Development Of Rapid And Highly Sensitive Immunoassays For Organophosphorus Pesticides Multi-Residues Based On Mimotope

Organophosphorus pesticides（OPs）are widely used in agricultural production,with a wide spectrum of insecticides,a variety of species and other characteristics.And OPs often cause excessive pesticide residues due to large and non-standard use,which poses a serious threat to people’s health.Therefore,it is of great significance to develop rapid,sensitive and low-cost OPs detection technology.In this study,the OPs class-specific single chain antibody immuno-magnetic beads sc Fv-5-MBs was used as the target,and the mimotopes of OPs were screened from the phage display random peptide library.Then,the best phage-mimotope was modified and enzyme-labeled phage-mimotopes were prepared.Finally,the direct competitive-phage magnetic chemiluminescence enzyme-linked immunoassays（DC-PMCLEIAs）of OPs multi-residues were established using immunomagnetic beads and enzyme-labeled phage-mimotopes,which could achieve a highly sensitive and rapid detection of OPs multi-residues.The main results are as follows:（1）The gene of 9-mer random peptide was inserted into p IT2-p8 vector and transformed into Escherichia coli TG1.And the phage display 9-mer random peptide library was obtained after rescued by helper phage KM13.The library capacity was 6.0×10⁷.Using sc Fv-5-MBs as target and parathion-methyl（PM）solution as eluent,three rounds of panning and monoclonal screening were performed on the peptide library to obtain"primary"mimotopes.Amino acid sequence analysis of ten"primary"mimotopes showed that eight of them contained conserved"W-W"sequences near the N-terminal.To further improve the performance of the mimotope,a"secondary"phage display 10-mer random peptide library（X-W-W-X-X-X-X-X-X-X）was constructed based on the amino acid sequence of the"primary"mimotopes.Three rounds of panning and monoclonal screening were performed on the"secondary"peptide library.The results of Indirect competitive-phage magnetic ELISA（IC-PMELISA）showed that the phage polyclonal performance obtained from the"secondary"peptide library has been significantly improved compared with that from the"primary"peptide library.Amino acid sequence analysis of ten"secondary"mimotopes showed that eight of them contained conserved"W-W-W"sequences on the N-terminal.The sequences of"secondary"mimotopes were more consistent than that of"primary"mimotopes.The sensitivity of the"secondary"mimotopes were determined and the results showed that the mimotope p28 had the best performance.Under the optimum conditions,the IC₅₀value of p28 based IC-PMELISA for PM was 18.26 ng/m L.（2）The p28 gene was inserted into the KM13 helper phage genome,and the p3-displayed component phage KM13-p28 was obtained by phage amplification.The IC₅₀value of KM13-p28 based IC-PMELISA for PM was 13.12 ng/m L,which is more sensitive than that of rescue phage-p28 based IC-PMELISA.The p IT2-p8-Avi plasmid in E.coli TG1 was rescued by KM13-p28,and the phage KM13-p28-Avi displaying both p28 mimotope and Avi-Tag was obtained.On the one hand,after the biotinylated of KM13-p28-Avi,SA was labeled to the phage by high affinity of biotin and streptavidin（SA）,and then Biotin-BSA-HRP was labeled to SA to obtain an enzyme-labeled phage-mimotope（KM13-p28-SA-HRP）.On the other hand,KM13-p28-Avi and anti M13-HRP were mixed and incubated in a certain proportion to obtain an enzyme-labeled phage-mimotope（KM13-p28-SA-HRP）,and then the KM13-p28-SA-HRP was settled using PEG/Na Cl solution.The performance of the enzyme-labeled phage-mimotopes were determined by the direct competitive-phage magnetic ELISA（DC-PMELISA）.Under the optimum conditions,the IC₅₀values of PM determined by PMELISA based on KM13-p28-SA-HRP and KM13-p28-anti M13-HRP were 12.82 and13.78 ng/m L,respectively.The sensitivity of DC-PMELISA is similar to that of IC-PMELISA based on KM13-p28,but it has fewer operating steps and requires shorter detection time.（3）The immunomagnetic beads and enzyme-labeled phage-mimotopes based DC-PMCLEIAs for OPs multi-residues were established.The IC₅₀value of DC-PMCLEIA based on KM13-p28-SA-HRP for 30 OPs was 1.35-28.96 ng/m L,the cross-reaction rate was 15.92-341.48%,the limit of detection（LOD）was 0.06-2.57 ng/m L,and the average IC₅₀value and cross-reaction rate was 9.05 ng/m L and 95.71%,respectively.The IC₅₀value of DC-PMCLEIA based on KM13-p28-anti M13-HRP for 30 OPs was 1.28-41.79 ng/m L,the cross-reaction rate was 12.63%-412.50%,the LOD was 0.06-1.44 ng/m L,and the average IC₅₀value and cross-reaction rate was 10.32 ng/m L and 100.51%,respectively.The sensitivity and cross-reactivity of both DC-PMCLEIAs can better meet the demand for multi-residue detection of organophosphorus pesticides.In addition,to verify the accuracy of DC-PMCLEIA,the recoveries of four OPs added to cucumber,lettuce and cabbage samples were determined.The results showed that,the recoveries of DC-PMCLEIA based on KM13-p28-SA-HRP and KM13-p28-anti M13-HRP were between 85.40%-107.14%and 82.40%-116.40%,respectively,and the coefficients of variation were 0.54%-6.91%and 0.48%-3.48%,respectively,which indicated that the two DC-PMCLEIAs had good accuracy.Finally,the two kinds of DC-PMCLEIA and GC-MS/MS were used to parallel analysis of cucumber samples spiked with four kinds of OPs,and the correlation analysis of the results showed that the two DC-PMCLEIA had good reliability.