Research On Magnetic Beads Synchronous Enrichment And Purification And Immunoassay Of Mycotoxins In Agricultural Products

Mycotoxins are small molecule secondary metabolites produced by fungi such as Aspergillus and Fusarium.There are a wide variety of mycotoxins.Currently,there are more than 400 known mycotoxins,of which more than 30 are harmful to human life and health,including teratogenic,carcinogenic and mutagenic.Mycotoxin contamination may occur in the production,storage,transportation and processing of crops,covering a wide range.It is found that some fungi can produce multiple mycotoxins at the same time,and it is common to contaminate a variety of fungi and their toxins in agricultural products and food.Once entering human body,synergistic or combined toxin effects will occur.Therefore,rapid and highly sensitive simultaneous mycotoxin detection technology is urgently needed to detect contamination in time and avoid harm.However,the existing mycotoxin detection technology is affected by the substrate of agricultural products and has poor stability,and the synchronous extraction and enrichment technology of various mycotoxins is lacking.Therefore,it is urgent to develop efficient sample pretreatment method and stable and highly sensitive synchronous detection technology of mycotoxin,which are hot issues in the field of mycotoxin detection at present.In this study,aflatoxin（AFB₁）and zearalenone（ZEN）were taken as examples to explore the simultaneous extraction technology of multiple mycotoxins by immunomagnetic beads enrichment and purification,and to create the simultaneous detection technology of AFB₁and ZEN time-resolved fluorescence immunochromatography,providing technical support for the supervision of mixed contamination of multiple mycotoxins in agricultural products.The main contents of this study are as follows:（1）The simultaneous extraction technology of aflatoxin B₁and zearalenone in agricultural products by immunomagnetic beads enrichment and purification was established to provide a feasible pre-treatment method for the establishment of simultaneous rapid detection methods for a variety of mycotoxins in agricultural products.Using two monoclonal antibodies against AFB₁and ZEN as specific targeting elements,immunomagnetic beads for simultaneous enrichment and purification of AFB₁and ZEN were developed.The extraction reagent type,loading buffer condition,amount of immunomagnetic beads,affinity adsorption reaction time and elution volume of samples were optimized.High performance liquid chromatography（HPLC）was used to verify the recoveries of AFB₁and ZEN pretreatment methods.The recoveries of AFB₁and ZEN in blank matrix ranged from 73.49%to 105.60%,with relative standard deviations（RSD）less than 4.5%.The results showed that the established synchronous pretreatment method had good recovery and repeatability,good purification effect,simple operation and high recovery,and could be used for simultaneous enrichment and purification of AFB₁and ZEN mycotoxins in agricultural products.（2）The simultaneous detection technology of AFB₁and ZEN time-resolved fluorescence immunochromatography was established to provide technical support for the supervision of mixed contamination of multiple mycotoxins in agricultural products.Immunofluorescence probes were prepared by coupling two monoclonal antibodies against AFB₁and ZEN with carboxylated europium fluorescent microspheres,respectively.Time-resolved fluorescence immunoassay strips for simultaneous detection of AFB₁and ZEN were established by optimizing the type of sustained-release solution,antibody labeling amount,immunoprobe dosage and concentration of coated antigen.The limits of detection for AFB₁and ZEN were 0.027μg/kg and 0.050μg/kg,respectively.The recoveries of corn and peanut were 87.27%to 100.13%,and the relative standard deviations（RSD）were less than 3.7%.This method was compared with HPLC method to detect the contents of AFB₁and ZEN in actual samples,and the results showed that the two methods had similar detection results.The results showed that this method has the characteristics of good stability,high sensitivity,fast and convenient,and can meet the needs of rapid detection of a variety of mycotoxins in agricultural products.