| The applications of pesticides insured a significant grain production increase and have made outstanding contribution to the maximization of resource utilization rate all around the world. Even though the pesticides using played an important role in solving hunger problem,the soil and water pollution and the human health problems caused by the pesticides misuse should not be underestimated.Pollution from residual pesticides, especially organophosphorus pesticides, has attracted attention in recent years. Consequently, many techniques have been used for determination of pesticide residues. Although these detection methods have high sensitivity and accuracy, they suffer from complex sample pretreatment processes, portability limitations because of instrument size, lengthy, run times and high costs, the traditional detection methods are not suitable for rapid, on-site testing. Therefore, it is necessary to establish a rapid and simple technique for pesticide residue detection. In this study, the BELISA detection method was established by the combination of both molecular imprinting technique and ELISA technique.The key results are showed as follows:(1) The synthesis of organophosphorus pesticides general hapten and the preparation of enzyme conjugate. 4-(dimethoxyphosphorothioylamino) butanoic acid were synthesized by O,O-dimethyl phosphorochloridothioate and 4-aminobutyric acid in NaOH ice-bath condition,the product was obtained by diethyl ester washing and extraction under rotary evaporation.Enzyme conjugates were prepared by the connection of 4-(dimethoxyphosphorothioylamino)butanoic acid with horseradish peroxidase(HRP) by active ester method and glutaraldehyde method.(2) The preparation of hydrophilic molecularly imprinted film. The imprinted film that can selectively recognize trichlorfon and acephate was synthesized directly on the surface of a96-well plate using 4-(dimethoxyphosphorothioylamino) butanoic acid as the template molecule, acrylamide as the functional monomer and ethylene glycol dimethacrylate(EGDMA) as the cross-linker. The novel imprinted film was evaluated by scanning electron microscopy(SEM), thermogravimetric analysis(TGA), and the static and kinetic adsorptionexperiments. The results showed that this novel imprinted film had flat and smooth surfaces and high thermostability.(3) Optimization of the BELISA conditions and establishment of direct competitive BELISA procedure. The optimization and detection of trichlorfon and acephate were tested respectively. The enzyme conjugate was diluted to 1:5000 before use, 5% methanol PBS at pH 7.37 was chosen as the working solution for trichlorfon and 5% methanol PBS at pH 6.30 was chosen as the working solution for acephate. Under optimal conditions, standard curves were obtained using the imprinted film as the artificial antibody with trichlorfon and acephate standard solution concentrations between 60000 μg L-1 and 0.6 μg L-1 in 5% methanol PBS solution. From the standard curves, the sensitivity(IC50) of the biomimetic enzyme-linked immunosorbent assay(BELISA) method was 12.0 mg L-1 for trichlorfon, and 30.0 mg L-1 for acephate. The limit of detection(IC15) was 8.0 μg L-1 for trichlorfon and 12.0 μg L-1 for acephate. The developed method was applied to the determination of the trichlorfon and acephate in the spiked asparagus samples with recoveries ranging from 72.1 to 92.0% for trichlorfon and 70.0 to 85.0% for acephate. |
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