**5. Advanced techniques for detection of mycotoxin based biosensor**

The integration of bioreceptors, nanomaterials, and different read-out techniques is capable of accomplishing the rapid, sensitive, and multiplexed detection of mycotoxins. In this section, the advanced applications of different read-out biosensors, including optical, EC, mass-sensitivity, and surface-enhanced Raman spectroscopy biosensors, integrated with the bio-receptors above and nanomaterials, are discussed (**Figure 2**).

#### **5.1 Electrochemical biosensors**

A biosensor is an analytical device that incorporating a bio-component or bio-receptor such as isolated enzymes, whole cell, tissues, aptamers with a suitable transducing system to detect chemical compound [30]. Measurement of the signal is generally electrochemical for biological, and this bio-electrochemical serves as transduction component in electrochemical biosensors. The biological reaction generates change in signal for conductance or impedance, measurable current or change accumulation, which can be measured by conductometric, potentiometric, or amperometric techniques [31]. The interaction between the target molecule and the electrical signal of bio-component produced can be measured.

Electrochemistry has been widely used in various fields, due to their high selectivity and sensitivity, high signal-to-noise ratio, simplicity, miniaturization, low cost, robust to liquid samples and more feasible for on-site application [20]. The electrochemical technique requires a reference, auxiliary, and a working electrode. Two exciting compounds are analyzed using compound biosensors that have interest

#### **Figure 2.**

*The applications of different read-out biosensors integrated with bioreceptors and nanomaterials.*

for nutritional food quality and contaminant such as toxin or pathogen that supposed not to be found in food products [30]. Selection of suitable working electrode is a crucial part of successful electrochemical measurement either by modification in working electrode materials or traditional metals such as mercury or gold [32].

Due to the widely occurring co-contamination of mycotoxins in raw food materials, Lu and Gunasekaran [33] designed and fabricated of an electrochemical immunosensor for simultaneous detection of two mycotoxins, fumonisin B1 (FB1) and deoxynivalenol (DON), in a single test. A dual-channel three-electrode electrochemical sensor pattern was etched on a transparent indium tin oxide (ITO)-coated glass via photolithography and was integrated with capillary-driven polydimethylsiloxane (PDMS) microfluidic channel. The achieved detection limits found 97 and 35 pg./mL, respectively. Besides, Nieto et al. [34] A third-generation enzymatic biosensor were developed to quantify sterigmatocystin (STEH). It was based on a glassy carbon electrode modified with a composite of the soybean peroxidase enzyme (SPE) and chemically reduced graphene oxide. A third-generation enzymatic biosensor to quantify STEH in corn samples spiked with the mycotoxin. The biosensor was based on glassy carbon (GC) electrode modified with a composite of SPE and chemically reduced graphene oxide (CRGO). The biosensor was also used to determine STEH in corn samples inoculated with Aspergillus flavus, which is an aflatoxins fungus producer. The biosensor showed a linear response in the concentration range from 6.9 × 10<sup>−</sup><sup>9</sup> to 5.0 × 10<sup>−</sup><sup>7</sup> mol L<sup>−</sup><sup>1</sup> . The limit of detection was 2.3 × 10<sup>−</sup><sup>9</sup> mol L<sup>−</sup><sup>1</sup> for a signal: noise ratio of 3:1.

#### **5.2 Aptasensor**

The aptamer is referred to the Latin word, aptus means "to fit," which relationship between aptamers and their target look like "lock-and-key" theory [35]. Aptamers usually single-stranded RNA or DNA, which consist of 2–60 nucleotides, which specifically bind to the target, including organic molecules and cells. Aptasensors referred to biosensors using aptamers as biorecognition element and aptasensor were described in 1996 [36] which had been used in multiple sensing applications.

