Rapid and sensitive detection of mycotoxins by advanced and emerging analytical methods: A review

Abstract Quantification of mycotoxins in foodstuffs is extremely difficult as a limited amount of toxins are known to be presented in the food samples. Mycotoxins are secondary toxic metabolites, made primarily by fungal species, contaminating feeds and foods. Due to the presence in globally used grains, it is an unpreventable problem that causes various acute and chronic impacts on human and animal health. Over the previous few years, however, progress has been made in mycotoxin analysis studies. Easier techniques of sample cleanup and advanced chromatographic approaches have been developed, primarily high‐performance liquid chromatography. Few extremely sophisticated and adaptable tools such as high‐resolution mass spectrometry and gas chromatography–tandem MS/MS have become more important. In addition, Immunoassay, Advanced quantitative techniques are now globally accepted for mycotoxin analysis. Thus, this review summarizes these traditional and highly advance methods and their characteristics for evaluating mycotoxins.

ultraviolet, fluorescence, photomultiplier, ion mobility, and tandem mass spectrophotometry, fourier transforms near infrared, adsorptive stripping voltammetry, and their lower detection limits, and sensitivity of different types of matrice has been reviewed. For the determination of mycotoxins, traditional quantitative methods viz. chromatography, immunological, and the advanced methods viz. ultrahigh-performance liquid chromatography, fluorescence polarization immunoassay, nanoparticle-based methods, microfluidics, and phage display methods have been discussed extensively in this review.

| Aflatoxin
Aflatoxins (AFs) are difuranocoumarins derivatives (Figure 1a Ali et al., 2005). The four important AFs found are Aflatoxin B 1 (AFB 1 ), Aflatoxin B 2 (AFB 2 ), Aflatoxin G 1 (AFG 1 ), and Aflatoxin G 2 (AFG 2 ) and can be differentiated according to their fluorescence under UV light (green or blue) and comparative chromatographic movement during thin-layer chromatography. Apart from major AFs, AFM 1 , a hydroxylated metabolite of AFB 1, frequently found in milk and milk based baby foods.

| Citrinin
Several species of Aspergillus, Monascus, and Penicillium are responsible for the production of Citrinin (Figure 1f). Among Aspergillus species, A. niger is reported to be mainly involved in the production of citrinin. Citrinin is a polyketide mycotoxin. It has a conjugated, planar structure which produces its natural fluorescence (the highest fluorescence is produced by a nonionized citrinin molecule at pH 2.5; Vazquez et al., 1996). Quantitative methods such as high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and LC-MS/MS have been compared for citrinin detection in red fermented rice samples, and it was observed that LC-MS/MS displayed better results in terms of limit of detection (LOD) and quantification compared to that of HPLC-FLD (Ji et al., 2015).

| Deoxynivalenol and deoxynivalenol-3glucoside
Deoxynivalenol (DON) is a major trichothecenes, one of several fusarium species mycotoxins. Maize, wheat, oats, barley, rice, and other grains are often contaminated in the field or during processing. DON can be converted to deoxynivalenol-3-glucoside (DON-3G) called as masked mycotoxin by plant detoxification (Dong et al., 2017). Methods like LC-MS/MS are developed to detect the both DON and DON-3G in the bakery products (Generotti et al., 2015). Similarly, Johny et al. (2019) have developed high-resolution LC-MS method to detect DON-3G exposed fish and in plant-based fish feed. The LOD was obtained 176 µg/kg for DON-3G in salmon, zebrafish, and fish feed.

