedical diagnostics (Doussineau et al., 2009). Recently, a lot of work has been done on optical pH-sensitive micro- (Borisov et al., 2008a; Doussineau et al., 2009; Schulz et al., 2009 ; Hornig et al., 2008) and nano-sensors (Allard & Larpent, 2008; Burns et al., 2006; Gao et al., 2005; Gao et al., 2007; Kim et al., 2006; Peng et al., 2007; Sun et al., 2006;). Here, the latter will be discussed,

> Response time

Leaching Operational

given

given

given

signal decrease

leaching

leaching

the dye

1s 30%

lifetime

not given < 5%

5 weeks < 4.5%

Effect of ionic strength

signal decrease

signal decrease

>6 months not given (Kim et

not given not given (Peng et

not given not given (Burns

pKa

not given not given (Sun et

>6 months change in

Ref.

(Gao et al., 2005)

(Gao et al., 2007)

al., 2006)

al., 2007)

et al., 2006)

(Allard & Larpent, 2008)

al.,2006)

since they are less than 100 nm in size. Table 1 summarizes pH nanosensors.

RITC2 5.0-10.0 5 min not

Fluorescein 4-8 5 min not

NVP4 4-8 not given not

FITC 6.4 not given no

0.05 pH units

FITC6 4-7

Polystyren FITC 5.6 few s no

Polyacrylamide AF7 5.8-7.2 0.4 s 9% of

dynamic range

leaching, operational lifetime, interferences, etc.

Material Indicator pKa or

Sol-gel (TEOS1)

Sol-gel (TEOS)

Sol-gel (ICTES3)

Sol-gel (TEOS, APTES5)

Sol-gel (TEOS, APTES)

1TEOS – Tetraethoxysilane

7AF – Aminofluorescein

2RITC - Rhodamine-β-Isothiocyanate 3ICTES - (3-isocyanatopropyl)triethoxysilane)

4NVP - Naphtalenylvinyl-pyridine 5APTES – Aminoproplytriethoxysilane 6FITC - Fluorescein isothiocyanate

Table 1. Nanosensors/probes for pH.

**3.4.1 Nanosensors for pH** 

hydrophobic silica precursor (Tapec et al., 2002), using water-soluble dextran-moleculeconjugated dyes, and synthesizing in acidic conditions (Frasco & Chaniotakis, 2009). The unique advantage of the w/o microemulsion method is that it produces highly spherical and monodisperse NPs of various sizes. It also permits the trapping of a wide variety of inorganic and organic dyes as well as other materials, such as luminescent quantum dots.

#### **3.3 Core-shell systems for sensing chemistry**

The core-shell nanostructures based on polymers and sol-gel materials are well known (Arduini et al., 2007; Burns et al., 2006; Frigoli et al., 2009; Sounderya & Zhang, 2008). The versatility matrix allows the sensing chemistry to be incorporated either into the core or into the shell of a bead, which for some NSs can slightly tune the sensitivity and helps to minimize interferences (Borisov et al., 2008a). Moreover, the encapsulation of two different fluorescent dyes in the core-shell structure, one analyte-sensitive dye and other analyteinsensitive incorporated into the core of the bead used as reference, allows ratiometric detection. This method overcomes the problems (fluctuations in light source intensity of the instrument, photobleaching of the analyte-sensitive dye, drifts in the optoelectronic setup and background fluorescence) associated with intensity-based measurements. Different approaches can be used to prepare ratiometric sensors, as shown in Fig. 3.

Fig. 3. Types of ratiometric approaches: a) ratiometric indicator dye is immobilized in the NP; b) indicator and reference dye are immobilized inside the NP; c) reference dye is immobilized (covalently attached or doped) in the NP, and the indicator dye is covalently bound to the NP surface; d) in the core-shell system, the reference dye is immobilized in the core and the indicator dye is immobilized in the shell.

#### **3.4 Sensors based on polymer and sol-gel nanoparticles**

In the following section the recently developed photoluminescence-based optical chemical NSs will be described, highlighting the most important sensor characteristics, such as the indicator and matrix used, dynamic range, response time, limit of detection (LOD), indicator leaching, operational lifetime, interferences, etc.

#### **3.4.1 Nanosensors for pH**

14 Advances in Chemical Sensors

hydrophobic silica precursor (Tapec et al., 2002), using water-soluble dextran-moleculeconjugated dyes, and synthesizing in acidic conditions (Frasco & Chaniotakis, 2009). The unique advantage of the w/o microemulsion method is that it produces highly spherical and monodisperse NPs of various sizes. It also permits the trapping of a wide variety of inorganic and organic dyes as well as other materials, such as luminescent quantum dots.

The core-shell nanostructures based on polymers and sol-gel materials are well known (Arduini et al., 2007; Burns et al., 2006; Frigoli et al., 2009; Sounderya & Zhang, 2008). The versatility matrix allows the sensing chemistry to be incorporated either into the core or into the shell of a bead, which for some NSs can slightly tune the sensitivity and helps to minimize interferences (Borisov et al., 2008a). Moreover, the encapsulation of two different fluorescent dyes in the core-shell structure, one analyte-sensitive dye and other analyteinsensitive incorporated into the core of the bead used as reference, allows ratiometric detection. This method overcomes the problems (fluctuations in light source intensity of the instrument, photobleaching of the analyte-sensitive dye, drifts in the optoelectronic setup and background fluorescence) associated with intensity-based measurements. Different

Fig. 3. Types of ratiometric approaches: a) ratiometric indicator dye is immobilized in the NP; b) indicator and reference dye are immobilized inside the NP; c) reference dye is immobilized (covalently attached or doped) in the NP, and the indicator dye is covalently bound to the NP surface; d) in the core-shell system, the reference dye is immobilized in the

In the following section the recently developed photoluminescence-based optical chemical NSs will be described, highlighting the most important sensor characteristics, such as the

core and the indicator dye is immobilized in the shell.