**91**

*Recent Biosensors Technologies for Detection of Mycotoxin in Food Products*

Advantages using aptamers are aptamers can provide high stability and affin-

ity. Aptamers also provide simplicity, low cost, and excellent batch-to-batch reproducibility. Aptasensor can attract massive attention because of excellent binding constant toward most mycotoxins. The critical step in the design of biosensors is immobilization of aptamers because this factor can affect the affinity of the aptamer for target and long-term stability for real sample. There are several immobilization strategies affect the used for aptasensor development. Firstly, the adsorption or π-π interaction between DNA bases aptamer and graphene oxide (GO)-modified interfaces [37]. The covalent linkage of the aptamer to the carboxylic acid group that presents on surface or nanomaterial [38] and thiolated binding aptamers to CdTe quantum dots (QDs) or Au-based materials [39]. Besides, affinity binding based on biotin-streptavidin or other affinity interaction [40, 41] and hybridization of partially complementary single-stranded DNA which immobilized on surface or nanoparticle [42]. Duan et al. [43] developed multicolor quantum dot nanobeads for simultaneous qualitative immunochromatographic detection of mycotoxins (ZEN, OTA, and FB1) in corn samples with detection limits reached up

Immunosensors are devices based on the detection of analyte-antibody interaction. Three main groups have been developing, which are luminescent or colorimetric sensors, surface plasmon resonance, and electrochemical sensors. The sensor usually combined with simple methanol–water for the extraction of a mycotoxin from food samples. Colorimetric and luminescent are based on the visible or UV light transformation into an analytical signal [44]. A colorimetric sensor developed for AFB1 detection using direct competitive ELISA principle. The color was detected and measured with spectrometer by reading absorbance at 620 nm. According to Garden and Strachen [45], this method could detect AFB1 as low as 0.2 ng/mL in artificially contaminated food material as compared to the sensitivity

Surface plasmon resonance (SPR) is an optical phenomenon which used for measure changes on the surface of thin metal films (Au or Ag) under condition total internal reflection [46]. The sensitivity of SPR sensors and microtiter plate ELISAs were compared for detection of AFB1 using same immunoreagents, which are a polyclonal antibody and AFB1-BSA conjugate. As a result, the SPR sensor (3.0– 49 ng/mL) is a more sensitive but narrow and linear range of detection compared to ELISA (12–25,000 ng/mL) [47]. Electrochemical immunosensor for mycotoxin are based on competitive ELISA principle, which electrochemical transducer allows detection redox directly [44]. Pemberton et al. [48] in their study, a calibration plot AFB1 obtained over the concentration range from 0.15 to 2.5 ng/mL, which give

OTA is small molecules that possess one epitope and no more than one antibody can bind due to their small molecular size. This molecule was detected using a competitive assay rather than a sandwich assay format. The competitive assay is based on the competition of immobilized antigen and a free antigen for the antibody in solution. One of the critical parameters to determine the sensitivity and limit of detection (LOD) is antibody concentration. The excessive antibody in solution may cause more antigen needed to create a measurable difference in signal. Therefore, to increase the binding capacity, protein conjugate such as SPR sensor development was used which the OTA either directly conjugated to BSA or PEG. The sensitivity increased with decreasing antibody concentration because the PEG-linked surface needs less initial antibody concentration for efficient analysis. Pirincci et al. [49]

*DOI: http://dx.doi.org/10.5772/intechopen.89022*

to 5, 20, and 10ng/mL within 10min, respectively.

detection limit around 0.15 ng/mL in buffer solution.

**5.3 Immunosensor**

of a microtitre plate ELISA.

*Recent Biosensors Technologies for Detection of Mycotoxin in Food Products DOI: http://dx.doi.org/10.5772/intechopen.89022*

Advantages using aptamers are aptamers can provide high stability and affinity. Aptamers also provide simplicity, low cost, and excellent batch-to-batch reproducibility. Aptasensor can attract massive attention because of excellent binding constant toward most mycotoxins. The critical step in the design of biosensors is immobilization of aptamers because this factor can affect the affinity of the aptamer for target and long-term stability for real sample. There are several immobilization strategies affect the used for aptasensor development. Firstly, the adsorption or π-π interaction between DNA bases aptamer and graphene oxide (GO)-modified interfaces [37]. The covalent linkage of the aptamer to the carboxylic acid group that presents on surface or nanomaterial [38] and thiolated binding aptamers to CdTe quantum dots (QDs) or Au-based materials [39]. Besides, affinity binding based on biotin-streptavidin or other affinity interaction [40, 41] and hybridization of partially complementary single-stranded DNA which immobilized on surface or nanoparticle [42]. Duan et al. [43] developed multicolor quantum dot nanobeads for simultaneous qualitative immunochromatographic detection of mycotoxins (ZEN, OTA, and FB1) in corn samples with detection limits reached up to 5, 20, and 10ng/mL within 10min, respectively.