| Mycotoxins toxicity and their adverse effects
Mycotoxins are the most hazardous among food and feed contami- and OTA under Group 2B (Bhat, Rai, & Karim, 2010). AFs are hepatotoxic and proven as a hepatocarcinogenic agents (Mishra & Das, 2003). AFs are immunosuppressive, teratogenic, and mutagenic in nature. Only Ochratoxin is potentially as important as AFs among the Aspergillus toxins. The main target organ is the kidney. OTA is a nephrotoxin for all animal species tested so far and is most likely to be toxic to humans (Creppy, 1999). It causes neurotoxicity and hepatotoxicity and affects blood coagulation and immunosuppressive carcinogenic agent. In all animal species studied, citrinin also acts as a nephrotoxin, but its acute toxicity varies among different species (Carlton & Tuite, 1977). It also causes hepatotoxicity and genotoxicity (Group 3, IARC). ZEA (group 3, IARC) has strong estrogenic effects, and trichothecenes (Group 3, IARC) can inhibit protein synthesis, induce immune-modulatory effects, alimentary toxic aleukia (Sudakin, 2003). Patulin generally found in apples and in unfermented apple juice (Trucksess & Tang, 1999). In general, toxicity of PATs is related to acute and subacute toxicity, genotoxicity, embryotoxicity, and teratogenicity (Puel, Galtier, & Oswald, 2010).
The United States, the European Commission, and many other countries have established a tolerable daily intake and maximum residue levels (MRLs) for AFB 1 , OTA, FBs, ZEA, DON (Group 3), and trichothecene (T-2, HT-2) toxins in different foodstuffs. In case of AFs, the limit is set in the range of 2-4 μg/kg in cereals, dried products, and peanuts (European Commission (EC), 2010).
For FBs and OTA, the limit has been set to 200-1,000 μg/kg in cereals and cereal-based products, and 2-10 μg/kg in cereals, wine, coffee, cheese, and cocoa, respectively. ZEA limits range from 20 to 100 μg/kg in cereals and cereal products. For DON and PAT have regulatory limits 200-500 μg/kg in cereals and cereal products and 10-50 μg/kg in apple and concentrate, respectively. Food and Drug Administration (FDA) has set the regulatory guidelines for major mycotoxins in food and feed. The United States has set MRLs as 20 μg/kg AFs in different food commodities like maize, wheat, rice, and peanut, 0.5 μg/kg AFM 1 in milk and milk products, 2,000-4,000 μg/kg FBs in maize and maize products, 1,000 μg/kg DON in cereals and cereal products, and 50 μg/kg PAT in apple and apple juices (FDA, 2002). Due to the common occurrence of regulated mycotoxins (AFs, ZEA, DON, FBs, OTA), their toxic nature has posed a risk to human and animal health, therefore demanding a solution for the protection of fauna. Although some toxins have not been regulated such as alternaria, sporidesmins, endophyte mycotoxins, sterigmatocystin, and phomposins, their toxigenic potential has been assessed in various studies (Nieto, Granero, Zon, & Fernández, 2018;Woudenberg, Groenewald, Binder, & Crous, 2013).
Regulated and unregulated mycotoxins and their toxicity were summarized in (Tables 1 and 2).
For better understanding of the global effect of mycotoxin contamination, accurate, more rapid, and highly sensitive methods are essential for routine identification and detection of these compounds. The diverse nature of the matrice, target, environment, time requirements, detection levels, and accessibility of appropriate technology are considered to be challenging. For developing an effective, precise, and sensitive method for mycotoxins analysis, a great deal of attention should be paid to the matrice effect. The matrice effect is the combined effect of all sample components other than an analyte of interest on quantification. If a particular element can be defined as having an effect, it is called interference. The matrice effect can be observed as a loss or increase in response and therefore results in estimation or overestimation of mycotoxin. The matrice effect therefore affects the precision, accuracy, and sensitivity of the analytical method. For mycotoxin analysis, the matrice effect is very important, since mycotoxins, themselves, are of various chemical entities and present in various sample matrices.
Thus, investigation and detection of mycotoxin contamination in foods and feeds have been a vital center of international and national activities over the years. For accurate and rapid determination of these mycotoxins in unprocessed cereals and cereal-based products, sensitive, analytical methods are highly relevant to the toxicological implications to animals and humans and highly desirable in order to measure risk of exposure, further to confirm regulatory levels fixed by the United States, European Union, or different international organizations. Analysis of mycotoxins usually requires toxin extraction from the matrice with a suitable extraction solvent, a cleanup procedure in order to remove interfering elements from the extract, and lastly, determination/detection of the toxin by appropriate analytical instrumentation.

