Study On Fluorescent And Colorimetric Immunoassay Based On Cerium Ions Triggering For Detection Of Ochratoxin A

Ochratoxin A（OTA）is a secondary metabolite produced by Aspergillus species and Penicillium species.It exists ubiquitously in agricultural products and daily food items and has a variety of toxicological effects.It has been categorized as a Group 2B carcinogen for humans by the International Agency for Research on Cancer（IARC）.Therefore,the European Union（EU）and China has been specified the minimum detection concentrations for OTA as low as 2-10 ng/m L in various products.Strict control of OTA detection levels in food and the construction of novel sensitive detection methods are essential to ensure human health and safety.In this study,three immunoassays were developed based on enzyme-linked immunoassay（ELISA）,cerium ion as triggering,and combining with high sensitivity of fluorescence analysis and visualization of colorimetric analysis in optical sensing.The specific contents are as follows:1.An indirect competitive enzyme-linked immunoassay（ic-ELISA）method was established to detect OTA.The linear regression equation was y=50781 x+9.11,correlation coefficient（R²）=0.998,and the linear range(IC₂₀-IC₈₀)was 1.2～36.2 ng/m L.The lowest detection limit(LOD,IC₁₀)was 0.95 ng/m L,which meets the requirement of the minimum detection limit of OTA.The method has good specificity without cross reaction with other mycotoxins.The recoveries of corns,rice and oats were 100.78%～111.45%,95.57%～104.75%,94.09%～113.74%,respectively,which were in accordance with the American Association of Analytical Chemists（AOAC）.2.A dual-readout immunoassay was developed based on fluorescence signal enhanced by aggregation induced emission（AIE）effect of Ce³⁺triggered Au nanoclusters（Au NCs）and colorimetric signal generated by Tetramethylbenzidine（TMB）oxidation of Ce⁴⁺.By designing the reaction conditions at room temperature,the fluorescence method can obtain higher fluorescence signal output based on AIE effect,and the colorimetric method can realize field detection based on smartphone reading RGB values.The dual-readout modes were applicable to both laboratory environment and field detection environment,and the fluorescence and colorimetric signals were applicable to different sample substrates,and can also be mutually verified by the two signal output modes,improve the applicability of the method.The linear regression equation of fluorescence method is y=2.199 x+8.648,R²=0.995.The LOD was 0.62 ng/m L,and the linear range was 2.89～34.72 ng/m L.The linear regression equation of colorimetric method is y=0.154 x-1.239,R²=0.99.The LOD was 0.81 ng/m L,and the linear range was 10～45ng/m L.Two assays both meets the requirement of the minimum detection limit of OTA.Dual-readout immunoassay showed good specificity,without cross-reaction with other mycotoxins,and the results were consistent with commercial ELISA kit.The recoveries of fluorescence method and colorimetric method were 94.4%-107.8%and 93.7%-106.9%respectively,which were in accordance with AOAC and it has good correlation with commercial ELISA kit.3.An in-situ ratiometric fluorescent immunoassay was developed for detection of OTA based on Ce⁴⁺oxidation and DHAA catalysis,while competitively reacting O-Phenylenediamine（OPD）.The in-situ ratiometric fluorescent immunoassay provided a control signal similar to the C-line of lateral flow immunoassay.The in-situ synthesis reduces the fluorescent background interference of the reporter,which has intrinsic correction ability without blank control to avoid false positives in ELISA.The linear regression equation is y=22.494-1.156 x,R²=0.982.The linear range was 4～16 ng/m L,and the LOD was 1.35 ng/m L,which meets the requirement of the minimum detection limit of OTA.The method has good specificity without cross-react of other mycotoxins.The recoveries of real samples were 91.65%～105.44%,which was in accordance with AOAC.