**2. Optical biosensors**

Optical biosensors are those which can sense phenomena related to the interaction of micro‐ organisms with the analytes and correlate the observed optical signal to the concentration of target compounds, based on the measurement of photons involved in the process, rather than electrons, as in the afore mentioned techniques. More specifically, optical detection is based on the measurement of luminescence, fluorescence, colour changes, by the measure‐ ment of absorbance, reflectance or fluorescence emissions that occur in the ultraviolet (UV), visible, or near-infrared (NIR) spectral regions.

Even though the first biosensor reported, the Clark´s amperometric enzyme electrode for glucose [1] is dated of 1962, the first fiber-optic biosensor was described only in 1975, by Lubbers and Opitz [11, 12] and, since then, with the incredible rapidity of new detection techniques development, various optical biosensors have been demonstrated. Among opti‐ cal properties, fluorescence is by far the most often exploited one by a significant number of techniques, mostly based on parameters such as intensity, lifetime, anisotropy, quenching efficiency, non-radiative and luminescence energy transfer, and so on.

Although fluorescent biosensors have been reported since the late nineties, [13- 15] very of‐ ten there is a confusion between labelling and the description of a biosensor, which turns the exact moment of the fluorescent biosensor proposal not very clear to point. Nevertheless, amazing development ondetection methods in biosensors, their construction, even at nano‐ scale, and applications have been reported in the last decade. [16- 22]

#### **2.1. Classes of optical biosensors**

Due to the enormous quantity of biosensors already proposed, it is a very hard task to classi‐ fy them all. Nevertheless, for our objective, it is convenient to classify them into two classes, not based on the detection method, which, of course, must be an optical method, but on the recognition one. With it in mind, optical biosensors can be sub-classified as:


With respect to the detection mechanism, biosensors can be of fluorescence, phosphores‐ cence, reflection, UV/Vis/IR absorbance, lifetime, which is the characteristic measured by Förster Resonant Energy Transfer (FRET) based biosensors, responsible for the wide range of applications in which biosensors had acted lately. Also, refractive indexes changes can be detected by several and modern techniques, such as interferometry and Surface Plasmon Resonance, which had opened a new and promising field for biosensor research. Further in‐ formation about them will be presented later in this chapter.

Among all optical properties exploited in biosensors construction, the most common meth‐ od of detecting and quantifying biological compounds is still based on the fluorescence ac‐ tivity due to the fact that fluorescent properties of most organic fluorophores are susceptible to environment changes, which is indispensable to sensing applications. The most important advantage of these biosensors is that they are proposed for general use, thus, they provide the possibility of multiple compounds detection within a single device, they are able to per‐ form remote sensing and they are ease to build. Nevertheless, there are some requirements for the construction of the fluorescent recognition unit that must be attended, regarded to structure of the fluorophore and to its photophysical activity.

When proposing a biosensor, the hardest task is to address the right fluorophore to compose it, the one that will provide the needed answer. This is because there is a number of fluores‐ cent probes which can be applied on biosensing and a number of device designs that can be proposed within the same objective.

Since in sensing applications, detection is based on changes of fluorescent responses of a particular fluoroprobe, when it is inserted into the analyte environment and interacts with it by a variety of mechanisms, it is of extremely importance to profoundly understand the photophysical behavior of the system and the fluorescence parameters that will be determi‐ nant in detection. Once this step is achieved, a biosensor can be built aiming to countless ap‐ plications, which include immunoassays, nucleic acid detection, cellular and sub-cellular labeling, resonance energy transfer studies, diagnostic assays and disease monitoring and treatment.

Most of fluoroprobes used in biosensors present an environment-dependent luminescent be‐ havior. Therefore, attention must be given to the environment characteristics that may inter‐ fere with the photophysics of fluoroprobe-analyte, such as pH dependence of the luminescent response; self-quenching at high concentrations of fluoroprobes; susceptibility to photo-bleaching; short excited state fluorescent lifetimes, which may confer low sensibili‐ ty to salvation relaxation; small Stokes shifts, which favors self-absorption effects and unde‐ sired luminescent emission shifts and short-term stability when in presence of water or in aqueous medium [23, 24].

#### **2.2. Fluorescent biosensors**

**2. Optical biosensors**

114 State of the Art in Biosensors - General Aspects

visible, or near-infrared (NIR) spectral regions.

**2.1. Classes of optical biosensors**

methods.

Optical biosensors are those which can sense phenomena related to the interaction of micro‐ organisms with the analytes and correlate the observed optical signal to the concentration of target compounds, based on the measurement of photons involved in the process, rather than electrons, as in the afore mentioned techniques. More specifically, optical detection is based on the measurement of luminescence, fluorescence, colour changes, by the measure‐ ment of absorbance, reflectance or fluorescence emissions that occur in the ultraviolet (UV),

Even though the first biosensor reported, the Clark´s amperometric enzyme electrode for glucose [1] is dated of 1962, the first fiber-optic biosensor was described only in 1975, by Lubbers and Opitz [11, 12] and, since then, with the incredible rapidity of new detection techniques development, various optical biosensors have been demonstrated. Among opti‐ cal properties, fluorescence is by far the most often exploited one by a significant number of techniques, mostly based on parameters such as intensity, lifetime, anisotropy, quenching

Although fluorescent biosensors have been reported since the late nineties, [13- 15] very of‐ ten there is a confusion between labelling and the description of a biosensor, which turns the exact moment of the fluorescent biosensor proposal not very clear to point. Nevertheless, amazing development ondetection methods in biosensors, their construction, even at nano‐

Due to the enormous quantity of biosensors already proposed, it is a very hard task to classi‐ fy them all. Nevertheless, for our objective, it is convenient to classify them into two classes, not based on the detection method, which, of course, must be an optical method, but on the

**•** probing biosensors: this class consists on biosensors that have based their activity in dif‐ ferences of interactions between analyte and recognition element, ruled by affinities. This behavior leads to changes in the optical response that can be measured by several optical

**•** reacting biosensors: in this class, their optical responses are related to chemical processes, such as chemisorptions, catalytic reactions of any kind, formation of new chemical bonds and so on. These chemical (and definitive changes) can also generate optical changes

With respect to the detection mechanism, biosensors can be of fluorescence, phosphores‐ cence, reflection, UV/Vis/IR absorbance, lifetime, which is the characteristic measured by Förster Resonant Energy Transfer (FRET) based biosensors, responsible for the wide range of applications in which biosensors had acted lately. Also, refractive indexes changes can be detected by several and modern techniques, such as interferometry and Surface Plasmon

efficiency, non-radiative and luminescence energy transfer, and so on.

scale, and applications have been reported in the last decade. [16- 22]

recognition one. With it in mind, optical biosensors can be sub-classified as:

which are detectable by countless optical methods.

