**2. Principles and evolution of biosensing techniques**

Biosensors are devices that can perform the measurement of a physiological activity in living organisms or that are constructed upon biological components. They determine chemical or biological analytes in systems where the minimum human intervention is present. They generate optical and/or electrochemical signals that are transduced by a variety of transducers, and depending on their operation, biosensors are classified.

To fulfill an application, biosensors can be constructed by using a wide variety of bioreceptors, that can deliver distinct types of signals and the choice of transducers and interfaces will respond for their selectivity and sensibility, as well as their configuration versatility and possibility of miniaturization. Techniques such as voltammetry, amperometry, potentiometry, among others, are exploited to transduce electrochemical signals, whereas fluorescence, light absorption or light reflectance in the ultraviolet (UV), visible, or near-infrared (NIR) spectral regions can have their intensity or lifetime changes determined to efficiently detect optical responses in optical biosensors. Also, there is a variety of biosensors that can explore dual or multiple transducers to deliver electrical and luminous signals that can be interpreted together or separately, giving rise for more versatile and usable biosensors.

Independent of their usage and characteristics, the basic configuration of the biosensors is similar. They must be composed of a sample holder, which is adapted to the sample physical characteristics; a biological recognition element, which must

**69**

surgery.

*Dynamic Luminescent Biosensors Based on Peptides for Oxygen Determination*

be highly selective; and a physical transducer to generate a measurable signal, a signal processor, and an interface that is able to communicate the data to the operator. The types of biosensor that can be constructed are based on the recognition elements employed, which can be any biological system, from antibodies to microbes and cells. It is selected considering the information to be obtained and the physical characteristics of the biosensor, which in its turn, determines the durability and the

The specificity and the sensitivity of biosensors have been the concern of several

scientists due to the variety of biochemical processes that need to be evaluated and followed, the increase of accuracy and reproducibility of measurements that is fundamental for the spread of usage of such devices, and due to the need for miniaturizing and automatizing devices, in order to turn them more applicable to

In special, optical biosensors can be widely applied if they accompanied the development of the spectroscopic and microscopic technique development to improve their transducing methods, signal processing, and interface. If these components are well developed, optical biosensors and, in special, fluorescent biosensors can present high sensitivity, perform real-time measurements with high frequency of detection, which enable them to find application in diagnostics and therapeutics, with the right transducers, it is possible to image disease progression and to monitor the organism response to therapeutics, and also, they can be thought for drug discovery programs development, as well as for clinical evaluation of new drugs [1]. In recent years, recognition elements based on graphene had been widely used due to their excellent electrical and optical properties. In biosensing, materials based on graphene had promoted efficient detection of biomarkers and have proportioned an important technological advance, due to the perspective of developing new and interesting materials, such as graphene-like 2D materials and

the impressive single-atom-thick layers of van der Waals materials [2].

molecule, enabling a single-molecule analysis [3].

Most of the fluorescent biosensors are small molecules that are arranged on a receptor that identifies a specific target, and its fluorescent response is readily transduced. In this case, the signal recognition is based on the distinct fluorescence emitted by the biosensor in the presence of the analyte, which can be metabolites, proteins, ions, or antibodies. These biosensors are based on the steady-state fluorescence that the device can produce. They find effective application for early biomarker detection, for instance, in clinical diagnostics and ordinary biochemical processes, as well as for tissue imaging and, as an extrapolation, in image guided

Biosensors based on time-resolved fluorescence are promising because they are able to promote improvements on selectivity, specificity, and sensibility, becoming ultrasensitives, capable of determining minimal local variations of the analyte concentration, and can be combined to several other analytical techniques. In this context, there are the nanopores, which are highly sensitive biosensors, able to detect the analyte at the range of nanomolar of concentration, due to their characteristic structure of a nanometer scale pore that is similar to the size of the target

Any of the fluorescence properties of the recognition element, namely, the intensity, wavelength, anisotropy or lifetime, can be exploited in optical sensing. One straightforward mechanism to consider is collisional quenching, in which a fluorophore has its fluorescence quenched upon collision with the analyte molecule. Nevertheless, the most encountered and relevant mechanism in sensing is the Förster Resonance Energy Transfer (FRET), which occurs via long range dipole-dipole interaction when an energy donor, in an excited state, and an acceptor are brought into close proximity, but not necessarily into contact. In FRET, the

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

distinct regions of a living organism.

more applicable processes to construct the biosensor.

#### *Dynamic Luminescent Biosensors Based on Peptides for Oxygen Determination DOI: http://dx.doi.org/10.5772/intechopen.84143*

be highly selective; and a physical transducer to generate a measurable signal, a signal processor, and an interface that is able to communicate the data to the operator. The types of biosensor that can be constructed are based on the recognition elements employed, which can be any biological system, from antibodies to microbes and cells. It is selected considering the information to be obtained and the physical characteristics of the biosensor, which in its turn, determines the durability and the more applicable processes to construct the biosensor.

