**4.4. Electrochemical biosensors**

The first biosensor based on cholinesterase (ChE) inhibition for detection of nerve agents was developed by G.Guilbaut in 1962 [34]. Since then, many other enzymes have been used in biosensor for detecting and quantifying a huge realm of parameters.

Other important enzyme used in biosensors is the acetylcholinesterase (AchE). The principal biological role of AchE is the termination of the nervous impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine [35]. The AFB1 inhibits AChE by binding at the peripheral site, located at the entrance of the active site (at the tryp‐ tophane residue) [36]. Even though, there other enzymes, as butyrylcholinesterase (BChE) that are also used for detection of AFB1, AChE is preferred because it is more sensitive than BChE for this purpose [37].

Biosensors based on the amperometric method allow the detection of low aflatoxin concen‐ tration. For example, acetylcholinesterase (AchE) is measured using a choline oxidase am‐ perometric biosensor [38]. In this example the decrease in the amperometric activity of AchE has a direct relationship with the quantity of aflatoxins. This method is commonly used when the aflatoxin concentration is too low, and it cannot be detected with the spectropho‐ tometric method.

#### **4.5. Biosensors that combine techniques**

Commonly, polymerase chain reaction (PCR) has been used to accurately detect low num‐ bers of different pathogens with multiple sets of primers. But, important disadvantages of PCR are: the inhibition of the polymerase enzyme by the contaminants from the sample; dif‐ ficulties in quantification; false positives resulting from the detection of naked nucleic acids; and non-viable microorganisms or contamination of samples in the laboratory. Biosensors are useful tools that provide a rapidly detection of the presence and amount of microorgan‐ isms in any given environments [26]. Thus, the mixture of different techniques might over‐ come the exposed problem. For example, in [39] there was a decrease of contaminants by coupling PCR with a piezoelectric biosensor.

There are biosensors that combine biological and physical/physicochemical transducers (SPR, piezoelectric, acoustic, and amperometric biosensors). The related problems for these biosensors are: chemical/physical stability of the transducers in the biological samples, the difficulty in production of highly specific antibodies, poor signal, etc. Such problems are of‐ ten overcome by: coating the surface to make the transducer compatible with the biological samples; using of highly specific monoclonal antibodies; and incorporating amplification steps to generate stronger signals [26].

Latest researches on nanomaterials, such as carbon nanotubes, metal nanoparticles, nano‐ wires, nanocomposite and nanostructurated materials reveal to be a key points in the design of the near future biosensing systems with applications in aflatoxin detection [40].

The aforementioned methods to quantify aflatoxins present several disadvantages, for in‐ stance those based on chromatography, however they have laborious and time-consuming process [41]. Therefore, a pathway to improve AFs detection is through biosensors. This term was first used by Cammaann in 1977 [42], who defined it as a device that enables the identification and quantification of the interest sample (e.g. water, air, food, solutions, among others). Nevertheless, the main characteristic in a biosensor is the biological recogni‐ tion element that is capable to create a response of interest. Such element can be an anti‐ body, an antigen or an enzyme [43].

There are many kinds of biosensors applied to detection aflatoxins, however they majorly work in conjunction with immunochemical methods. Such junctions are based on the high affinity of antigen-antibody interaction and have the aim of increasing the sensitivity and decreasing the detection time of the toxic element [41].

#### **4.6. Immunochemical**

Among the optical techniques used in biosensors it can be found: non linear optics (based on surface plasmon resonance) [30], resonant mirror, fiber-optics [31], complementary metal ox‐ ide semiconductors, fluorescence/phosphorescence [32], reflectance, light scattering, chemi‐

Such advantages, plus their easy operation and wide detection capacity, have made of opti‐

The first biosensor based on cholinesterase (ChE) inhibition for detection of nerve agents was developed by G.Guilbaut in 1962 [34]. Since then, many other enzymes have been used

Other important enzyme used in biosensors is the acetylcholinesterase (AchE). The principal biological role of AchE is the termination of the nervous impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine [35]. The AFB1 inhibits AChE by binding at the peripheral site, located at the entrance of the active site (at the tryp‐ tophane residue) [36]. Even though, there other enzymes, as butyrylcholinesterase (BChE) that are also used for detection of AFB1, AChE is preferred because it is more sensitive than

