Study On Bio-barcode Fluorescence Immunoassay Of Detection Of Imidacloprid

In this paper,a polyclonal antibody against imidacloprid was prepared,and ELISA and bio-barcode immunoassay methods based on this polyclonal antibody were established to achieve the quantitative detection of imidacloprid in agricultural products.An imidacloprid hapten was prepared by using imidacloprid andβ-mercaptopropionic acid as raw materials.The prepared hapten was coupled with ovalbumin（OVA）to prepare coating antigen and coupled with bovine serum albumin（BSA）to prepare immunogen respectively by activated ester method.New Zealand white rabbits were immunized with immunogen,and polyclonal antibodies（p Abs）with the best performance were selected for subsequent experiments.This ELISA was established with the prepared antibody.The optimal amount of coating antigen is 0.01μg/well,the optimal dilution of antibody is 1﹕8000,and the PBS（p H=7.4）is selected as dilution solution.The assay sensitivity（IC50）is2.88μg/L,and the limit of detection(LOD,IC_l5)is 0.23μg/L.A bio-barcode fluorescence immunoassay was developed based on the prepared polyclonal antibody above.Firstly,colloidal gold solution（Au NPs）with uniform particle size and good dispersion was synthesized by trisodium citrate reduction method.Polyclonal antibodies were conjugated with Au NPs by electrostatic adsorption,and thiolated oligonucleotide chains（SH-ss DNA-6-FAM）were conjugated with Au NPs by freezing-thaw method to prepare fluorescent immunoprobes.The preparation conditions of the immunoprobe（p H value,PEG content,freezing time,DTT concentration,unchain time）and the working parameters of the immunoprobe（molar ratio of ss DNA﹕Au NPs,amount of Abs,coating amount and dilution ratio of immunoprobes）were optimized.The sensitivity(IC₅₀),LOD(IC_l5)and linear range(IC₂₀-IC₈₀)of the method are 1.44μg/L,0.016μg/L and 0.071-15.13μg/L,respectively.The LOD of bio-barcode fluorescence immunoassay established in this paper is 14times lower than that of ELISA using the same antibody.Moreover,the bio-barcode fluorescence immunoassay established in this paper can effectively reduce the cross-reaction of structural analogues of imidacloprid,which is in good agreement with the results of high performance liquid chromatography（HPLC）.The bio-barcode fluorescence immunoassay provides a new strategy for the detection of imidacloprid.