#### **5.3 Immunosensor**

*Mycotoxins and Food Safety*

for nutritional food quality and contaminant such as toxin or pathogen that supposed not to be found in food products [30]. Selection of suitable working electrode is a crucial part of successful electrochemical measurement either by modification in working electrode materials or traditional metals such as mercury or gold [32]. Due to the widely occurring co-contamination of mycotoxins in raw food materials, Lu and Gunasekaran [33] designed and fabricated of an electrochemical immunosensor for simultaneous detection of two mycotoxins, fumonisin B1 (FB1) and deoxynivalenol (DON), in a single test. A dual-channel three-electrode electrochemical sensor pattern was etched on a transparent indium tin oxide (ITO)-coated glass via photolithography and was integrated with capillary-driven polydimethylsiloxane (PDMS) microfluidic channel. The achieved detection limits found 97 and 35 pg./mL, respectively. Besides, Nieto et al. [34] A third-generation enzymatic biosensor were developed to quantify sterigmatocystin (STEH). It was based on a glassy carbon electrode modified with a composite of the soybean peroxidase enzyme (SPE) and chemically reduced graphene oxide. A third-generation enzymatic biosensor to quantify STEH in corn samples spiked with the mycotoxin. The biosensor was based on glassy carbon (GC) electrode modified with a composite of SPE and chemically reduced graphene oxide (CRGO). The biosensor was also used to determine STEH in corn samples inoculated with Aspergillus flavus, which is an aflatoxins fungus producer. The biosensor showed a linear response in the

*The applications of different read-out biosensors integrated with bioreceptors and nanomaterials.*

to 5.0 × 10<sup>−</sup><sup>7</sup>

The aptamer is referred to the Latin word, aptus means "to fit," which relationship between aptamers and their target look like "lock-and-key" theory [35]. Aptamers usually single-stranded RNA or DNA, which consist of 2–60 nucleotides, which specifically bind to the target, including organic molecules and cells. Aptasensors referred to biosensors using aptamers as biorecognition element and aptasensor were

described in 1996 [36] which had been used in multiple sensing applications.

for a signal: noise ratio of 3:1.

mol L<sup>−</sup><sup>1</sup>

. The limit of detection was

**90**

2.3 × 10<sup>−</sup><sup>9</sup>

**Figure 2.**

**5.2 Aptasensor**

concentration range from 6.9 × 10<sup>−</sup><sup>9</sup>

mol L<sup>−</sup><sup>1</sup>

Immunosensors are devices based on the detection of analyte-antibody interaction. Three main groups have been developing, which are luminescent or colorimetric sensors, surface plasmon resonance, and electrochemical sensors. The sensor usually combined with simple methanol–water for the extraction of a mycotoxin from food samples. Colorimetric and luminescent are based on the visible or UV light transformation into an analytical signal [44]. A colorimetric sensor developed for AFB1 detection using direct competitive ELISA principle. The color was detected and measured with spectrometer by reading absorbance at 620 nm. According to Garden and Strachen [45], this method could detect AFB1 as low as 0.2 ng/mL in artificially contaminated food material as compared to the sensitivity of a microtitre plate ELISA.

Surface plasmon resonance (SPR) is an optical phenomenon which used for measure changes on the surface of thin metal films (Au or Ag) under condition total internal reflection [46]. The sensitivity of SPR sensors and microtiter plate ELISAs were compared for detection of AFB1 using same immunoreagents, which are a polyclonal antibody and AFB1-BSA conjugate. As a result, the SPR sensor (3.0– 49 ng/mL) is a more sensitive but narrow and linear range of detection compared to ELISA (12–25,000 ng/mL) [47]. Electrochemical immunosensor for mycotoxin are based on competitive ELISA principle, which electrochemical transducer allows detection redox directly [44]. Pemberton et al. [48] in their study, a calibration plot AFB1 obtained over the concentration range from 0.15 to 2.5 ng/mL, which give detection limit around 0.15 ng/mL in buffer solution.