| E X TR AC TI ON AND PRECLE ANING ME THODS
Primary extraction (Pascale, 2009) is necessary for the determination of mycotoxins from the different matrices like (wheat, maize, peanut, etc.). Selection of a suitable solvent is required for all the extraction procedures (Gilbert & Vargas, 2003). The choice of solvent primarily depends on the type of analyte. Generally determination of mycotoxins from solid feeds and food requires organic solvents. The two recommended methods are solvent extraction and solid-phase extraction (SPE).

| Solvent extraction method
Solvent extraction is a process to distinguish compounds based on their relative solubility in two different immiscible liquids, usually an organic solvent and water. Solvents possessing low dielectric constants (tendency to be immiscible with water) are good at extracting nonpolar compounds for example mycotoxins. To decrease the respective miscibility, appropriate solvents such as acetonitrile or methanol must be mixed with water in the presence of salts. The polar analytes selectively move into the polar organic phase from the aqueous phase. The following factors like polarity, solvent power (selectivity), and reactivity should be considered while selecting a particular solvent system. The main disadvantage of the solvent extraction method is poor selectivity of most solvents, and the final extract obtained is often colored and viscous.

| Solid-phase extraction method
Solid-phase extraction is considered to be significant for sample preparation in mycotoxins analysis. The application of SPE is basically determined by the sorbent consumed in the extraction column. Currently, an enormous number of solvents are accessible, and the commonly used group of sorbents include polymers, porous/graphitized Carbon, chemically modified silica gel, and selective sorbents (immunosorbents, molecularly imprinted polymers).
Most preferably used selective solid phase is those dependent on immunoaffinity recognition, where the target mycotoxin acts as an antigen, and solid phase possesses a targeted antibody. The choice of pretreatment method depends on many parameters such as the availability of analytical instrumentation, template, and target. Mycotoxins are small organic molecules which have different solubility in different solvents, so further cleanup methods are required.

| Immunoaffinity column
Immunoaffinity column (IAC), a system based on antigen-antibody interaction, has some advantages, including limited mycotoxin loss and total removal of interfering substances. Therefore, the use of IAC as a cleanup technique could greatly improve the accuracy of subsequent analysis compared to SPE extraction,. The formulation of specific antibody solid-phase materials plays an important role in extraction procedure (Şenyuva & Gilbert, 2010;Tessini et al., 2010).

| New absorbents
Many advanced nanomaterials, including carbon nanomaterials and magnetic carbon nanomaterials, have been used for mycotoxin determination. The main advantage of carbon nanomaterials is due to their high adsorption ability (Wang, Liu, Lu, & Qu, 2014). Graphene oxide (GO) and multi-walled carbon nanotubes (MWCNTs) are the example of Nanomaterial used as absorbent. GO was recently used in preconcentration of the extraction of AFs from traditional Chinese proprietary medicines for the first time (Ran, Chen, Ma, & Jiang 2017). MWCNTs have been shown to adsorb type A trichothecenes and were therefore used as SPE sorbents in maize, wheat, and rice to purify and enrich mycotoxins (Dong et al., 2015). However, only AFs, ZEA, and the four trichothecenes of type A (T-2, HT-2, DAS, and NEO) were studied. Many other types of mycotoxins are awaiting evaluation of the appropriate nanomaterials. Lehotay et al. (2007) reported QuEChERS (quick, easy, cheap, effective, rugged, and safe) solid-phase extraction method to detect twenty pesticides in 3 matrices (grapes, lettuces, and oranges) at levels ranging from 10 to 1,000 ng/g. Azaiez, Giusti, Sagratini, Mañes, and Fernández-Franzón (2014) also reported a method to detect mycotoxin in dried fruits using quenchers extraction. This method is preferred over other methods due to its ability of simultaneous extraction of multiple mycotoxins, less solvent utilization, cost-effectiveness, quick, and lower detection limit than EU regulations.