In fluorescent biosensors, a particularly interesting characteristic that have been more and more exploited is the effect that local interactions can cause on fluoroprobe´s electronic ex‐ cited states, even when no chemical changes are included. With this respect, mechanisms of controlling and orienting these effects are proposed and studied. For example, non-radiative energy transfers leading to a more selective system or a more efficient luminescent response, achieved by combining common organic fluorophores with metallic nanoparticules, metallic complexes or with nanostructures of carbon or peptides have presented interesting results. In our previous work, with leadership of Prof. Alves, [25] diphenylalanine peptide nano‐ tubes were physically modified with a fluoroprobe containing a polar head, 1-pyrenyl-car‐ boxylic acid, and their steady-state and dynamic fluorescence responses were determined (fig.2). Also, the mechanism of interaction between peptides to form the supramolecular structure and their interaction with the fluorophore were studied and revealed. By computa‐ tional simulations, it was shown that the nanotube formation and the fluorophore adsorp‐ tion are governed by π-stacking interactions, which makes of electrostatic interactions essential. Moreover, since the connection of nanostructured materials, especially biomateri‐ als such as peptides, with solid well-structured surfaces make the manipulation of such structures an interesting alternative for device fabrication. Although the afore mentioned work focused on the supramolecular chemistry of nanostructure obtainment and the role of pH conditions to ensure the right nanotube dimensions, it rose the perspective of construct‐ ing an electronic device for biological recognition. Aiming at the application of this nano‐ structured peptides to a biosensor construction, it is of great importance to determine whether the interaction of the peptide nanostructure with the probable substrates can influ‐ ence the optical response measured. The first step is to determine the nature of the interac‐ tion between the fluorescent peptide nanotubes with Indium-Tin Oxide (ITO) electrode, elected as a good candidate to act as anode in a future biosensor device.The modified Phe-Phe nanotube was then deposited on ITO electrode and the time-resolved fluorescence of this system was detailed studied.

Since the amphoteric ITO is a transparent conductive oxide of low electrical resistivity (10-6 to 10-4Ω.cm) and small bandgap (of 3.3 eV), high physical-chemical stability and good sur‐ face morphology, it is widely used as electrode in a variety of devices, which includes sen‐ sors. When applied to biosensors, ITO can interact with protein residues due to –OH groups on its surface, which enables the adsorption of carboxylic and amine residues via hydrogen bonds.

By combining the final fluorescent nanotubes with ITO electrodes, a non-radiative resonant energy transfer (FRET) was detected between the thin layer of mixed oxide and the fluoro‐ phore doping in nanotube surface. In fact, the results show that when pyrenyl-doped nano‐ tubes are formed at neutral and higher pH ranges, FRET occurs, leading to a small excitedstate lifetime of pyrenyl moieties, but, at lower pH ranges, the excited states of pyrenyl moieties are stabilized and the lifetime rises. This energy transfer process is favored, in this case, by the strong electrostatic interaction between the charged nanotube and the charge transporter ITO of dipole-dipole induced type. When nanotubes are formed in low pH rang‐ es, the final structure presents an overall superficial positive charge, due to protonation of carboxylic and amine groups of peptide residues and carboxylic groups of the fluorophore, which is responsible for a less effective electrostatic interaction between combined peptide structure and 1- pyrenyl-carboxylic acid moieties and the ITO surface. These results contri‐ bution laid in terms of the supramolecular control of the structures, showing that they can be designed and actually obtained as desired and many aspects of biosensing activity can be exploited in the same device conception. In this example, both a fluorescent-based biosensor for local environment monitoring and a FRET-based fluorescent biosensor can be designed, depending only on the approach.

New Insights on Optical Biosensors: Techniques, Construction and Application http://dx.doi.org/10.5772/52330 117

**Figure 2.** Epifluorescence images (200 times increased) and respective fluorescence spectra of Phe-Phe nanotubes samples obtained at of 0.07% m/m of 1-pyrenyl-carboxilic acid and at distinct pH ranges, deposited over glass and glass covered by ITO substrates.

#### **2.3. FRET-based fluorescent biosensors**

tubes were physically modified with a fluoroprobe containing a polar head, 1-pyrenyl-car‐ boxylic acid, and their steady-state and dynamic fluorescence responses were determined (fig.2). Also, the mechanism of interaction between peptides to form the supramolecular structure and their interaction with the fluorophore were studied and revealed. By computa‐ tional simulations, it was shown that the nanotube formation and the fluorophore adsorp‐ tion are governed by π-stacking interactions, which makes of electrostatic interactions essential. Moreover, since the connection of nanostructured materials, especially biomateri‐ als such as peptides, with solid well-structured surfaces make the manipulation of such structures an interesting alternative for device fabrication. Although the afore mentioned work focused on the supramolecular chemistry of nanostructure obtainment and the role of pH conditions to ensure the right nanotube dimensions, it rose the perspective of construct‐ ing an electronic device for biological recognition. Aiming at the application of this nano‐ structured peptides to a biosensor construction, it is of great importance to determine whether the interaction of the peptide nanostructure with the probable substrates can influ‐ ence the optical response measured. The first step is to determine the nature of the interac‐ tion between the fluorescent peptide nanotubes with Indium-Tin Oxide (ITO) electrode, elected as a good candidate to act as anode in a future biosensor device.The modified Phe-Phe nanotube was then deposited on ITO electrode and the time-resolved fluorescence of

Since the amphoteric ITO is a transparent conductive oxide of low electrical resistivity (10-6 to 10-4Ω.cm) and small bandgap (of 3.3 eV), high physical-chemical stability and good sur‐ face morphology, it is widely used as electrode in a variety of devices, which includes sen‐ sors. When applied to biosensors, ITO can interact with protein residues due to –OH groups on its surface, which enables the adsorption of carboxylic and amine residues via hydrogen

By combining the final fluorescent nanotubes with ITO electrodes, a non-radiative resonant energy transfer (FRET) was detected between the thin layer of mixed oxide and the fluoro‐ phore doping in nanotube surface. In fact, the results show that when pyrenyl-doped nano‐ tubes are formed at neutral and higher pH ranges, FRET occurs, leading to a small excitedstate lifetime of pyrenyl moieties, but, at lower pH ranges, the excited states of pyrenyl moieties are stabilized and the lifetime rises. This energy transfer process is favored, in this case, by the strong electrostatic interaction between the charged nanotube and the charge transporter ITO of dipole-dipole induced type. When nanotubes are formed in low pH rang‐ es, the final structure presents an overall superficial positive charge, due to protonation of carboxylic and amine groups of peptide residues and carboxylic groups of the fluorophore, which is responsible for a less effective electrostatic interaction between combined peptide structure and 1- pyrenyl-carboxylic acid moieties and the ITO surface. These results contri‐ bution laid in terms of the supramolecular control of the structures, showing that they can be designed and actually obtained as desired and many aspects of biosensing activity can be exploited in the same device conception. In this example, both a fluorescent-based biosensor for local environment monitoring and a FRET-based fluorescent biosensor can be designed,

this system was detailed studied.

