Quantitative Detection Of Mycotoxins By Fluorescence Immunochromatography Based On Aggregation-induced Emission Microspheres

Mycotoxins are a class of secondary metabolites produced by fungi,which are ubiquitous in crops.Most mycotoxins are carcinogenic,teratogenic and mutagenic,which cause serious harm to human life and health.Therefore,the detection of mycotoxins is of great significance to ensure people’s life and health.Immunochromatography is widely used in the field of food safety detection because of its simple,portable,economical and efficient advantages,and it is a common method for rapid detection of mycotoxins.However,traditional colloidal gold immunochromatographic strips have poor sensitivity of detection,which cannot meet the increasingly stringent detection requirements of mycotoxins.The development of new labeled probes with higher signal intensity is one of the important strategies to improve the detection sensitivity of immunochromatographic assay（ICA）.Compared with colorimetric probes,fluorescent probes have higher signal output ability.However,the traditional fluorescent materials have the defect of aggregation caused quenching（ACQ）,which leads to the low signal intensity of the existing fluorescent probes and cannot meet the needs of high sensitivity detection.On the contrary,the photoluminescence of aggregation-induced emission（AIE）materials is significantly enhanced in the aggregate state and solid state,which provides an opportunity to solve the problem of traditional fluorescent ACQ.Based on this,this study carried out a series of studies on the preparation of AIE probe and its application in fluorescence immunochromatography,and on this basis,the feasibility and practicability of this method for the detection of mycotoxins in food samples are discussed as follows:（1）In this study,the AIE dye with orange-yellow emission was embedded in the polymer microspheres by the microemulsion method,and the AIE microspheres（AIEMs）with orange-yellow fluorescence were successfully prepared.Subsequently,this study used the AIEMs as fluorescent signal probes to construct a highly sensitive fluorescence immunochromatographic assay（AIEMs-ICA）for the detection of ochratoxin A（OTA）in corn samples.Under the optimal experimental conditions,the regression equation of the AIEMs-ICA established in this study for OTA detection was y=0.2055x^-0.467,R2=0.982.The half-maximal inhibitory concentration(IC₅₀)was as low as 0.149 ng m L^-1,and the limit of detection（LOD）was 0.042 ng m L^-1.The average recoveries of OTA in corn samples were 82.60%～113.14%,and the coefficient of variation（CV）was 1.26%～11.57%,which indicated that the AIEMs-ICA method had good precision and accuracy.In addition,the developed AIEMs-ICA did not cross-react significantly with other mycotoxins.In conclusion,the AIEMs-ICA method constructed in this study can be used for rapid screening detection of OTA and other mycotoxins in food samples.（2）Multiple immunochromatography can achieve the simultaneous detection of multiple targets,which not only improves detection efficiency and reduces the detection costs,but also enables more abundant sample information to be obtained.It is of great significance for the prevention and control of multiple mycotoxins contamination in food samples.Therefore,in this study,AIE microspheres with red,green and yellow fluorescence were used as probes of T1,T2 and C line of test strips,respectively,and a double immunochromatographic assay（DICA）was successfully established for simultaneous detection of aflatoxin B₁（AFB₁）and zearalenone（ZEN）in corn and wheat.This method used chicken Ig Y as an independent C line to achieve accurate quantitative detection of AFB₁ and ZEN by PLI_T/PLI_C ratio.Under the optimal detection conditions,the quantitative equation of AFB₁ and ZEN in corn matrix is as follows:y=-0.166ln（x）+0.0059（R2=0.9839）and y=-0.215ln（x）+0.1031（R2=0.9775）.Their LOD were 4.6 and 25 pg m L^-1,respectively.The quantitative equations in wheat matrix were y=-0.161ln（x）+0.0227（R2=0.9809）and y=-0.197ln（x）+0.117（R2=0.9845）.Their LOD were 4.3 and 19 pg m L^-1,respectively.The recovery rates of intra-assay and inter-assay were in the range of87.26%to 112.93%,indicating that the method has good precision and accuracy.These results indicate that the multicolor immunochromatographic strips established in this study can be used for simultaneous and accurate quantitative detection of a variety of mycotoxins.