Micro Array For Screening Aptamers Of Organophosphorus Pesticides

In recent years,the food safety problem caused by chemical pollution in our country is very serious,which seriously affects the health and safety of the people.Chemical pollution mainly includes pesticide,veterinary drug,antibiotic and residues of various toxic chemical additives.Organophosphorus pesticides have become the main cause of food safety because of their broad spectrum,high efficiency,rapidity and commercialization.Therefore,how to effectively solve the food safety problem caused by pesticide residues is an important issue facing food researchers.The aptamer provides a new research direction for the detection of organophosphorus pesticides because of its easy modification,low cost and easy storage.In this paper,the standard 0P3 of organophosphorus pesticides was used as the research object,and the micro-array technology was used to screen 2?3 aptamers with high affinity of OP3,thus establishing a new sensitive,efficient and rapid detection method.The work is as follows:(1)Exploring the effect of the length of the aptamer sequence on the binding of the target OP3,and the conclusion that the fixed sequence at both ends of the aptamer has a certain auxiliary effect on the correct folding by the micro-thermal migration experiment(MST);(2)In the early stage,simple structure prediction and molecular docking simulation were performed on the aptamer and OP3 molecule using tools such as mfold and SYBYL,and the modification scheme of the aptamer was roughly determined by combining the sequence information;(3)The aptamer was placed in a liquid environment and combined with 0P3 for pre-experiment.The fluorescent signal was detected by a microplate reader,and six aptamers with good affinity were screened,namely PES05,PES11,PES17,PES19,PES20 and PES21;(4)According to the design scheme,the preparation of the microarray chip and the hybridization work were carried out by using the FAM-labeled aptamer.During the optimization,the experimental conditions such as spotting solution and spotting concentration were optimized,and finally two aptamers were obtained,which were respectively PES11 and PES19;(5)Further optimizing the aptamer modification scheme,designing and synthesizing the complementary sequence of Cy3 marker,forming a competition system with OP3,and screening three aptamers with high affinity,namely PES05,PES11 and PES19.