116 State of the Art in Biosensors - General Aspects

depending only on the approach.

bonds.

FRET is often exploited in biosensors due to the variety of systems that can present this ef‐ fect and to the quantity of sensing elements that can be employed in the biosensor construc‐ tion. An example is the use of colloidal luminescent semiconductor nanocrystals, the quantum dots (QDs), to biosensors. These fluorescent compounds are very well known as fluorescent labels for a series of studies, in particular, for imaging of biological and non-bio‐ logical systems. [26-28] Nevertheless, they also give rise to more robust biosensors, since their unique fluorescent properties can overcome the organic-based fluorophores liabilities.

The major property that inform about the occurrence of non-radiative energy transfer proc‐ ess is changes in the fluorophoreexcited state lifetimes. Due to the variety of compounds that can have their excited state disturbed, several detection techniques can be applied, sev‐ eral device architectures only dependent on the application, rather than in the detection technique, generating biosensors with significant sensitivity and selectivity enhancement, which can be even at the one single molecule limit. FRET is also essential to the conductive polymers, DNA, aptamer or protein-based biosensors and all these possible architectures make use of the interpretations of luminescent signals that reveals several mechanisms for FRET. Indeed, FRET is a resonant process that depends on several conditions of the environ‐ ment where it takes place. In a short description, this non-radiative excitation energy trans‐ fer occurs always that a donor chromophore and an acceptor become close to each other and their electronic energy levels interact, with some pre-requisites. As described by Förster [29], FRET is determined by a long range dipole–dipole interaction between the donor and the acceptor and his formulations for these events are widely applied from solutions to solid systems, which contain the chromophores of biochemical interest. As an advantage, FRET offers an experimental approach to determine molecular distances through luminescent spectral measurements, which correspond to the efficiency of energy transfer between a do‐ nor and an acceptor located at two distinct specific sites, with separation limited to a range of 10–80 Å. Because of the sensitiveness of this technique corresponds to the inverse sixth power dependence of the transfer efficiency to the donor-acceptor distance (equation 1), FRET is assumed to consist of a sensitive technique for detection of global structural altera‐ tions. Förster formalism assumes that donor and acceptor are stationary in the timescale of their electronic excited-states lifetimes and, as a consequence, the donor-acceptor separation is static, giving a single distance between them. Nevertheless, the dynamic nature of large systems such as proteins and polymers cannot be ignored and the distances between them are expressed as a distribution. Förster mechanism involves an inductive resonance transfer in which the excitation process creates an electric field around the donor, due its charge transport. As a second oscillator, the acceptor, come closer to the donor, it inductively oscil‐ ates and, if it occurs with the adequate frequency, the energy of the donor is transferred to the acceptor. The energy transfer is maximum when both oscillators are similar. To observe this phenomenon, it is necessary that electronic transitions of both donor and acceptor are permitted and then, that coulombic interactions, such as dipole-dipole, which are distance dependent by a factor of R-3, occur. This leads to a probability of occurrence of FRET propor‐ tional to R-6. Förster predicts that the energy transfer occurs if there is a coupling between transitions and radiation field, at a rate constant (kDA) given by:

$$k\_{DA} = \frac{9000k^{\circ} \ln 10}{128\pi^{\circ} n^{\circ} N\_A \pi\_{DA} r^6} \mathbf{f} \frac{F\_D \varepsilon\_A \langle \nu \rangle}{\nu^4} d\nu \tag{1}$$

In which *k2* describes donor-acceptor dipole relative orientation; *NA* is the Avogadro Con‐ stant; *FD* stands for the donor corrected fluorescence intensity; *εA* is the acceptor molar ex‐ tinction coefficient; *τDA* is the donor fluorescence lifetime; *r* is the distance between donor and acceptor; *n*, the refractive index and *ν* the wavenumber.

When the probability of FRET occurrence is 50%, the distance in which it takes place is a reference distance, called Förster Distance (R0), defined as the distance in which the FRET rate KDA is equivalent to the fluorescence rate of the donor in the absence of the acceptor τ<sup>D</sup> -1. They are related by:

$$k\_{DA} = \frac{1}{\tau\_D} \left(\frac{R\_0}{r}\right)^{\theta} \tag{2}$$

When R = R0, KDA = 1/τ<sup>D</sup>

#### **2.4. DNA – based fluorescent biosensors**

FRET is determined by a long range dipole–dipole interaction between the donor and the acceptor and his formulations for these events are widely applied from solutions to solid systems, which contain the chromophores of biochemical interest. As an advantage, FRET offers an experimental approach to determine molecular distances through luminescent spectral measurements, which correspond to the efficiency of energy transfer between a do‐ nor and an acceptor located at two distinct specific sites, with separation limited to a range of 10–80 Å. Because of the sensitiveness of this technique corresponds to the inverse sixth power dependence of the transfer efficiency to the donor-acceptor distance (equation 1), FRET is assumed to consist of a sensitive technique for detection of global structural altera‐ tions. Förster formalism assumes that donor and acceptor are stationary in the timescale of their electronic excited-states lifetimes and, as a consequence, the donor-acceptor separation is static, giving a single distance between them. Nevertheless, the dynamic nature of large systems such as proteins and polymers cannot be ignored and the distances between them are expressed as a distribution. Förster mechanism involves an inductive resonance transfer in which the excitation process creates an electric field around the donor, due its charge transport. As a second oscillator, the acceptor, come closer to the donor, it inductively oscil‐ ates and, if it occurs with the adequate frequency, the energy of the donor is transferred to the acceptor. The energy transfer is maximum when both oscillators are similar. To observe this phenomenon, it is necessary that electronic transitions of both donor and acceptor are permitted and then, that coulombic interactions, such as dipole-dipole, which are distance dependent by a factor of R-3, occur. This leads to a probability of occurrence of FRET propor‐ tional to R-6. Förster predicts that the energy transfer occurs if there is a coupling between

transitions and radiation field, at a rate constant (kDA) given by:

and acceptor; *n*, the refractive index and *ν* the wavenumber.

