Study On Lateral Flow Immunoassay For Multi-residue Detection Of Pesticides In Leek

Leeks are rich in nutrients,is a popular consumer favorite vegetables.There is a large area planted in China.The production process of leek is prone to stem blight,gray mold,leek maggots and other pests and diseases,control with a variety of pesticides,pesticide residue exceeds the standard and the use of excess scope of the problem,causing widespread concern,but also become the national focus of governance of vegetable species in recent years.Rapid detection is the first time to find a powerful grasp of the problem,based on the specific combination of antigen antibody colloidal gold immunochromatography technology for time-sensitive,sensitive,convenient and low-cost has become a mainstream technology for rapid detection of contaminants,is used for rapid on-site screening of agricultural products/food safety.However,due to the small molecular weight of chemical pesticides and the difficulty of antibody preparation,resulting in a small number of covered species,coupled with sample matrix interference and bottlenecks in multi-residue detection,existing technical methods are in urgent need of innovation to meet the demand for simultaneous field rapid detection of multi-residues of pesticides in fruits and vegetables.In the paper,six pesticides,including carbofuran,acetamiprid,imidacloprid,carbofuran,pyrimethanil and carbendazim in leeks,were used as research objects.Based on the design,synthesis and preparation of haptens and antibodies,the matrix effects of chlorophyll and organic sulfides in leek on colloidal gold immunochromatography were investigated,and the mechanism of their effects were thoroughly studied.By optimizing the pretreatment method to reduce or eliminate the matrix effect,and using multiple T-line strategy to develop colloidal gold test strips,combined with our group’s self-developed immunochromatographic image recognition system,to build a good accuracy,high sensitivity,intelligent pesticide multi-residue rapid detection technology methods.To achieve high throughput,intelligent detection of pesticide multi-residues in leeks to provide a theoretical basis and methodological reference to effectively ensure the quality and safety of leeks.The main research contents are as follows:1.In this study,1,2-dimethylcyclopropane-1,2-dicarbonylimine was selected as the characteristic structure of putrescine hapten,containing four carbon arms of carboxyl group instead of chlorine atom,to synthesize putrescine hapten.The putrescine hapten was coupled with BSA and OVA,respectively,using the active ester method.The ELISA sensitivity IC₅₀ was 86.57 ng/m L and the detection limit IC₂₀was 14.42 ng/m L.The cross-reactivity rate of putrescine and its structural analogues was less than 1%,which has good specificity.The excellent performance of the monoclonal antibody to putrescine provides the core material for further construction of the colloidal gold immunochromatographic method,which can meet the demand for the detection of putrescine MRLs.2.In this study,based on the construction of homologous coating colloidal gold immunochromatographic method,the sensitivity of the colloidal gold immunochromatographic method for putrescine was increased by 10-fold by changing the coating original on the T-line.The substrate effects and influence mechanism of chlorophyll and organosulfide in leek on the detection of pesticide residues of putrescine,acetamiprid,imidacloprid,carbofuran,pyrimethanil and carbendazim by colloidal gold immunochromatography were further investigated.The results showed that both chlorophyll and organic sulfide had effects on the detection results,and chlorophyll was the main interfering factor.Chlorophyll affects the accuracy of colloidal gold immunochromatographic assay results by influencing the affinity of antigen and antibody,which in turn affects the accuracy of colloidal gold immunochromatographic assay results.Through molecular docking and molecular dynamics simulations,it was shown that chlorophyll mainly affects the Coulombic forces to make the antigen-antibody binding of putrescine more stable and stronger affinity,in which 43LYS,107TRP and141ARG of putrescine antibody are the key amino acids that play a stabilizing role.The effect of chlorophyll on antigen-antibody affinity was studied mechanistically to provide a theoretical basis for minimizing the matrix effect of colloidal gold immunochromatography.In this study,we optimized the sample pretreatment steps,diluted the substrate 3000 times to reduce the specific binding,added formic acid to degrade chlorophyll to minimize the matrix effect,and used multiple T-line strategy to develop colloidal gold test strips,combined with the group’s self-developed immunochromatographic image recognition system and other antibodies prepared to construct a semi-quantitative detection of pyrimethanil,acetamiprid,carbofuran,3-OH carbofuran,and carbaryl at the same time.OH carbofuran,carbofuran and carbendazim.The multiplex T-line colloidal gold immunochromatographic method achieved a cross-reactivity rate of 73%for 3-OH carbofuran and could meet the requirements of the definition of carbofuran residues in GB 2763-2021"Maximum Residue Limits of Pesticides in Food".The limits of detection of pyrimethanil,acetamiprid,carbofuran,3-OH carbofuran,carbaryl and carbendazim in leek substrate were 18.96,18.72,12.63,15.48,115.32and 9.42μg/kg,respectively,with the average recoveries of 71%-92%and the RSDs of 5%-18%.The results were in good agreement with the LC-MS/MS assay.It showed that the method has good sensitivity,accuracy,precision and specificity,and can achieve accurate,high-throughput and intelligent rapid detection of pesticide multi-residues in leeks.