Study Of Chemiluminescence Immunoassay For Trace Detection Of Carbaryl

Chemiluminescence immunoassay is a microanalytical technique that combines high sensitivity chemiluminescence with highly specific immunoreactivity.Chemiluminescent enzyme immunoassay uses horseradish peroxidase to label antigen or antibody,and luminol reaction is added to the system to produce a luminescent reaction.Because of its strong specificity,high sensitivity,and trace detection,it is widely used in the detection of pesticide residues in the matrix.Carbaryl is a typical representative of carbamate pesticides.It has moderate toxicity and is prone to residue in the environment and agricultural production process,which is a serious threat to human health.Therefore,the rapid and efficient chemiluminescence immunoassay method for the development and establishment of carbaryl not only provides technical support for the rapid detection of carbamate pesticides,but also provides reference for other pesticide immunoassays.The main research contents of this paper are as follows:1.CarbarylchemiluminescenceHRP-Luminol-H₂O₂systemwasstudied.SelectingHRP-labeled hapten with specific adsorption,and further optimizing and comparing the sensitivity,stability and detection limit of luminescence system,a direct competitive chemiluminescence immunoassay method with high sensitivity and specificity of carbaryl residues was established.Optimize RLUmax/IC₅₀0 values by square matrix method to determine the optimal working concentration of direct competition CLEIA antibody and enzyme-labeled hapten.The results showed that in the HRP-Luminol-H₂O₂ system,the optimal antibody coating concentration of carbaryl was 5?g/L,and the optimal enzyme hapten reaction concentration was 2.5?g/L.The concentration of carbaryl and its inhibition rate were fitted.The regression equation was y=8.1149 ln?x?+39.883,R²=0.9703,IC₅₀0 was 3.48?g/L,detection limit was 0.25?g/L,linear range was 0.25?g/L³10.3?g/L.Using the established CLEIA analysis method,the recovery test?0.05 mg/kg,0.1mg/kg,0.2 mg/kg?was carried out in three substrates of tomato,apple and citrus.The results showed that the average recovery rate?n=5?was 77.0%¹19.8%,relative standard deviation?RSDs?was 10.7%¹8.6%,which proved that the method can meet the technical requirements of carbaryl detection.2.Indirect competitive chemiluminescence immunoassay for the establishment of carbaryl.Chess titration was used to optimize and compare the dilution ratios of OVA-hapten concentration,antibody and enzyme-labeled secondary antibody to obtain the optimal HRP-catalyzed luminol luminescence system.The results showed that the OVA-hapten coating concentration was 1.0?g/L,the antibody was diluted 32000 times,and the enzyme-labeled secondary antibody was diluted 3000 times.The fitting regression equation was y=8.5552 ln?x?+40.912,R²=0.9796,IC₅₀0 was 2.89?g/L,the detection limit was 0.23?g/L,and the linear range was 0.23?g/L to 481.0?g/L.Addition recovery test?0.05 mg/kg,0.1 mg/kg,0.2 mg/kg?was carried out in tomato,apple and citrus.The average recovery?n=5?was 78.0%¹14.1%,relative standard deviation?RSDs?ranged from 10.1%to 11.8%.Compared with the direct competitive immunoassay method of the first part,the established indirect competitive chemiluminescence immunoassay method has the advantages of high sensitivity and wide linear range,which can better meet the requirements of the detection of the pesticide residue of the carbaryl.3.To verify the accuracy and reproducibility of the established CLEIA method,the QuEChERS-GC-MS/MS method was used to add recovery tests for tomato,apple and citrus samples.The results showed that the recovery of GC-MS/MS analysis method was80.0%¹07.0%,and the relative standard deviation?RSDs?was 3.0%⁷.6%.Compared with this method,the CLEIA method established in this study is accurate.Although the repeatability of CLEIA is weaker than the GC-MS/MS method,it is low in cost,easy to operate,and suitable for rapid detection on site.