Study On A Magnetic Particles-Chemiluminescence Immunoassay For Multi-Residue Detection Of Dimethoxy Organophosphorus Pesticides

Organophosphorus pesticides（OPs）are the most widely used insecticides in the world,posing serious environmental problems due to their structures dimethoxy and diethoxy.There was a survey for nearly 20 shops in Wuhan pesticide market which found that Trichlorfon,Dichlorvos,Methidathion,Malathion,Phenthoate,Fenitrothion,Fenthio-n,Dimethoate,Omethoate and Chlorpyrifos-methyl with the structure of dimethoxy are currently used.Increasing public concern over pesticide contamination of food and environment has urged the demands for broader and stricter pesticide monitoring.However,conventional detection methods cannot qualify the needs rapid detection and there is a real need to develop fast,easy-to-use,robust,sensitive,and cost-effective field analytical techniques.This study made it possible by establishing a magnetic particles-based chemiluminescence immunoassay for multi-residue detection of organophosphorus pesticides.（1）S-carboxyethyl-O,O-dimethyl dithio phosphate was synthesised as general haptens for dimethoxy organophosphorus pesticides.Preparation of hapten had two steps purified by column chromatography with yield 11.5%,the eluent was methanol:chloroform=1:8.Infrared identification results showed that the product had dimethyl and carboxyl which was inline with the structure of hapten.Mass spectrum identification results showed that ESI-MS（m/z）213.2[M-H]^-,indicating a molecular weight consistent with that of hapten.Hapten were prepared successfully.（2）The chemiluminescent markers were synthesised by the reaction of O,O-dimethyl thiophosphoryl chloride and luminol,which had common structure of dimethoxy organophosphorus pesticides and shine group of luminol.The product was purified by column chromatography with yield 20.1%,the eluent was ethyl acetate:petroleum ether=1:6.Ultraviolet spectrum showed that product and luminol both had three characteristic absorption peak.The absorption peak of product moved towards long wave because of-（OCH₃）₂.Infrared identification results showed that the product had dimethoxy and luminol group.Mass spectrum identification results showed that ESI-MS（m/z）300.3[M-H]^-which was consistent with the molecular weight of chemiluminescent markers.Chemiluminescent markers were prepared successfully and used for subsequent experiments.（3）The O-dimethyl thiophosphoryl chloride chemiluminescent markers produced chemiluminescence in alkaline conditions and put up a good performance.The stronger the system alkalinity and the higher concentration of hydrogen peroxide,The greater the luminous intensity.Chemiluminescent markers exhibited a good linear relationship when the concentration between 10-160mg/L.The standard curve equation was y=252.755x+5729.167,and the correlation coefficient R was 0.9984.The solid markers were stored at 4℃refrigerator.（4）The polyclonal antibody against dimethoxy organophosphorus pesticides was coupled with four kinds of magnetic particles respectively.The order of coupling rate of four types of immune magnetic beads was:gold magnetic particles,isothiocyanate root,amino terminal and carboxyl terminal magnetic particle.The antibody coupling rate of gold magnetic particles was the highest above 80%.Under 4℃,the stability of gold magnetic particles immune magnetic beads was the best and the decomposition rate was lowest.Gold magnetic particles were used as the carrier of antibodies for subsequent experiments.（5）A magnetic particles-based chemiluminescence immunoassay for multi-residue detection of organophosphorus pesticides in agricultural products was established.The method was optimized conditions by single-factor experiment.Results showed that the optimal concentration of gold magnetic particles-immune magnetic beads and chemiluminescent markers were 175μg/L and 40μg/L respectively,the optimum p H for 9,the concentration of hydrogen peroxide was 0.4 mmol/L,the reaction time was 50min.Under the optimum reaction conditions,performance of the pesticide detection method was evaluated including linear,the minimum detection limit,precision,specificity,matrix effect and accuracy.The results showed that between the concentration of 10-250μg/L,Dichlorvos,Methyl chlorpyrifos and Fenitrothion showed a good linear relationship with a correlation coefficient of more than 0.99 while the linear of Dimethoate was poor.The detection limit was 8.98μg/L,7.74μg/L,10.04μg/L and 6.17μg/L respectively.It had good precision with intra-assay precision 3.4%-6.7%and inter-assay precision 6.5%-8.5%.The cross-reactivity test results showed that the cross reaction rate of Trichlorfon and Malathion with dimethoxy structure was over 95%and Chlorpyrifos and Triazophos with diethoxy structure was less than 10%.There was no cross reaction with Chlorothalonil because of structure difference.The method had good specific identification for dimethoxy organophosphorus pesticides.To reduce the impact of actual testing of the sample matrix on the test results,the samples were diluted twice.In samples spiked recovery test,the recovery was 87.7%-110.7%and the relative standard deviations was 4.9%-15.7%.It showed good accuracy and was used in the detection of agricultural products such as vegetables and fruits.