**3. ROS and oxygen sensing**

One very important class of compounds that play a major role in regulating biological processes, which also have a close relationship with the differentiated metabolism profile of tumor cells, is the reactive oxygen species, ROS, and for that reason, they receive significant attention in sensing/biosensing research. ROS are very reactive free radicals that act as electron acceptors, thus being strong oxidizing agents, which react with any neighboring molecule in order to attain a stable configuration. Hydrogen peroxide, the superoxide anion, and the singlet excited states of oxygen are examples of ROS. These molecules are produced physiologically mainly as a by-product of oxygen metabolism during electron transfer events in respiratory chain processes. Since they are highly reactive, ROS are harmful for the cells, and antioxidant enzymes located in the cytosol and mitochondria are responsible for a delicate regulation process that control the oxidative stress generated by ROS. Despite their toxic effects, moderate levels of ROS play a role in vital biological processes, such as biological signaling, chemical defense, biosynthetic reactions, etc. [12]. For instance, in biological signaling, the ROS act as secondary messengers in cellular adhesion, spreading, and migration, thus governing the proliferation and, ultimately, the survival of cells [13].

*Biosensors for Environmental Monitoring*

fluorescence intensity and decay time of the donor are decreased, as depicted in the Jablonski diagram and spectrum models in **Figure 1**. The energy transfer efficiency in FRET depends on the distance between donor and acceptor: when the donoracceptor pair is in between 20 and 60 angstroms apart, which is called the Förster radius, the efficiency of energy transfer is around 50%. The efficiency also depends on the spectral overlap between the absorption spectrum of the acceptor and the emission spectrum of the donor: the greater the overlap, the more efficient the

*Illustration of the components and function of biosensors based on fluorescence spectroscopy transducing.*

Since FRET does not require contact between the electronic clouds of donor and acceptor, it can occur over macromolecular distances. This is one of the reasons, along with energy transfer efficiency, responsible for the high sensitivity of FRETbased sensors. Low concentrations of analyte that would result in great distances from the donor-acceptor pair would most likely involve FRET rather than collisional quenching, which requires physical contact, in order to bring about a change in the FRET process necessary for sensing. Besides, since the donor and acceptor in a FRET sensor do not need to be bound molecules, it simplifies the design of the fluorophore because the donor is not required to be intrinsically sensitive to the

A great variety of FRET sensors and biosensors can be found in the literature. The major advantages that make them special are high sensitivity and fast response. Besides, due to the biocompatibility of FRET-based biosensors, allied to the inherent sensitivity of optical sensing techniques, they are becoming ubiquitous in clinical applications and in the field of biomedical research. A recent example is a fluorescent peptide/GO sensor containing a fluorophore-labeled peptide sequence that proved versatile for measuring the activity of different protein kinases. Kinases are group of proteins that regulates intracellular phosphorylation pathways. Several deceases such as cancer, diabetes, Alzheimer's, etc. are related to anomalous activity of kinase proteins. In their sensor, the fluorophore-labeled peptide sequence is cleaved by the carboxypeptidase, in the absence of phosphorylation, and is separated from the GO, resulting in recovery

analyte and can be chosen according to the desired light source [4].

**70**

of fluorescence [5].

process.

**Figure 1.**

Multicellular organisms also maintain a homeostasis of O2 levels by regulating the distribution of O2 according to the demands of each organ. In this context, the cells also respond to hypoxia condition, a state of insufficient levels of O2 necessary for maintaining nominal cellular function, in an effort to adapt to such condition and ensure its survival. This condition is always present in tumor cells, and understanding the cell mechanisms of coping with hypoxia is crucial for understanding tumor growth and survival. For that to work, the cells have a sensing mechanism for O2. The hypoxia-inducible factors HIF-1 and HIF-2 are oxygen sensitive transcriptional complexes that mediate the response of cells to O2 levels [14].