| DE TEC TI ON TECHNI QUE S
UV absorbance and fluorescence characteristics of mycotoxins have been utilized for their detection and quantification. Various detectors, namely, UV, fluorescence, laser-induced fluorescence (LIF), mass spectrometry (MS), and photomultipliers (PTM). have been used for quantitative determination of mycotoxins.

| Ultraviolet absorption
Ultraviolet-visible spectroscopy is a type of absorption spectroscopy. It has been reported that all the AFs have a molar

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Inhibition of important cellular functions such as spindle formation during mitosis and the intracellular transport of lipids. Distortions of cell nucleus shape plus apparent disruptions to membrane systems within the cell. Battilani et al. (2011) absorptivity of 20,000 cm 2 /mol exhibiting maximum absorption at 360 nm (Akbas & Ozdemir, 2006). Experimental data suggested that the detection limit of AFs can be improved by the selection of appropriate method for extraction and cleanup procedure (Ali et al., 2005;Göbel & Lusky, 2004). The sensitivity of UV system is not enough to detect AFs in trace levels (Alcaide-Molina et al., 2009) since its limit of detection reaches up to only micro molar ranges (Couderc, Caussé, & Bayle, 1998). Hence, fluorescence (FL) techniques have gained more popularity for AFs detection.

| Fluorescence
Fluorescence is an important parameter for the analysis and characterization of molecules that emit energy at specific wavelengths. It has been reported that almost every AF exhibits a maximum absorption at 360 nm (Akbas & Ozdemir, 2006). Different techniques for AFs detection associated with fluorescence are illustrated in (Table 3).

| Fluorescence spectrophotometer
It has been used to analyze AFs in cereals, mainly in peanuts. The fluorometric method can quantify AFs from 5 to 5,000 μg/kg in less than 5 min (Herzallah, 2009). Fluorometric derivatization is required for better analysis of AFs for enhancement of their fluorescence. The detection limit for AFs in this case is also slightly higher than the limit set by EU (4 μg/kg). Urraca, Marazuela, and Moreno-Bondi (2004) have reported a method to analyze ZEA and α-zearalenol in wheat samples and swine feed. The LOD achieved was 6 ng/g for ZEA in wheat samples and swine feeds. For α-zearalenol, LOD achieved was 3, 4 ng/g in wheat and swine feed, respectively. Fluorescence detection provides better accuracy and higher precision in the broad concentration range.

| High-performance liquid chromatography coupled with fluorescence detection
There is no doubt that fluorescence detectors are the most sensitive among all the advanced current HPLC sensors. It enables to detect the presence of even a single analyte molecule in the flow cell.
Usually, the sensitivity of fluorescence is 10-1,000 times better than that of UV detector for higher UV absorbing materials due to which this technique is used regularly in the measurement of specific fluorescent compounds present in the samples. (Coix lacryma-jobi)seed. The technique is based on the use of methanol/water (80/20) for fast ultrasonic solid-liquid removal, followed with the cleanup of the IAC, photochemical derivatization, and HPLC-FLD. The detection limit for mycotoxins ranged from 0.01 to 0.04 μg/ kg, which was noted to be lower than the tolerance levels set by the European Union (EU). This approach offers many advantages over recent practices, including sensitive detection and rapid separation.

| High-performance liquid chromatography coupled with photodiode array
To obtain spectral profiles from molecular mixtures or chromato- has been demonstrated to detect up to 4 ng analytes in 1 g samples with the use of a fluid chromatography instrument (Taheri et al., 2012). Results suggested that the method described above was more resilient than other methods for mycotoxin detection.