**3.4 Sensors based on polymer and sol-gel nanoparticles** 

approaches can be used to prepare ratiometric sensors, as shown in Fig. 3.

**3.3 Core-shell systems for sensing chemistry** 

The determination of pH has great importance for a wide range of applications, e.g., in the medical, environmental and biotechnological fields. The most common pH sensors are electrochemical devices (glass electrodes). Although they can be reliable analytical tools in many situations, their use for intracellular measurements is limited. In the field of biology and medicine, sensors able to monitor pH in real time are in great demand, as they can be used for the general understanding of biological processes and for biomedical diagnostics (Doussineau et al., 2009). Recently, a lot of work has been done on optical pH-sensitive micro- (Borisov et al., 2008a; Doussineau et al., 2009; Schulz et al., 2009 ; Hornig et al., 2008) and nano-sensors (Allard & Larpent, 2008; Burns et al., 2006; Gao et al., 2005; Gao et al., 2007; Kim et al., 2006; Peng et al., 2007; Sun et al., 2006;). Here, the latter will be discussed, since they are less than 100 nm in size. Table 1 summarizes pH nanosensors.


1TEOS – Tetraethoxysilane

2RITC - Rhodamine-β-Isothiocyanate

3ICTES - (3-isocyanatopropyl)triethoxysilane)

4NVP - Naphtalenylvinyl-pyridine

5APTES – Aminoproplytriethoxysilane

6FITC - Fluorescein isothiocyanate

7AF – Aminofluorescein

Table 1. Nanosensors/probes for pH.

Optical Chemical Sensors: Design and Applications 17

either in typical microparticles type (Borisov et al., 2008a; Waich et al., 2008) or in recent NP type (Borisov et al., 2009; Cywinski et al., 2009), consists of a dye entrapped in a matrix with a high permeability to oxygen. Borisov et al., 2009, showed that different polymer NPs (size 44-55 nm), depending on their gas permeability, embedded with iridium (III) coumarin, could be used as oxygen NSs. However, the characterisation of these sensors should be included. Cywinski et al., 2009, prepared ratiometric polystyrene-based oxygen NSs (20.9 ± 7.1 nm). Platinum(II)mesotetra(pentafluorophenyl)porphine was used as an indicator dye, whereas N,N-bis(1-hexylheptyl)perylene-3,4:9,10-bis-(dicarboximide) was used as a

Table 2 summarizes the recently developed ion NSs. Lead (Pb2+) is one of the most toxic heavy metals, and it is particularly dangerous in terms of its effects on children. Therefore, the determination of lead in low concentrations is important. Arduini et al., 2007, developed a fluorescent nanosensor for the detection of Pb2+ ions. Silica core-shell NPs were produced from 50 nm diameter "pure" silica cores and a 5-nm-thick shell in which the indicator dansyl dye was covalently linked to a silica network. Then the surface of the shell was functionalized with (mercaptopropyl) triethoxysilane. Surface thiol groups play a key role in the recognition of the analyte. Selectivity tests show that Cu2+ is a serious interferent. On the other hand, no effect is observed after the addition of Zn2+, Ca2+, and Mg2+, while Ni2+ and

Copper (Cu2+) is an essential trace element that is required in various biological processes (hemopoiesis, metabolism, growth, and immune system (Uauy et al., 1998)). On the other hand, excess Cu2+ concentrations are extremely toxic. The detection of trace amounts of copper ions is of increasing importance in light of its environmental and biomedical implications. NSs for the detection of Cu2+ ions typically combine two components: a metal chelating molecule designed to bind the target ion selectively and a fluorophore as a readout system. The chelating molecules and the fluorophore are both covalently linked on to the surface of the NP (Brasola et al., 2003; Ramazzo et al., 2005) or the fluorophore is entrapped inside the NP (Méallet-Renault et al., 2004; Frigoli et al., 2009). The use of two different metal chelating ligands, picolinamide (Brasola et al., 2003) and (2-pyridinmethyl) glycinamide (Ramazzo et al., 2005), and the same fluorescent dye (danslyamide) for the surface modification of silica NPs were reported. Méallet-Renault et al., 2004, described the concept of associating an efficient fluorescent reporter, a BODIPY derivative, and an excellent metal-chelating receptor, cyclam, in polymer NPs. The hydrophobic dye is entrapped within the particle core and the receptor, covalently linked to the polymer backbone, is mainly located near the surface. The detection is based on the quenching of

fluorescence by energy transfer from the dye to the receptor-target complex.