The specificity and the sensitivity of biosensors have been the concern of several scientists due to the variety of biochemical processes that need to be evaluated and followed, the increase of accuracy and reproducibility of measurements that is fundamental for the spread of usage of such devices, and due to the need for miniaturizing and automatizing devices, in order to turn them more applicable to distinct regions of a living organism.

In special, optical biosensors can be widely applied if they accompanied the development of the spectroscopic and microscopic technique development to improve their transducing methods, signal processing, and interface. If these components are well developed, optical biosensors and, in special, fluorescent biosensors can present high sensitivity, perform real-time measurements with high frequency of detection, which enable them to find application in diagnostics and therapeutics, with the right transducers, it is possible to image disease progression and to monitor the organism response to therapeutics, and also, they can be thought for drug discovery programs development, as well as for clinical evaluation of new drugs [1]. In recent years, recognition elements based on graphene had been widely used due to their excellent electrical and optical properties. In biosensing, materials based on graphene had promoted efficient detection of biomarkers and have proportioned an important technological advance, due to the perspective of developing new and interesting materials, such as graphene-like 2D materials and the impressive single-atom-thick layers of van der Waals materials [2].

Most of the fluorescent biosensors are small molecules that are arranged on a receptor that identifies a specific target, and its fluorescent response is readily transduced. In this case, the signal recognition is based on the distinct fluorescence emitted by the biosensor in the presence of the analyte, which can be metabolites, proteins, ions, or antibodies. These biosensors are based on the steady-state fluorescence that the device can produce. They find effective application for early biomarker detection, for instance, in clinical diagnostics and ordinary biochemical processes, as well as for tissue imaging and, as an extrapolation, in image guided surgery.

Biosensors based on time-resolved fluorescence are promising because they are able to promote improvements on selectivity, specificity, and sensibility, becoming ultrasensitives, capable of determining minimal local variations of the analyte concentration, and can be combined to several other analytical techniques. In this context, there are the nanopores, which are highly sensitive biosensors, able to detect the analyte at the range of nanomolar of concentration, due to their characteristic structure of a nanometer scale pore that is similar to the size of the target molecule, enabling a single-molecule analysis [3].

Any of the fluorescence properties of the recognition element, namely, the intensity, wavelength, anisotropy or lifetime, can be exploited in optical sensing. One straightforward mechanism to consider is collisional quenching, in which a fluorophore has its fluorescence quenched upon collision with the analyte molecule. Nevertheless, the most encountered and relevant mechanism in sensing is the Förster Resonance Energy Transfer (FRET), which occurs via long range dipole-dipole interaction when an energy donor, in an excited state, and an acceptor are brought into close proximity, but not necessarily into contact. In FRET, the

*Biosensors for Environmental Monitoring*

techniques.

brane interactions.

analytical methods.

biosensors are classified.

monitoring of physiological conditions. Sensing methods of remarkable sensitivity, reliability, and selectivity based on fluorescence spectroscopy dominates the field of sensing and biosensing. DNA sequencing and fragment analysis, fluorescence staining for bioimaging and fluorescence immunoassays are all based on fluorescence

The countless possibilities of combinations of biorecognition element, support matrix, and the transducing method in biosensors constituted of nanomaterials make it possible to design versatile and selective biosensors. In this review, particular attention is centered on luminescent biosensors based on the Förster resonance energy transfer, or FRET, biosensing transducing method, which encompasses a huge variety of biosensors, due to their unique sensitivity, selectivity, and fast response. For this reason, FRET-based sensors have enabled, for example, intracellular monitoring of ROS kinetics and oxygen sensing, which is vital for elucidating how tumor cells respond to treatment, in order to develop better therapeutic strategies. Before FRET sensors were introduced, these practices were hampered by difficulties and unreliability in real-time monitoring intracellular ROS. In fluorescence bioimaging, thanks to its high spatial and temporal resolution, it is becoming possible to probe in real time biological elements and processes, such as enzymatic reactions, protein/protein, protein/nucleic acid, protein/substrate, and biomem-

In the context of environmental monitoring, with the possibility of miniaturization of biosensors based on nanomaterials, it is becoming possible to perform fast and accurate field analysis and real-time surveillance of analytes relevant in the assessment of water quality. A variety of FRET-based biosensors for water pollutants, such as heavy metal ions, pesticides, antibiotics, and halogenated compounds, is reported, some of them capable of detecting concentrations in the pico and nanomolar scales. Such sensitivity levels are far from reach with conventional

Biosensors are devices that can perform the measurement of a physiological activity in living organisms or that are constructed upon biological components. They determine chemical or biological analytes in systems where the minimum human intervention is present. They generate optical and/or electrochemical signals that are transduced by a variety of transducers, and depending on their operation,

To fulfill an application, biosensors can be constructed by using a wide variety of bioreceptors, that can deliver distinct types of signals and the choice of transducers and interfaces will respond for their selectivity and sensibility, as well as their configuration versatility and possibility of miniaturization. Techniques such as voltammetry, amperometry, potentiometry, among others, are exploited to transduce electrochemical signals, whereas fluorescence, light absorption or light reflectance in the ultraviolet (UV), visible, or near-infrared (NIR) spectral regions can have their intensity or lifetime changes determined to efficiently detect optical responses in optical biosensors. Also, there is a variety of biosensors that can explore dual or multiple transducers to deliver electrical and luminous signals that can be interpreted together or separately, giving rise for more versatile and usable

Independent of their usage and characteristics, the basic configuration of the biosensors is similar. They must be composed of a sample holder, which is adapted to the sample physical characteristics; a biological recognition element, which must

**2. Principles and evolution of biosensing techniques**

**68**

biosensors.