| Laser-induced fluorescence screening method
Laser-induced fluorescence coupled with HPLC is a sensitive and powerful technique used to detect AFs at subpicogram levels.

| Photomultipliers
Since fluorescence detection technique is concised to fluorescence, there is a need for other methods to detect mycotoxins.

| Ion-mobility spectrometry
The ion-mobility spectrometry is a technique used to label chemicals that depend on the velocity achieved by the gas-phase ions in the presence of an electrical field. The working of ion-mobility spectrometry (IMS) is similar to that of Fourier Transform Near Infrared (FT-NIR). The advantages of IMS include low detection limit, simple, fast response and cost-effective. Khalesi, Sheikh-Zeinoddin, and Tabrizchi (2011)

| Mass spectrometry/Tandem mass spectrometry
Mass spectrometry is an analytical technique that sorts the ions depends on their mass to charge ratio and ionizes chemical species. Tandem mass spectrometry (MS/MS) has an advantage in chromatographic peak detection. Mass spectrometry perfor- showed LODs in the range of 0.05-10 μg/L. The risk assessment research showed that people are not exposed to mycotoxins using tea beverages.

| Fourier Transform Near Infrared spectrometry
This technique depends on the absorbance quantity of the light emitted by the sample whose wavelength differs in the range of

| TR ADITIONAL QUANTITATIVE ME THODS
The commonly used chromatographic methods for mycotoxins de-

| Chromatography methods
Numerous chromatographic methods are available for the quantification of mycotoxins. Traditional TLC is considered as an effective screening method for mycotoxins and has gained great significance due to low cost, simple instruments and fluorescent spots under UV, though it has poor accuracy and low sensitivity, making quantification difficult. TLC is widely accepted as an approved reference method for the determination of AFs; it has been replaced with HPLC for quantitative analysis of mycotoxins. Caputo et al. (2014) reported the development in TLC detection for OTA analysis. It was noted that when 2 µl was dropped onto the TLC plate, and 0.2 µg of OTA could be detected. This method shows better sensitivity than UV lamp and shows limit of detection as like LC methods as less as parts per billion (μg/kg).

| Liquid chromatography
Liquid chromatography has been developed to overcome the limita-

| High-performance liquid chromatography and ultrahigh-performance liquid chromatography (UHPLC)
High-performance liquid chromatography (HPLC) has been evolved, since the late 1960s. HPLC is considered as the common chromatographic method with a wide range of detection approaches (Vail and Homann, 1990 (Table 3). Iha, Barbosa, Heck, and Trucksess (2014) and Iha, Barbosa, Okada, and Trucksess (2011) detected OTA and AFM 1 in human milks and dairy products using fluorescence method linked with HPLC. Electrochemical and fluorescence detection is the two sensitive detection modes applied for quantitative studies in HPLC. Sensitive intensities of these amalgam techniques are much superior than conventional fluorescence.
A simple liquid-liquid microextraction was applied to concentrate the toxin at low concentration (ng/L)and to reduce solvent requirements. The major limitations of HPLC methods are portability and practical issues based on the matrice effect, sample type, sample preparation, and choice of calibration. Therefore, there is a need for further analytical methodologies.

| Gas chromatography-Mass spectrometry (GC-MS)
GC mainly depends on differential partitioning of analytes be-  with an immunoaffinity column sample preparation using the same antibody used by Klarić, Cvetnić, Pepeljnjak, and Kosalec (2009) and was found to be extremely sensitive at 0.02 μg/L. In order to recover high sensitivity, most researchers concentrated on modifying the normal ELISA protocol. ELISA formats (such as direct, indirect, competitive, and sandwich) are recognized as an excellent and accurate for screening the mycotoxins, but the procedure is somewhat time-consuming, not ideal for field testing and requires  (2011) specialist plate readers. Therefore, a transduction system was integrated with appropriate molecular recognition elements (immunochemical) that favored for portable and field analysis. Different analytical methods for OTA, FB 1 , patulin, trichothecenes detection described in (Tables 4-6).