OTA is small molecules that possess one epitope and no more than one antibody can bind due to their small molecular size. This molecule was detected using a competitive assay rather than a sandwich assay format. The competitive assay is based on the competition of immobilized antigen and a free antigen for the antibody in solution. One of the critical parameters to determine the sensitivity and limit of detection (LOD) is antibody concentration. The excessive antibody in solution may cause more antigen needed to create a measurable difference in signal. Therefore, to increase the binding capacity, protein conjugate such as SPR sensor development was used which the OTA either directly conjugated to BSA or PEG. The sensitivity increased with decreasing antibody concentration because the PEG-linked surface needs less initial antibody concentration for efficient analysis. Pirincci et al. [49]

described that the OTA-sensitive QCM sensor was developed by direct immobilization of OTA to the sensor surface.

## **5.4 Molecularly imprinted polymer (MIP)**

Molecular imprinted polymer (MIP) is a method which is described as a method that highly efficient in producing functional material that able to mimic natural recognition entities, such as antibodies and biological receptors [50] which equipped with particular identification characteristics. In 2009, an electrochemical sensor was built by Pardieu et al. [51] for the method of detection. Thus, this method is used to recognize a specific element for template molecule detection.

MIP is used in various field of application to recognize biological and chemical molecules including amino acids and proteins [52], nucleotide derivatives, pollutants, drugs and foods [53]. Molecularly imprinted polymer method had been applied in chromatography for HPLC and GC, Solid phase extraction, Chemical sensor systems, catalysis, drug delivery, antibodies, and receptors system [54]. The formation of a complex between an analyte and the functional monomer determines the Molecularly imprinted polymer. A three-dimensional polymer network is formed due to the presence of a significant excess of a cross-linking agent [55]. A specific recognition site is formed which complementary in shape, size, and chemical functionality to the template molecule as the template being removed from the polymer after the polymerization process occurs as shown in the figure. The recognition phenomena occur when the intermolecular interactions such as hydrogen bonds, dipole–dipole, and ionic interactions between the template molecule and the functional groups present in the polymer matrix. This method is used due to their high selectivity and affinity for the target molecules. Therefore, the recognized polymer will bind to the template molecule only selectively.

The molecularly imprinted materials have excellent physical and chemical characteristics. The materials can resist high physical and chemical reaction against external degrading factor. Thus, the molecularly imprinted polymer is stable against mechanical stress, high temperature, and pressure, resistant against treatment with acid, base, or metal ions, and also stable in a wide range of solvents [56]. Sellergren firstly reported the application of MIP in solid phase extraction in 1994. Generally, the MIP as a sorbent was recognized as an accurate, selective, and sensitive pre-treatment method in detecting trace amounts of chemicals in the matrix. The application of MIP in solid phase extraction is used for veterinary residues, pesticides residue, illegal drugs, mycotoxins, and persistent organic pollutants had been published.

#### **5.5 Optical biosensors**

Biosensors can be divided into different groups, which are electrochemical, optical, thermometric, piezoelectric, or magnetic [57]. Somehow, the optical biosensor is the most preferred among the other methods. This is because it has powerful analytical techniques which have a high specification, sensitivity, small size, and cost-effectiveness [58, 59]. An optical biosensor is a device which is selective and sensitive that can detect deficient levels of chemicals and biological substances and for the measurement of molecular interactions in situ and in real time [60].

