Study On Fluorescence Immunochromatographic Detection Of Multi-target Mycotoxins By Using Europium Nanoparticles

Mycotoxins are a class of natural toxic metabolites or secondary metabolites produced by mold.Affected by natural factors,mycotoxins are widely present in agricultural products and feed.Even if the concentration is very low,they can still enter the human body through eating and cause great harm to the human body.Both citrinin and zearalenone are one of the mycotoxins and may coexist in a single agricultural product,posing a greater potential threat.Therefore,the EU and China and other countries have clearly stipulated the legal limit values of these two mycotoxins.In this paper,based on the establishment of multi-target colloidal gold immunochromatographic detection method and the replacement of antibody labeling materials,the multi-target fluorescence immunochromatographic method of using europium was developed for simultaneous detection of citrinin and zearalene.The results obtained are as follows:1.To synthesize zearalenone artificial antigen by carbodiimide method,it was necessary to first prepare zearalenone hapten,namely ZEN-CMO.The ZEN-CMO was used as the starting material to couple carrier protein BSA/HSA to form the immunogen ZEN-HSA and the coated proto-ZEN-BSA,respectively.Due to the carboxyl group of citrinin penicillin's chemical structure is functional group,which cannot be destroyed.Therefore,the ccitrinin artificial antigen was synthesized by formaldehyde addition method.Using CIT as the starting material,the carrier protein BSA/HSA was subjected to CIT to synthesize the immunogen CIT-HSA and the coated original CIT-BSA,respectively.2.The synthetic immunogens ZEN-HSA and CIT-HSA were used to immunize Babl/c mice by conventional animal immunoassay protocols.In terms of both potency and sensitivity,Z-1 mice and C-2 mice were selected for impact immunization for subsequent cell fusion.After screening by subcloning technology,three hybridoma cell lines capable of secreting stable antibodies against ZEN and four hybridoma cell lines capable of secreting stable antibodies against CIT were selected to produce mouse ascites,and purified by caprylic acid-ammonium sulfate method to obtain specific antibodies.The purified antibodies were identified,and antibodies 8B3 and 4B9 with the best potency and sensitivity were selected from the corresponding antibodies of ZEN and CIT hybridoma cell lines.3.Using the obtained ZEN and CIT specific monoclonal antibodies,a multi-target colloidal gold immunochromatographic detection technique was prepared.The results of simultaneous detection of ZEN and CIT by this method:the visual detection limit of ZEN was 50 ng/m L,the IC₅₀value was 9.05 ng/m L,and the instrument detection limit was 1.66 ng/m L;the visual detection limit of CIT was 25ng/m L,The IC₅₀value was 4.29 ng/m L,and the instrument detection limit was 0.72ng/m L.In the confirmation experiment of the specific detection of the test strip,seven mycotoxins were verified.Except for the two mycotoxins,ZEN and CIT,there was no obvious cross-reaction with the other five mycotoxins.colloidal gold immunochromatographic detection technique can simultaneously and sensitively detection of ZEN and CIT.4.Based on the preparation of multi-target colloidal gold immunochromatographicteststrips,themulti-targetfluorescent immunochromatographic test strips was prepared by replacing the labeling material colloidal gold with europium nanoparticles with better characteristics.Under the best experimental conditions,the visual detection limit of ZEN is 5 ng/m L,the IC₅₀value was 0.755 ng/m L,and the instrument detection limit was 0.078 ng/m L.The visual detection limit of CIT was 2.5 ng/m L,and the IC₅₀value was 0.353 ng/m L,the detection limit of the instrument was 0.035 ng/m L.The test strip was verified by specific cross-reaction,which could simultaneously detect ZEN and CIT without cross-reaction to other mycotoxins.After verifying the addition of recovery experiments,within the Intra-assay of the spiked concentration,the average recovery of CIT was 86.3%to 109.0%,and the RSD was less than 7.6%.In comparison,the average recovery of ZEN was 88.7%to 114.4%,and the RSD was less than 4.2%.For Inter-assay,the average recovery of CIT was 96.5%to 111.6%,and the RSD was less than 9.9%.In comparison,the average recovery of ZEN was 86.6%to 110.9%,and the RSD was less than 6.1%,which indicates that the multi-target fluorescent immunochromatographic test strip established in this study could accurately simultaneously detect ZEN and CIT simultaneously.In this paper,the hybridoma cell technology was used to prepare two specific monoclonal antibodies against ZEN and CIT.Multi-target colloidal gold immunochromatographic detection technology and multi-target fluorescent immunochromatographic detection technology were established for ZEN and CIT.The simultaneous detection of two mycotoxins provides more sensitive,accurate and reliable method.