Biosensors based on the amperometric method allow the detection of low aflatoxin concen‐ tration. For example, acetylcholinesterase (AchE) is measured using a choline oxidase am‐ perometric biosensor [38]. In this example the decrease in the amperometric activity of AchE has a direct relationship with the quantity of aflatoxins. This method is commonly used when the aflatoxin concentration is too low, and it cannot be detected with the spectropho‐

Commonly, polymerase chain reaction (PCR) has been used to accurately detect low num‐ bers of different pathogens with multiple sets of primers. But, important disadvantages of PCR are: the inhibition of the polymerase enzyme by the contaminants from the sample; dif‐ ficulties in quantification; false positives resulting from the detection of naked nucleic acids; and non-viable microorganisms or contamination of samples in the laboratory. Biosensors are useful tools that provide a rapidly detection of the presence and amount of microorgan‐ isms in any given environments [26]. Thus, the mixture of different techniques might over‐ come the exposed problem. For example, in [39] there was a decrease of contaminants by

There are biosensors that combine biological and physical/physicochemical transducers (SPR, piezoelectric, acoustic, and amperometric biosensors). The related problems for these biosensors are: chemical/physical stability of the transducers in the biological samples, the difficulty in production of highly specific antibodies, poor signal, etc. Such problems are of‐ ten overcome by: coating the surface to make the transducer compatible with the biological

cal biosensors useful tools for the detection of dangerous organisms as aflatoxins.

in biosensor for detecting and quantifying a huge realm of parameters.

luminescence, and refractive index [33].

298 Aflatoxins - Recent Advances and Future Prospects

**4.4. Electrochemical biosensors**

BChE for this purpose [37].

**4.5. Biosensors that combine techniques**

coupling PCR with a piezoelectric biosensor.

tometric method.

These kinds of sensors use mainly immunological receptor units such as antibodies or anti‐ gens, and detection methods as optic effects ( e.g. fluorescence and plasmon resonance), electrochemical, or acoustical readout [44]. The majorly of these techniques are comprised of three main steps: First, the extraction of the aflatoxin from de complex mixtures of materials in which it is found; then, the purification of the sample for removing pollutants; and final‐ ly, the detection and quantification of the toxins [45].

The main challenges of these types of biosensors are the design and construction of proto‐ types which minimize their handling. Besides, they must use the best immunochemical techniques, with the aim to generate automated sensors that replace the existing large, complex, cumbersome, and chemical laboratory analysis systems. Such immunochemical biosensors would offer the benefit of an increasingly developing of modular design that would permit the rapid substitution of other reagents to detect different toxics with the same platforms [45].

In [45] is reported a biosensor that it is based in the property of fluorescence. This fluores‐ cence system consists on an arc lamp that generates a microsecond flash and a lens that fo‐ cuses in the radiation into the sample. Such sample was previously treated, with process shown on the Figure 3 which in turn shows the three main steps before the antigen detection with the automated process placed in the arrows. Then the detection consists in using a filter which allows the passing of UV radiation, around 365 nm. This wavelength excites the fluo‐ rescence of aflatoxins.

that the biosynthesis of aflatoxins has been extensively studied, and more than 25 genes ar‐

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DNA biosensor has given out rapid and accurate measurements of aflatoxins in milk or dai‐ ry products [41]. The novel contribution of such system is its measurement technique based on electrochemical impedance spectroscopy (EIS) to analyze compounds that have restricted

The EIS method in recent years has become a powerful tool for evaluating many biochemi‐ cal and biophysical processes. The biosensor's characterization and fabrication can be gener‐ ated through EIS. Moreover, with the interaction between enzyme–substrate, biomolecule which have no reaction sequence after binding (such as antigen–antibodies), and DNA, among others, charge transfer changes occurred after affinity interactions can also be moni‐

To identify the aflatoxin M1A, it is necessary the ss-HSDNA, which was specifically bounded to this aflatoxin. It is necessary to immobilize the ss-HSDNA on gold electrodes with the help of cysteamine and gold nanoparticles. The differences between before and after binding of aflatoxin M1 to the HSDNA probe can be analyzed with a cyclic voltammetry and IES. An aflatoxin M1 calibration curve was prepared by considering the differences in electron trans‐

However, in the most of the cases, the sample needs a pretreatment. For milk case, in order to remove the milk fat, the sample was centrifuged. Then three completely separated phases were obtained. The layer at the top was the fat; the cream was at the center; and the fat free milk was at the bottom. This last phase was used for the experiment as a sample, in order to