In which *k2*

118 State of the Art in Biosensors - General Aspects

They are related by:

When R = R0, KDA = 1/τ<sup>D</sup>

*kDA* <sup>=</sup> <sup>9000</sup>*<sup>k</sup>* <sup>2</sup>

128*π* <sup>5</sup>

*ln*10

*F <sup>D</sup>εA*(*ν*)

describes donor-acceptor dipole relative orientation; *NA* is the Avogadro Con‐

*<sup>ν</sup>* <sup>4</sup> *dυ* (1)

*<sup>r</sup>* )<sup>6</sup> (2)


*<sup>n</sup>* <sup>4</sup>*<sup>N</sup> <sup>A</sup>τDAr* <sup>6</sup> *<sup>∫</sup>*

stant; *FD* stands for the donor corrected fluorescence intensity; *εA* is the acceptor molar ex‐ tinction coefficient; *τDA* is the donor fluorescence lifetime; *r* is the distance between donor

When the probability of FRET occurrence is 50%, the distance in which it takes place is a reference distance, called Förster Distance (R0), defined as the distance in which the FRET rate KDA is equivalent to the fluorescence rate of the donor in the absence of the acceptor τ<sup>D</sup>

> *kDA* <sup>=</sup> <sup>1</sup> *τD* ( *R*0

Despite specific requirements of the systems of interest, these assumptions can be applied to any measurements that can identify the energies of the electronic transitions involved and the excited states lifetimes. In DNA-based biosensors, high sensitivity detection and real time information are crucial. In their work, Liu and Bazan [30] proposed homogeneous bio‐ sensor assays, which were based on the detection of distinct luminescent responses of a wa‐ ter-soluble conjugated polymer and took advantage of its characteristic of self-assemble to improve the biosensor capability over those employing small molecules. In this biosensor, the interaction between the oligonucleotide hybridized with a cationic polythiophene and a single-stranded DNA or a double-stranded DNA in the presence of cationic poly(fluoreneco-phenylene) leads to conformational changes on polymer backbone and to changes in the fluorescent response, as the cationic poly(fluorene-co-phenylene) acts as donor in the fluo‐ rescence energy-transfer assay and, hence, to a signal amplification(fig.3). In the presence of the single-stranded DNA, the positively charged polymer interacts with it, but without ener‐ gy transfer and only emission from the poly(fluorene-co-phenylene) is detected. When inter‐ acting with the double-stranded DNA, emission from the poly(fluorene-co-phenylene) decreases and emission from the hybridized oligonucleotide is observed. The signal trans‐ duction is then controlled by specific electrostatic interactions.

**Figure 3.** Scheme of the transduction mechanism on Liu´s conductive polymer FRET-based biosensor for DNA detec‐ tion. Reprinted (adapted) with permission from [30]. Copyright (2004) American Chemical Society.

The approach of employing oligonucleotides in the transduction process of biosensors has become very attractive and popular in such a way that a new class of biosensor has arose: the *aptamer-based* ones.

#### **2.5. Aptamer-based biosensors**

Although most of the aptamer-based biosensor utilizes optical methods for detection, it is not exclusive. In fact, they consist of a versatile tool for biosensors, since they behave as effi‐ ciently as antibodies, selectively interact with the target and consist of innovative ap‐ proaches for biosensor construction. There are numbers of aptamers that can be selected from the Systematic Evolution of Ligands by Exponential (SELEX) enrichment, which con‐ sists of an *in vitro* iterative process of adsorption, recovery and re-amplification of singlestranded DNA combinatorial lists. This routine of select an aptamer is necessary due to the specificity of their interactions and the variety of biosensors that can be proposed.

In a recent work, Yildirim et al. [31] showed an environmental application for aptamerbased optical biosensors. In their approach, β-estradiol 6-(O-carboxy-methyl)oxime-BSA was covalently immobilized on an optical fiber surface to develop an aptamer-based biosen‐ sor for rapid, sensitive and highly selective detection of 17β-estradiol, a endocrine disrupt‐ ing compound that is a common water pollutant. In an indirect competitive detection approach, samples of 17β-estradiol were premixed with a fluorescence-labeled DNA aptam‐ er. In the sensor surface, a higher concentration of 17β-estradiol led to a less intense fluores‐ cence of the labeled aptamer, by creating a dose-response curve of 17β-estradiol, with a detection limit as low as 0.6 ng mL-1.

#### **2.6. Quantum dots-based fluorescent biosensors**

Some of the quantum dot´s photophysical properties overcome by several orders of magni‐ tude those of common fluorophores. For example, they present very broad absorption spec‐ tra, from UV region towards blue-visible region, corresponding to a large wavelength range; their molar extinction coefficients are of hundred times larger than those of small organic fluorophores and can reach values of several millions. [32] Also, they present the ability of tuning their photoluminescence as a function of the core size, which turn possible assign a determined quantum dot for an application.[33, 34] This possibility becomes a great advant‐ age of quantum dots in comparison to organic luminescent polymers, which cannot have their behavior well predicted only by their chain size: it is important to know their solubility and the interaction forces that act in a given system, since their final photophysical proper‐ ties are intimately related to their chain conformation and, so, to inter and intrachain energy transfer processes.

Regarding to the photoluminescent properties of quantum dots, their tunable fluorescence combined to the very broad absorption lead to a large effective stokes shifts and to the prob‐ ability of an efficient excitation of a mixed population of quantum dots, at a single wave‐ length, several nanometers delocalized from their fluorescent maximum. The characteristics of size-tunable luminescence and of broad absorption spectra make of quantum dots suita‐ ble for multi-color (or, as usually called multiplexed) immunoassays. Their photostability and sensitivity also make them good options for a number of imunoassays, especially be‐ cause they provide flexibility to the analytical techniques [35, 36].

