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High Throughput Detection Methods for Multiplex Mycotoxins

Jianlin Li1*, Yang Deng1, Yan Liu1, Zhi Ding1, Yichen Li1, Yanhao Jin1, Xuerui Zhu1, Tingting Cai1, Tiesong Zheng1* and Wei Li2

1Department of Food Science and Engineering, Nanjing Normal University, Nanjing 210024, China

2Department of Electronic and Electrical Engineering, The University of Sheffield, Sheffield, S3 7HQ, United Kingdom

*Corresponding Authors:
Jianlin Li
Department of Food Science and Engineering
Nanjing Normal University, Nanjing 210024, China
Tel: +86 25 83598286
[email protected]

Tiesong Zheng
Department of Food Science and Engineering
Nanjing Normal University, Nanjing 210024, China
E-mail: [email protected]

Received Date: July 19, 2017; Accepted Date: August 17, 2017; Published Date: August 23, 2017

Citation: Li J, Deng Y, Liu Y, Ding Z, Li Y, et al. (2017) High Throughput Detection Methods for Multiplex Mycotoxins. Toxicol Open Access 3:131. doi: 10.4172/2476-2067.1000131

Copyright: © 2017 Li J, et al. This is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use,distribution, and reproduction in any medium, provided the original author and source are credited.

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Mycotoxins are toxic low-molecular-weight compounds which produced by the metabolism of certain fungi species. Due to their toxicity, chemistry stability, diversity and co-occurrence in agriculture products, it is urgently needed to develop the rapid, simple and effective detection technique methods to monitor and prevent mycotoxin contamination in whole food chain. This article gives a review about high throughput detection methods for multiplex mycotoxins which mainly includes chromatographic instrument techniques, microarray chip, suspension array, lateral flow biosensors, surface plasmon resonance (SPR) and nanoparticle-based biosensors. The advantages and disadvantages and the key steps of them have been discussed. The insight and evaluation of the technique progress were given, which would be helpful to further develop this filed.


Mycotoxins; High-throughput detection; Research advance


AFB1-2: Aflatoxin B1-2; AFG1-2: Aflatoxin G1-2; AFM1: AflatoxinM1; DON: Desoxynivalenol; FB1: Fumonisin B1; OTA: Ochratoxin A; ZON: Zearalenone; HT-2: HT-2 Toxin; CIT: Citrinin; ZEN: Zearalanone; NIV: Nivalenol; NEO: Neosolaniol; DAS: Diacetoxyscirpe-nol; FUS-X: Fusarenon-X; 3-ADON: 3- Acetyldeoxynivalenol; 15-ADON: 15-Acetyldeoxynivalenol; TMB: 3,3′, 5,5′-tetramethylbenzidine; QDs: Quantum Dots; HRP: Horseradish Peroxidase; SPR: Surface Plasmon Resonance; Ispr: Imaging SPR; HPLC: High Performance Liquid Chromatography; GC-MS: Gas Chromatography and Mass Spectrometry; LC/MS/MS: Liquid Chromatography and Mass Spectrometry; UPLC-MS/MS: Ultra- Performance Liquid Chromatography-Tandem Mass Spectrometry; IAC: Immunoaffinity Columns; LOD: Limits of Detection; LOQ: Limit of Quantitation; ELISA: Enzyme-Linked Immunosorbent Assay; ESI: Electrospray Ionisation; APCI: Atmospheric Pressure Chemical Ionization; FAO: The Food and Agriculture Organization; FLD: Fluorescence Detector; UV: Ultraviolet Detector; Xmap: Multi Analyte Profiling; QuEChERS: Quick, Easy, Cheap, Effective, Rugged and Safe; SPE: Solid-Phase Extraction; LE: Liquid-Liquid Extraction; SPDE: Solid Phase Dispersion Extraction; PLE: Pressurized Liquid Extraction; SPCMs: Silica Photonic Crystal Microspheres.


Mycotoxins are toxic low-molecular-weight compounds which produced by the metabolism of certain fungi species. Nowadays, hundreds of mycotoxins have already been isolated and identified and the most common toxin-producing genera are Aspergillus , Penicillium , Alternaria and Fusarium fungi which correspondingly produced aflatoxins (AFs), ochratoxins (OTA), patulin (PAT), and fusarium toxins [1]. Most of the mycotoxins have been identified as carcinogenic, mutagenic, and teratogenic agents. Many countries and regions have set maximum limits tolerable for the mycotoxins in food and feed to protect human and animal health [2].

Mycotoxins often appear in cereal crops, stored cereals and feedstuff, which can easily enter the food chain when the agricultural commodities are harvested, stored, transported and processed in the absence of proper conditions. The Food and Agriculture Organization (FAO) estimated that about 25% of the world’s food crops are contaminated by mycotoxins, resulting in annual losses of about 1 billion metric tons of food and food products equivalent to about 5 billion dollars per year in Unite States and Canada [3-5]. The mycotoxins display the properties of chemical and thermal stability, which result in that they are difficult to remove once they contaminate the food chain. Therefore, the first priority is to develop the rapid, simple and effective detection technique methods to prevent the mycotoxin contamination from food chain, especial for the developing countries.

