Novel Immunoassays For Mycotoxins Based On Magnetic Separation Technology

Mycotoxin contamination in agricultural environment was a serious threat to food quality and consumer life and health.With the continuous improvement of detection standards for mycotoxins,as well as the severe current situation of low concentration contamination and simultaneous occurrence of multiple mycotoxins in agricultural environmental samples,it was of great practical significance to research and develop ultra-high sensitivity,more efficient immunoassay technology that can simultaneously detect multiple mycotoxins.In this study,zearalenone（ZEN）and aflatoxin B₁（AFB₁）were used as the research objects,and three novel immunoassays were established by using magnetic separation and multiple signal amplification strategies,which were applied to the assessment of two mycotoxins in real agricultural environment and agricultural by-products.The development of this study was of great reference value for the ultra-high sensitivity,simplicity,high efficiency and rapid simultaneous immune analysis of ZEN and AFB₁.This thesis mainly included the following three aspects:1.Research of ZEN immunoassay based on BAS signal amplification and magnetic separationWhen the magnetic probe and ZEN and ZEN monoclonal antibody competed for immune reaction,the signal was amplified by the biotin-streptavidin system（BAS）to establish ZEN BAS magnetic immunoassay method（BAS-MBI）.The limit of detection（LOD）of this BAS-MBI for ZEN was 0.098 ng m L^-1,the IC₅₀was 0.71 ng m L^-1,and the detection range was 0.098-5.16 ng m L^-1.The cross-reaction rates with structural analogs were all lower than 12.0%.This method was used to detect 405samples of agricultural and its by-products in the Chinese market.The positive levels of ZEN were between 1.1-1100.0 ng g^-1.More than 20%of the positive samples exceeded the maximum level of contamination,showing a high detection rate and a high level of contamination.The results showed that the method could be applied to the investigation and evaluation of ZEN in real samples sensitively,rapidly.2.Research of AFB₁ultra-sensitive immuno-PCR assay based on bio-barcode and magnetic separationAFB₁monoclonal antibody,captured DNA and barcode DNA were successively modified on 20 nm colloidal gold surface to prepare colloidal gold composite probe.AFB₁competitive antigen and magnetic bead were coupled to prepare magnetic probe.After competitive immune reaction and magnetic separation,the bio-barcode was disintegrated with dithiothritol（DTT）and detected by real-time quantitative PCR.The LOD of this method was 0.01 pg m L^-1（480 times lower than ELISA）,the IC₅₀was 0.072 pg m L^-1,and the detection range was 0.01 to 100 pg m L^-1.At the same time,this method showed good specificity,accuracy and precision.The established AFB₁bio-barcode immuno-PCR assay could achieve ultra-high sensitivity detection at pg m L^-1level,and the detection efficiency was significantly improved through the magnetic separation.3.Research of simultaneous fluorescence immunoassay for ZEN and AFB₁based on oligonucleotide and magnetic separationTwo magnetic probes were prepared by conjugating ZEN and AFB₁competitive antigens with magnetic beads,respectively.Two fluorescent probes were prepared by conjugating oligonucleotide modified cyanine fluorescein Cy5（Cy5）,6-carboxyl fluorescein（6-FAM）and corresponding monoclonal antibodies with colloidal gold,respectively.After immune reaction,free Cy5 and 6-FAM were obtained by DTT dissociation,and the fluorescence intensity of the two toxins was detected simultaneously to realize the simultaneous detection of the two toxins.LOD values of ZEN and AFB₁were 0.378 pg m L^-1and 0.043 pg m L^-1（529 and112 times lower than ELISA）,IC₅₀values were 30.7 pg m L^-1and 26.2 pg m L^-1,respectively.The detection ranges were 0.654-1438.8 and 0.215-3190.1 pg m L^-1,respectively.The specificity and accuracy of the method were evaluated well.The results showed that the fluorescence magnetic immunoassay based on oligonucleotides could realize the simultaneous detection of ZEN and AFB₁at the pg m L^-1level,which had a good reference value for the simultaneous analysis of multi-component substances.