**4. FRET applied to environmental biosensing**

Due to the outstanding selectivity and sensitivity of optical biosensors, especially those based on FRET transducing, they meet the requirement of trace and even ultratrace detection of a great variety of environment pollutants, such as pesticides, antibiotics, halogenated contaminants [20], etc., which represent a major concern of the modern era due to the threat they pose to ecosystems and human health. In addition to this, the portability offered by the possibility of miniaturization of biosensor platforms enables the fast and low-cost field analysis, which is not possible through expensive conventional analytical methods like chromatography, mass spectrometry, and others.

Aptamer FRET sensors, or aptasensors, for instance, comprise a class of versatile and very sensitive biosensors, capable of detecting concentrations in nano and picoscales. Ultrasensitive FRET aptasensors for trace detection of metal ions [21, 22] and antibiotics [23] are reported. In a multiplexed detection system for Pb(II), Hg(2), and Ag(II) ions, the binding of an ion or multiple ions to DNA sequences triggers the DNA self-assembly. Subsequently, a cascade FRET event results in a fluorescent spectrum that can be interpreted as a fingerprint of the presence of a single or multiple metal ions [21]. In the sensor for the kanamycin antibiotic, an aptamer sensor, upconversion nanoparticles bound to an aptamer for kanamycin act as energy donor and the graphene as acceptor, in which the FRET is blocked in the presence of kanamycin, resulting in fluorescence. An impressive lower detection limit of 9.0 picomolar concentration is reported in aqueous buffer solution [23]. Indeed, by designing the suitable aptamer, versatile and selective FRET sensors for countless targets can be constructed.

Quantum dot and nanoparticle biosensors of equally impressive sensitivity for molecules of a wide range of sizes, from ions to large proteins, temperature, pH,

**75**

fuel cell [34].

*Dynamic Luminescent Biosensors Based on Peptides for Oxygen Determination*

and oxygen, also have potential application in environmental monitoring [24–26]. Most recent examples include a sensor for edifenphos fungicide, comprised of a ZnS QD conjugated to a single-stranded DNA aptamer immobilized on a graphene oxide sheet. In this sensor, FRET occurs between the QD and graphene sheet, and the fluorescence quenching is proportional to analyte concentration. Interestingly, their sensor showed remarkable selectivity, even when comparing to other pesticides of similar molecular structure [27]. In a chlorophyll-containing carbon QD of tunable fluorescence for organophosphate pesticide determination, the fluorescence of the QD is quenched via a FRET process by gold nanoparticles when the analyte is not present [28]. In another recent contribution, Luo et al. constructed a highly sensitive fluorescent sensor of Au/Ag nanoparticles containing rhodamine B for the detection of organophosphorus pesticides, which showed a

of Au/Ag core-shell nanoparticles for the detection of arsenic has a lower detection limit of 0.1 ppb (parts per billion) [29]. Another FRET sensor constituted of Au upconversion nanoparticles for fluoroquinolones detection showed a sensitivity

Other prominent biosensors for environmental surveillance are the nanophotonic biosensors, which are devices constituted of biological receptor layers immobilized onto the core surface of a waveguide, to detect evanescent waves [31]. Their functioning mechanism is based on the exposure of the waveguide surface to the analyte, resulting in a biochemical interaction that promotes a local change in the optical properties of the waveguide transducer, which can be detected, and its amplitude is modulated by the concentration of the analyte. An advantage of photonic biosensors is that they can be integrated to lab-on-a-chip platforms,

Oxygen sensing is not limited to the biomedical field. It is also a valuable analyte in environmental monitoring. Determination of O2 levels in aqueous ecosystems, such as rivers and lakes, is a common routine for evaluating the habitability conditions of these waters. Biochemical oxygen demand (BOD) and chemical oxygen demand (COD) analyses are the standard quantitative analytical methods applied for that purpose. BOD is the amount of dissolved oxygen demanded by aerobic microorganisms to decompose organic matter in a given water sample during an incubation time, usually 5 days. BOD expresses the concentration of consumed oxygen during this time period. In rivers polluted with high levels of organic waste, aerobic bacteria consume the dissolved O2 during decomposition of organic matter, which results in a drastic reduction of available O2 that aquatic animals need to survive. Analogous to BOD, the COD is a more general method, which gives the amount of oxygen needed for oxidation of any chemically oxidizable material, apart