Core-shell-type dual-fluorescent NPs (NPs) in the 16-nm-diameter range with a selective ligand (cyclam) attached to the surface and two fluorophores, 9,10-diphenyl-anthracene (donor, D) and pyrromethene PM567 (acceptor, A), embedded within the polymer core were synthesized (Frigoli et al., 2009). Dual-dye-doped fluorescent NPs permit the design of cascade FRET-mediated sensing devices. The two sequential FRET processes, the energy transfer from D to A, and quenching of the resulting sensitized emission of A by copper complexes that are formed at the NP surface, provide an efficient means to sense

reference dye.

**3.4.3 Nanosensors for ions** 

Co2+ produce much less quenching than Pb2+.

Several sol-gel pH NSs were prepared using a microemulsion procedure (Allard & Larpent, 2008; Gao et al., 2005; Gao et al., 2007; Kim et al., 2006; Peng et al., 2007; Sun et al., 2006) and one by Stöber (Burns et al., 2006). Gao et al., 2007, designed pH sensors using the doping technique for the immobilization of indicators in sol-gel NPs. Although leaching can be a serious problem, in this case it was not tested. The pH dynamic range of sensing NPs (60 ± 4 nm) is between 5.0 and 10.0 (Gao et al., 2005) and between 4.0 and 8.0 for NSs (30 ± 4 nm) based on indicator Fluorescein (Gao et al., 2007). The response time (5 minutes) for both sensors is relatively long compared to other pH NSs (Table 1), even compared to macrosized pH optical sensors (Jerónimo et al., 2007).

Kim et al., 2006, designed ormosil NPs (33±6 nm) in which the ratiometric pH-responsive dye is covalently immobilized. Peng et al., 2007, and Burns et al., 2006, developed twofluorophore-immobilized NPs sensors. The first author used fluorescein isothiocyanate as an indicator dye that was covalently crosslinked with the network formed by the hydrolysis of tetraethoxysilane and aminoproplytriethoxysilane, while tris(2,2′-bipyridyl) dichlororuthenium(II) hexahydrate (reference) was entrapped simultaneously inside the same NSs (42 nm) by electrostatic interaction. The second author synthesized a nanosensor (70 nm) via a modified Stöber method, incorporating a reference (tetramethylrhodamineisothiocyanate) and an indicator dye (fluorescein isothiocyanate) covalently bound to the matrix in the coreshell architecture. In this case the pH sensor is composed of a shell of covalently bound indicator-dye molecules surrounding a core of sequestered, covalently bound, reference-dye molecules. What is interesting is that although both authors used covalent immobilization, Peng et al., 2007, reported an extensive dye leakage.

Two different approaches have been used for the development of polymeric ratiometric pH nanosensors. In the first case a reference dye (Rhodamine B derivate) and a pHsensitive dye (Aminofluorescein) were covalently embedded in polyacrylamide NPs (50 nm) (Sun et al., 2006). Allard & Larpent, 2008, presented a pH sensor based on polystyrene NPs, in the 20 nm range, with a hydrophobic reference dye (1,9 diphenylanthracene) embedded within the particle and the indicator dye (Fluorescein isothiocyanate) covalently attached to the surface. In this case the pH sensor may retain many of the drawbacks related to free fluorescent dyes because the indicator molecules are not protected from the cellular environment. It would be interesting to know, if the fotostability of the covalently attached indicator to the NP surface is improved compared to the free indicator.

#### **3.4.2 Nanosensors for oxygen**

Oxygen is undoubtedly one of the most important analytes. In particular, a determination of the dissolved oxygen is of utmost importance in many fields of science and technology (Papkovsky & O'Riordan, 2005), including biotechnology (Kostov et al., 2001), biology (Schmaelzlin et al., 2005), marine science (Schroeder et al., 2007), and in medicine (Kimura et al., 2007). Recent interest in methods for measuring dissolved oxygen has been focused mainly on optical sensors, due to their advantages over conventional amperometric electrodes in that they are faster and do not consume oxygen (McDonagh et al., 2002).

The principle behind the operation of these sensors is the reduction in the luminescent intensity as a consequence of the oxygen quenching of the emitting state. The sensor optode, either in typical microparticles type (Borisov et al., 2008a; Waich et al., 2008) or in recent NP type (Borisov et al., 2009; Cywinski et al., 2009), consists of a dye entrapped in a matrix with a high permeability to oxygen. Borisov et al., 2009, showed that different polymer NPs (size 44-55 nm), depending on their gas permeability, embedded with iridium (III) coumarin, could be used as oxygen NSs. However, the characterisation of these sensors should be included. Cywinski et al., 2009, prepared ratiometric polystyrene-based oxygen NSs (20.9 ± 7.1 nm). Platinum(II)mesotetra(pentafluorophenyl)porphine was used as an indicator dye, whereas N,N-bis(1-hexylheptyl)perylene-3,4:9,10-bis-(dicarboximide) was used as a reference dye.