Generally, co-occurrence of multiplex mycotoxins is often found in the same sample because a single species of fungi can produce several toxic metabolites, or several species can be present simultaneously, producing different toxins [5]. In addition, various types of mycotoxins also often are present in mixtures of different raw material samples, such as breakfast cereals, flours and bread [5]. These co-occurrence multiplex mycotoxins show additional or synergistic toxic effects [5-10]. It is very necessary to rapidly high throughput screen or detect the multiplex mycotoxins in the samples for accurate risk assessment of food safety. Here, high throughput detection or screening method refers to that it can simultaneously and rapidly detect or screen multiplex target molecules.

At present, the multiplex mycotoxin detection or screening methods mainly included chromatographic instrument techniques, microarray chip, suspension array, lateral flow biosensors, SPR and nanoparticlebased biosensors (Figure 1). The article will give a short review about these detection methods for multiplex mycotoxins.


Figure 1: The scheme of the high throughput detection methods for multiplex mycotoxins.

Chromatographic Instrument Techniques

Chromatographic instrument techniques mainly include high performance liquid chromatography (HPLC) [11], gas chromatography and mass spectrometry (GC-MS [12], GC- MS/MS [13]), liquid chromatography and mass spectrometry (LC/MS/MS [14]) and other instrumental analysis methods. Generally, chromatographic detection methods are often used for qualitative and quantitative analysis for mycotoxin confirmatory analyses. These techniques, especial for HPLC-MS/MS, are becoming mainstream methods for rapid screening of multiplex mycotoxins because of their high sensitivity, high accuracy, good reproducibility and short detection time.


HPLC is one of the most used techniques for qualitative and quantitative of mycotoxin detection, which used different extraction, purification and sensitive detection system equipped with ultraviolet detector (UV), fluorescence detector (FLD), differential detector, photodiode array and ammeter. For example, Chan et al. have established an automated HPLC method with immune affinity column purification and FLD for the simultaneous detection of aflatoxins (AF) and ochratoxin A (OTA) [15]. Limits of detection (LOD) were estimated as 0.2 μg/kg for OTA and AFB1, AFB2, AFG1 and AFG2 and the recovery is 72%~101% [15]. Ainiza et al. developed a reverse-phase HPLC and fluorescence detector (FLD) with a photochemical derivatisation system for simultaneous determination of AFs and OTA using amulti-mycotoxin immunoaffinity column purification. The recovery is 72%~100% for AFs and 73%~113% for OTA. The LOD was 0.1 μg/kg for AFB1/AFG1, 0.05 μg/kg for AFB2/AFG2 and 0.1 μg/kg for OTA [16]. Kong et al. developed a HPLC method with ultrasoundassisted solid-liquid extraction and immunoaffinity column clean-up coupled with on-line post-column photochemical derivatizationfluorescence detection for simultaneous multi-mycotoxin determination of AFs and OTA. The recovery and LOD showed the satisfied results in 13 edible and medicinal nutmeg samples [17]. Recently, Xu et al. set up an ultra HPLC coupled with photo-diode array detection method for the simultaneous determination of deoxynivalenol and its acetylated derivatives with quick, easy, cheap, effective, rugged and safe (QuEChERS) solid-phase extraction (SPE) purification, which has the LOD (21.7-57.4 μg/kg) and quantitation (72.3-191.4 μg/kg) for deoxynivalenolsin in wheat flour and rice [18].HPLC could provide a reliable, high sensitivity, high resolution and good reproducibility results for multiplex mycotoxins. However, the main problem of multiplex mycotoxin analysis is not detection but samples pretreatment for obtaining a selective and fast extraction of mycotoxins from the matrix [19]. Therefore, most of them required complex pretreatment of samples such as multi-mycotoxin immunoaffinity column purification, C18 column purification or supercritical fluid extraction, and post-column derivatization which limited their application in practical. QuEChERS extraction and new SPE purification system for HPLC methods for detection of multiplex mycotoxin should be widely developed and practiced. The detailed sample pretreatment for mycotoxins from matrix can be seen in reference [5].

GC and GC-MS

Though GC and GC-MS are cheaper than HPLC and HPLC-MS, GC and GC-MS have been less widely used for multiplex mycotoxin analysis comparing with HPLC and HPLC-MS. Only a few of the references about GC and GC-MS for multiplex mycotoxin analysis were reported, which may be ascribed to the complicated sampling procedures such as derivatization although methods without derivatization have been reported.