Biosensors based on piezoelectric effect are commonly used for aflatoxins detection because they have the property of providing sensitive measurements in air as well as in liquids. This kind of biosensors, based on piezoelectric quartz crystal (PQC), is usually combined with most of the above mentioned methods, like immunosensors with cells, bacteria, proteins (in‐

Between the different existing kinds of immunosensors, the PQC has been extensively ap‐ plied to biorecognition sensing due to its advantages of cost-effectiveness, direct detection, experimental simplicity, and real-time output. The principle of these sensors is based in the fact that the quartz is used as transducer, its resonance frequency changes with the change

In [49] the author reports a DNA-based piezoelectric biosensor with the aim of detecting a PCR-amplified 248-bp fragment of the aflD gene of *A. flavus and A. parasiticus* involved in the conversion from norsolorinic acid to averantin. Such biosensor was used for the analysis of DNA fragments coming from the amplification of DNA extracted from reference strains of *A. parasiticus.* Originally it was designed with the objective of researching about the influ‐

ranged in a 70-kbp gene cluster were identified [49].

fer resistances before and after aflatoxin M1 binding [41].

avoid any possibly negative effect of fat on the EIS.

cluding antibody or antigen), DNA and so on [50].

in the mass, according to the Sauerbrey equation.

**4.8. Piezoelectric biosensors**

catalytic interaction activity such as aflatoxins.

tored with EIS.

**Figure 3.** immunochemical-based capture, purification and detection process modify by [45].

After the excitation it is necessary the monitoring of the fluorescence response. This moni‐ toring is carried out by a second lens that captures some of the light emitted by the sample through a photomultiplier tube and a filter centered around 455 nm. This is the wavelength where these AFs fluorescence. This device detects concentrations from 0.1 o 50 ppb in less than 2 minutes using a sample volume of 1 ml [45].

Another method used in conjunction with immunological techniques is named optical waveguide lightmode spectroscopy (OWLS). This technique is based in the precise measure‐ ment of the resonance angle of polarized laser light, diffracted by a grating in coupled into a thin waveguide. The incoupling resonance effect is very sensitive; such effect depends on the optical parameters of the sensor and the refractive index of the covering sample medi‐ um. The intensity effect response is carried out by a photodiode with the aim of determining the refractive index of the resonance incloupling angle detected with high accuracy [31].

There is another versatile technique named self-assambly structures that are considered as promising noble nanoscale systems with a several numbers of applications (solar cells, data storage, and biosensors). With this process it is possible to create biomarkers to exploit the absence of ligands on these nanoparticles surface that enhances the possibility of working better with molecules [46].

The self-assembly nanoparticles of nickel and gold are widely used for biosensing applica‐ tions due to their biocompability, high surface to volume ratio, strong adsorption, fast elec‐ tron transfer, enhanced sensitivity, high selectivity, and large detection range [47].

#### **4.7. Centralized testing of DNA**

Due to the necessity of creating simpler and more user-friendly methods for aflatoxins de‐ tection, it has been developed centralized testing of DNA. This method allows the early de‐ tection of genes associated with human diseases [48]. In this case, it is interesting to denote that the biosynthesis of aflatoxins has been extensively studied, and more than 25 genes ar‐ ranged in a 70-kbp gene cluster were identified [49].

DNA biosensor has given out rapid and accurate measurements of aflatoxins in milk or dai‐ ry products [41]. The novel contribution of such system is its measurement technique based on electrochemical impedance spectroscopy (EIS) to analyze compounds that have restricted catalytic interaction activity such as aflatoxins.

The EIS method in recent years has become a powerful tool for evaluating many biochemi‐ cal and biophysical processes. The biosensor's characterization and fabrication can be gener‐ ated through EIS. Moreover, with the interaction between enzyme–substrate, biomolecule which have no reaction sequence after binding (such as antigen–antibodies), and DNA, among others, charge transfer changes occurred after affinity interactions can also be moni‐ tored with EIS.

To identify the aflatoxin M1A, it is necessary the ss-HSDNA, which was specifically bounded to this aflatoxin. It is necessary to immobilize the ss-HSDNA on gold electrodes with the help of cysteamine and gold nanoparticles. The differences between before and after binding of aflatoxin M1 to the HSDNA probe can be analyzed with a cyclic voltammetry and IES. An aflatoxin M1 calibration curve was prepared by considering the differences in electron trans‐ fer resistances before and after aflatoxin M1 binding [41].