| Microplate reader
Microtiter readers can detect the intensity of fluorescence or chemiluminescence and optical absorbance. Microtiter plates possess the unique characteristic of binding proteins evenly (e.g., antibodies or antigens contrary to AFs or secondary antibody).

| Lateral flow strip
Lateral flow strip assay for immunochromatography has fascinated excessive concern in current years. This technique is based on the Lateral flow strip assay has several benefits, such as simple step procedures, manageable setup, and quantity of target analytes can be detected straight with the bare eyes and rapid on-site detection (5-15 min), low cost and less interference due to chromatography separation.

| NE W DE TEC TI ON ME THODS FOR MYCOTOXINS QUANTITATIVE ANALYS IS
In the recent years, with the fast advancement of detection technologies and introduction of biotechnology, the detection technology of mycotoxins has grown rapidly. Some of the new technologies that have been applied for the detection of mycotoxins are elaborated in the following sections.

| Ultrafast liquid chromatography connected with tandem mass spectrometry (UFLC-MS/MS)
Ultrafast performance liquid chromatography (UFLC) has made substantial improvements in column technology to accomplish a dramatic increase in speed, resolution, and in separation performance that do not hinge on pressure as in liquid chromatography.

| Fluorescence polarization immunoassay
Time-resolved fluorescence immunoassay (TRFIA) is a novel analytical method that has been established since 1980s. This technique uses trivalent rare-earth metal ions (Eu3+, Tb3+, Sm3+, Dy3+) as tracers. Rare-earth ions-chelator-antigen chelates are prepared by mixing rare-earth ions, antigen, and chelator. The tested antigen and labeled antigen compete for the antibody to form immune complexes, and the rare-earth metal ion presents in the antigen-antibody binding portion of immune complexes is responsible for the fluorescence.
Hence, the intensity of fluorescence radiated from the metal ion can be measured by TRFIA (Hagan & Zuchner, 2011). Huang et al. (2009) created TR-FIA for AFB 1 and OTA utilizing Eu and Sm as a marker correspondingly. In this reaction, antigen-protein was treated onto micro titer plates followed by the addition of sample and antibody (Monoclonal Abs for OTA and Polyclonal Abs for AFB 1 ) after which distinct labeled second antibody was added. This TR-FIA was verified and displayed a LOD of 0.02 μg/L and 0.05 μg/L for AFB 1 and OTA, respectively. Multi-analyte immunoassay is possible using two different markers. This technique provides a wide detection range, good reproducibility, high sensitivity, and prolonged luminescence in comparison with the conventional fluorophore. TRFIA is a quick, simple, economic, and stable technology that can be used to identify mycotoxins in a huge number of samples. Further improvement has been done by researchers to combine immunochemical recognition elements and Raman spectroscopy method. Chauhan et al. (2015) used Raman spectroscopy for AFB 1 detection. Li, Wen, et al. (2016) has developed multi-analytes immunoassays and have gained high consideration due to their low sample consumption and short assay times and reduced detection costs per assay. In optimum circumstances, the LOD with MWFPIA was 17.8, 331.5, and 242.0 μg/kg for T-2 toxin, FB 1, and DON, respectively, giving adequate sensitivity for these three contaminants in maize as fixed by the EU. The overall period of analysis and sample preparation was noted to be less than with better sensitivity reduce the detection interval, and targets of interest will result in a simple procedure which can be completed effortlessly.

| Nanoparticles based detection methods
Gold nanoparticles were used to enhance the traditional ELISA method. Label-free sensor has the same sensitivity to a typical Simple sample preparation, inexpensive equipment, high sensitivity, simultaneous analysis of multiple samples, suitable for screening. ELISA has the added advantages of not having to use radioisotopes (radioactive substances) or an expensive radiation counter (radiation counter) Cross-reactivity with related mycotoxins, matrix interference problems, possible false-positive/ negative results