Ryu et al. used GC-MS to simultaneously quantitatively detect seven trichothecene mycotoxins after trimethyl silyl derivatization [20]. Nielsen et al. developed a fast method for simultaneously screening of trichothecences in species of Fusarium , Stachybotrys , Trichoderma and Memnoniella by micro scale extraction and derivatized with pentafluoropropionic anhydride or heptafluorobuturyl imidazole [21]. Rodríguez-Carrasco et al. established GC-MS/MS method to determine 15 mycotoxins and metabolites in human urine with salting-out assisted acetonitrile-based extraction, which displayed a range of 72-109% recoveries and limit of quantitation ranged from 0.25 to 8 μg/kg [22]. Cegielska-Radziejewska et al. established the simultaneous determination of seven trichothecences (T-2, HT-2, DAS, DON, 3-ADON, 15-ADON and NIV) by GC-MS in feed mixtures for broiler chickens [23]. Ibáñez-Vea et al. developed a validated method for the simultaneous determination of eight type-A and type-B trichothecenes in barley by simultaneous extraction sample with acetonitrile-water (84:16), cleaning up with Multisep columns, derivatization with pentafluoropropionic anhydride and imidazole and GC-MS analysis [24]. Escrivá et al. recently reported a method for the simultaneous determination of seven trichothecenes, neosolaniol (NEO), diacetoxyscirpe-nol (DAS), deoxynivalenol (DON), nivalenol (NIV), fusarenon-X (FUS-X), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) in feed samples with N,Obis( trimethylsilyl) acetamide+trimethylchlorosilane+N-trimethylsilyimidazole (3:2:3) as the derivatization reagent under extracting and purifying samples with acidified mixture of acetonitrile/water [25]. LOQs were between 1 and 10 μg/kg for all studied trichothecenes and recoveries ranged from 62% to 97% [25].

These GC and GC-MS methods for multiplex trichothecene mycotoxins provided the lower LODs, greater selectivity, higher precision and inexpensive analytical performance. However, some mycotoxin specific problems such as non-linearity of calibration curves, poor repeatability, matrix induced over-estimation, and memory effects from previous sample injections, which make them still limited in narrower analytical scope [26].


Over the last years, a significant number of papers for analysis of multiplex mycotoxins have focused increasingly on HPLC-MS, HPLCMS/ MS and UPLC-MS/MS. Ionization is an important procedure for Liquid chromatographic/mass spectrometric (LC-MS) methods, especially for different food matrix. After atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) have been realized since the mid to late 90s, LC-MS methods have been rapidly developed [27]. LC-MS/LC-MS/MS/UPLC-MS/MS methods for multiplex mycotoxin analysis were listed in Table 1, which showed that ESI interface has been used in majority of LC-MS/MS/UPLCMS/ MS. ESI interface is mainly well suited for the analysis of polar compounds, whereas other atmospheric pressure ionization (API) interfaces like APCI and atmospheric pressure photo ionization (APPI) are highly effective for the analysis of medium and low-polar substances [5]. These multiplex mycotoxin analysis techniques could provide high sensitive, selective, rapid and reliable quantification and confirmation at the low concentration. These methods rely mainly on multiple mycotoxin parallel or sequential sample preparation strategies of one sample followed by separate analysis of each isolated class of mycotoxins in a single run [41].

