Rapid Detection Methods Of Organophosphorus Pesticides Residues Based On Broad-specificity Antibodies Recognition

As broad-spectrum and highly effective insecticide,Organophosphorus(OPs)have been frequently used to ensure the quality of agricultural products.However,due to their wide variety and illegal abuse,OPs have posed great threats to food safety,human health and environmental protection,and thus have faced severe challenges in the detection of their residues.The detection method of large instrument is complicated in operation and requires high specialization,which is difficult to meet the daily monitoring needs of OPs.Most of the rapid detection methods are convenient to carry and easy to operate,which is conducive to popularization and application.Among them,immunoassay has become one of the mainstream rapid detection methods because of its high efficiency and stability.Therefore,this paper used broad-specificity antibody of organophosphorus pesticides(Abs)as the core identification element to explore three rapid detection methods with different principles,which could meet different OPs rapid detection requirements.The main research contents were as follows:(1)In order to better meet the requirements of OPs rapid preliminary screening detection under laboratory conditions,response surface methodology was used to optimize the working conditions of the indirect competitive ELISA assay.Firstly,the optimal working concentration combination of organophosphorus pesticide general structural antigen and Abs was studied.Then,on the basis of single factor test,the response surface optimization design experiment was carried out.The modified optimum operating conditions(ion concentration of 0.04 M,p H value of 7.89,methanol concentration of 2.9%)were used to test the standard.It showed a stable and reproducible detection result,with a sensitivity of 1.28 ng/m L.The detection sensitivity of the Abs to different OPs had little difference,and the detection sensitivity of parathion,phosphomebium,queathion and methyl parathion were 0.72,3.39,4.62 and 4.88 ng/m L,respectively.So,it could be applied to the preliminary screening and detection of a large number of samples in the laboratory.(2)In order to meet the need of rapid trace detection of OPs,a novel immunoelectrochemical sensor was prepared with Prussian-Blue(PB)as conductive substrate.In order to strengthen the effective binding between antibody and PB,the gold labeled probe was prepared by coupling Abs with Au NPs.In order to simplify the process of sensor preparation,the electrodeposition composite film was prepared on the electrode surface by one-step electrodeposition method in the mixed solution of gold labeled probe and PB.Under the optimal experimental conditions,the concentration of OPs showed a good linear relationship with the corresponding electrochemical response value(R~2=0.9918),and the detection limit was 0.003 ng/m L.The recoveries of actual samples were 98.05%～102.05%.Therefore,when the concentration of the target substance in the sample was low,the sensor could be used for detection.(3)In order to reduce the professional threshold of OPs detection and further improve the detection efficiency,a time-resolved fluorescent paper-based sensor was prepared by fluorescence labeling the Sheep anti-mouse antibody(Sa MIg G).An indirect time-resolved fluorescent probe was fabricated by connecting Abs with europium oxide latex microspheres via Sa MIg G.This strategy could effectively amplify the signal and save the amount of target antibody.After the detection,the fluorescence intensity at the detection line could be monitored qualitatively with the help of the ultraviolet light source with the wavelength of 365 nm.The screened positive sample images could be analyzed by the software Image J to complete the quantitative detection.The construction and working conditions of the sensor were optimized,and the detection limit could reach 0.53 ng/m L.The sensor could be used stably in the test of vegetable sample matrix.The sensor had the advantages of low production cost,easy portability,simple operation,and could be used by non-professionals or a large number of samples for rapid detection.The above studies provided a variety of detection schemes for OPs detection,which could basically meet the rapid detection needs of different populations and different scenarios.