Micro plate reader
It improves simple ELISA method by reducing the coating, blocking, and competition time. It can reach a higher sensitivity than ELISA Not portable and convenient device for field application Lateral flow strip One-step assay, no washing step necessary, fast and low cost, low sample volume, simple test procedure Qualitative or semi quantitative results, imprecise sample volume reduces precision Immunosensor Immunosensors have the following advantages: portability due to their small scale, high selectivity and sensitivity, quick detection, and cheap materials, no cleanup procedure Cross-reactivity with related mycotoxins, variation in reproducibility and repeatability, due to small sizes of most the mycotoxin, it is difficult to develop antibody against them; skilled personnel are required to handle the sophisticated equipment

Fluorescence polarization immunoassay
Multi-analyte immunoassay is feasible, wide detection range, long-lived luminescence in comparison with conventional fluorophore Background interference in sample, longer incubation time is required for better reproducibility Nano particle based methods The traditional ELISA method is enhanced by gold nanoparticles, Multiple mycotoxins detection using a competitive immunoassay format Difficult to synthesize and not cost-effective Molecular imprinting (MIP) Cleanup, easy operation, low cost, stable, reusable, high affinity and selectivity toward the target molecule, Polymers are cost-effective to synthesized and store for several years at room temperature Poor selectivity, large volume of organic solvents, and long extraction time is required Microarray technology High-throughput screening miniaturized, multiplexed, and parallel processing method Not common because of their variability and reproducibility, intensive labor requirement merchantable competitive ELISA kit. Another label-free process was revealed by Xu, Liu, Li, and Ying (2013) who used gold nanorods treated with antibodies to determine AFB 1 . Jodra, López, and Escarpa (2015) used an equivalent technique to encapsulate magnetically labeled elements conveying particular antibody enzyme composite, and electrochemical method was used for AFB 1 detection.
Surface plasmon resonance (SPR) is an example of an optical detection method that happens when a polarized light hits a prism enclosed by a thin metal (gold) layer. In a few circumstances (polarization, incidence angle, and wavelength), nonbounded electrons at the level of the biochip absorb incident photons and change them into surface plasmon waves. The SPR imaging (SPRi) technique makes SPR method a stage advance. The CCD camera is used to visualize the whole chip and is a sensitive label-free method. This arrangement permits the biochips to be organized in an array setup in which every active site provides SPR information instantaneously.
Detection of multiple toxic mycotoxins is really important to control food quality. A gold nanoparticle (AuNP) intensified SPRi chip was reported to analyze numerous mycotoxins by a competitive immunoassay setup . Highly sensitive and specific immediate analysis is attained for three characteristic mycotoxins comprising OTA, ZEA, and AFB 1 with low detection limits of 30, 15, and 8 pg/ml, respectively. SPRi is an innovative device for simultaneous numerous analysis with better accuracies even though problems occur due to minute extents of mycotoxins with single epitope for an unresponsive competitive immunoassay and restricted sensitivity due to the instrumental restraint.

| Lateral flow immunochromatographic assay detection method
Lateral flow immunochromatographic assay (LFICA) technique has been extensively used for the analysis of mycotoxins in feeds, foods, and agronomic goods because of its low cost, simplicity, and speed.
The modern developments of LFICA with various nanomaterials labeled in the detection of mycotoxins were overviewed and prospected (Xie, Yang, Kong, Yang, & Yang, 2015). Detection sensitivity and specificity were enhanced continuously, and the limit of detection