LC-MS Mycotoxins Matrix Sample preparation Column Ionisation/ion selection LOD/LOQ
Recovery (%) References
UPLC-MS/MS 6 mycotoxins Wheat flour and rice QuEChERS-SPE filter BEH C18 ESI 21.7-57.4 80-104.4 [18]
UPLC-MS/MS 36 mycotoxins wines QuEChERS BEH C18 ESI 0.1 70-120 [28]
LC-MS FB1,HFB1 Corn products LEP and C18 cleanup 60-RP B ESI 5 54.6-97.4 [29]
LC-MS/MS A,B-Tichothecenecs,zearalenone maize LEP-MycoSep®#226, 227 cleanup RP-18 ESI 0.3 30-99 [30]
UPLC-MS   17 mycotoxins Corn feed, peanuts LEP-MycoSep®#226, cleanup BEH C18 ESI 0.01-0.7 70.7-119 [31]
LC-MS/MS 24 mycotoxins Sweet pepper LEP, NH2-SPE column, C18-SPE column  C18 ESI 0.32-42.48 76-110 [32]
UPLC-MS/MS 11 mycotoxins Maize,whea, rice, oat, barley, rye, sorghum, millet LEP-nylon filter BEH  C18 ESI 0.1 70-110 [33]
LC-MS/MS 31 mycotoxins Wheat, barley, oats the accelerated solvent extraction Inertsil ODS-EP ESI 1-1250    51-122 [34]
UPLC-MS/MS 12 mycotoxins beer SPE with C18 BEH C18 ESI 0.02-0.14 70-106 [35]
LC-MS/MS 99 mycotoxins Home dust LE Gemini® C 18 ESI 0.005-2200 <50for half of samples [36]
LC-MS/MS 87 mycotoxins bread LE Gemini® C 18 ESI 0.02-225 55-287 [37]
LC-MS/MS DON,ZON and their metabolites Cereal-based food, beer LE-C18-SPEcleanup, LE-Immunoaffinity, LE-MycoSep226 RP-C 18 ESI 0.5-50 89-119 [38]
UPLC-MS/MS 5 mycotoxins traditional Chinese medicines LE-cleanup cartridge; 0.22μm filter HSS T3 ESI  0.29-0.99   88.5-119.5 [39]
LC-MS/MS DON,ZEN,T-2,HT-2 Wheat and biscuit LE-Immunoaffinity C18 ESI 0.03-0.33 78-109 [40]
LC-MS/MS Six mycotoxins Wheat and maize LE-Immunoaffinity, Ultrasonic LE-syringe filter RP-C 18 pressure chemical ionization (APCI) 1.5-4.7 35-175 [41]
LC-MS/MS Six mycotoxins urine LE-Immunoaffinity C18 ESI 0.01-2.2 62-96 [42]
LC-MS/MS Eleven mycotoxins Cereal samples LE-0.22μm filter C18 ESI APCI 0.01-20 76.8-108.4 [43]
UPLC-MS/MS 11 mycotoxins Wheat, rice, oat, maize, barley LE-0.22μm filter C18 ESI APCI 0.01-25 83.5-107.3 [44]
LC-MS/MS AFB1, AFB2, AFG1, AFG2 Lotus seeds LE- Immunoaffinity Zorbax SBC18 ESI 0.003-0.007 66.3-110 [45]
LC-MS/MS Monacolins and citrinin Red fermented rice LE- 045μm filter Zorbax SBC18 ESI 0.0005 98.3-99.4 [46]
UPLC-MS/MS 12mycotoxins pig urine salting-out assisted LLE C18 ESI 0.07-3.3 70-108 [47]
UPLC-MS/MS 4 Trichothecenes potato LE-PriboFast 270 column-0.22μm filter BEHC18 ESI 0.002-0.005 77.97-113.28 [48]
UPLC-MS/MS 6mycotoxins Feed LE-carbon nanorubes SPE-0.22μm filter BEHC18 ESI 0.2-0.29 95.3-107.2 [49]
UPLC-MS/MS AFB1,OTA, ZON,ZOL Milk LE-Mycosep226 filter BEHC18 ESI 0.003-0.015 87-109 [50]
LC-MS/MS Trichothecenes deoxynivalenol chicken LE-HLB cartridge 0.22μm filter C18 ESI 0.16-2.07 73.7-106.4 [51]
LC-MS/MS 16mycotoxins Dried fruit QuEXhERS-C18- 0.22μm filter C18 ESI 0.08-15 60-135 [52]
LC-MS/MS 19mycotoxins Biscuits SPE- 0.2μm filter C18 ESI 0.04-80.2 63-107 [53]
LC-MS/MS 12mycotoxins Maize, wheat, corn LE- Immunoaffinity C18 ESI 0.2-0.5 63-88 [54]
LC-MS/MS 18mycotoxins wheat solid phase dispersion extraction (MSPD) - 0.2μm filter C18 ESI 1.0-250 68-89 [55]
UPLC-MS/MS 7 mycotoxins Dried fruit Pressurized liquid extraction(PLE)-C18 PFP column heated (H-ESI) 2-110 83-103 [56]
LC-MS/MS 21mycotoxins coffee beverages LE- 0.22μm filter C18 ESI 0.02-39.64 72-97 [57]
LC-MS/MS 15mycotoxins eggs QuEChERS C18 ESI 0.2-5 67.5-105.4 [58]
LC-MS/MS 8mycotoxins  Apple, orange, cherry and tomato SPE-0.22μm membrane filter C18 ESI 01-May 74.2-102.4 [59]
LC-MS/MS 26mycotoxins durum wheat grain LE- 0.22μm filter C18 ESI Feb-70 59-110 [60]
UPLC-MS/MS 12 mycotoxins Areca catechu LE- 0.22μm filter C18 ESI 0.1-20 85-115 [61]
LC-MS/MS 5mycotoxins layer feed LE- MycoSep 227 C18 ESI APCI 0.9-7.5 50-63 [62]
UPLC-MS/MS 8 mycotoxins Feed LE- Immunoaffinity BEH C18 ESI 0.006-0.12 91.2-104.1 [63]
LC-MS/MS 7mycotoxins vegetable oil LE-MSPD-0.2μm filter C18 ESI APCI 0.04-2000 87.9-106.6 [64]
LC-MS/MS 7mycotoxins Wheat, corn, peanut LE-Immunoaffinity -0.2μm membrane filter C18 ESI APCI 0.04-0.4 95.3-103.3 [65]

Table 1: Overview on LC-MS/LC-MS/MS/UPLC-MS/MS for mutiplex mycotoxin analysis.

For LC-MS/MS multiplex mycotoxin analysis, the most important and critical steps still is sample pretreatment and sample cleanup. Liquid-liquid extraction (LE), solid phase extraction (SPE), solid phase dispersion extraction (SPDE), pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) [66] have been used for sample extraction. Currently, the most common extraction solvents employed are methanol/water and acetonitrile/water, although acidified acetone and ethyl acetate/acetonitrile/water have been reported to a lesser extent [67]. Water could increase the solvent enter into the samples and help the release of mycotoxins and increase the extraction efficiency [5,67]. As the improvement of environmental protection and healthful consciousness, organic solutions, especial for hazardous and poisonous solutions, have been avoided as far as possible to use during sample pretreatment. QuEChERS sample preparation approach has been applied in simultaneous extraction of multiplex mycotoxins. However, QuEChERS protocols are different formula as the different chemicals and often are inherently inefficient and reduce the sensitivity of the analytical method [1].