Biosensors for oxygen and organic pollutants sensing for environmental surveillance are evolving rapidly due to many advantages they offer over the traditional methods of BOD and COD analyses, such as faster and more accurate results and the possibility of online and real-time monitoring of water quality [32–34]. One recent contribution in this field includes a microbial fuel cell biosensor for real-time BOD analysis that was tested for urine sensing. The device emits a sound alarm whenever the concentration of the analyte exceeds a given concentration threshold and is self-powered by the electroactive microorganisms of the microbial

Regarding oxygen sensing, both electrochemical and optical biosensors of noticeable sensitivity are found in the literature. In a recent electrochemical biosensor, peptide micro/nanostructures are self-assembled with a complex of copper

in fruit and water samples [25]. A similar biosensor

. Fluoroquinolones are a class of antibiotics that have become

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

detection limit of 1.8-pg ml<sup>−</sup><sup>1</sup>

serious water contaminants [30].

enhancing their application possibilities.

from organic matter [32].

of 0.19–0.32 ng ml<sup>−</sup><sup>1</sup>

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

and oxygen, also have potential application in environmental monitoring [24–26]. Most recent examples include a sensor for edifenphos fungicide, comprised of a ZnS QD conjugated to a single-stranded DNA aptamer immobilized on a graphene oxide sheet. In this sensor, FRET occurs between the QD and graphene sheet, and the fluorescence quenching is proportional to analyte concentration. Interestingly, their sensor showed remarkable selectivity, even when comparing to other pesticides of similar molecular structure [27]. In a chlorophyll-containing carbon QD of tunable fluorescence for organophosphate pesticide determination, the fluorescence of the QD is quenched via a FRET process by gold nanoparticles when the analyte is not present [28]. In another recent contribution, Luo et al. constructed a highly sensitive fluorescent sensor of Au/Ag nanoparticles containing rhodamine B for the detection of organophosphorus pesticides, which showed a detection limit of 1.8-pg ml<sup>−</sup><sup>1</sup> in fruit and water samples [25]. A similar biosensor of Au/Ag core-shell nanoparticles for the detection of arsenic has a lower detection limit of 0.1 ppb (parts per billion) [29]. Another FRET sensor constituted of Au upconversion nanoparticles for fluoroquinolones detection showed a sensitivity of 0.19–0.32 ng ml<sup>−</sup><sup>1</sup> . Fluoroquinolones are a class of antibiotics that have become serious water contaminants [30].

Other prominent biosensors for environmental surveillance are the nanophotonic biosensors, which are devices constituted of biological receptor layers immobilized onto the core surface of a waveguide, to detect evanescent waves [31]. Their functioning mechanism is based on the exposure of the waveguide surface to the analyte, resulting in a biochemical interaction that promotes a local change in the optical properties of the waveguide transducer, which can be detected, and its amplitude is modulated by the concentration of the analyte. An advantage of photonic biosensors is that they can be integrated to lab-on-a-chip platforms, enhancing their application possibilities.

Oxygen sensing is not limited to the biomedical field. It is also a valuable analyte in environmental monitoring. Determination of O2 levels in aqueous ecosystems, such as rivers and lakes, is a common routine for evaluating the habitability conditions of these waters. Biochemical oxygen demand (BOD) and chemical oxygen demand (COD) analyses are the standard quantitative analytical methods applied for that purpose. BOD is the amount of dissolved oxygen demanded by aerobic microorganisms to decompose organic matter in a given water sample during an incubation time, usually 5 days. BOD expresses the concentration of consumed oxygen during this time period. In rivers polluted with high levels of organic waste, aerobic bacteria consume the dissolved O2 during decomposition of organic matter, which results in a drastic reduction of available O2 that aquatic animals need to survive. Analogous to BOD, the COD is a more general method, which gives the amount of oxygen needed for oxidation of any chemically oxidizable material, apart from organic matter [32].

Biosensors for oxygen and organic pollutants sensing for environmental surveillance are evolving rapidly due to many advantages they offer over the traditional methods of BOD and COD analyses, such as faster and more accurate results and the possibility of online and real-time monitoring of water quality [32–34]. One recent contribution in this field includes a microbial fuel cell biosensor for real-time BOD analysis that was tested for urine sensing. The device emits a sound alarm whenever the concentration of the analyte exceeds a given concentration threshold and is self-powered by the electroactive microorganisms of the microbial fuel cell [34].

