Selection And Application Of Aptamers For Organophosphorus Pesticides

Organophosphorus pesticides residues in food,water and environment are seriously harmful to human health.The development of rapid detection technology is a major strategic requirement to protect human health,which is of great significance.Biometric materials are the core of rapid and accurate detection technology,and the development of novel biometric materials is the key technology for the development of rapid detection technology.This work intends to improve the aptamer screening method based on magnetic beads-systematic evolution of ligands by exponential enrichment(MB-SELEX)to develop a novel biometric material nucleic acid aptamer for screening phoxim,methyl parathion and other substances,and apply it to construct fast and convenient fluorescence or electrochemical sensors.The main contents of this thesis are as follows.1.Selection of phoxim aptamer and construction of fluorescence sensor.Using the improved MB-SELEX screening method,the aptamer PHO-APT3 for specific recognition of phoxim was obtained,and the dissociation constant was 0.93±0.25μM.Based on the modified aptamer PHO-APT3s,a competitive fluorescent aptasensor was constructed by modifying fluorescein at the 5’end of the aptamer and the quenching agent at the 3’end of the complementary strands.This sensor had a linear relationship in the range of 0.1-5μM(R~2=0.992).The limit of detection(LOD)was 34.35 n M,which had the advantages of convenient operation,fast reaction speed and low price.Considering the priority recognition of aptamers and the reduction of background signal,we also constructed a fluorescent aptasensor based on multiple complementary strands and nano-gold(Au NPs).The sensor had a lower background and a wider range of fluorescence intensity,and the detection error was small.The sensor has a linear relationship in the range of 0.05-5μM(R~2=0.996),and the LOD was 29.69 n M.The results showed that the selected phoxim aptamers had the characteristics of specific capture of phoxim in complex real environment and have important practical value.2.Selection of methyl parathion aptamer and construction of electrochemical aptasensor.Based on the existing selection experience,the quantitative method of elution rate was used to monitor the selection process.A methyl parathion aptamer(MPapta-6)with high affinity and specificity was obtained,the Kd was 39.66±14.73μM.The electrochemical aptasensor based on electrochemical aptasensor based on functionalized black phosphorus nanosheets was fabricated.Under the optimum conditions,the sensor had a good linear relationship in the range of 1-10~5p M(R~2=0.996)and the LOD was as low as 0.49 p M.This aptasensor had the advantages of high sensitivity,good specificity and fast response.