Based On MnO₂ Nanosheets-mediated Fluorescence And Colorimetric Immunoassay For Detection Of Zearalenone

Zearalenone（ZEN）is a secondary metabolite produced by Fusarium,which is commonly found in cereals and their derivatives.It can cause acute and chronic poisoning of animals,lead to abnormal reproductive function and even death,and cause economic losses to livestock farms.Due to the widespread presence of ZEN and its harmful effects,there is an urgent need to develop a simple and sensitive method to detect ZEN.In this paper,three immunoassays were constructed for the highly sensitive detection of ZEN in cereals and based on the specific reaction between antigen and antibody.The main research elements are as follows:1.Development of an indirect competitive ELISA（ic-ELISA）for detection of ZENAn indirect competitive ELISA method for the detection of ZEN was constructed using BSA-ZEN,Mc Ab,and Ig G-HRP.The method has a minimum detection limit of0.22 ng/mL for ZEN and a detection range of 0.24～0.66 ng/m L.The recoveries of the corn samples ranged from 95.95%～103.91%in accordance with the American Association of Analytical Chemists（AOAC）,demonstrating the accuracy and reliability of the method.2.Establishment of a fluorescent immunoassay based on MnO₂ nanosheets triggering ox VB1 luminescenceTo further improve the sensitivity of the detection method,a FLISA method based on MnO₂ nanosheets triggering ox VB1 luminescence was constructed for the rapid and sensitive detection of ZEN in cereals by combining MnO₂ nanosheets with a cheap and easily available fluorescent substrate VB1.The established FLISA method has a low minimum detection limit of 15.5 pg/m L for ZEN and a linear range of 100～700 pg/m L.The method is nearly 14 times more sensitive compared to conventional ic-ELISA.The recoveries in the actual sample spiked recovery experiments ranged from 94.24%to108.26%in accordance with the AOAC regulations.3.Establishment of a dual-mode immunoassay based on MnO₂ nanosheets and7-hydroxycoumarinTo solve the problem of unstable single fluorescent signal,a colorimetric and fluorescence dual-mode immunoassay of MnO₂ nanosheets and 7-hydroxycoumarin was constructed based on the internal filtration effect（IFE）and static burst effect（SQE）as well as the colorimetric effect of MnO₂ nanosheets.The minimum detection limit of this assay was 0.103 ng/m L for colorimetric detection of ZEN and 0.097 ng/m L for fluorometric detection of ZEN.The dual-mode immunoassay showed good linearity with ZEN concentrations of 1～10 ng/m L,while the recoveries in the actual sample spiked recovery experiments ranged from 96.65%to 139.38%in accordance with the AOAC regulations.The dual-mode immunoassay has extensive detection adaptability and the accuracy of mutual calibration of detection results.The three immunoassay methods constructed in this study can be used for the sensitive detection of ZEN in corn samples,and all meet the requirements of the maximum allowable concentration of ZEN in the national standard.