#### **3.4.3 Nanosensors for ions**

16 Advances in Chemical Sensors

Several sol-gel pH NSs were prepared using a microemulsion procedure (Allard & Larpent, 2008; Gao et al., 2005; Gao et al., 2007; Kim et al., 2006; Peng et al., 2007; Sun et al., 2006) and one by Stöber (Burns et al., 2006). Gao et al., 2007, designed pH sensors using the doping technique for the immobilization of indicators in sol-gel NPs. Although leaching can be a serious problem, in this case it was not tested. The pH dynamic range of sensing NPs (60 ± 4 nm) is between 5.0 and 10.0 (Gao et al., 2005) and between 4.0 and 8.0 for NSs (30 ± 4 nm) based on indicator Fluorescein (Gao et al., 2007). The response time (5 minutes) for both sensors is relatively long compared to other pH NSs (Table 1), even compared to macro-

Kim et al., 2006, designed ormosil NPs (33±6 nm) in which the ratiometric pH-responsive dye is covalently immobilized. Peng et al., 2007, and Burns et al., 2006, developed twofluorophore-immobilized NPs sensors. The first author used fluorescein isothiocyanate as an indicator dye that was covalently crosslinked with the network formed by the hydrolysis of tetraethoxysilane and aminoproplytriethoxysilane, while tris(2,2′-bipyridyl) dichlororuthenium(II) hexahydrate (reference) was entrapped simultaneously inside the same NSs (42 nm) by electrostatic interaction. The second author synthesized a nanosensor (70 nm) via a modified Stöber method, incorporating a reference (tetramethylrhodamineisothiocyanate) and an indicator dye (fluorescein isothiocyanate) covalently bound to the matrix in the coreshell architecture. In this case the pH sensor is composed of a shell of covalently bound indicator-dye molecules surrounding a core of sequestered, covalently bound, reference-dye molecules. What is interesting is that although both authors used covalent immobilization,

Two different approaches have been used for the development of polymeric ratiometric pH nanosensors. In the first case a reference dye (Rhodamine B derivate) and a pHsensitive dye (Aminofluorescein) were covalently embedded in polyacrylamide NPs (50 nm) (Sun et al., 2006). Allard & Larpent, 2008, presented a pH sensor based on polystyrene NPs, in the 20 nm range, with a hydrophobic reference dye (1,9 diphenylanthracene) embedded within the particle and the indicator dye (Fluorescein isothiocyanate) covalently attached to the surface. In this case the pH sensor may retain many of the drawbacks related to free fluorescent dyes because the indicator molecules are not protected from the cellular environment. It would be interesting to know, if the fotostability of the covalently attached indicator to the NP surface is improved compared

Oxygen is undoubtedly one of the most important analytes. In particular, a determination of the dissolved oxygen is of utmost importance in many fields of science and technology (Papkovsky & O'Riordan, 2005), including biotechnology (Kostov et al., 2001), biology (Schmaelzlin et al., 2005), marine science (Schroeder et al., 2007), and in medicine (Kimura et al., 2007). Recent interest in methods for measuring dissolved oxygen has been focused mainly on optical sensors, due to their advantages over conventional amperometric electrodes in that they are faster and do not consume oxygen (McDonagh et al., 2002).

The principle behind the operation of these sensors is the reduction in the luminescent intensity as a consequence of the oxygen quenching of the emitting state. The sensor optode,

sized pH optical sensors (Jerónimo et al., 2007).

Peng et al., 2007, reported an extensive dye leakage.

to the free indicator.

**3.4.2 Nanosensors for oxygen** 

Table 2 summarizes the recently developed ion NSs. Lead (Pb2+) is one of the most toxic heavy metals, and it is particularly dangerous in terms of its effects on children. Therefore, the determination of lead in low concentrations is important. Arduini et al., 2007, developed a fluorescent nanosensor for the detection of Pb2+ ions. Silica core-shell NPs were produced from 50 nm diameter "pure" silica cores and a 5-nm-thick shell in which the indicator dansyl dye was covalently linked to a silica network. Then the surface of the shell was functionalized with (mercaptopropyl) triethoxysilane. Surface thiol groups play a key role in the recognition of the analyte. Selectivity tests show that Cu2+ is a serious interferent. On the other hand, no effect is observed after the addition of Zn2+, Ca2+, and Mg2+, while Ni2+ and Co2+ produce much less quenching than Pb2+.

Copper (Cu2+) is an essential trace element that is required in various biological processes (hemopoiesis, metabolism, growth, and immune system (Uauy et al., 1998)). On the other hand, excess Cu2+ concentrations are extremely toxic. The detection of trace amounts of copper ions is of increasing importance in light of its environmental and biomedical implications. NSs for the detection of Cu2+ ions typically combine two components: a metal chelating molecule designed to bind the target ion selectively and a fluorophore as a readout system. The chelating molecules and the fluorophore are both covalently linked on to the surface of the NP (Brasola et al., 2003; Ramazzo et al., 2005) or the fluorophore is entrapped inside the NP (Méallet-Renault et al., 2004; Frigoli et al., 2009). The use of two different metal chelating ligands, picolinamide (Brasola et al., 2003) and (2-pyridinmethyl) glycinamide (Ramazzo et al., 2005), and the same fluorescent dye (danslyamide) for the surface modification of silica NPs were reported. Méallet-Renault et al., 2004, described the concept of associating an efficient fluorescent reporter, a BODIPY derivative, and an excellent metal-chelating receptor, cyclam, in polymer NPs. The hydrophobic dye is entrapped within the particle core and the receptor, covalently linked to the polymer backbone, is mainly located near the surface. The detection is based on the quenching of fluorescence by energy transfer from the dye to the receptor-target complex.

