Establishment Of Immunoassay For Zearalenone Based On TMB Mediated Etching Of Au NBPs

Zearalenone（ZEN）is a non-steroidal secondary metabolite mainly produced by Fusarium fungi,it is often found in cereal crops contaminated by Fusarium molds,and may be contaminated in the links of grain harvest,storage,transportation,processing and sale.ZEN,with estrogen-like activity,has reproductive toxicity,genetic toxicity,cytotoxicity and immunotoxicity,which can cause damage to the human reproductive system,immune system,liver and kidney and other major metabolic organs.Therefore,it is of great practical significance to establish a sensitive,fast,efficient and low-cost ZEN detection method to control ZEN contamination and ensure food safety.The detection method for ZEN is the traditional instrumental analysis methods that high performance liquid chromatography（HPLC）,liquid chromatogram-tandem mass spectrometry（LC-MS）and thin layer chromatography（TLC）,and immunological analysis methods.Traditional instrumental analysis has the characteristics of accuracy,reliability and good repeatability of analysis results,but it also has the shortcomings of complex sample pretreatment,tedious operation,long detection time and high cost,which cannot be applied to the rapid screening of large-scale samples in the field.Immunoassay has gradually become the main method for large-scale sample screening in the field because of its rapid efficiency,simple operation and low cost.Three immunoassays for ZEN were established using ZEN monoclonal antibodies（McAb）,gold nanopyramid（Au NBPs）and manganese dioxide nanosheets（MnO₂NS）.1.An indirect competitive ELISA（ic-ELISA）method for ZEN.The BSA-ZEN coated in the microplate and ZEN in the sample competitively bind with McAb After washing,the BSA-ZEN～McAb～Ab₂-HRP ternary immune complex was formed.Under the catalysis of HRP,H₂O₂ reacted with TMB to form oxidized TMB with blue color.After the sulfuric acid was added to stop the reaction,the system showed yellow color.The absorption peak at 450 nm was obvious,and the analysis results were obtained by reading OD₄₅₀ with the multifunctional microplate reader.The calibration equation was Y=6.56X-0.139,R²=0.9864.The linear range(IC₁₅-IC₈₅)was between0.04 ng/mL and 0.15 ng/mL,and the detection limit(IC₁₀)was 0.036 ng/mL.The average recovery of corn meal samples was between 97%and 116%.2.Multicolor immunosensor based on ALP and MnO₂NS-TMB mediated Au NBPs etching.Based on ic-ELISA system,TMB was catalyzed by MnO₂NS to TMB⁺,and it was converted into TMB²⁺under acidic condition.With the participation of CTAB,Au NBPs was etched by TMB²⁺,which results in the change of the geometric shape of Au NBPs and the blue shift of the local surface plasmon resonance（LSPR）absorption peak of Au NBPs.The dephosphorylation of ascorbate phosphate（AAP）can be catalyzed by Ab₂-ALP to produce ascorbic acid（AA）,which can degrade MnO₂ NS,inhibit the oxidation of TMB,and reduce the LSPR absorption peak offset（Δλ）of Au NBPs.The concentration of ZEN is proportional to theΔλvalue of Au NBPs.After the optimization of detection conditions,the standard curve was obtained,and the calibration equation was Y=137.486X-9.153（Y=Δλ,R²=0.9785）.In the range of0.1～2 ng/mL,the linear relationship was good,the detection limit was 0.01 ng/mL,when ZEN concentration was 0.1 ng/mL,the naked eyes can still distinguish the significant change of solution color.The average recovery of corn meal samples was between 109.9%and 125.2%.3.Multicolor immunosensor based on HRP-TMB mediated Au NBPs etching.Based on the ic-ELISA system,the reaction of H₂O₂ and TMB were catalyzed by HRP to produce TMB²⁺.In the presence of CTAB,Au NBPs was etched by TMB²⁺,which changes the shape and size of Au NBPs,and causes the LSPR absorption peak of Au NBPs to shift.It also shows visible color changes.Quantitative analysis of ZEN was performed by reading the analysis results with multifunctional microplate reader,and semi-quantitative analysis of ZEN was performed by reading the results with naked eyes.The calibration equation of the standard curve was as follows:Y=6.16-139.50X(Y=Δλ,X=lg C_ZEN,R²=0.9799).It has a good linear relationship in the range of 0.02～0.8 ng/mL,and the detection limit is 0.01 ng/mL.At a concentration of 0.02 ng/mL,a significant change in the color of the solution was still discernable with the naked eyes.The average recovery of corn meal samples was between 89.50%and 106.50%.The three immunoassays for ZEN established in this study have the characteristics of rapid and high sensitivity.The ic-ELISA method has the advantages of mature technology,simple operation and low cost.The multicolor immunosensor based on ALP and MnO₂NS-TMB mediated Au NBPs etching and multicolor immunosensor based on HRP-TMB mediated Au NBPs etching have the advantages of higher sensitivity,more remarkable detection signal and naked eyes semi-quantitative detection than the ic-ELISA.All the three detection methods can meet the requirements of the residue limit of ZEN in grains,and the average detection recovery rate can meet the standards of AOAC,which has a certain potential for popularization and application.