Selection Of Aptamers For New Organophosphorus Pesticide OP3 Based On Magnetic Beads-SELEX

Organophosphorus pesticides have been widely used in agricultural production in China because of their good efficacy and easy variety.However,with the increase of productivity,the unscientific use and abuse of organophosphorus pesticides has become more and more serious,which resulted in the excessive pesticide residues in the food and environment,and even posing a threat to human health.Therefore,there is an urgent need to develop a new technology to achieve rapid and sensitive detection of organophosphorus pesticide residues in food and the environment.Aptamers have great application potential in the detection of organophosphorus pesticide residues due to their high affinity,low cost,wide target range,and easy chemical modification.Therefore,it is necessary to screen aptamers that can recognize a variety of organophosphorus pesticides,and lay a foundation for the establishment of a method based on aptamers to rapidly and sensitively detect organophosphorus pesticide residues.In this study,aptamers of organophosphorus pesticide OP3 were obtained based on magnetic bead-SELEX technology,and their properties were characterized.The main work is as follows:1?The candidate targets OP3 and OP4 were coupled with BSA and OVA respectively to obtain four coupling products:OP3-BSA,OP3-OVA,OP4-BSA and OP4-OVA.The UV spectrum means was performed to confirm whether the coupling was successful,then the coupling ratios of the four coupling products were obtained and compared by MALDI-TOF MS.It was found that OP3-BSA had the highest coupling ratio and was more advantageous for selection,so the organophosphorus pesticide OP3 was selected as the target for this study.2?The OP3-BSA with the highest coupling ratio was immobilized on the amino magnetic beads for positive selection,and the BSA was immobilized on the amino magnetic beads for counter selection.The OP3-BSA-Beads and the primary single-stranded DNA library were mixed and reacted for 45 min at room temperature,after which the unbound oligonucleotides were removed and the bound oligonucleotides were eluted with an elution buffer at 95 ?.The eluted ssDNA was subjected to PCR amplification,and then the amplified double-stranded DNA was denatured into ssDNA to make a secondary library for the next round of selection.A total of 19 rounds of selection were performed and six rounds of counter selection were performed to remove ssDNA with affinity for BSA.Through the determination of the enrichment of ssDNA with affinity for OP3 in each round of library,the 14th round of library was selected for high-throughput sequencing,and mfold was used for secondary structure simulation of the sequences.After analysis and comparison,a total of 16 aptamers were obtained.3?The affinities of the 16 sequences were preliminary characterized,and then the dissociation constants(Kd)of the four aptamers PES 11,PES 19,PES 20 and PES 21 with the highest affinity were measured.The Kd values of the above aptamers were respectively 276±13.4 nM,28.99±4.57 nM,28.85±3.30 nM and 45.30 ±4.24 nM,indicating that all four aptamers had high affinity for OP3.4?The binding properties of the above four aptamers to OP3,fenthion,parathion,parathion-methyl,fenitrothion,fensulfothion and phoxim were evaluated and they were found to have the ability to bind to at least two kinds of the above pesticides.In addition,aptamer PES 11 could bind to all of the above seven pesticides.