Optical methods, such as colorimetric, fluorescent, chemiluminescent, and surface plasmon resonant strategies, are proper techniques for mycotoxins detection due to their simplicity, rapidity, reliability, and high sensitivity. An optical biosensor is a system which combined various entities in a single system such as sampling, a biosensor, a system for replenishing information, and a data analysis system which to implement a biological model that provides information for human

**93**

*Recent Biosensors Technologies for Detection of Mycotoxin in Food Products*

or machine [57]. The biosensor systems are developed by crucial attributes, which are the integration of fluidics, electronics, separation technology, and biological sub-systems. An optical biosensor is a compact analytical device, having a biological sensing element, integrated or connected to an optical transducer system. In this method, the analyte of interest that binds to the complementary optical bio-recognition element is recognized as immobilized on a suitable optical substrate [61]. An electronic signal is produced which the magnitude of the frequency is proportional that correspond to the concentration of an analyte or a group of analytes, to which the element will bind is the objective of optical biosensors [62]. Meanwhile, enzyme, substrate, antibody, and nucleic acids are used as the primary biological materials in optical biosensor technology [57]. The detection usually relies on an enzyme system which converts the analytes to products catalytically and can be oxidized or reduced

Optical biosensing has two general modes, which are label-free and labelbased. For label-free mode, the interaction of the analyzed material with the transducer will generate a detectable signal. On the contrary, the use of the label and the optical signal then generated by a colorimetric, fluorescent, or luminescent method are involved in label-based sensing [63]. The usage of optical biosensor depends on the different fields of use. This is because it has own requirements in term of measuring analysis, required precision of output, the sample concentration required, the time taken to complete the probe, the time necessary to prepare

and reuse the biosensor, and the cleaning requirements of the system [57].

In the food industry, this method is used for the direct detection of bacteria in products. Optical biosensor used to detect the changes of refractive indices as the cell bind to the receptor, which is immobilized on the transducer [49]. The advantages of using optical biosensors are their speed, immunity of signal to electrical or magnetic interference. Besides, it is highly sensitive, reproducible, and simple-tooperate analytical tools. Somehow, some instrumentation involved in this method high in cost. Nabok et al. [4] reviewed the recent progress in the development of novel optical biosensing technologies for the detection of mycotoxins indirect assay

There are a variety of enzymes such as cholinesterase, urease, glucose oxidase

Based on the previous research, it is proven that AChE is inhibited by the AFB1 from binding at the external site, which is located at the active site gorge entrance located at the tryptophan residue. The inhibitory effect of the AFB1 can be seen by its action where the toxin blocks the entrance to the active site so that the substrate

and more that have been applied in an enzymatic inhibition analysis and this method is pretty standard [64]. According to Puiu et al. [65], Acetylcholinesterase (AChE) is the most commonly used enzyme, and the reason is it is susceptible toward mycotoxin which is becoming the preferred method for mycotoxin detection. This statement is also supported by [66], which stated that biosensors for Aflatoxin B1 (a type of mycotoxin) or AFB1, in short, is developed by using AChE due to the inhibitory effect of AFB1 to AChE enzymatic activity. Also, the inhibitory effect of mycotoxin is a reversible process due to the non-covalently binding nature to the enzyme [67]. Soldatkin et al. [68] stated that aflatoxin showed the highest sensitivity toward enzymatic inhibition method among the other groups of toxins. A past study conducted by Egbunike and Ikegwuonu [69] also suggests that usage of cholinesterase in biosensor method as the biological component is usable as AFB1 detector as aflatoxicosis has been reported to be correlated with a significant reduc-

*DOI: http://dx.doi.org/10.5772/intechopen.89022*

with either specific antibodies or aptamers.

tion of acetylcholine turnover in rat brain.

**5.6 Enzymatic inhibition**

at a working electrode.

#### *Recent Biosensors Technologies for Detection of Mycotoxin in Food Products DOI: http://dx.doi.org/10.5772/intechopen.89022*

*Mycotoxins and Food Safety*

tion of OTA to the sensor surface.

