Screening And Detection Research Of Aptamer Capture-SELEX For Food Small Molecule Hazard

A series of problems affecting human health caused by the residual pollution of small molecular hazards in food has always been the focus of global attention.In recent years,some immunoassay technologies that do not require the use of large instruments and biologically active small molecule recognition probes have played an important role in the detection of food small molecule hazards.Aptamer is a new type of biological recognition molecule.Compared with antibody,aptamer not only realizes the specific recognition of small molecule hazards,but also has the advantages of easy synthesis and stable properties.It shows great advantages and application potential in food safety detection.However,there is no report on the screening of aptamers for?-cyhalothrin and ethyl carbamate.This paper focuses on the capture SELEX screening technology,and uses this improved technology to screen aptamers that can specifically recognize?-cyhalothrin and ethyl carbamate.Finally,the performance identification and preliminary detection analysis of aptamers are carried out.The main conclusions are as follows:(1)In this study,the improved Capture-SELEX strategy was applied to screen aptamers of?-cyhalothrin and fix the random ss DNA library onto magnetic beads.Then,?-cyhalothrin was added for incubation,and the affinity sequences which could bind to?-cyhalothrin were competitively captured from magnetic beads.After 14 rounds of screening,the aptamer which can specifically recognize?-cyhalothrin was obtained and named LCT-1(K_d=50.64±4.33nmol·L^-1).Subsequently,molecular docking technology was used to analysis the recognition mechanism between LCT-1 and?-cyhalothrin.It is found that?-cyhalothrin and LCT-1 aptamer were mainly combined by hydrophobic interaction and hydrogen bonding force.Next,the affinity sequence LCT-1 was truncated and named LCT-1-39.The results of molecular docking showed that?-cyhalothrin was bound in the hydrophobic cavity of the three-dimensional structure of LCT-1-39 aptamer through hydrophobic interaction.The lowest binding free energy is-8.1 kcal·mol^-1.The binding and dissociation constant of?-cyhalothrin with LCT-1-39 aptamer was 10.27±1.33 nmol·L^-1?(2)In this paper,the aptamer of ethyl carbamate was screened by Capture-SELEX technology.After 12 rounds of screening,the aptamer which can specifically recognize ethyl carbamate was obtained and named EC1(K_d=49.75±9.42 nmol·L^-1).We constructed the binding model of EC1 aptamer with ethyl carbamate by molecular simulation software and found that ethyl carbamate was bound in the hydrophobic cavity of three-dimensional structure of EC1through hydrophobic interaction.Ethyl carbamate is surrounded by side chain bases G-8,T-9,A-26,G-28,and G-29,in which G-8,G-28,and G-29 form stable hydrogen bonds with small molecules of ethyl carbamate.(3)We used LCT-1 and LCT-1-39 aptamers as recognition molecules to establish aptasensors for the determination of?-cyhalothrin based on cationic compound PDDA mediated Au NPs aggregation.The detection limits were 0.0197?g·ml^-1 and 0.0186?g·ml^-1with good repeatability and reproducibility.The aptasensors can be used for the rapid detection of?-cyhalothrin residues in agricultural products.In conclusion,the capture SELEX technology has been further improved,and the improved screening strategy has been successfully used to screen aptamers with high affinity,which can specifically recognize?-cyhalothrin and ethyl carbamate.This work not only provides new ideas for the detection of?-cyhalothrin and ethyl carbamate residues in food,but also further expands the application scope of aptamer technology in the detection of small molecule hazards,and also provides reference and technical support for the screening of other small molecule hazards in food.