Sample cleanup is often needed to remove any impurities/ interfering material in addition to concentrating the extract prior to analysis and quantification although injection of crude extracts without purification step has been proposed, especial for confirmatory analytical methods. SPE columns, immunoaffinity columns and Mycosep®/Multisep® purification have been usually applied in multiplex mycotoxin analysis. SPE columns are one of the most commonly applied methods of cleanup, such as Florisil, C8, C18 reversed phase and aluminum oxide [67]. The targets were often retained on the surfaces of SPE columns and remove the impurities, then eluted by solvent solution. In some SPE purification protocols, impurities were retained on the surfaces of SPE columns and the eluent was the targets. SPE purification could be affected by pH, type of solvent and ionic strength of sample [5]. Immunoaffinity columns (IAC) could provide the high specificity enrichment for mycotoxins by their antibodies bound to the surfaces of columns. However, the antibodies may lose their activities when the organic solvent is used as extraction solution and eluent. In addition, they are expensive and few commercial companies have produced IAC for the multiplex mycotoxin analysis. Most of the IAC used in references were prepared by authors in their lab. Mycosep®/Multisep® purification are another well-established method for multiplex mycotoxins. Mycosep®#226 and #227 were often used for purification for multiplex mycotoxin analysis [30,32,38,50,62]. These columns comprise different adsorbents (e.g., charcoal and ionexchange resins), which can adsorb proteins, fats and pigments of samples no requirement for activation, washing and elution.

The matrix effects caused by co-elution of matrix components should be carefully considered because they can result in suppression or, more rarely, enhancement of the ionization efficiency of the analyte affecting the accuracy and precision of the results, especial for injection of crude extracts [33,68]. Matrix-matched standards calibration has accurately quantified multiplex mycotoxins in different food matrix without need to use isotope-labelled internal standards [33].

UPLC-MS/MS is being used increasingly in multiplex mycotoxin analysis because its short analysis time, superior resolution and more sensitivity compared with HPLC-MS/MS [5]. However, the cost of analysis is the highest among the MS methods.

Array-based Biosensors for Multiplex Mycotoxin Analysis

Array-based biosensors mainly include DNA microarray, protein microarray and suspension array, which are powerful tools for multiplex target analysis in parallel. The remarkable properties of these techniques lie in its high throughput, automation and integration for analysis equipment. DNA and protein microarray techniques mainly composed of spotting robot, hybridization chamber and chip scanner. DNA microarray usually is applied in high throughput detection for multiplex mycotoxin biosynthesis genes [69]. Protein microarray based on specificity reaction of antibodies and antigens has been used to detect multiplex mycotoxins. Wang et al. established simultaneous determination method for six mycotoxins using indirect competitive immunoassay on a protein microarray by immobilization of complete antigens of six mycotoxins on agarose-modified glass slides [6]. It is reported that the LOD in drinking water were 0.01, 0.24, 15.45, 15.39, 0.05, and 0.01 ng/mL for AFB1, AFM1, DON, OTA, T-2 and ZEN, respectively. The recovery ratios in drinking water ranged from 80%-120%. Though microarray techniques could provide a high throughput and sensitive determination multiplex targets in sample in parallel, their application in practice is limited because of the expensive equipment.

Suspension array techniques have been developed for more than ten years and shown a powerful platform for multiplex mycotoxin analysis because of its high flexibility, fast reaction and good repeatability for detection [70-75]. Unlike planar microarray using the coordinate of positions for encoding different probe molecules, most of suspension array techniques employed the encoding polymer microspheres with spectra as support carriers. The most of multiplex detection principle of suspension arrays based on fluorescence dye immunoassay is seen in Figure 2. The key techniques of suspension array are the encoding and decoding techniques. The most prominent suspension array system comes from Luminex Corporation (Austin, Texas, USA), which has been applied in high throughput screening for multiplex mycotoxins as the mainstream equipment Table 2. The Luminex suspension array Multi Analyte Profiling (xMAP) systems mainly depend on the fluorescent dye encoding techniques for encoding different microspheres and flow cytometer technique for high throughput decoding and reading the signals of targets on the different microspheres. Luminx xAMP system can allow simultaneous measurements of up to 100 different biomolecular interactions in a single well. Compared with the planar microarray, the kinetics of molecular reaction on the surfaces of microspheres has been great improved because of the rotational motion of microspheres in reaction solution. In addition, the suspension array system could easily integrate with sample pretreatment (such as magnetic microspheres for enrichment, purification and detection). More importantly, its cost is not more than one-tenth of planar microarray. Therefore, the suspension array system has great potential in application for multiplex mycotoxin detection.


Figure 2: The multiplex detection principle of suspension arrays based on fluorescence dye [79].

However, suspension array systems encoding with fluorescent dyes are encountering some problems. For example, the fluorescence dyes tend to be quenched or bleached and the optic system are complicated [79,81]. Our group established the easier, simpler and more flexible suspension array system which is based on silica photonic crystal microsphere (SPCM) encoding with their structure colors for multiplex mycotoxin analysis [79-82]. SPCMs for multiplex mycotoxin analysis showed a wide detection linear range and pg/mL LOD [79,81]. The three-dimensional porous microsphere has a large internal surface,bright structure colors and easily been fabricated and manipulated in common lab which make them much more advantages over common solid microspheres. Main suspension arrays for multiplex mycotoxin analysis were summarized in Table 2.