**5.4 Molecularly imprinted polymer (MIP)**

described that the OTA-sensitive QCM sensor was developed by direct immobiliza-

Molecular imprinted polymer (MIP) is a method which is described as a method that highly efficient in producing functional material that able to mimic natural recognition entities, such as antibodies and biological receptors [50] which equipped with particular identification characteristics. In 2009, an electrochemical sensor was built by Pardieu et al. [51] for the method of detection. Thus, this method is

MIP is used in various field of application to recognize biological and chemical molecules including amino acids and proteins [52], nucleotide derivatives, pollutants, drugs and foods [53]. Molecularly imprinted polymer method had been applied in chromatography for HPLC and GC, Solid phase extraction, Chemical sensor systems, catalysis, drug delivery, antibodies, and receptors system [54]. The formation of a complex between an analyte and the functional monomer determines the Molecularly imprinted polymer. A three-dimensional polymer network is formed due to the presence of a significant excess of a cross-linking agent [55]. A specific recognition site is formed which complementary in shape, size, and chemical functionality to the template molecule as the template being removed from the polymer after the polymerization process occurs as shown in the figure. The recognition phenomena occur when the intermolecular interactions such as hydrogen bonds, dipole–dipole, and ionic interactions between the template molecule and the functional groups present in the polymer matrix. This method is used due to their high selectivity and affinity for the target molecules. Therefore, the recognized polymer will bind to the template molecule only selectively. The molecularly imprinted materials have excellent physical and chemical characteristics. The materials can resist high physical and chemical reaction against external degrading factor. Thus, the molecularly imprinted polymer is stable against mechanical stress, high temperature, and pressure, resistant against treatment with acid, base, or metal ions, and also stable in a wide range of solvents [56]. Sellergren firstly reported the application of MIP in solid phase extraction in 1994. Generally, the MIP as a sorbent was recognized as an accurate, selective, and sensitive pre-treatment method in detecting trace amounts of chemicals in the matrix. The application of MIP in solid phase extraction is used for veterinary residues, pesticides residue, illegal drugs, mycotoxins, and persistent organic pollutants had been published.

Biosensors can be divided into different groups, which are electrochemical, optical, thermometric, piezoelectric, or magnetic [57]. Somehow, the optical biosensor is the most preferred among the other methods. This is because it has powerful analytical techniques which have a high specification, sensitivity, small size, and cost-effectiveness [58, 59]. An optical biosensor is a device which is selective and sensitive that can detect deficient levels of chemicals and biological substances and for the measurement

Optical methods, such as colorimetric, fluorescent, chemiluminescent, and surface plasmon resonant strategies, are proper techniques for mycotoxins detection due to their simplicity, rapidity, reliability, and high sensitivity. An optical biosensor is a system which combined various entities in a single system such as sampling, a biosensor, a system for replenishing information, and a data analysis system which to implement a biological model that provides information for human

used to recognize a specific element for template molecule detection.

**92**

**5.5 Optical biosensors**

of molecular interactions in situ and in real time [60].

or machine [57]. The biosensor systems are developed by crucial attributes, which are the integration of fluidics, electronics, separation technology, and biological sub-systems. An optical biosensor is a compact analytical device, having a biological sensing element, integrated or connected to an optical transducer system. In this method, the analyte of interest that binds to the complementary optical bio-recognition element is recognized as immobilized on a suitable optical substrate [61]. An electronic signal is produced which the magnitude of the frequency is proportional that correspond to the concentration of an analyte or a group of analytes, to which the element will bind is the objective of optical biosensors [62]. Meanwhile, enzyme, substrate, antibody, and nucleic acids are used as the primary biological materials in optical biosensor technology [57]. The detection usually relies on an enzyme system which converts the analytes to products catalytically and can be oxidized or reduced at a working electrode.

Optical biosensing has two general modes, which are label-free and labelbased. For label-free mode, the interaction of the analyzed material with the transducer will generate a detectable signal. On the contrary, the use of the label and the optical signal then generated by a colorimetric, fluorescent, or luminescent method are involved in label-based sensing [63]. The usage of optical biosensor depends on the different fields of use. This is because it has own requirements in term of measuring analysis, required precision of output, the sample concentration required, the time taken to complete the probe, the time necessary to prepare and reuse the biosensor, and the cleaning requirements of the system [57].