It has been reported that the level of ROS in the cells is related to the activity of HIF-α factor. However, elucidation of cell metabolism related to or governed by ROS, as well as the cellular oxygen sensing mechanisms, has been hampered by the difficulties in tracking intracellular ROS kinetics with reliability using fluorescent dyes. FRET-based sensors have demonstrated to overcome these obstacles [14–16]. Guzy et al. suggested that mitochondria functions as an O2 sensor and signal hypoxia-induced HIF stabilization by releasing ROS to the cytosol. To confirm this debated hypothesis, they fabricated a reliable and accurate redox-sensitive FRET protein sensor for intracellular ROS determination that consists of a cyan and yellow fluorescent protein as the donor-acceptor pair linked by a redox-regulated HPS33 protein domain. Oxidation of the protein domain by ROS causes a conformational change, which alters the donor-acceptor distance required for FRET efficiency, thus leading to fluorescence recovery [14]. More recently, Bernardini et al. also used this same sensor configuration for monitoring the kinetics of ROS in cultured cells of mouse carotid body, seeking to elucidate the role of a NADPH oxidase subunit on cell response to O2 levels [15].

Overall, cyan and yellow fluorescent protein FRET sensors containing an internal metabolite-binding protein are successfully used for monitoring biomolecules in real time, metabolite dynamics, protein interaction, and signal transduction, due to the fast response ability of these sensors. Just like in aptamer sensors, the choice of the right metabolite-binding protein is the key for obtaining a highly selective FRET sensor for the desired target [16].

Apart from reactive oxygen species, the O2 itself is also an important indicator of biological processes, including the monitoring of decease progression. A variety of optical biosensors for oxygen determination are found in the literature, given the importance of this molecule in the study of biological systems.

Recently, fluorescence biosensors based on QDs are proving their potential for monitoring tumor activity, thanks to advantages such as the ability to penetrate solid tumor cells, high photoluminescence quantum yields, photostability, and the other photophysical properties already mentioned. Oxygen-sensitive phosphorescent molecules are particularly interesting for detecting oxygen in biological systems, because it is noninvasive and has high spatiotemporal resolution [17].

The underlying energy transfer mechanism involved in oxygen sensing is mostly a triplet-triplet annihilation process. After absorption of light, the sensing molecule is excited to a singlet excited state, S1. Subsequently, it can return to the ground state, S0, through fluorescence or undergo intersystem crossing to a triplet excited state, T1, and then return to the S0 state by phosphorescence. Alternatively, in a competing process, the molecule in the T1 state can interact with molecular oxygen in the ground triplet state via collisional quenching. When this happens, a triplettriplet annihilation process occurs, which is characterized by generation of excitedstate singlet oxygen, as illustrated in the Jablonsky diagram in **Figure 2**. This process involving the O2 is also called a photosensitization effect.

**73**

FRET process.

**Figure 2.**

*molecular oxygen.*

*Dynamic Luminescent Biosensors Based on Peptides for Oxygen Determination*

also decreased. The oxygen concentration can be determined in a steady-state or time-resolved manner, in which the intensity or lifetime decrease of the quenched molecule is related to the concentration of oxygen by the Stern-Volmer kinetics of

*Jablonsky energy diagram illustrating a T-T annihilation process involving a photosensitizer molecule and* 

In this expression, I0 and I are the fluorescence intensities in the absence and presence of O2, respectively, τ0 is the natural fluorescence lifetime of the molecule in the absence of O2, τ is the lifetime at a given oxygen concentration, [Q ], and kq is

However, intersystem crossing to T1 state is generally a slow process, and in order to achieve an oxygen sensor of high sensitivity, the population of the triplet state must be maximized. The addition of heavy metal atoms can circumvent this limitation by increasing spin-orbit coupling, which favors the forbidden transition between electronic states of different spin multiplicities. Still, once in the T1 state, the phosphorescence competes with energy transfer to O2, which further limits the sensitivity of the sensor. The coupling of an oxygen-responsive phosphorescent molecule with a QD results in a sensor with enhanced sensitivity toward oxygen. FRET readily occurs between the molecule and QD, which form the donor-acceptor pair, and emission becomes oxygen-dependent due to the analyte interference in the

In addition to FRET, QD sensors can also function through Dexter exchange interactions, another mechanism of nonradiative energy transfer between an acceptor-donor pair, which involves the donation of an electron from the LUMO orbital of the donor followed by the transfer of an electron from the acceptor HOMO orbital to the HOMO of the donor. Unlike the FRET, Dexter interactions require physical contact between donor and acceptor. Both mechanisms can compete depending on the degree of spectral overlap. It has been found that the size of QD determines the predominant energy transfer mechanism in QD-based sensors: smaller QD, which exhibit low spectral overlap and high orbital overlap with the donor, favors Dexter energy transfer, whereas in larger QDs, FRET dominates [17]. Belonging to the realm of QD-based sensors for oxygen, self-assembled sensors

have interesting advantages including ease of preparation and allow easy finetuning of donor-acceptor ratio. In general, these sensors are comprised of a metal ion bound to functional groups such as imidazole, amines, etc. and an acceptor molecule attached to the functional group. For instance, Lemon et al. managed to red shift the emission of a CdSe QD sensor reported previously in order to increase

<sup>τ</sup> = 1 + *kq* τ0[Q](1) (1)

I0 <sup>I</sup> or\_\_ τ0

*DOI: http://dx.doi.org/10.5772/intechopen.84143*

collisional quenching, as shown below.

the bimolecular quenching rate constant [4].