However, in the most of the cases, the sample needs a pretreatment. For milk case, in order to remove the milk fat, the sample was centrifuged. Then three completely separated phases were obtained. The layer at the top was the fat; the cream was at the center; and the fat free milk was at the bottom. This last phase was used for the experiment as a sample, in order to avoid any possibly negative effect of fat on the EIS.

#### **4.8. Piezoelectric biosensors**

**Figure 3.** immunochemical-based capture, purification and detection process modify by [45].

than 2 minutes using a sample volume of 1 ml [45].

300 Aflatoxins - Recent Advances and Future Prospects

better with molecules [46].

**4.7. Centralized testing of DNA**

After the excitation it is necessary the monitoring of the fluorescence response. This moni‐ toring is carried out by a second lens that captures some of the light emitted by the sample through a photomultiplier tube and a filter centered around 455 nm. This is the wavelength where these AFs fluorescence. This device detects concentrations from 0.1 o 50 ppb in less

Another method used in conjunction with immunological techniques is named optical waveguide lightmode spectroscopy (OWLS). This technique is based in the precise measure‐ ment of the resonance angle of polarized laser light, diffracted by a grating in coupled into a thin waveguide. The incoupling resonance effect is very sensitive; such effect depends on the optical parameters of the sensor and the refractive index of the covering sample medi‐ um. The intensity effect response is carried out by a photodiode with the aim of determining the refractive index of the resonance incloupling angle detected with high accuracy [31].

There is another versatile technique named self-assambly structures that are considered as promising noble nanoscale systems with a several numbers of applications (solar cells, data storage, and biosensors). With this process it is possible to create biomarkers to exploit the absence of ligands on these nanoparticles surface that enhances the possibility of working

The self-assembly nanoparticles of nickel and gold are widely used for biosensing applica‐ tions due to their biocompability, high surface to volume ratio, strong adsorption, fast elec‐

Due to the necessity of creating simpler and more user-friendly methods for aflatoxins de‐ tection, it has been developed centralized testing of DNA. This method allows the early de‐ tection of genes associated with human diseases [48]. In this case, it is interesting to denote

tron transfer, enhanced sensitivity, high selectivity, and large detection range [47].

Biosensors based on piezoelectric effect are commonly used for aflatoxins detection because they have the property of providing sensitive measurements in air as well as in liquids. This kind of biosensors, based on piezoelectric quartz crystal (PQC), is usually combined with most of the above mentioned methods, like immunosensors with cells, bacteria, proteins (in‐ cluding antibody or antigen), DNA and so on [50].

Between the different existing kinds of immunosensors, the PQC has been extensively ap‐ plied to biorecognition sensing due to its advantages of cost-effectiveness, direct detection, experimental simplicity, and real-time output. The principle of these sensors is based in the fact that the quartz is used as transducer, its resonance frequency changes with the change in the mass, according to the Sauerbrey equation.

In [49] the author reports a DNA-based piezoelectric biosensor with the aim of detecting a PCR-amplified 248-bp fragment of the aflD gene of *A. flavus and A. parasiticus* involved in the conversion from norsolorinic acid to averantin. Such biosensor was used for the analysis of DNA fragments coming from the amplification of DNA extracted from reference strains of *A. parasiticus.* Originally it was designed with the objective of researching about the influ‐ ence of different parameters, such as amplicon concentration, dilution, and PCR specificity on the biosensor's response.

oratory; the biosensors must be the way to create embedded systems with the aim to detect the aflatoxins *in vivo*. Because of this, it is necessary to automate the whole process including the pretreatment of the sample to generate more efficient systems and manageable and bet‐