**Figure 1.**

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

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 process.

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

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 of fluorescence [5].

**71**

Section 4.

**3. ROS and oxygen sensing**

and, ultimately, the survival of cells [13].

*Dynamic Luminescent Biosensors Based on Peptides for Oxygen Determination*

make them ideal for bioimaging applications as fluorescent probes [6].

Quantum dots (QDs) comprise another class of intensively researched materials for biosensing application due to their advantages such as high photostability and large extinction coefficient. Additionally, their broad absorption spectra and the possibility of tuning emission wavelength make them suitable as sensors based on FRET energy transfer, in which the spectral overlap is important for efficiency of the process, and, ultimately, the sensitivity of the biosensor. Their broad absorption spectrum also enables selective excitation of QD donors without exciting the acceptors and allows excitation of different donors at once, making them useful for multiplexing applications. Their high luminescence and nanoscale dimensions also

Despite their versatility and large scope of possibilities for biosensing, one major

Similarly to graphene QDs, the high efficiency of energy transfer from dyes to graphene oxide, GO, along with GO's intrinsic properties, opened up a new avenue for designing a lot of FRET-based biosensors. Thanks to pi stacking and hydrogen bonding interactions, GO is capable of strong binding with biomolecules, such as fluorescent dyes, which are quenched by GO via the FRET process. In the past few years, a number of GO-based biosensors using DNA as a probe are reported [9, 10]. As a special case of FRET-based biosensors, there is the bioluminescence resonance energy transfer (BRET) principle, which has been used to produce new and ultrasensitive biosensors. In these biosensors, a bioluminescent enzyme is the energy donor and a specific fluorescent molecule, chosen by spectral overlapping, acts as an acceptor. This process is extensively used to monitor and study molecular interactions between proteins and other metabolites, in vitro and in living cells [11].

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

disadvantage of QDs fabricated with inorganic materials is their toxicity, which limits their clinical applications. Graphene and other carbon-based QDs, unlike the conventional heavy-metals DQs, are biocompatible, environment friendly, and easier to prepare [7, 8], and for that reason, they have gained considerable attention. Among the most recent examples of CQD-based is a fluorescent CQD biosensor for hydrogen peroxide detection and simultaneous monitoring of the acetylcholinesterase reaction system [7]. A great number of QD-based biosensors for environmental monitoring are found in the literature. A few recent contributions are described in

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

#### *Dynamic Luminescent Biosensors Based on Peptides for Oxygen Determination DOI: http://dx.doi.org/10.5772/intechopen.84143*

Quantum dots (QDs) comprise another class of intensively researched materials for biosensing application due to their advantages such as high photostability and large extinction coefficient. Additionally, their broad absorption spectra and the possibility of tuning emission wavelength make them suitable as sensors based on FRET energy transfer, in which the spectral overlap is important for efficiency of the process, and, ultimately, the sensitivity of the biosensor. Their broad absorption spectrum also enables selective excitation of QD donors without exciting the acceptors and allows excitation of different donors at once, making them useful for multiplexing applications. Their high luminescence and nanoscale dimensions also make them ideal for bioimaging applications as fluorescent probes [6].

Despite their versatility and large scope of possibilities for biosensing, one major disadvantage of QDs fabricated with inorganic materials is their toxicity, which limits their clinical applications. Graphene and other carbon-based QDs, unlike the conventional heavy-metals DQs, are biocompatible, environment friendly, and easier to prepare [7, 8], and for that reason, they have gained considerable attention. Among the most recent examples of CQD-based is a fluorescent CQD biosensor for hydrogen peroxide detection and simultaneous monitoring of the acetylcholinesterase reaction system [7]. A great number of QD-based biosensors for environmental monitoring are found in the literature. A few recent contributions are described in Section 4.

Similarly to graphene QDs, the high efficiency of energy transfer from dyes to graphene oxide, GO, along with GO's intrinsic properties, opened up a new avenue for designing a lot of FRET-based biosensors. Thanks to pi stacking and hydrogen bonding interactions, GO is capable of strong binding with biomolecules, such as fluorescent dyes, which are quenched by GO via the FRET process. In the past few years, a number of GO-based biosensors using DNA as a probe are reported [9, 10].

As a special case of FRET-based biosensors, there is the bioluminescence resonance energy transfer (BRET) principle, which has been used to produce new and ultrasensitive biosensors. In these biosensors, a bioluminescent enzyme is the energy donor and a specific fluorescent molecule, chosen by spectral overlapping, acts as an acceptor. This process is extensively used to monitor and study molecular interactions between proteins and other metabolites, in vitro and in living cells [11].