\_\_

Consequently, a quenching of fluorescence or phosphorescence (depending on what radiative process is being monitored) is observed. The lifetime is *Dynamic Luminescent Biosensors Based on Peptides for Oxygen Determination DOI: http://dx.doi.org/10.5772/intechopen.84143*

**Figure 2.**

*Biosensors for Environmental Monitoring*

Multicellular organisms also maintain a homeostasis of O2 levels by regulating the distribution of O2 according to the demands of each organ. In this context, the cells also respond to hypoxia condition, a state of insufficient levels of O2 necessary for maintaining nominal cellular function, in an effort to adapt to such condition and ensure its survival. This condition is always present in tumor cells, and understanding the cell mechanisms of coping with hypoxia is crucial for understanding tumor growth and survival. For that to work, the cells have a sensing mechanism for O2. The hypoxia-inducible factors HIF-1 and HIF-2 are oxygen sensitive transcrip-

It has been reported that the level of ROS in the cells is related to the activity of HIF-α factor. However, elucidation of cell metabolism related to or governed by ROS, as well as the cellular oxygen sensing mechanisms, has been hampered by the difficulties in tracking intracellular ROS kinetics with reliability using fluorescent dyes. FRET-based sensors have demonstrated to overcome these obstacles [14–16]. Guzy et al. suggested that mitochondria functions as an O2 sensor and signal hypoxia-induced HIF stabilization by releasing ROS to the cytosol. To confirm this debated hypothesis, they fabricated a reliable and accurate redox-sensitive FRET protein sensor for intracellular ROS determination that consists of a cyan and yellow fluorescent protein as the donor-acceptor pair linked by a redox-regulated HPS33 protein domain. Oxidation of the protein domain by ROS causes a conformational change, which alters the donor-acceptor distance required for FRET efficiency, thus leading to fluorescence recovery [14]. More recently, Bernardini et al. also used this same sensor configuration for monitoring the kinetics of ROS in cultured cells of mouse carotid body, seeking to elucidate the role of a NADPH

Overall, cyan and yellow fluorescent protein FRET sensors containing an internal metabolite-binding protein are successfully used for monitoring biomolecules in real time, metabolite dynamics, protein interaction, and signal transduction, due to the fast response ability of these sensors. Just like in aptamer sensors, the choice of the right metabolite-binding protein is the key for obtaining a highly selective FRET

Apart from reactive oxygen species, the O2 itself is also an important indicator of biological processes, including the monitoring of decease progression. A variety of optical biosensors for oxygen determination are found in the literature, given the

Recently, fluorescence biosensors based on QDs are proving their potential for monitoring tumor activity, thanks to advantages such as the ability to penetrate solid tumor cells, high photoluminescence quantum yields, photostability, and the other photophysical properties already mentioned. Oxygen-sensitive phosphorescent molecules are particularly interesting for detecting oxygen in biological systems, because it is noninvasive and has high spatiotemporal resolution [17].

The underlying energy transfer mechanism involved in oxygen sensing is mostly a triplet-triplet annihilation process. After absorption of light, the sensing molecule is excited to a singlet excited state, S1. Subsequently, it can return to the ground state, S0, through fluorescence or undergo intersystem crossing to a triplet excited state, T1, and then return to the S0 state by phosphorescence. Alternatively, in a competing process, the molecule in the T1 state can interact with molecular oxygen in the ground triplet state via collisional quenching. When this happens, a triplettriplet annihilation process occurs, which is characterized by generation of excitedstate singlet oxygen, as illustrated in the Jablonsky diagram in **Figure 2**. This process

Consequently, a quenching of fluorescence or phosphorescence (depending on what radiative process is being monitored) is observed. The lifetime is

tional complexes that mediate the response of cells to O2 levels [14].

oxidase subunit on cell response to O2 levels [15].

importance of this molecule in the study of biological systems.

involving the O2 is also called a photosensitization effect.

sensor for the desired target [16].