Suspension array Microspheres Mycotoxins Matrix  Detection principle LOD/LOQ (μg/kg) Recovery (%) References
Luminex 200 system Luminex microspheres ZEN, FB1, DON, AFB1 Corn,  wheat, feedstuff direct competitive fluorescent immunoassay 0.51 92.3- 115.5 [9]
BD FACSArray™ Bioanalyzer BD FACSArray™ microspheres AFB1, OTA, FB1, DON, ZEA, T-2 Wheat, pea direct competitive fluorescent immunoassay 0.01-75.73 80-110 [70]
Luminex100 system paramagnetic microspheres AFB1, OTA, FB1, DON, ZEA, T-2 Feeds indirect competitive fluorescent immunoassay 0.29-6.7 - [73]
Luminex100 Luminex microspheres OTA,FB1 Corn, oat indirect competitive fluorescent immunoassay pg <50 [74]
Luminex100 system Luminex microspheres AFB1, T-2, DON,  ZON corn and peanut indirect competitive fluorescent immunoassay pg/mL 80.16-117.65 [76]
Imaging planar bead array analyser MagPlex, Beads, Invitrogen Dynal AFB1,OTA, ZON,DON,T-2,FB1 direct competitive fluorescent immunoassay 2.5-1000 - [77]
Luminex100 FLEXMAP 3D paramagnetic microspheres Luminex FB1,OTA,ZEN Wheat, maize direct competitive fluorescent immunoassay 0.7-1270 - [78]
microarray scanner Silica photonic crystal microsphere AFB1, FB1, Citrinin(CIT) Wheat, peanut, corn direct competitive fluorescent immunoassay 0.5-1pg/mL 74.7-127.9 [79]
microarray scanner Silica photonic crystal microsphere FB1, OTA Wheat, rice corn direct competitive fluorescent aptamer 0.16-0.25pg/mL 76.58-116.38 [80]
multifunctional microplate reader Silica photonic crystal microsphere AFB1,FB1,OTA Wheat, rice corn indirect competitive chemiluminescent immunoassay 0.73-1.19pg/mL 63.5-121.6 [81]
multifunctional microplate reader Silica-hydrogelphotonic crystal microsphere AFB1,FB1,OTA Wheat, rice corn indirect competitive chemiluminescent immunoassay 0.4-2.1 pg/mL 74.96-104.87 [82]
microarray scanner Silica photonic crystal microsphere AFB1, FB1, OTA Wheat, rice corn Direct fluorescent aptamer 3.96fg/mL-11.04 pg/mL 71.20-113.19 [83]

Table 2: Overview on suspension arrays for multiplex mycotoxin analysis.

These xAMP systems have given 2-6 mycotoxins analysis results and most of them depended on the immunoassay principles combining fluorescence and chemiluminescence techniques. Theoretically, the microsphere-based arrays could simultaneously detect hundreds of mycotoxins. However, these systems may be limited by the antibody preparation and antibody quantities of mycotoxins. Fortunately, the antibodies of the common mycotoxins can easily be obtained from commercial company. Therefore, it is possible that these systems replace the traditional enzyme-linked immunosorbent assay (ELISA) methods because of their obvious advantages.

Recently, aptamer techniques on the microsphere array carriers have been established to detect the multiplex mycotoxins [80,83]. The principles of aptamer techniques on microsphere array carriers were based on the changes of fluorescence signal before and after mycotoxins binding to their aptamers (Figure 3). The methods are designed as one-step detection for multiplex mycotoxins. They have a great potential for replacing the immunoassay methods for multiplex mycotoxin analysis because they are simple, easily operated, high sensitive and cost-effective.


Figure 3: The aptamer fluorescence signal recovery screening for multiplex mycotoxins [83].

Multiplex lateral flow biosensors analysis for mycotoxins

Lateral flow biosensors are self-operating devices that perform rapid assays on a membrane or gel in a chromatographic manner from a single sample addition [84]. The one-step lateral-flow methods often use colloidal gold-labelled antibodies as the indicator visible to the naked eye and specific reagents to bind its antigen immobilized on the membrane surface. Multiplex lateral flow techniques are based on the traditional single-plex lateral flow biosensor for the multiplex analyte detection. Generally, artificial antigens of multi-mycotoxins were immobilized on the surface of membrane or gel and the competitive immunoassay protocol was performed on the surface of membrane among the targets and artificial antigens to binding to labelled mycotoxin antibodies (Figure 4). Compared with HPLC-MS/MS methods, though the number of mycotoxins for multiplex analysis is limited, lateral flow methods are simple, rapid and low cost and could provide qualitative, semiquantitative or quantitative visual on-site testing. These techniques have been rapidly developed recently because they allow the operation and interpretation for the non-skilled personnel. For example, Kolosova et al. developed a qualitative onestep test for simultaneous assay for DON and ZEA using colloidal gold-labelled monoclonal antibodies [85]. It can provide the cut-off levels of 1500 and 100 μg/kg for DON and ZEA, respectively. Burmistrova et al. used alkaline phosphatase labelled antigens to develop colors for non-instrumental multiplex semiquantitative and qualitative detection of OTA, ZEN and FB1 in wheat, and maize samples [86]. Kong et al. established a gold nanoparticle-based semiquantitative and quantitative ultrasensitive paper sensor for the detection of twenty mycotoxins [87]. Other multiplex lateral flow analysis for mycotoxins were summarized in the Table 3.


Figure 4: The principle of multiplex lateral low immunoassay for mycotoxin determination [2].

