Peptidomimetics Of Imidacloprid Selected From Phage Display Peptide Libraries And Its Application In Immunoassay

Imidacloprid,a broad-spectrum neonicotinoid insecticide,is widely used worldwide.However,owing to its significant use in environment systems,it may cause high risk of pesticide residue,which could pose serious risks for ecological security and human health.Therefore,it is of great significance to develop efficient and convenient methods.Immunoassays are practical tools for the detecting residual pesticides with the advantages of fast,simple,sensitive and low-cost,and it is widely used in pesticide residue detection.The immunoassays with a competitive format are generally used to detect small molecular weight compounds(including pesticide).At present,pesticide competitors are usually prepared by chemical synthesis and trial and error process,obtaining such compounds is often time-consuming and it is impossible to predict whether the competitor is useful.However,phage display peptide technology can easily screen the best competitors and has the advantages of simple operation,short cycle,low cost and less pollution,and the isolated phage is easy to prepare and purify in large quantities,which accelerate the development of immunoassays.In this study,we isolated phage-borne peptides that against imidacloprid by using phage display technology.And three rapid immunoassays were developed by using these competitors.It has been successfully applied to the detection of imidacloprid in the environment and agricultural products.1.We isolated two phage-borne peptides that bind to monoclonal antibody against imidacloprid from the libraries of Ph.D.-C7C and Ph.D.-12 by biopanning.Two sequences of HSLWMASPMPGY(L12-1)and QIFTSSPMPAMV(L12-2)were isolated.We used phage ELISA to determine the sensitivity of the two phage-displayed peptides as competitors to imidacloprid,and then choose the more sensitive L12-2 as heterogeneous competitor for the establishment of immunoassays.2.We developed a phage enzyme-linked immnosorbent assay(P-ELISA)by using the phage-borne peptides that bind to monoclonal antibody against imidacloprid.The standard curve was established by optimizing the concentrations of coating antibody,phage-borne peptide and working buffer.Under the optimum conditions,the P-ELISA had the IC50 of 0.067 ng/mL,linear ranges(IC10-IC90)of 0.024-0.40 ng/mL and limits of detection(LOD,IC10)of 0.024 ng/mL.The cross-reactivity revealed that the proposed immunoassays demonstrated no significant CR with most of the analogues except for imidaclothiz(100%)and clothianidin(34%).The average recovery of spiked in paddy water,soil,wheat,rice,apple,and cucumber was 70.1%-102.1%and RSD was 3.3%-10.2%,which met the requirements for the detection of pesticide residue.There is a good correlation with high performance liquid chromatography(HPLC)in the detection of soil and rice samples,these results showed that the phage-displayed peptide can be used to develop higher sensitivity heterogeneous competitive immunoassay,and the immunoassay had satisfactory accuracy and precision and can be used to detect imidacloprid in the environment and agricultural products.3.DTTA-Eu3+was conjugated with anti-M13 pAb and goat anti-rabbit IgG to prepare the fluorescence tracers.The P-TRFIA directly developed by using Eu3+-labeled anti-M13 pAb was named P-TRFIA-1 and Indirectly developed by using Eu3+-labeled goat anti-rabbit IgG was named P-TRFIA-2.Under the optimal conditions,the standard curves were established for the detection of imidacloprid.The IC50,LOD and linear ranges of P-TRFIA-1 were 0.085,0.020 and 0.020-2.3 ng/mL respectively,while 0.056,0.017 and 0.017-0.24 ng/mL for P-TRFIA-2.The two established P-TRFIAs demonstrated no significant CR with most of the analogues except for imidaclothiz and clothianidin.The mean recoveries of spiked in paddy water,soil,wheat,rice,apple,and cucumber of P-TRFIA-1 and P-TRFIA-2 ranged from 75.7%-99.6%and 71.2%-105.1%,with RSDs of 1.9%-9.1%and 5.2%-11.8%,respectively.The results showed that the immunoassays had satisfactory accuracy and precision,the established fluorescent immunoassays method were rapid and sensitive in the detection of imidacloprid.4.We fused the phage-borne peptide to NanoLuc and mNeonGreen to generate novel fluorescent biosensors,and designed a bioluminescence resonance energy transfer(BRET)systems by using NanoLuc as the fluorescent donor and mNeonGreen as the receptor.By the process of construction of the vector,expression,purification and verification of the recombinant fluorescent biosensors,we successfully prepared four fluorescent biosensors that the order of the phage-borne peptide varied.After analyzing the relative affinity to antibody of the individual fluorescent biosensors,we selected Sensor-1 that showed the highest affinity to develop N-HFIA and HFIA.Under the optimal conditions,the N-HFIA had the IC50 of 0.0605 ng/mL,linear ranges(IC10-IC90)of 0.00513-0.745 ng/mL and limits of detection(LOD)of 0.00513 ng/mL,while the HFIA was 4.02 ng/mL,0.517-25.4 ng/mL and 0.517 ng/mL,respectively.The two established fluorescence immunoassay demonstrated no significant CR with most of the analogues except for imidaclothiz and clothianidin.The average recoveries of spiked in paddy water,soil,wheat,rice,apple,and cucumber of N-HFIA and HFIA ranged from 71.2%-96.5%and 70.6%-99.1%,with RSDs of 2.6%-10.0%and 3.0%-10.1%,respectively.The results in the detection of soil and rice show that the correlation relationship between the established N-HFIA,HFIA and HPLC are close to 1,indicated that the established two methods have high accuracy and reliability.And the homogeneous reaction does not need complicated steps such as coating,blocking,and washing,it has simple operation,which provides a new method for the rapid detection of imidacloprid.