Core-shell-type dual-fluorescent NPs (NPs) in the 16-nm-diameter range with a selective ligand (cyclam) attached to the surface and two fluorophores, 9,10-diphenyl-anthracene (donor, D) and pyrromethene PM567 (acceptor, A), embedded within the polymer core were synthesized (Frigoli et al., 2009). Dual-dye-doped fluorescent NPs permit the design of cascade FRET-mediated sensing devices. The two sequential FRET processes, the energy transfer from D to A, and quenching of the resulting sensitized emission of A by copper complexes that are formed at the NP surface, provide an efficient means to sense

Optical Chemical Sensors: Design and Applications 19

et al., 2004)

et al., 2002)

et al., 2007)

Kopelman, 2005)

et al., 2003)

not given pH (Park

not given K+ (Sumner

et al., 2009)

8MPS – (mercaptopropyl)triethoxysilane 9DAP - 9,10-diphenyl-anthracene 10PM567 – pyrromethene

Analyte Material Indicator LOD (M) Response time Leaching Operational lifetime Interferences Ref. Pb2+ Sol-gel (TEOS, MPS8) Dansyl-

amide 1.1×10-6 not given not given >30 days Cu2+, Co2+, Ni2+

(Arduini et al., 2007) Cu2+ Sol-gel Dansyl-

amide not given not given not given not given Ni2+ (Brasola et al., 2003) Cu2+ Sol-gel Dansyl-

amide 3.0×10-8 not given not given not given Ni2+ (Ramazzo et al., 2005) Cu2+ latex BODIPY-

ciklomat 5×10-9 not given no leaching not given no (Méallet-Renault et al., 2004) Cu2+ Styren DAP9-

20×10-9 not given no >6 months no (Frigoli

PM56710-

ciklomat

Zn2+ Polyacry

Newport

4×10-6

<4 s intensity

signal drop for

3% aft. 2 days

TSQ12 2.6×10-7 not given not given not given Cu2+ Cd2+ (Teolato

1×10-6 10 min not given not given Cu(I) (Sumner &

lamide

Zn2+ Sol-gel

(TEOS)

Fe3+ Polyacry

Alexa

lamide

Mg2+ Polyacry

lamide

Ca2+ Sol-gel

(TEOS)

343

Fluor 448

Coumarin

340×10-6

<4 s intensity

signal drop for

less than 11.8

% after 24-h

signal drop for

not given pH (Hun & Zhang,

2007)

17% after 12-h

calcein 7.48×10-8 intensity

Green

Cu2+ Sol-gel QlOEt11 3.8× 10-7 not given not given not given Zn2+ (Zheng

Table 2. Nanosensors/probes for ions.

12TSQ - 6-methoxy-8-(p-toluensulfonamido)-quinoline

11QlOEt - N-(quinoline-8-yl)-2-(3-triethoxysilyl-propylamino)-acetamide

copperions by monitoring the quenching of the acceptor dye upon irradiation at the donor excitation wavelength. This sensor is highly selective towards other divalent cations. Zheng et al., 2010, have developed a Cu2+ sensor based on a fluorescent ligand (N- (quinoline-8-yl)-2-(3-triethoxysilyl-propylamino)-acetamide) that acts as both a binding and readout system.

Zinc (Zn2+) is the second most abundant trace element in humans after iron. Most of the Zn2+ found in the body is bound to proteins such as carbonic anhydrase or zinc finger proteins. Zinc has been implicated in Alzheimer's and Parkinson's disease (Cuajungco & Lees, 1997; Frederickson et al., 2000) and as a neuromodulator (Choi & Koh, 1998). Since the full extent of zinc's purpose in the body remains unclear, the development of nanosensors is needed to further elucidate the role of zinc in vivo. Sumner et al., 2002, designed a polyacrylamide PEBBLE sensor, containing immobilized Newport Green as the indicator dye and Texas Red-dextran as the reference dye. The maximum response to zinc (percentage increase in the "intensity ratio") is 50%, which is less than observed with the Newport Green dye in solution (increase of approx. 250%). It is obvious that the matrix is affecting the indicator dye's sensitivity, but is on the other hand preventing the indicator dye from reacting to non-specific bindings of proteins (ex. bovine serum albumin). This is a prime advantage of the PEBBLEs compared to free dyes, especially for cellular analysis. Teolato et al., 2007, selected the 6-methoxy-8-(p-toluensulfonamido)-quinoline (TSQ) as the active unit for the preparation of Zn2+ sensing silica NPs via the Stöber–Van Blaaderen procedure. The Coumarin 5 was used as a reference dye covalently linked to a silica network. The sensor suffers from the Cu2+ interferences and, to a minor extent, from Cd2+.

Iron (Fe3+) is an integral metal ion in several physiological processes involving electron transfer and oxidation. Numerous enzymes use Fe3+ as a cofactor for hydroxylation, oxygen transport, DNA synthesis, etc. (Frausto da Silva & Williams, 1991). Because of iron's vital role in a number of proteins, deficiency during the developmental stages can lead to an irreversible loss of motor skills and has been linked to behavioral alterations in irondeficient rats (Burdo & Connor, 2003). The accumulation of iron in the central nervous system has been implicated in a number of diseases. Therefore, the development of sensors that allow in-situ measurements would be beneficial. A PEBBLE sensor based on Alexa Fluor 448 (indicator dye) and Texas Red (reference) immobilized in polyacrylamide NPs for Fe3+ detection has been introduced (Sumner & Kopelman, 2005). What is surprising is that the reference dye is quenched by 20% in the presence of 1 µM copper.