Mycotoxins Matrix Membrane material Color development Sample clean-up LOD or Cut-off level (μg/kg)    References
AFB1,ZEA,DON Wheat, maize nitrocellulose membrane Colloidal gold nanoparticles without 1,50 and 60   [2]
DON, ZEA wheat Hi-Flow Plus 75 Colloidal gold without 1500,100 for DON and ZEA [85]
OTA, FB1,ZEN, Wheat, maize, silage Immunodyne ABC 5-bromo-4-chlor-3-indolyl-phosphate/nitro-blue-terazolium glass microfiber filter 2.5,50,1000 for OTA,  ZEN, FB1 [86]
ZEAs, DONs,T-2s, AFs,    FBs Cereal smaples Nitrocellulose (NC) high flow-plus Colloidal gold nanoparticles  without 0.25-1,5-500,1-10,0.5-2.5 and 5-25 for ZEAs, DONs,T-2s,AFs, FBs [87]
AFB1, OTA, DON,  ZON,FB1 corn, wheat, peanut polyvinylidene fluoride 3,3’,5,5’-tetramethylbenzidine (TMB) without 20,60,1000,20 and 250 for AFB1, ZON,DON, OTA, FB1                    [88]
AFB1,OTA,ZEA Maize, rice, peanut nitrocellulose membrane Colloidal gold nanoparticles without LOD 0.25, 0.5,  and 1for AFB1,   OTA,  ZEA      [89]
ZEA, DON, T-2/HT-2, FB1 /FB2 Wheat, maize nitrocellulose membrane Colloidal gold nanoparticles without Qualitative          [90]
ZEN,FB1 Wheat, corn nitrocellulose membrane Colloidal gold nanoparticles without 0.35and5.23ng/mL For ZEN and FB1    [91]
T-2,HT-2 wheat polyethylene filters or sepharose gel TMB without 0.55 and 1.7ng/g        [92]
AFB1, OTA, ZEN, DON, maize, peanuts, and cassava flour sepharose 4B,Immunodyne ABC membranes TMB C18 clean-up sorbent 3,1,250,1000,200 for sepharose 4B 3,5,700,175 for  membrane For OTA,AFB1,DON and  ZEN [93]
FB1,DON,T2,ZEA Wheat, oats, maize nitrocellulose membrane Colloidal gold nanoparticles without 280,400,1400 ,3200 and for 80,400,1400 ZEA,T2,DON And FB1 in maize and wheat/oats [94]
AFB1,ZEA, OTA Corn, rice, peanut nitrocellulose membrane Colloidal gold nanoparticles without 0.10-0.13,0.42-0.46,0.19-0.24for AFB1, ZEA  OTA [95]

Table 3: Overview on lateral flow biosensors for multiplex mycotoxin analysis.

The color development mainly depended on the colloidal gold nanoparticles and enzyme substrate chromogenic reagents, which could provide the qualitative response and the quantitative results by means of a colorimetric reader. For example, Song et al. developed qualitative and semiquantitative triplex lateral flow immunoassay with LOD of 0.05,1 and 3 μg/kg for AFB1, ZEA and DON, respectively. The strip reader gave the relative optical density between the signal of a positive sample and the blank, which allow establish the calibration curves for the three mycotoxins [2]. 3, 3’, 5, 5’-tetramethylbenzidine (TMB) was often used as color development of horseradish peroxidase (HRP) which catalyzes TMB to produce blue color. Although the colloidal gold nanoparticles and TMB have shown good sensitivity and low limits of detection for color development, different signal enhancement strategies have been developed to significantly improve LOD, sensitivity and stability of the system, which is strongly related with the development of nanomaterial techniques. Graphene, quantum dots, nano-silver and magnetic nanoparticles have been introduced in the system and showed the stable, enhanced signal and wide detection linear ranges [96]. In addition, except nitrocellulose membrane as the support substrate, the three-dimensional porous smart nanomaterial may bring the field to a new level.

Nevertheless, the lateral flow devices still have some problems when they have been applied in the real samples. As the increase of analysis kinds of mycotoxins in the single device, the large of reagents will be consumed in the system [84]. Most of the systems just give qualitative or semiquantitative response by naked-eye and the false-positive or false-negative results are often reported. In addition, unspecific adsorptions coming from different matrix compounds often occur in the membrane or gel, which could result in invalid or failed detection.