In Pinwattana et al. [37] work, quantum dots were conjugated to a secondary anti-phospho‐ serine antibody in a heterogeneous sandwich immunoassay, acting as labels and generated amplified electrochemical signals, analyzed by square-wave voltammetry, which is not an optical technique, but it demonstrates the amplitude of analytical methods that the choice of quantum dots as actives in biosensors permit. Their experiments consisted of the addition of the model phosphorylated protein, bovine serum albumin, to a primary bovine serum albu‐ nmin antibody-coated polystyrene microwells, followed by the addition of a quantum dot labeled anti-phosphoserine antibody. This quantum dot label was then removed by acid at‐ tack and the free label was detected, leading to current responses that were proportional to the concentration of the phosphorylated bovine serum albumin.

stranded DNA combinatorial lists. This routine of select an aptamer is necessary due to the

In a recent work, Yildirim et al. [31] showed an environmental application for aptamerbased optical biosensors. In their approach, β-estradiol 6-(O-carboxy-methyl)oxime-BSA was covalently immobilized on an optical fiber surface to develop an aptamer-based biosen‐ sor for rapid, sensitive and highly selective detection of 17β-estradiol, a endocrine disrupt‐ ing compound that is a common water pollutant. In an indirect competitive detection approach, samples of 17β-estradiol were premixed with a fluorescence-labeled DNA aptam‐ er. In the sensor surface, a higher concentration of 17β-estradiol led to a less intense fluores‐ cence of the labeled aptamer, by creating a dose-response curve of 17β-estradiol, with a

Some of the quantum dot´s photophysical properties overcome by several orders of magni‐ tude those of common fluorophores. For example, they present very broad absorption spec‐ tra, from UV region towards blue-visible region, corresponding to a large wavelength range; their molar extinction coefficients are of hundred times larger than those of small organic fluorophores and can reach values of several millions. [32] Also, they present the ability of tuning their photoluminescence as a function of the core size, which turn possible assign a determined quantum dot for an application.[33, 34] This possibility becomes a great advant‐ age of quantum dots in comparison to organic luminescent polymers, which cannot have their behavior well predicted only by their chain size: it is important to know their solubility and the interaction forces that act in a given system, since their final photophysical proper‐ ties are intimately related to their chain conformation and, so, to inter and intrachain energy

Regarding to the photoluminescent properties of quantum dots, their tunable fluorescence combined to the very broad absorption lead to a large effective stokes shifts and to the prob‐ ability of an efficient excitation of a mixed population of quantum dots, at a single wave‐ length, several nanometers delocalized from their fluorescent maximum. The characteristics of size-tunable luminescence and of broad absorption spectra make of quantum dots suita‐ ble for multi-color (or, as usually called multiplexed) immunoassays. Their photostability and sensitivity also make them good options for a number of imunoassays, especially be‐

In Pinwattana et al. [37] work, quantum dots were conjugated to a secondary anti-phospho‐ serine antibody in a heterogeneous sandwich immunoassay, acting as labels and generated amplified electrochemical signals, analyzed by square-wave voltammetry, which is not an optical technique, but it demonstrates the amplitude of analytical methods that the choice of quantum dots as actives in biosensors permit. Their experiments consisted of the addition of the model phosphorylated protein, bovine serum albumin, to a primary bovine serum albu‐ nmin antibody-coated polystyrene microwells, followed by the addition of a quantum dot labeled anti-phosphoserine antibody. This quantum dot label was then removed by acid at‐

cause they provide flexibility to the analytical techniques [35, 36].

specificity of their interactions and the variety of biosensors that can be proposed.

detection limit as low as 0.6 ng mL-1.

120 State of the Art in Biosensors - General Aspects

transfer processes.

**2.6. Quantum dots-based fluorescent biosensors**

Since quantum dots are usually obtained from organometallic precursors, they are poorly water-soluble and this is, in some cases, an issue to biosensing, since it seems improbable that, in these conditions, a quantum dot will interact with a biosystem. Nevertheless, there are several methods available to efficiently exchange or functionalize their native organic li‐ gands with a desired ligand that can better both solubility and bioconjugation potential, by either chemical or physical processes. One of the most common modifications is to attach a biomolecule to a functional group on the quantum dot surface, which can be amines, carbox‐ yls or even thiols. In this matter, amines andcarboxyls can be easily modified by 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, a common reactant. Thiol groups are usual sites for maleimide.

The quantum dot surface modification can also be conducted by direct interaction with the biomolecule. In this case, interaction forces balance may govern the stability and the yield of the modified quantum dot. Examples are metal-affinity between polyhistidine appended proteins and Zinc atoms present on quantum dot´s structure, which leads to coordination of the biomolecule to the metallic center [38, 39] or dipole interactions between thiol groups of cysteine residues with sulfur atoms present in the surface.[40, 41] These modifications can transform quantum dots into efficient elements to immunoassays applications. Examples were presented by Puchades et al. In their review, [42] they showed that by electing a quan‐ tum dot, taking into account its size and by coating it with a variety of substances, from anti‐ bodies to silica, the quantum dot can by assigned to a specific immunoassay. For example, immunoassays which employ fluorescence spectroscopy as analytical technique had used distinct quantum dots as labels, such as quantum dots conjugated to antibodies, covered by biotin or bare quantum dots, while lanthanide-quantum dots were assigned to time-re‐ solved fluorescence experiments and streptavidin-modified quantum dots were used in ei‐ ther in fluorescent or chemiluminescent experiments.

Such a variety of methods to adapt the inorganic nanoparticles to immunoassays leads to an‐ other classification, with respect to the exploited mechanism. In this sense, immunoassays are classified *as non-competitive* or *sandwich assays*, when involving an analytical path in which the antigen in the sample is bound to the antibody site and a second labeled antibody is bound to the antigen, resulting in a response that is directly proportional to the concentration of the ana‐ lyte; and as *competitive immunoassays*, when involving the competition of the antigen in the sample and the labeled antigen to bind to specific antibodies, resulting in a response that is in‐ versely related to concentration. In a brief comparison, direct assays are faster, once they em‐ ploy only one antibody, eliminating the secondary antibody cross-reactivity. In contrast, indirect immunoassays are much more sensitive.

As a competitive immunoassay example, Ding et al. [43] coated a microtiter platewith ovalbumin haptenand observed the decrease of the fluorescenceat higheranalyte concen‐ trations. This indirect immunoassay was used to determine sulfamethazine residuesin chicken-muscle tissues with alimit of detection of 1 ng/mL for the immunoassay. Compet‐ itive assays also contemplate those using fluorescent probes as internal standards, such as quantitative assays.

As an example of non-competitive detection strategies is the new indirect immunoassay pro‐ posed by Li et al., [44] in which quantum dot fluorescent labels were combined to enzyma‐ ticchemiluminescentlabels. This system was used to simultaneously detect three cancer markers in human serum and thelimits of detection were the same for all markers, in the ng/mL range. Seeking for a new strategy that to be applied to the construction of a biosensor for most distinct application, they coupled the quantum dot to other two chemiluminescent enzymes, creating a hybrid multiplexed detection system for lung cancer.