Changes in intracellular ion concentrations accompany many processes in living cells, including transport, signalling, and enzyme function (Nuccitelli, 1994). A magnesiumsensitive PEBBLE, which contains immobilized Coumarin 343 as the indicator dye, and Texas Red-dextran as a reference dye, has been reported (Park et al., 2003). The interference from calcium is a major complication of current strategies aimed at the measurement of measuring magnesium concentrations in biological environments (Gotoh et al., 1999). The results show that Coumarin 343 is insensitive to Ca2+. A fluorescence-based calcium nanosensor was described (Hun & Zhang, 2007) that exploits silica NPs doped with calcein as an indicator dye for Ca2+ determination in blood serum. It is surprising that the effect of pH on the fluorescent intensity of the nanosensor was not tested in the clinically important pH range (6.6-7.8).


copperions by monitoring the quenching of the acceptor dye upon irradiation at the donor excitation wavelength. This sensor is highly selective towards other divalent cations. Zheng et al., 2010, have developed a Cu2+ sensor based on a fluorescent ligand (N- (quinoline-8-yl)-2-(3-triethoxysilyl-propylamino)-acetamide) that acts as both a binding

Zinc (Zn2+) is the second most abundant trace element in humans after iron. Most of the Zn2+ found in the body is bound to proteins such as carbonic anhydrase or zinc finger proteins. Zinc has been implicated in Alzheimer's and Parkinson's disease (Cuajungco & Lees, 1997; Frederickson et al., 2000) and as a neuromodulator (Choi & Koh, 1998). Since the full extent of zinc's purpose in the body remains unclear, the development of nanosensors is needed to further elucidate the role of zinc in vivo. Sumner et al., 2002, designed a polyacrylamide PEBBLE sensor, containing immobilized Newport Green as the indicator dye and Texas Red-dextran as the reference dye. The maximum response to zinc (percentage increase in the "intensity ratio") is 50%, which is less than observed with the Newport Green dye in solution (increase of approx. 250%). It is obvious that the matrix is affecting the indicator dye's sensitivity, but is on the other hand preventing the indicator dye from reacting to non-specific bindings of proteins (ex. bovine serum albumin). This is a prime advantage of the PEBBLEs compared to free dyes, especially for cellular analysis. Teolato et al., 2007, selected the 6-methoxy-8-(p-toluensulfonamido)-quinoline (TSQ) as the active unit for the preparation of Zn2+ sensing silica NPs via the Stöber–Van Blaaderen procedure. The Coumarin 5 was used as a reference dye covalently linked to a silica network. The sensor

Iron (Fe3+) is an integral metal ion in several physiological processes involving electron transfer and oxidation. Numerous enzymes use Fe3+ as a cofactor for hydroxylation, oxygen transport, DNA synthesis, etc. (Frausto da Silva & Williams, 1991). Because of iron's vital role in a number of proteins, deficiency during the developmental stages can lead to an irreversible loss of motor skills and has been linked to behavioral alterations in irondeficient rats (Burdo & Connor, 2003). The accumulation of iron in the central nervous system has been implicated in a number of diseases. Therefore, the development of sensors that allow in-situ measurements would be beneficial. A PEBBLE sensor based on Alexa Fluor 448 (indicator dye) and Texas Red (reference) immobilized in polyacrylamide NPs for Fe3+ detection has been introduced (Sumner & Kopelman, 2005). What is surprising is that

Changes in intracellular ion concentrations accompany many processes in living cells, including transport, signalling, and enzyme function (Nuccitelli, 1994). A magnesiumsensitive PEBBLE, which contains immobilized Coumarin 343 as the indicator dye, and Texas Red-dextran as a reference dye, has been reported (Park et al., 2003). The interference from calcium is a major complication of current strategies aimed at the measurement of measuring magnesium concentrations in biological environments (Gotoh et al., 1999). The results show that Coumarin 343 is insensitive to Ca2+. A fluorescence-based calcium nanosensor was described (Hun & Zhang, 2007) that exploits silica NPs doped with calcein as an indicator dye for Ca2+ determination in blood serum. It is surprising that the effect of pH on the fluorescent intensity of the nanosensor was not tested in the clinically important

suffers from the Cu2+ interferences and, to a minor extent, from Cd2+.

the reference dye is quenched by 20% in the presence of 1 µM copper.

and readout system.

pH range (6.6-7.8).

8MPS – (mercaptopropyl)triethoxysilane

9DAP - 9,10-diphenyl-anthracene

10PM567 – pyrromethene

11QlOEt - N-(quinoline-8-yl)-2-(3-triethoxysilyl-propylamino)-acetamide 12TSQ - 6-methoxy-8-(p-toluensulfonamido)-quinoline

Table 2. Nanosensors/probes for ions.