In the food industry, this method is used for the direct detection of bacteria in products. Optical biosensor used to detect the changes of refractive indices as the cell bind to the receptor, which is immobilized on the transducer [49]. The advantages of using optical biosensors are their speed, immunity of signal to electrical or magnetic interference. Besides, it is highly sensitive, reproducible, and simple-tooperate analytical tools. Somehow, some instrumentation involved in this method high in cost. Nabok et al. [4] reviewed the recent progress in the development of novel optical biosensing technologies for the detection of mycotoxins indirect assay with either specific antibodies or aptamers.

## **5.6 Enzymatic inhibition**

There are a variety of enzymes such as cholinesterase, urease, glucose oxidase and more that have been applied in an enzymatic inhibition analysis and this method is pretty standard [64]. According to Puiu et al. [65], Acetylcholinesterase (AChE) is the most commonly used enzyme, and the reason is it is susceptible toward mycotoxin which is becoming the preferred method for mycotoxin detection. This statement is also supported by [66], which stated that biosensors for Aflatoxin B1 (a type of mycotoxin) or AFB1, in short, is developed by using AChE due to the inhibitory effect of AFB1 to AChE enzymatic activity. Also, the inhibitory effect of mycotoxin is a reversible process due to the non-covalently binding nature to the enzyme [67]. Soldatkin et al. [68] stated that aflatoxin showed the highest sensitivity toward enzymatic inhibition method among the other groups of toxins. A past study conducted by Egbunike and Ikegwuonu [69] also suggests that usage of cholinesterase in biosensor method as the biological component is usable as AFB1 detector as aflatoxicosis has been reported to be correlated with a significant reduction of acetylcholine turnover in rat brain.

Based on the previous research, it is proven that AChE is inhibited by the AFB1 from binding at the external site, which is located at the active site gorge entrance located at the tryptophan residue. The inhibitory effect of the AFB1 can be seen by its action where the toxin blocks the entrance to the active site so that the substrate cannot enter to participate to the catalytic site result in the choline unable to exit as proposed by the steric blockade model [70]. Based on the observation in the study conducted by Hansmann et al. [71], their results lead them to two findings. The first observation is the addition of AFB1 in the binding site of the active site did not fulfill the description for inhibitory activity, and this suggests that the AFB1 does not slide to the catalytic site. As for the second observation, mutation of Trp321 to alanine in Dm-AChE put a stop on the inhibitory activity at 10 μM concentration, and AFB1 at a concentration of 100 μM does not inhibit Hu-BuChE enzymatic activity. Also, the researchers assumed that AFB1 could not enter into the active site due to its relatively big size, especially when considering the hydrophilic shell might be further increased in size. Due to this condition, aflatoxin is grouped as a ligand which binds on the external site of the cholinesterase [72].

#### **5.7 Mimotope**

Mimotope or also known as peptide-displaying phage or synthetic peptides [73] is now one of the most reliable methods that are used to identify epitopes which are detected by monoclonal antibodies which are antibodies that made by the same immune cell is given that they are clones of one single parent cell. Next, the usage of mimotope in mycotoxin detection involves the usage of peptides which are identified to be structurally not identical to the original epitope of mycotoxin but at least have the properties to mimic the epitope by binding to the antibodies [74]. Generally, this method shared instead of the same concept with enzymatic inhibition, which in this case, the mimotope will be the one that elicits antibody. Also, this method is beneficial when the original epitopes (example from a mycotoxin) are hard to be isolated and at the same time only available in minimal amount [75]. The first assay that using mimotope for detection is being done by Yuan et al. [76], where a mimotope is used to identify the mycotoxin deoxynivalenol.

A study has been conducted by Sellrie et al. [74] which aims to describe a competitive immunoassay for identification of hapten fluorescein by utilizing a monoclonal anti-fluorescein antibody B13-DE1 and a mimotope peptide which act by binding to the antibody. Based on their findings, the peptide mimotope was conjugated to horseradish peroxidase (HRP) which is then competing for binding to monoclonial antibody B13-DE1 with fluorescein. Based on the result, they have proven that mimotopes can be used to utilization in simple yet sensitive immune assays in order to quantitatively identify and determine substance with low molecular weights. As for the reliability and reproducibility, the assay was proved by validation data and found to be in the range which is described in the literature for conventional competitive immunoassays by Wild [77].