#### *2.6.1. The Surface Enhanced Resonance-Raman Scattering applied to quantum dots biosensors*

It is noteworthy that quantum dots can be applied to surface-enhanced resonance Raman scattering (SERS) measurements as a powerfultool for ultrasensitive analysis, since unlike fluorescence techniques, SERS-active groups do not self-quench. An example is Han et al. work, [45] where fluorescein was used as Raman probe for a microtiter plate covered by an‐ tigen (human IgG) samples of unknown concentration. In their experiments, a solution of fluorescein-conjugated antibody was added to the sample and fluorescein SERS spectrum was recorded with a limit of detection of 0.2 ng mL-1 in a several-days stable device.

#### **2.7. Labeled and labeled-free optical biosensors**

Among optical biosensors that can be constructed based on this spectroscopic technique, there are those classified as labeled-fluorescent biosensor, as the above mentioned examples, but there is also a evolving rinsing class of optical label-free biosensors [46]. The most cru‐ cial distinction between these techniques is that label-free biosensors can directly evaluate the properties of the system, instead of the fluorescent response of a labeled material to the effect of its local environment. In their review, Fan et al. [46] make a clear distinction be‐ tween labeled and label-free optical biosensors, with respect to techniques of detection, sam‐ ple preparation, sensibility and versatility. They mentioned that although there are some great differences between fluorescence-based and label-free detection techniques, both are widely used in optical sensors construction.These distinct characteristics of the label-free de‐ vices make of these optical biosensors the most versatile among all types of sensing technol‐ ogies that are only able of label-free detection, as in the case of surface acoustic wave and quartz crystal microbalance technologies. It also discusses Raman, refractive index and ab‐ sorbance as detection methods. These approaches enable the optical detection to be yet more versatile, enabling the construction of a series of other biosensors, only by specifying even more de detection technique. It is not unusual that an optical label-free biosensor mixes more optical structures to enhance its sensing performance. For example, among the refrac‐ tive index optical biosensors, they can be:

**•** Surface plasmon resonance

The first work that employed the plasmon resonance phenomenon to sensing was devel‐ oped by Liedberg et al. in 1983.[47] Since then, this remarkable technique has been widely employed in many biochemical and biotechnological fields and greatly developed. Several types of biorecognition elements are currently used, depending on the application. This technique is so versatile that have been employed in a wide range of processes, including food and environmental monitoring and clinical analyses.

In simple words, surface plasmon is a charge density wave over a metallic surface. In the case that a thin layer of a metal is deposited on glass, there must be distinct dielectric con‐ stants on both faces of the film: the one in contact with glass surface and the other in contact with air. Then, a charge density oscillation occurs at these interfaces, leading to the phenom‐ enon occurrence. It is observed as a sharp minimum of the light reflectance when the inci‐ dent angle is changed, leading to a very important sensitivity to refractive indexes variations. These systems can be excited by some methods, as the waveguide coupling, where a dielectric and a metal are positioned over the substrate, generating a waveguiding layer, which creates an interface between metal and the waveguide. Light, therefore, propa‐ gates in this waveguide through totalinternal reflection, giving rise to an evanescent field at the interface, which excites the surface plasmon wave. [48, 49] Although this is a very popu‐ lar technique, there are other methods to excite the surface plasmon wave that can lead to better detection limits, such as prism coupling. Nevertheless, waveguide coupling consists of an alternative due to easily combine to other optical components.

Some advances of these techniques had been presented in the last few years. In their work, Sacarano et al.[50] presented the SPR imaging technique, or *"SPR microscopy"* as the "most attractive and powerful advancement of SPR-based optical detection", which presents the advantage of coupling the sensitivity of the SPR measurements with the spatial capabilities of imaging. In this approach, the entire biochip surface is visualizedin real time, which, as a perspective, might enable experiments based on the continuous monitoring of immobilized spot arrays, with controlled size and shape and with no need of labeling.

**•** Interferometry:

itive assays also contemplate those using fluorescent probes as internal standards, such as

As an example of non-competitive detection strategies is the new indirect immunoassay pro‐ posed by Li et al., [44] in which quantum dot fluorescent labels were combined to enzyma‐ ticchemiluminescentlabels. This system was used to simultaneously detect three cancer markers in human serum and thelimits of detection were the same for all markers, in the ng/mL range. Seeking for a new strategy that to be applied to the construction of a biosensor for most distinct application, they coupled the quantum dot to other two chemiluminescent

enzymes, creating a hybrid multiplexed detection system for lung cancer.

*2.6.1. The Surface Enhanced Resonance-Raman Scattering applied to quantum dots biosensors*

was recorded with a limit of detection of 0.2 ng mL-1 in a several-days stable device.

**2.7. Labeled and labeled-free optical biosensors**

tive index optical biosensors, they can be:

**•** Surface plasmon resonance

It is noteworthy that quantum dots can be applied to surface-enhanced resonance Raman scattering (SERS) measurements as a powerfultool for ultrasensitive analysis, since unlike fluorescence techniques, SERS-active groups do not self-quench. An example is Han et al. work, [45] where fluorescein was used as Raman probe for a microtiter plate covered by an‐ tigen (human IgG) samples of unknown concentration. In their experiments, a solution of fluorescein-conjugated antibody was added to the sample and fluorescein SERS spectrum

Among optical biosensors that can be constructed based on this spectroscopic technique, there are those classified as labeled-fluorescent biosensor, as the above mentioned examples, but there is also a evolving rinsing class of optical label-free biosensors [46]. The most cru‐ cial distinction between these techniques is that label-free biosensors can directly evaluate the properties of the system, instead of the fluorescent response of a labeled material to the effect of its local environment. In their review, Fan et al. [46] make a clear distinction be‐ tween labeled and label-free optical biosensors, with respect to techniques of detection, sam‐ ple preparation, sensibility and versatility. They mentioned that although there are some great differences between fluorescence-based and label-free detection techniques, both are widely used in optical sensors construction.These distinct characteristics of the label-free de‐ vices make of these optical biosensors the most versatile among all types of sensing technol‐ ogies that are only able of label-free detection, as in the case of surface acoustic wave and quartz crystal microbalance technologies. It also discusses Raman, refractive index and ab‐ sorbance as detection methods. These approaches enable the optical detection to be yet more versatile, enabling the construction of a series of other biosensors, only by specifying even more de detection technique. It is not unusual that an optical label-free biosensor mixes more optical structures to enhance its sensing performance. For example, among the refrac‐

The first work that employed the plasmon resonance phenomenon to sensing was devel‐ oped by Liedberg et al. in 1983.[47] Since then, this remarkable technique has been widely

quantitative assays.