**72**

*Jablonsky energy diagram illustrating a T-T annihilation process involving a photosensitizer molecule and molecular oxygen.*

also decreased. The oxygen concentration can be determined in a steady-state or time-resolved manner, in which the intensity or lifetime decrease of the quenched molecule is related to the concentration of oxygen by the Stern-Volmer kinetics of collisional quenching, as shown below.

$$\frac{\mathbf{I}\_0}{\mathbf{I}} \text{or} \frac{\boldsymbol{\pi}\_0}{\boldsymbol{\pi}} = \mathbf{1} + kq \,\, \boldsymbol{\pi}\_0 \, [\mathbf{Q}] \, \{\mathbf{1}\} \tag{1}$$

In this expression, I0 and I are the fluorescence intensities in the absence and presence of O2, respectively, τ0 is the natural fluorescence lifetime of the molecule in the absence of O2, τ is the lifetime at a given oxygen concentration, [Q ], and kq is the bimolecular quenching rate constant [4].

However, intersystem crossing to T1 state is generally a slow process, and in order to achieve an oxygen sensor of high sensitivity, the population of the triplet state must be maximized. The addition of heavy metal atoms can circumvent this limitation by increasing spin-orbit coupling, which favors the forbidden transition between electronic states of different spin multiplicities. Still, once in the T1 state, the phosphorescence competes with energy transfer to O2, which further limits the sensitivity of the sensor. The coupling of an oxygen-responsive phosphorescent molecule with a QD results in a sensor with enhanced sensitivity toward oxygen. FRET readily occurs between the molecule and QD, which form the donor-acceptor pair, and emission becomes oxygen-dependent due to the analyte interference in the FRET process.

In addition to FRET, QD sensors can also function through Dexter exchange interactions, another mechanism of nonradiative energy transfer between an acceptor-donor pair, which involves the donation of an electron from the LUMO orbital of the donor followed by the transfer of an electron from the acceptor HOMO orbital to the HOMO of the donor. Unlike the FRET, Dexter interactions require physical contact between donor and acceptor. Both mechanisms can compete depending on the degree of spectral overlap. It has been found that the size of QD determines the predominant energy transfer mechanism in QD-based sensors: smaller QD, which exhibit low spectral overlap and high orbital overlap with the donor, favors Dexter energy transfer, whereas in larger QDs, FRET dominates [17].

Belonging to the realm of QD-based sensors for oxygen, self-assembled sensors have interesting advantages including ease of preparation and allow easy finetuning of donor-acceptor ratio. In general, these sensors are comprised of a metal ion bound to functional groups such as imidazole, amines, etc. and an acceptor molecule attached to the functional group. For instance, Lemon et al. managed to red shift the emission of a CdSe QD sensor reported previously in order to increase its sensitivity at physiological O2 pressure by pairing Pd(II) porphyrins, which emits at 690 nm, with the CdSe core-shell QDs that emit at 519 nm. The QD was chosen to maximize spectral overlap with Pd(II) porphyrin absorption, thereby increasing FRET efficiency to 94%, greatly improving sensitivity [18]. Later on, other authors have used Au(III) corroles to shift the emission even further to the NIR [19]. Red emitting sensors are interesting for biomedical applications due to the greater penetration of red light into organic tissues and less scattering. These contributions reveal the versatility of QD-based sensors, which can be easily designed and adjusted to fulfill the desired purpose.

Fluorescence microscopy is another common technique in biological and clinical fields for visualizing intracellular structures, both in vitro and in vivo, which is based on the staining of a cell with a fluorescent probe. It is also possible to determine intracellular concentration of analytes of interest and monitor reactions. However, one major problem of microscopy based on steady-state intensity is the intensity dependence on the probe concentration. The difficulty in knowing the probe concentration within the different regions of the cell impedes quantitative measurements with reliability. Fluorescence lifetime imaging, on the other hand, circumvents this problem because the lifetime of the fluorophore probe is independent on its concentration. Therefore, variations in lifetime due to interactions of the probe with biomolecules can be correlated to analyte concentration regardless of the probe concentration. For this reason, high fidelity images with improved contrasts can be achieved. Lifetime imaging is employed, for example, in intracellular oxygen sensing, which is not possible via any microscopic method based on intensity measurements [4].