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Aflatoxins measurement usually implies complex, expensive and slow methods. However, this determination can be carried out taking into account the response of aflatoxins to deter‐ minate electrical stimulus. These methods are called electrochemical, where immunosensors are applied to determine the presence of aflatoxins in a sample. Usually, these sensors are composed by two screen-printed-electrodes (SPE), the first one is made of graphite, plati‐ num, or gold; and it is known as working, active, or measuring electrode. The second elec‐ trode is the reference and is commonly made of Ag/AgCl. In general, this technique involves two basic steps. In the first, the immunosensor working electrode is coated with an anti‐ body; after an incubated time, the sample that contains the aflatoxins is added to this elec‐ trode, while the left one reacts for a determinate time; finally, a conjugated of aflatoxins and enzymes is added to the electrode, it is then when the competitive reaction begins. In this reaction, free aflatoxins compete to link to antibodies present in the working electrode. After a stabilization time, the measuring electrode is removed from the sample and rinsed with a buffer solution. The second step implies to apply an electrical potential (commonly 100 mV) to the electrode, which changes its electrical conductivity according with the aflatoxins con‐ centration. After sampling the electrode; an increase or reduction in the electric current flow will appear according with the concentration of aflatoxins in the sample. This technique has received improvements; disposable immunosensors have been reached for measurement of aflatoxins M1 (AFM1) directly in milk following a simple centrifugation step but without di‐ lution or other pretreatment steps. Exhibiting a good working range, comparable to the ones obtained in buffer; linearity between 30 and 240 ng/ml making it useful for AFM1 monitor‐ ing in milk (maximum acceptable level of AFM1 in milk is 50 ppt) [52]. It is easy to notice that electrochemical techniques offer some advantages over traditional methods for aflatox‐ ins determination, among which it can be found: reliability, low cost, *in-situ* measurements, fast processes, and easier methodology than common chromatography techniques through a

Other improvements to this methodology involve the analysis of thermal stability given that the conductivity properties of materials also change with temperature variations and not on‐ ly for the aflatoxins concentration in the electrode. SPEs with platinum as substrate for the working electrode have been used to achieve long-term stability. Probes have shown that this type of electrodes maintain a good biorecognition affinity for antibodies on its layer and a decrease in the detected signal of less than 10% after two weeks inside a refrigerator (5 °C) and less than 22% at laboratory temperature (25 °C), values that allow partial usability for

ter aflatoxin detection biosensors.

**5. Miscellaneous methods**

similar performance

**5.1. Electrochemical methods for aflatoxins determination**

An important point of these kinds of devices is that the crystal only can be used for 25 meas‐ urements without losing sensitivity; this is because the devices that work with mechanical effects are majorly affected with the use. This is the reason for coupling the PCR protocol and the DNA piezoelectric biosensor. After its characterization with synthetic oligonucleoti‐ des, the piezoelectric-DNA biosensor led to the clear identification and quantification of contaminated feed samples with aflatoxins [49].

## **4.9. Optoelectronic**

The principle of the optical waveguide light-mode spectroscopy (OWLS) technique is the measurement of the resonance angle of polarized laser light, diffracted by a grating and in‐ coupled into a thin waveguide. Incoupling resonance occurs at very precise angles depend‐ ing on the optical parameters of the sensor chips and the complex refractive index of the covering sample medium. The intensity of the incoupled light guided within the waveguide layer by multiple internal reflections is measured with a photodiode [31].The refractive in‐ dex is determined from the resonance incoupling angle detected at high precision. Such in‐ dex allows the determination of layer thickness and coverage of the adsorbed or bound material with high sensitivity. This method allows the construction of both chemical and bi‐ osensors. Therefore, it can be applied for direct sensing of various types of biomolecules.

Other optical based biosensor uses a high-tech semiconductor material–silicon for the effi‐ cient accuracy registration of narrow spectral bands or specific wavelengths. This biosensor is used for detecting and quantifying aflatoxins that are commonly found in a variety of ag‐ ricultural products.

Based on the above mentioned techniques it was developed a structure with two oppositely directed potential barriers, the total current conditioned by these barriers depended on both, the external voltage and the wavelength of the absorbed radiation. A modification in these parameters resulted in the obtaining of high-accuracy data of aflatoxins contaminants in food and provender in natural conditions [51].

Detection and identification of harmful organisms, such as aflatoxins, in a cost and time ef‐ fective way is a challenge for the researchers. Biosensors have proved to be useful tools for detection and quantification of such organisms. These sensors have advantages such as: fast response, relative easiness of use, a huge realm of applications, and flexibility for combina‐ tion of techniques. Such advantages are derived from the involvement of multidisciplinary research activities. But, even though the vast research on biosensors, it is still needed the in‐ jection of economical funds to locate them in the commercial market, and impulse their use in real applications.

The research on biosensor has the aim to develop, at low cost an analytical approach simpler and faster. Being an alternative improving the classical techniques. It is necessary that the improvement of the processes is focused in autonomous measurement, in order to avoid, as much as possible, the human error. The mostly classical measurements are linked to the lab‐ oratory; the biosensors must be the way to create embedded systems with the aim to detect the aflatoxins *in vivo*. Because of this, it is necessary to automate the whole process including the pretreatment of the sample to generate more efficient systems and manageable and bet‐ ter aflatoxin detection biosensors.