Optical Chemical Sensors: Design and Applications 21

A nonenzymatic sensor for glucose based on amino-functionalized luminescent silica NPs (LSNPs) doped with the europium(III) mixed complex, Eu(TTA)3phen with 2 thenoyltrifluoroacetone (TTA) and 1,10-phenanthroline(phen) was reported (Gao et al., 2009). Lapresta et al., 2009, developed a fluorescent polyacrylamide nanosensor for the determination of non-steroidal, anti-inflammatory drug naproxen. The fluorescent monomer (E)-4-[4-(dimethylamino)styryl]-1-[4-(methacryloyloxymethyl)benzyl]-pyridinium chloride (mDMASP) was used as an indicator. Upon indicator immobilization in polymer NPs the sensitivity and LOD were reduced. The high LOD thus enables those NSs only to be used in wastewater or industrial routine tests. For the detection of dipicolinic acid, a chemical marker for bacterial spores, silica NSs have been developed based on Eu-complex (Ai et al., 2009) and Tb-complex (Taylor & Lin, 2009). In both cases the EDTA ligand is covalently attached to the surface of silica NPs doped with reference dyes, such as fluorescein isothiocyanate (Ai et al., 2009) or Ru-complex (Taylor & Lin, 2009). Interferences resulting from the nonselective binding of aromatic ligands (benzoic acid, m-phthalic acid, o-phthalic acid) to a Tb-complex should be tested. Moreover, it is surprising that a Ru-complex, a common indicator for oxygen sensors (Li et al., 2007; Wu et al., 2010; Zhang et al., 2008) has been used as a reference, but the possible effect of oxygen on its fluorescence properties has

The design of an optical sensor is primarily governed by the application for which the sensor is to be developed. And because of the existence of many optical principles as well as of many sensor components that can be altered, optical chemical sensors (OCSs) can be applied to a huge number of applications. OCSs may either be very simple and cost-effective devices or enable advanced multisensor applications. In part by the sensitive layers, it is selectivity, sensitivity, stability, and reversibility that are the requirements for sensor systems that must be provided. Besides, the user normally expects the following sensor characteristics: a relatively high signal-to-noise ratio, short response times, low limits of

However, the most critical limitations of optical sensors result from unavailable or inadequate (unspecific) molecular probes. The detection limit that in certain cases is expected to lie in the 10-13 to 10-14 mol/L concentration range could also be improved. A preferred solution for improving the selectivity would be the use of sensor arrays (Paolesse et al., 2011), rather than a single sensor. It additionally turns out that most of the improvements that can be expected in optical detection methods are in the area of sensitive

Another step in achieving improvements in sensor characteristics is the use of nanoscale materials for OCS, which has emerged as one of the most important research areas over the past decade. Nanomaterials exhibit highly tunable size- and shape-dependent chemical and physical properties show a unique surface chemistry, thermal and electrical properties, a high surface area and a large pore-volume per mass unit area. Because of their unique and advantageous features they can help to improve the sensitivity, response time and detection limit of sensors. Nevertheless, although zero-dimensional materials are applied in sensor design, the factors such as matrix, indicator and immobilization technique are still

detection, high sensitivity, low cost, a possibility for on-line, in-situ applications.

layers, which also have to provide reversibility and greater stability.

indispensable tools in achieving the desired sensor's characteristics.

not been tested.

**4. Summary and trends** 

#### **3.4.4 Nanosensors for other molecules**

The characteristics of NSs for the detection of glucose, naproxen and dipicolinic acid are summarized in Table 3.


13mDMASP - (E)-4-[4-(dimethylamino)styryl]-1-[4-(methacryloyloxymethyl)benzyl]pyridinium chloride

14cAMP - Adenosine 3:5-cyclic monophosphate

15EDTA - Ethylenediamine tetraacetic acid

Table 3. Nanosensors/probes for other molecules

The characteristics of NSs for the detection of glucose, naproxen and dipicolinic acid are

et al., 2009)

2009)

13mDMASP - (E)-4-[4-(dimethylamino)styryl]-1-[4-(methacryloyloxymethyl)benzyl]pyridinium

acid

Sol-gel

Eu-EDTA15

(TEOS,

complex

APTES)

dipicoli-nic

Sol-gel

Tb-EDTA

10.3×10-9

not given not given not given no (Taylor & Lin,

(TEOS,

complex

APTES)

acid

Analyte Material Indicator LOD (M) Response time Leaching Operational lifetime Interferences Ref. naproxen Polyacryl

0.5×10-3 not given not given not given cAMP14 (Lapresta et al., 2009) glucose Sol-gel (TEOS1, APTES5) Eu complex

4.4×10-6 20 min not given not given no in the 5% range of signal change (Gao et al., 2009) dipicoli-nic

0.2×10-9 30 s not given not given no (Ai

 -amide mDMASP13

**3.4.4 Nanosensors for other molecules** 

summarized in Table 3.

chloride

14cAMP - Adenosine 3:5-cyclic monophosphate 15EDTA - Ethylenediamine tetraacetic acid

