Mycotoxins are chemically diverse and capable of inducing a wide diversity of acute and chronic ‎symptoms, ranging from feed refusal to rapid death. Accurate detection and monitoring of ‎mycotoxins is an essential component of the prevention, diagnosis, and remediation of ‎mycotoxin-related issues in livestock and human food. Current trends in food analysis are ‎focusing on the application of fast, simple procedure needed, and low-cost biosensor ‎technologies that can detect with high sensitivity and selectivity different compounds associated ‎with food safety.‎

Mycotoxin

Fungal toxins are secondary metabolites, which can cause some diseases in living things known ‎as mycoses; meanwhile, dietary exposure to such metabolites produces the disease named ‎mycotoxicoses. Mycotoxins are known as secondary metabolites, produced from microfungi and ‎able to cause–effect human health as well as animals. Mycotoxins are commonly used as ‎antibiotics and growth promotants because of their unique characteristics in pharmacological ‎activity. Most of the mycotoxin are found as natural contaminant food, mainly in vegetable and ‎feed. Nut, cereals, oilseeds, dried fruits, spices, and food from animal origins for example milk, ‎egg, and meat are also may contain mycotoxin either outside or inside the product . A mycotoxin ‎is believed no function in the life of a producer cell, unlike primary metabolites . There are few ‎types of mycotoxin such as aflatoxins (AFs), zearalenone (ZEA), deoxnivalenol (DON), ‎ochratoxin (OTA) and T-2 toxin (trichothecene mycotoxin) which are a significant threat to the ‎life and health of human and live stocks . Mycotoxins are low molecular weight and thermal-‎stable secondary metabolite of toxic molds that belong to genera Aspergillus, Penicillium, ‎Alternaria, and Fusarium. These toxins are present in the mycelium and spore of the mold. ‎Mycotoxin may become a biological weapon in bioterrorism because of its acute and chronic ‎toxicities .‎

Advanced techniques for detection of mycotoxin based biosensor

‎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 . 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 . 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 . The electrochemical technique requires a ‎reference, auxiliary, and a working electrode. Two exciting compounds are analyzed using ‎compound biosensors that have interest for nutritional food quality and contaminant such as ‎toxin or pathogen that supposed not to be found in food products. 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.‎

Due to the widely occurring co-contamination of mycotoxins in raw food materials, Lu and ‎Gunasekaran 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. 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 × 10−9 to 5.0 × 10−7 mol L−1. The limit of detection was 2.3 × 10−9 mol L−1 for a signal: ‎noise ratio of 3:1.‎

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 . 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 which had been used in multiple sensing ‎applications.‎

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 . The ‎covalent linkage of the aptamer to the carboxylic acid group that presents on surface or ‎nanomaterial  and thiolated binding aptamers to CdTe quantum dots (QDs) or Au-based ‎materials . Besides, affinity binding based on biotin-streptavidin or other affinity interaction  and ‎hybridization of partially complementary single-stranded DNA which immobilized on surface or ‎nanoparticle . Duan et al. 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 10 ng/mL within 10 min, respectively.‎

‎3.‎  Immunosensor

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 . 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 , 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 . 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) . Electrochemical immunosensor for ‎mycotoxin are based on competitive ELISA principle, which electrochemical transducer allows ‎detection redox directly . Pemberton et al.  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.  described that the OTA-sensitive ‎QCM sensor was developed by direct immobilization of OTA to the sensor surface.‎

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  which equipped with particular identification characteristics. ‎In 2009, an electrochemical sensor was built by Pardieu et al.  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 , nucleotide derivatives, pollutants, drugs and foods . ‎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 . 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 . 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 . 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.‎  Optical biosensors

Biosensors can be divided into different groups, which are electrochemical, optical, ‎thermometric, piezoelectric, or magnetic . 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 . 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. 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 or ‎machine . 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 . 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 . Meanwhile, enzyme, substrate, antibody, and nucleic ‎acids are used as the primary biological materials in optical biosensor technology . 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 label-based. 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 . 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. 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  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-to-operate analytical tools. Somehow, some instrumentation involved in ‎this method high in cost. Nabok et al.  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.‎

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 . According ‎to Puiu et al. , 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 , 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 . Soldatkin et al.  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 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. Based on the ‎observation in the study conducted by Hansmann et al. , 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.‎

‎7.‎  Mimotope

Mimotope or also known as peptide-displaying phage or synthetic peptides  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 . ‎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 . The first assay that using mimotope for detection is being ‎done by Yuan et al. , were a mimotope is used to identify the mycotoxin deoxynivalenol.‎

A study has been conducted by Sellrie et al. 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.‎

Advanced techniques for detection of mycotoxin based biosensor

During the last few decades, consumers have become more aware of health and food quality, ‎consequently, research on food safety augmented. The variety of contaminants in many food ‎products requires the development of high-throughput, real-time, and portable detection ‎methods. The evaluation of the different mycotoxins residues in foodstuffs became an essential ‎factor in guaranteeing the products’ quality. Hence, it is essential to improve the analytical ‎standards to detect and quantify the presence of a mycotoxin. The operation procedure should be ‎simplified continuously for the convenience of users. The biosensor based nanotechnology can be ‎extensively used in food contaminants monitoring and eventually become effectively routine ‎analysis tools that could meet numerous challenges.‎