Other biosensor techniques for multiplex mycotoxin analysis

Other biosensor analysis systems for multiplex mycotoxins mainly include label-free techniques (such as surface plasmon resonance (SPR)) and nanoparticle-based biosensor techniques. SPR techniques are the typical optical label-free in real time biosensors for multiplex mycotoxins. SPR continuously monitors changes of refractive index of the biorecognition layer on the sensor surface. Nielen group developed a competitive inhibition immunoassay using the imaging SPR (iSPR) technique for the simultaneous detection of DON and ZEN, which showed 84 and 68 μg/kg for DON, 64 and 40 μg/kg for ZEN in maize and wheat samples, respectively [97]. Recently, they used the technique to detect DON and DON, ZEA, T-2, OTA, OTA, FB1and AFB1 on nanostructure chip surfaces for beer and barley samples [98,99]. The competitive immunoassay principle of iSPR for multiplex mycotoxin detection is seen in Figure 5. They reported the nanostructure chip could be regenerated for 450 cycles [98] and 60 cycles [99] after each cycle. The system can be used for in-field or at-line detection of DON in beer and barley without preconcentration, while OTA in beer requires an additional enrichment step [98]. The LODs in beer were 17 ng/mL for DON and 7 ng/mL for OTA [97]. The LOD in barley (in μg/kg) were determined to be 26 for DON, 6 for ZEA, 0.6 for T-2, 3 for OTA, 2 for FB1 and 0.6 for AFB1 [99]. The detection platform could provide a rapid and semi-quantitative screening for multiplex mycotoxins prior to LC-MS/MS [99]. Hu et al. developed a sensitive method for detection of AFB1, OTA and ZEN using iSPR with gold nanoparticles as signal amplification tags in gold chip surface [100]. The competitive immunoassay format was performed on the gold chip surface and then the secondary antibody-conjugated gold nanoparticles were used to bind with monoclonal antibodies for further amplification of the iSPR signal. They showed that the LODs were 8, 30 and 15 pg/mL for AFB1, OTA and ZEN and dynamic ranges covering three orders of magnitude. For the detection for multiplex mycotoxins, much more volume reagents are needed in the system. Furthermore, non-purification samples may result in false response because of other compounds in matrix. Therefore, the focus of the platform should be on miniaturization of the chip and integration of pretreatment of samples with detection.


Figure 5: The competitive immunoassay principle of iSPR for the multiplex mycotoxin detection [99].

Nanoparticle-based biosensor techniques for multiplex mycotoxin analysis have been rapidly developed due to the booming progress in nanomaterial field. Graphene, quantum dots, nano-silver, upconversion and magnetic nanoparticles have been designed as the labeled probe or color development for multiplex mycotoxin analysis. Immunoassay and aptamer principles have been developed to detect multiple mycotoxins. Wu et al. established multiplexed fluorescence resonance energy transfer aptasensor between upconversion nanoparticles and graphene oxide for the simultaneous determination of OTA and FB1 with the LOD of 0.02 ng/mL for OTA and 0.1 ng/mL for FB1 [101]. Chen et al. applied antigen-modified magnetic nanoparticles as biosensor probes and antibody-functionalized improved upconversion nanoparticles as signal probes to develop an ultrasensitive fluorescence biosensor, which showed the LOD of 0.001 ng/mL and detection linear range of 0.001-0.1 ng/mL for AFB1 and DON in peanut oils [102]. Saeger group used quantum dot (QD) nanolabels to develop the multiplex fluorescent immunosorbent simultaneous analysis method which has LODs of 3.2, 0.6, 0.2, 10 and 0.4 μg/kg for DON, ZEN, AFB1, T-2 and FB1 in maize [103]. Then they used [email protected]2 and silica-coated liposomes loaded with QDs as labels to establish the multiplex fluorescent immunosorbent assay with the LODs of 6.1 and 5.3, 5.4, and 4.1, 2.6, and 1.9 μg/kg for DON, ZEN and AFB1 [104], 16.2 and 18, 2.2 and 2.6 μg/kg for ZEN and AFB1 in maize and wheat. Zhang et al. designed a fluorescent aptasensor by DNA-scaffolded silver nanoclusters coupling with Zn-ion signal-enhancement for simultaneous detection of OTA and AFB1, which showed the LOD of 0.2 and 0.3 pg/mL for OTA and AFB1 in rice, corn, and wheat. This field has a great potential for developing the simple,rapid and sensitive sample pretreatment and detection kit for multiplex mycotoxins.


Co-occurrence of multi-mycotoxins in the plant source products and their synergistic toxic effects has been demonstrated in previous reports. Multiplex mycotoxin analysis has a vital significance in preventing human and livestock food chain to avoid their contaminations. LC-MS/MS equipped with ESI has been mainstream technique for multiplex mycotoxin analysis in confirmation analysis. UPLC-MS/MS method would be next generation technique for multiplex mycotoxin analysis because of their obvious advantages over LC-MS/MS in short analysis time, superior resolution and more sensitivity. Sample pretreatment and sample cleanup would still be focus for the chromatographic instrument techniques in multiplex mycotoxin analysis.

For the large quantities of the screening samples and on-site rapid detection, suspension array and lateral flow biosensor techniques for multiplex mycotoxins analysis will be simple, rapid, cost-efficient and practical. These techniques are strongly depended on the development of nanotechnology and specific probe molecules. The stability, sensitivity and specificity of them would be the key regions to break through. In addition, the integration between sample pretreatment and detection techniques would be paid more attention in these systems. As the development of aptamer techniques, the aptamer-suspension array or aptamer-lateral flow biosensor will insult in the simpler, cheaper, faster and more convenient methods for multiplex mycotoxin analysis. However, for analysis of the “masked” or conjugated multiplex mycotoxins, HPLC-MS/MS or UPLC-MS/MS is irreplaceable techniques and urgently needed to further research.


We gratefully acknowledge the financial supports from National Natural Science Foundation of China no.31471642 and no.31071542, Natural Science Foundation of Jiangsu Province no. BK20131398 and 100 Talents Program of Nanjing Normal University.

The authors have declared no conflict interest.


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