| "Food is the basic of the people, and security is the basic of the food", so food safety is one of the major issues which concern both people’s livelihood of the whole world and social harmonious. In recent years, food contamination is more and more serious and frequent, which not only result in huge economic loss, but also badly affect the human health. Mycotoxin is a kind of the major pollutants, which can enter the food chain through contaminated food, feed and the animals with these fed, and indirectly income into human body, eventually leading to be carcinogenic, teratogenic, mutagenic and other serious consequences.To date, the methods of analysis for many mycotoxins mainly include bioassay, chemical analysis (primarily Thin Layer Chromatography, i.e., TLC), instrumental analysis (mainly High Performance Liquid Chromatography, Gas Chromatography-Mass Spectroscopy, and Liquid Chromatography-Mass Spectroscopy), and immunoassay (among them, Enzyme-linked Immunosorbent Assay, i.e., ELISA as the most classic and common). However, these detection methods have some shortcomings, such as only a single target, complex sample preparation, tedious operation, large sample volume required, time-consuming duration and so on, which can not meet the demand for quantitative analysis. Therefore, the establishment of accurate, efficient, rapid, and high-throughput detection methods for simultaneously measuring multiple mycotoxins is the key problem which should be solved immediately.In our study, we chose several typical mycotoxin contaminants (including Aflatoxin B1: AFB1, Aflatoxin M1:AFM1, Deoxynivalenol:DON, Ochratoxin A:OTA, T-2Toxin:T-2, Zearalenone:ZEN) for analysis, and constructed two biochip detecting techniques, i.e., an protein immunochip for simultaneous, rapid and high-throughput detection of six different mycotoxins (AFB1, AFM1, DON, OTA, T-2and ZEN), and a suspension array technology for simultaneously detecting four mycotoxins (AFB1, DON, T-2and ZEN) in corn and peanut. The research content of this paper is described in detail as follows:1. High-throughput detection of six mycotoxins (including AFB1, AFM1, DON, OTA, T-2and ZEN) using immunochip technology In our research, agarose-modified glass slides were chose as carrier. The spotting conditions were that the relative humidity was adjusted to22%-37%, and the temperature maintained at24-26℃. The probes were spotted onto the chips using the contact printing, so that the probes can be coupled to the surface of the chips with the function of both physical absorption and covalent binding.Subsequently, the working concentrations of both six mycotoxin complete antigens (Ags) and the corresponding monoclonal antibodies (Abs) were optimized. Results obtained from our assays were ultimately determined that the optimal concentrations of AFT-BSA, AFM1-BSA, DON-BSA, OTA-OVA, T-2-BSA and ZEN-BSA were0.20,0.10,2.00,2.00,0.20and1.00mg/mL, respectively, and those of AFT-Ab, AFM1-Ab, DON-Ab, OTA-Ab, T-2-Ab and ZEN-Ab were1.00,0.50,1.00,0.10,1.00and1.00μg/mL, respectively. In addition, the specificity among the six Ags and Abs was detected, and the result showed that apart from AFT and AFM1, other four kinds have high specificity; the cross-reactive phenomenon of AFT and AFM1is the reason that they belong to the same type of material, and after comparison with the two cross-reactivities (CRs), we decided that AFT-BSA and AFT-Ab were selected for quantifying AFB1, and AFM1-BSA and AFM1-Ab were chose for quantifying AFM1.And then, the calibration curves for simultaneously detecting the six mycotoxins using the immunochip were plotted. The result showed that:the competitive trend was evident, and the linear coefficients R2were more than0.9774, the linear detection range of AFB1, AFM1, DON, OTA, T-2and ZEN when simultaneously detected were0.04-1.69,0.45-3.90,20.20-69.23,35.68-363.18,0.11-1.81and0.08-7.47ng/mL, respectively, up to2-3orders of magnitude; the IC50were0.31±0.04,1.49±0.21,34.54±1.30,134.06±11.75,0.49±0.05and1.54±0.22ng/mL, respectively; intra-assay coefficient of variation (CV) was lower than15%. All the above results primarily proved that the immunochip method could be fast, accurate, precise and high-throughput quantitative detection of the six mycotoxins. In addition, the approach has another advantage of being visually semiquantitative with our naked eyes, through the scanned hybridization singals with different colors.Eventually, the drinking water was chose as matrix for the spiked experiment of the six mycotoxins. Results showed that the recoveries of each target were in the range of 80%-120%.and CV was also lower than15%.2.Simultaneous detection of four mycotoxins(including AFB1,DON,T-2and ZEN)in corn and peanut using suspension array technologyFirstly,the four Ags(including AFT-BSA,DON-BSA,T-2-BSA and ZEN-BSA)were covalently coupled with carboxylated microshperes using EDC method. After that, one-component suspension array method was analyzed,including the optimal working concentration of between each mycotoxin Abs and Sec-Ab by chessboard titration,and single-channel competitive standard curve plotted.Results showed that:R2were more than0.9953,the linear detection range of AFB1,AFM1,DON,T-2and ZEN were0.001-0.20,0.02-13.10,0.27-11.85,0.03-0.33,0.02-0.26ng/mL,respectively,with1-2orders of magnitude；IC50were0.02±0.002,0.48±0.065,2.21±0.294,0.12±0.003and0.06±0.002ng/mL,respectively;LOD were0.05,4.94,107.50,11.58and29.78pg/mL,respectively;CV was lower than15%.Secondly,multi-eomponent suspension array method was established,including optimal concentration of four mycotoxin Abs and Sec-Ab by chessboard titration simultaneously,test of the specificity among the four Ags and Abs,exploring the way of adding samples,and drawing out multi-channel competitive standard curves.Results demonstrated that:R2were more than0.9819,the linear detection range of AFB1,DON,T-2and ZEN were0.04-380.24,0.44-3251.17,0.01-10.91and0.05-84.76ng/mL,respectively,with3-4orders of magnitude, which was better than that of single-channel detection；LOD were0.22±0.03,32.91±3.16,0.79±0.11and11.29±1.15pg/mL,respectively；CV was lower than15%.Thirdly,the suspension array assay was applied in corn and peanut for simultaneously detecting the four mycotoxins,including exploration of the preprocessing method in the actual samples,and plotting the multi-channel competitive standard curves,when the four targets were simultaneously detected in samples. Results displayed that:after simple extraction using the methanol/water solution,R2were more than0.9804,the linear detection range of AFB1,DON,T-2and ZEN in corn were0.07-593.31,0.71-1525.29,0.01-9.56and0.02-55.35ng/g,respectively,and in peanut were0.04-252.65,0.16-2283.88,0.01-11.02and0.18-35.72ng/g,respectively,up to3-4orders of magnitude;LOD in corn were0.42±0.06,193.09±13.57,0.50±0.05and0.45±0.05pg/g,respectively,and in peanut were0.64±0.09, 3.73±0.36,0.32±0.04and76.97±10.53pg/g, respectively; CV was lower than15%.Finally, the comparative experiment between suspension array method and HPLC was tested, including the establishment of the four targets using HPLC in national standard of China, as well as the validation of the method accuracy and precision. Results recealed that: recoveries of the two approaches were85.11%-119.26%and80.16%-117.65%, both in the range of80%-120%; CV was lower than15%.To sum up, the two biochip methods (i.e., immunochip and suspension array technology) can not only rapidly, simply, simultaneously and quantitatively detect multiple targets, but also have higher sensitivity and better stability. Our study provides new research mentality and reference in order to determine small molecule residue in food safety testing applications using biochip technology, and laid the foundation for the research and development of rapid detetction kit in commercialization. |
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