122 State of the Art in Biosensors - General Aspects

Based on the interferometry technique of improving analytical signals, some types of inter‐ ferometer-based biosensors were developed, such as Mach-Zehnder, Young's multi-channel and Hartman, among the most commonly used. They are based on the concept that a guided wave suffers a phase change when its evanescent field interacts with the sample. This inter‐ action produces an optical phase change that is quantitatively related to the sample. In these constructions, a sensitive biosensor must present a long interaction length between guided wave and sample. Although many are the interferometric components that can be employed in a biosensor construction, these are by far the most commonly found:

**•** Mach-Zehnder interferometer:

It is composed of beam splitter that divides a coherent, polarized single frequency of a laser beam into two branches. The first one is passed through a window that leads to the refer‐ ence branch of the interferometer. The second one passes through the detection branch win‐ dow, in which the evanescent field interacts with the sample. Both beams are kept apart by a thick coating layer. They recombine at the output, resulting in an interference pattern that is detected at the photodetector. This type of interferometer gives rise to excellent bulk refrac‐ tive index detection capability, but until now, there had not been much development of de‐ vices based on this interferometer.

**•** Young´s interferometer:

Similar to Mach-Zehnder interferometer, this is also based on the passage of a laser beam into a slit, reaching a splitter. The laser beam is divided into reference and sensing branches, but instead of recombining at the output, the optical output of the two branches combine to form interference fringes on a CCD detector. This improves the signal, giving information about spatial intensity distribution along the CCD.

**•** Hartman interferometer:

In this configuration, optical elements are placed over a planar waveguide, organized in strips. A laser beam enters the device by an input grating, reaching the optical elements composed of functionalized molecules and leaving the device by the output grating. [51] In‐ tegrated optics is positioned after the output, creating interference between pairs of func‐ tionalized strips.

**•** Backscattering interferometer:

The most common backscattering interferometers employ in their construction a simple op‐ tical arrangement composed of a coherent light source (ususally a low-power He-Ne or reddiode laser), a microfluidic path, and, of course, a phototransducer. The backscattering interferometry technique presents some advantages when compared to the above men‐ tioned techniques. Since it is based on microfluidic concepts, it shows comparable perform‐ ance as former interferometers, but using a much smaller sensing area,which permits a wide range of configuration. In this approach, the coherent laser beam is focused on a small sens‐ ing area, the interaction of the laser beam with the fluid-filled microchannelleads toan inter‐ ference pattern that is registered in the photodetector, which is sensible to the laser reflected intensity. When a biological sample is placed over the illuminated surface, a laser of distinct intensity is detected due to a phase change caused by the light reflection over this surface.

**•** Photonic Technologies

The photonic technology has been object of many research fields and of a very rapid im‐ provement, compared to other technologies. In this sense, there are many methods to em‐ ploy photonicprinciples, and a wide range of scientific and technological issues to apply it, resulting in several equipment proposals. The broad range of applications of photonic devi‐ ces permits to glimpse the importance of this emerging field. It is possible to incorporate dif‐ ferent types of lasers, dielectric waveguide structures and photodetectors in a variety of possible equipments, that enables the perspective of explore from ultraviolet to far infrared, extending the fundamental research approach and the application possibilities, that can ex‐ plain why photonics application, in other potential technologies, has grown in such impres‐ sive way.

In the biosensor perspective, many materials and concepts of application have been devel‐ oped and a wide range of the proposed devices employ optical fiber and waveguides, as well as photonic crystals. Here some characteristics of such devices are pointed.

**•** Optical fiber

detected at the photodetector. This type of interferometer gives rise to excellent bulk refrac‐ tive index detection capability, but until now, there had not been much development of de‐

Similar to Mach-Zehnder interferometer, this is also based on the passage of a laser beam into a slit, reaching a splitter. The laser beam is divided into reference and sensing branches, but instead of recombining at the output, the optical output of the two branches combine to form interference fringes on a CCD detector. This improves the signal, giving information

In this configuration, optical elements are placed over a planar waveguide, organized in strips. A laser beam enters the device by an input grating, reaching the optical elements composed of functionalized molecules and leaving the device by the output grating. [51] In‐ tegrated optics is positioned after the output, creating interference between pairs of func‐

The most common backscattering interferometers employ in their construction a simple op‐ tical arrangement composed of a coherent light source (ususally a low-power He-Ne or reddiode laser), a microfluidic path, and, of course, a phototransducer. The backscattering interferometry technique presents some advantages when compared to the above men‐ tioned techniques. Since it is based on microfluidic concepts, it shows comparable perform‐ ance as former interferometers, but using a much smaller sensing area,which permits a wide range of configuration. In this approach, the coherent laser beam is focused on a small sens‐ ing area, the interaction of the laser beam with the fluid-filled microchannelleads toan inter‐ ference pattern that is registered in the photodetector, which is sensible to the laser reflected intensity. When a biological sample is placed over the illuminated surface, a laser of distinct intensity is detected due to a phase change caused by the light reflection over this surface.

The photonic technology has been object of many research fields and of a very rapid im‐ provement, compared to other technologies. In this sense, there are many methods to em‐ ploy photonicprinciples, and a wide range of scientific and technological issues to apply it, resulting in several equipment proposals. The broad range of applications of photonic devi‐ ces permits to glimpse the importance of this emerging field. It is possible to incorporate dif‐ ferent types of lasers, dielectric waveguide structures and photodetectors in a variety of possible equipments, that enables the perspective of explore from ultraviolet to far infrared, extending the fundamental research approach and the application possibilities, that can ex‐ plain why photonics application, in other potential technologies, has grown in such impres‐

vices based on this interferometer.

124 State of the Art in Biosensors - General Aspects

about spatial intensity distribution along the CCD.

**•** Young´s interferometer:

**•** Hartman interferometer:

**•** Backscattering interferometer:

**•** Photonic Technologies

sive way.

tionalized strips.