Table 3. Nanosensors/probes for other molecules

A nonenzymatic sensor for glucose based on amino-functionalized luminescent silica NPs (LSNPs) doped with the europium(III) mixed complex, Eu(TTA)3phen with 2 thenoyltrifluoroacetone (TTA) and 1,10-phenanthroline(phen) was reported (Gao et al., 2009). Lapresta et al., 2009, developed a fluorescent polyacrylamide nanosensor for the determination of non-steroidal, anti-inflammatory drug naproxen. The fluorescent monomer (E)-4-[4-(dimethylamino)styryl]-1-[4-(methacryloyloxymethyl)benzyl]-pyridinium chloride (mDMASP) was used as an indicator. Upon indicator immobilization in polymer NPs the sensitivity and LOD were reduced. The high LOD thus enables those NSs only to be used in wastewater or industrial routine tests. For the detection of dipicolinic acid, a chemical marker for bacterial spores, silica NSs have been developed based on Eu-complex (Ai et al., 2009) and Tb-complex (Taylor & Lin, 2009). In both cases the EDTA ligand is covalently attached to the surface of silica NPs doped with reference dyes, such as fluorescein isothiocyanate (Ai et al., 2009) or Ru-complex (Taylor & Lin, 2009). Interferences resulting from the nonselective binding of aromatic ligands (benzoic acid, m-phthalic acid, o-phthalic acid) to a Tb-complex should be tested. Moreover, it is surprising that a Ru-complex, a common indicator for oxygen sensors (Li et al., 2007; Wu et al., 2010; Zhang et al., 2008) has been used as a reference, but the possible effect of oxygen on its fluorescence properties has not been tested.

## **4. Summary and trends**

The design of an optical sensor is primarily governed by the application for which the sensor is to be developed. And because of the existence of many optical principles as well as of many sensor components that can be altered, optical chemical sensors (OCSs) can be applied to a huge number of applications. OCSs may either be very simple and cost-effective devices or enable advanced multisensor applications. In part by the sensitive layers, it is selectivity, sensitivity, stability, and reversibility that are the requirements for sensor systems that must be provided. Besides, the user normally expects the following sensor characteristics: a relatively high signal-to-noise ratio, short response times, low limits of detection, high sensitivity, low cost, a possibility for on-line, in-situ applications.

However, the most critical limitations of optical sensors result from unavailable or inadequate (unspecific) molecular probes. The detection limit that in certain cases is expected to lie in the 10-13 to 10-14 mol/L concentration range could also be improved. A preferred solution for improving the selectivity would be the use of sensor arrays (Paolesse et al., 2011), rather than a single sensor. It additionally turns out that most of the improvements that can be expected in optical detection methods are in the area of sensitive layers, which also have to provide reversibility and greater stability.

Another step in achieving improvements in sensor characteristics is the use of nanoscale materials for OCS, which has emerged as one of the most important research areas over the past decade. Nanomaterials exhibit highly tunable size- and shape-dependent chemical and physical properties show a unique surface chemistry, thermal and electrical properties, a high surface area and a large pore-volume per mass unit area. Because of their unique and advantageous features they can help to improve the sensitivity, response time and detection limit of sensors. Nevertheless, although zero-dimensional materials are applied in sensor design, the factors such as matrix, indicator and immobilization technique are still indispensable tools in achieving the desired sensor's characteristics.

Optical Chemical Sensors: Design and Applications 23

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Geigenberger, P. & Loehmannsroeben, H.-G. (2005). An optical multifrequency phase-modulation method using microbeads for measuring intracellular oxygen


**2** 

*Russia* 

**Optical Sensors Based on Opal Film and Silica** 

Ivan Boldov1, Natalia Orlova2, Irina Kargapolova2, Alexandr Kuchyanov1,

Chemical sensing using optics is under extensive research all over the world and many optical chemical sensors are finding increasing application in industry, environmental monitoring, medicine, biomedicine and chemical analysis (Baldini et al., 2006). Optical sensors can be used as fiber optics microsensors, as planar coatings in bioreactors, in microtiterplate format, in disposable single-shot device, and as planar membranes that can be imaged using sensing camera. The spectral range extends from the UV to the infrared, and from absorption to emission and to surface plasmon resonance (Narayanaswamy & Wolfbeis, 2004). Hence, a variety of schemes are conceivable. The purpose of this chapter is to introduce the concept of highly sensitive and selective optical sensors for different type

reagent based on opal film and silica nanoparticles modified with a functional dye.

integrate photonic devices on high functionality optical chips.

In the last two decades photonic crystals have much potential for creation of new optical functional materials (Joannopoulos et al., 2008). Photonic crystals are inhomogeneous media with a periodically varying relative permittivity. The capability of such periodic structures to form photonic band gaps changes the paradigm of controlling the propagation of light. For this reason, the features of the behavior of refracted light in the allowed bands and at the edge of the band gaps currently attract a lot of attention. A number of unusual properties of the propagation of light in photonic crystals such as the refraction of light in negative media (Foteinopoulou et al., 2003) and the effect of a superprism and the self-collimation of light (Kosaka et al., 1998) were recently revealed. It was proposed to use these effects for creating a supersensitive light beam splitter (Baba & Nakamura, 2002; Wu et al., 2003) and for controlling an optical flux (Chen et al., 2004). Moreover, schemes with photonic crystals can underlie supersensitive optical chemical sensors (Yakimansky et al., 2009). For such applications, it is important to determine and analyze regions with a strong angular dispersion in optical systems with photonic crystals and to study the behavior of light at the interface of photonic crystals with other optical media. Extension of sensory capabilities of photonic crystals by the chemical modification of silica nanoparticles with functional dyes offers promise as a high selective fluorescent sensor and many ways to miniaturize and

**1. Introduction** 

**Nanoparticles Modified with a Functional Dye** 

Vladimir Shelkovnikov2 and Alexandr Plekhanov1 *1Institute of Automation and Electrometry, Siberian Branch,* 

*2Novosibirsk Institute of Organic Chemistry, Siberian Branch,* 

*Russian Academy of Sciences,* 

*Russian Academy of Sciences,* 

