Study On Immunoassay Methods Of Three Typical Mycotoxins In The Agricultural Environment

Mycotoxins,as secondary toxic metabolites of fungi,have been found to cause serious pollution to many agricultural products and foods.Besides,mycotoxins also pollute the water environment mainly through the drainage and runoff discharged into the water environment of infected farmland and by human or animal feces or urine discharged into the environment after eating contaminated agricultural products.The pollution poses a serious threat to the agricultural environment and human health and there is an urgent need to develop efficient and accurate detection methods to detect mycotoxins in the agricultural environment.The immunoassay method can meet the above-mentioned needs,and has become a research hotspot in the rapid analysis of mycotoxins due to its simplicity,rapidity,high throughput and high sensitivity.At present,there are many reports on the detection methods of mycotoxins in agricultural products,but the rapid detection method of mycotoxins in the water environment has not yet been reported.In this study,three typical mycotoxins,deoxynivalenol（DON）,zearalenone（ZEN）and fumonisin B₁（FB₁）,were used as research objects,and three immunoassay methods combined with nanomaterials were constructed,and applied to the analysis and determination of agricultural environmental samples.（1）In this study,a new type of enzyme-labeled secondary antibody complex（HRP/Ab@ZIF-L）was prepared,namely horseradish peroxidation（HRP）,goat anti-mouse secondary antibody（Ab）and metal organic framework（ZIF-L）are fused in situ and applied to the ic-ELISA method to achieve high-sensitivity and rapid determination of mycotoxins ZEN in agricultural environments.Under optimal conditions,the limit of detection（LOD）of the proposed method reaches 0.5 ng/L,which is 126 times lower than that based on traditional enzyme-labeled secondary antibodies（HRP-Ab）.In addition,the proposed method shows excellent selectivity,and has a good linear detection of ZEN in the range of 0.5-0.476μg/L.The recovery rate of ZEN in spiked corn and wheat samples ranged from 84.50%to 96.70%,and the relative standard deviation was below 8.9%,indicating that the method has good accuracy and precision.（2）In this study,a method based on colloidal gold lateral flow immunoassay（LFIA）test strips was established for the simultaneous quantitative detection of DON and FB₁ in the agricultural environment.Under optimized conditions,LODs of DON and FB₁ after photographing and computer analysis were calculated to be 3.46 ng/m L and 2.65 ng/m L,respectively,which were compared to the LODs（DON:100 ng/m L;FB₁:25 ng/m L）with the naked eyes,the sensitivity is improved by about 29 times and10 times,respectively.Through the analysis of spiked water samples and agricultural products samples,the recovery rate reached 80.5%-114.3%and the relative standard deviation was below 9.75%,indicating that the method has good accuracy and precision.（3）In this study,an enhanced LFIA method based on gold core platinum shell structure（Au@Pt）nanoenzymes was established combined with smart phones and self-written Android applications（App）to make simultaneous detection for DON and ZEN more convenient,accurate and highly sensitive in the agricultural environment and realize the detection on site.The detection method uses the peroxidase mimic enzyme activity of Au@Pt and uses AEC as the color enhancement agent.After optimized conditions,the LODs of DON and ZEN are 2 ng/m L and 0.2 ng/m L,respectively.The LODs are 0.24 ng/m L and 0.04 ng/m L after After mobile phone quantification,respectively.At the same time,we also studied the two LFIA methods based on the colloidal gold and Au@Pt for simultaneous detection of DON and ZEN.In the former method,the LOD of DON and ZEN with the naked eyes were 100 ng/m L and 6 ng/m L,respectively;the LODs after quantification were 2.3 ng/m L and 0.3 ng/m L,respectively.The LODs of the latter method were separately 20 ng/m L and 3 ng/m L observed by naked eyes,and the LODs were 0.4 ng/m L and 0.13 ng/m L after quantification.Obviously,the newly constructed enhanced LFIA method has greatly improved sensitivity compared to that based on colloidal gold and Au@Pt itself.