Regarding oxygen sensing, both electrochemical and optical biosensors of noticeable sensitivity are found in the literature. In a recent electrochemical biosensor, peptide micro/nanostructures are self-assembled with a complex of copper

*Biosensors for Environmental Monitoring*

adjusted to fulfill the desired purpose.

intensity measurements [4].

mass spectrometry, and others.

**4. FRET applied to environmental biosensing**

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

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

Due to the outstanding selectivity and sensitivity of optical biosensors, especially those based on FRET transducing, they meet the requirement of trace and even ultratrace detection of a great variety of environment pollutants, such as pesticides, antibiotics, halogenated contaminants [20], etc., which represent a major concern of the modern era due to the threat they pose to ecosystems and human health. In addition to this, the portability offered by the possibility of miniaturization of biosensor platforms enables the fast and low-cost field analysis, which is not possible through expensive conventional analytical methods like chromatography,

Aptamer FRET sensors, or aptasensors, for instance, comprise a class of versatile and very sensitive biosensors, capable of detecting concentrations in nano and picoscales. Ultrasensitive FRET aptasensors for trace detection of metal ions [21, 22] and antibiotics [23] are reported. In a multiplexed detection system for Pb(II), Hg(2), and Ag(II) ions, the binding of an ion or multiple ions to DNA sequences triggers the DNA self-assembly. Subsequently, a cascade FRET event results in a fluorescent spectrum that can be interpreted as a fingerprint of the presence of a single or multiple metal ions [21]. In the sensor for the kanamycin antibiotic, an aptamer sensor, upconversion nanoparticles bound to an aptamer for kanamycin act as energy donor and the graphene as acceptor, in which the FRET is blocked in the presence of kanamycin, resulting in fluorescence. An impressive lower detection limit of 9.0 picomolar concentration is reported in aqueous buffer solution [23]. Indeed, by designing the suitable aptamer, versatile

and selective FRET sensors for countless targets can be constructed.

Quantum dot and nanoparticle biosensors of equally impressive sensitivity for molecules of a wide range of sizes, from ions to large proteins, temperature, pH,

**74**

that acts as the oxygen reduction catalyst, immobilized onto a glassy carbon. This biosensor showed a lower detection limit of 0.1 mg l<sup>−</sup><sup>1</sup> [35]. Most recent optical biosensors based on FRET transducing include a BOD biosensor chip and a ratiometric FRET sensor. In the biosensor chip for BOD analysis, an oxygen sensitive ruthenium complex coated with a polyethylene-polypropylene film permeable only by oxygen avoids the interference of pollutants from the sample. In this biosensor, the fluorescence intensity is correlated with oxygen concentration [36]. Another ratiometric FRET oxygen sensor consists of a Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6 pentafluorophenyl)-porphyrin oxygen probe entrapped in a copolymer matrix that is capable of real-time monitoring of extra-cellular O2 consumption by *E. coli* bacteria and Hela cells. This biosensor showed a sensitivity of 0.08 mg l<sup>−</sup><sup>1</sup> [37]. The coating of the sensing unit or its immobilization in a matrix selective to oxygen permeability is a commonly adopted strategy in the design of optical sensors for oxygen in order to ensure its selectivity. Additionally, transition metal complexes, especially those of ruthenium and platinum, have a long phosphorescence lifetime, a requirement for efficient energy transfer from the sensing unit to molecular oxygen through collisional quenching, as described in Section 3, necessary for achieving high levels of sensitivity [4, 38, 39].

Our group has also developed a colorimetric sensor for dissolved O2. Our sensor, comprised of a self-assembled peptide containing a fluorescent dye, is based on a FRET energy transfer between the constituents of the system that arises from the formation of a charge transfer complex. It showed remarkable sensitivity and selectivity toward dissolved O2, both in steady-state and time-resolved fluorescence measurements. This self-assembled sensing platform, which was tested in fish breeding environment and showed good reproducibility, might be useful in analytical methods for determination of O2 levels in polluted water samples.

Additionally, our material, when allied with an antioxidant drug used in cancer treatment, showed antioxidant activity by sensing singlet oxygen, as well as prooxidant behavior by generating that same reactive oxygen species when irradiated with light, which makes it promising for photodynamic therapy as well [40]. The singlet excited state of O2 is perhaps the most important of the ROS molecules. Due to its considerable lethal effect for cells, it is exploited in photodynamic therapy, an alternative approach for a number of cancers that has proven to be efficient and far less invasive and harmful than the side effects of conventional treatment protocols. It is based on the same photosensitization process as described earlier.