The two basic concepts of optical fiber based biosensors are the *Fiber Bragg´s grating* and the *long-term grating*. They differ from each other not in principle, but in construction. While the fiber Bragg´s grating concept requires the etching of the fiber (or grating) surface, followed by the physical pattern of the surface, the long-term grating is a configuration based on peri‐ odic grating of 100 µm to 1 mm, which make them much larger than the common Fiber Bragg´s gratings and confer them the advantage of an increased sensing to refractive index changes. Moreover, they are easier to build and can be customized by chemically removal of the coating. Either fiber Bragg´s or long term grating designs can lead to very high refractive index sensitivity and low detection limits, which consist of the most desired characteristics of promising biosensors.

**•** Optical waveguide

This elegant technique has been applied for biosensing in the last decade with a considera‐ ble success. Due to that, many structures of construction had been proposed usually direct‐ ed by the analyte of interest. In this concept, some popular structures are:


In this configuration, light suffers a total internal reflection in the boundaries of a curved interface between a high and a low refractive media. This process leads light to propa‐ gate in the circulating waveguide form or in the whispering gallery modes, as illustrated by Fan and cited by some other works therein.[46]Devices based on this technique can be constructed in a much smaller scale than those based on the former techniques with simi‐ lar sensing capability, which is their great advantage. They can be constructed in a num‐ ber of configurations, in which the microfabricated ring shaped, disk shaped or microtoroid shaped resonators on a chip, the stand-alone dielectric microspheres and the so called capillary-based opto-fluidic ring resonators are the most common examples. The chip-based ring resonators present some advantages which include the capability of opto‐ electronic integration, but, apart from the microtoroid configuration, they usually present problems of low quality factors (Q-factor), which are designated as all intrinsic and extrin‐ sic losses occurred in the optical resonant cavity system [52] and in this case, these prob‐ lems are related to their surface roughness. These types of ring resonators are very well presented and discussed in Fan´s review. [46]

**•** Photonic crystal

This class of biosensors is, in fact, an evolution of the optical fiber based ones. They are formed by photonic crystal microcavities, obtained by introducing a defect in periodically organized microstructured holes, usually of silica, by altering their dimensions. Some can be embedded with molecules, which are responsible for the occurrence of a change in the re‐ fractive index of the biosensor, leading to a detectable signal in the form of a spectral shift of the resonant wavelength of the photonic crystal cavity. Also, polymers can be used as a coat‐ ing layer for the photonic crystal cavities, as showed by Chakravarty et al., [53] which doped the photonic crystal microcavities with a quantum dot and coated it with anion-selective polymer. With this procedure, they were able to construct a sensor with good properties, such as a very specific and accurate detection for changes of perchlorate anions and calcium cations at submicro concentrations in solution, while Lee et al. [54] presented a photonic crystal suitable for protein and single particle detection. In their experimental and theoreti‐ cal work, they claimed their device achieved a sensing volume of 0.15 µm3 , and that it pre‐ sented a limit of detection as small as 1 fg. They also determined its performance for particles in the size range of a variety of viruses, using latex spheres as models.

In a recent work, Aroua et al.[55] have studied, also by experimental and theoretical means, a label-free biosensor in order to determine it characteristics, the field intensity and the reso‐ nant wavelength shift when the nanocavities of the photonic crystal are filled with blood plasma, water or dried air. With this protocol, they showed that the enhancement on sensi‐ tivity is related to the photonic crystal design parameters.

#### **2.8. Carbon nanotubes and graphene-based biosensors**

Some new materials had also found a great deal of applications, especially in biosensing, such as carbon nanotubes and lately, graphene. As for single-walled carbon nanotubes, (SWNTs), they are known to exhibit unique intrinsic properties, which include a semiconductive behav‐ ior and photophysical propertiesdependent of their structure. For example, nanotubes with some chirality, band gap fluorescence is observed, as well as strong resonance Raman scatter‐ ing.In this way, hybrid materials of SWCNTs and biomolecules is a way to obtain good materi‐ als for biosensing applications, since the fluorescence band-gap of SWNTs is highly sensitive to its environment and show shifts when the nanotube is in contact with other molecules.

In their work, Jin et al. [56] proposed the construction of a platform for selectively deter‐ mine the hydrogen peroxide efflux from living cells, in order to biosensing human carci‐ noma, in an array of fluorescent single-walled carbon nanotubes. In this biosensor, the carbon nanotubes have their fluorescence quenched when H2O2 is liberated by A431 hu‐ man epidermal carcinoma cells, in response to the epidermal growth factor. They show that this array is able to distinguish between peroxides originated on the cell membrane from other contributions.

Also to show the versatility of carbon nanotubes, Chen et al. [57]presented a sensitive meth‐ od for multiplexed protein detection by using functionalized single-walled carbon nano‐ tubes (SWNTs) as multicolor Raman labels. They claim that this method is a good alternative for standard fluorescence-based techniques since, unlike fluorescence, Raman de‐ tection benefits from the sharp scattering peaks of SWNTs with minimal background inter‐ ference. Also, it can be combined to surface-enhanced Raman scattering substrates, allowing protein detection sensitivity down to 1 x 10-15Mol L-1, which is three orders of magnitude mi‐ nor then the detection limit of fluorescence-based methods. They used these modified SWNT to Raman detection of human autoantibodies against proteinase 3, a biomarker for the Wegener's granulomatosisautoimmune disease, and by conjugating different antibodies to pure (12)C and (13)C SWNT isotopes, they had demonstrated the multicolor Raman pro‐ tein detection.

In their work,Morales-Narvaéz and Merkoçi[58] took advantage of graphene´sinnovative mechanical, structural (several graphenes present lattice-like nanostructures), electrical, thermal and opticalproperties. They employed graphene oxide (GO) as a biosensing plat‐ form due to its ability of nanoassemble in wire form when in presence of biomolecules, its processability in solution and due to its heterogeneous chemical and electronic structure, which confers to GO the ability to be used as insulator, semiconductor or semi-metal. Also, they presented graphene oxide as a universal highly efficient long-range quencher, with the perspective of been applied to several novel biosensing strategies.

Phan and Viet also worked on testing graphene application on biosensors by replacing car‐ bon nanotubes for graphene ribbons in biosensors,[59] which were able to sense the transi‐ tion of DNA secondary structure from the native right-handed form to the alternate lefthanded form.

Although studies ongraphene´s propertiesare still preliminary, it is thought as a promising platform for biosensing. In their review, Yang et al. [60] discuss all aspects of functionality, performance, properties, fabrication, handling and challenges of these carbon-based materi‐ als as part of biosensors. Ina critical analysis, they present the great opportunities yet to come with the use of these materials and point us what is necessary to have in mind when proposing a new architecture for biosensors and new materials to be employed. Neverthe‐ less, graphene application on bioelectronics is still controversial.