| Implementation of microfluidic "lab-on-a-chip" for the detection of mycotoxins in foods
An enormous determination has been dedicated to ultra-accurate and ultrafast quantitation of trace amount of mycotoxins in foodstuffs, and microfluidic devices have developed as a favorable up-to-date analytical platform. The awareness of microfluidic analytical platform develops from the theory of Total Analysis System (TAS), which tends to minimize and assimilate the essential phases for exploration of a sample onto a particular instrument. The microfluidic analytical platform, also recognized as Micro Total Analysis Systems (mTAS), additionally increases its usage thereby creating the entire arrangement of a research laboratory onto a distinct chip in micrometer level (Kovarik et al., 2013;Dittrich, Tachikawa, & Manz, 2006). As its term designates, microfluidics compacts with regulatory solutions of minute quantity (naturally in nanoliters) in micro scale passages (Squires & Quake, 2005).
Electrochemical-dependent detection technique is an outstanding method to be merged into the microfluidic LOC instruments because of its inbuilt ability for small scale without affecting performance depletion, better sensitivity and compatibility (Neagu, Perrino, Micheli, Palleschi, & Moscone, 2009;Yeh, Chen, Lin, Chang, & Lin, 2009). It has specific benefits as its reaction is not restricted by sample turbidity or optical path length (Hervás, López, & Escarpa, 2012). MS detection method is noteworthy for its sensitivity and fast speed. The combination of microfabricated devices with this instrument can attain a low limit of detection.
MS analysis is a label-free method joined with microfluidic immu- capillary electro migration microchip (CE chip) that are forced by capillary force and capillary electro migration respectively (Luppa, Sokoll, & Chan, 2001;Li et al., 2012). For illustration, a competitive immunoassay-microfluidic instruments have been evolved for the segregation and quantification of ZEA (Hervas, Lopez, & Escarpa, 2011). Molecular imprinting (MIP) is an alternative well-organized separation technique that has been combined into the microfluidic LOC system. MIP is an advanced template induced formation of specific recognition sites where the template guides the alignment and positioning of the substance in structural elements of a self-build machinery (Ulbricht, Matuschewski, Oechel, & Hicke, 1996). A MIP electrochemical sensor has been recently fabricated for the selective detection of T-2 toxin by introducing iron ions (Fe 3+ ) to enhance the chelation of the templates and metal ions (Gao et al., 2014).

| CON CLUS ION
This review summarizes the recent trends of developments in the methods of sample extraction, cleanup processes, detection technologies, quantitative methods, and also on the current research of fast and noninvasive detection methods. Sample pretreatment has continuously been a challenging step for analysis of mycotoxins in various food matrices. Sample preparation protocol often needs to be optimized to increase the extraction efficiency. Improvement in analytical chemistry and recent advances in immunochemistry have led to more specific, sensitive, simple, and rapid immunoassays that deliver quantitative and semiquantitative results on-site and have developed as the process of selection for routine analysis of mycotoxins in the field and storehouses. ELISA method has been used for the analysis of AFs (AFB 1 , AFB 2 , AFG 1, AFG 2 ) and OTA in rice and food stuffs; still, these approaches need confirmatory analysis using other vigorous procedures. It is worth noting that although several sensitive methods like the microplate reader and lateral flow strip have been mentioned in the analysis of mycotoxins based on immunochemical format, they require expertise and wellinstructed operators. Therefore, the quest for label-free, fast, and more sensitive tools based on immune-biosensor format continues.
Which can offer compact, lightweight, responsive, and reliable mycotoxin detection devices in the field.
Apart from typical antibodies, several new recognition components such as molecularly imprinted polymers and aptamers are applied in mycotoxin detections at pg/ml level. Efforts are continuing on optimizing aptasensors that bind to AFB 1 for detection in the field.
Nanoparticles and nanostructure-based analytical devices have high sensitivity and low detection limits and can be potentially used as por-

ACK N OWLED G M ENTS
The authors are grateful to the management of Vellore Institute of Technology, Vellore, Tamilnadu, India for providing the facilities for the presented review paper. Ms. Jyoti Singh is also thankful to the Vellore Institute of Technology for providing her an institutional research fellowship.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
Human and animal testing is unnecessary in our study.