Selection Of Aptamers Specific For Spinosad And Development Of Aptamer Sensors

Spinosad is a metabolite produced by the fermentation of Saccharopolyspora spinosa.As a new type of insecticide,it not only has the characteristics of low toxicity and environmental friendliness as a biological pesticide,but also can play a role as an efficient chemical pesticide.It has broad applications in the domestic and foreign pest control.Because the production mode of Spinosad is microbial fermentation,its low yield and high cost limit its application.In order to improve the yield by screening high-productive strains and optimization of fermentation conditions,the first thing is to build a fast and high-throughput detection method of Spinosad.Biosensors enable rapid detection of targets by transducing the changes in the combination of the identification element and the target into a measurable signal output.Among them,aptamers are good materials for identification elements due to their strong affinity and ease of synthesis,and have received widespread attention in the construction of biosensors that recognize small molecules.In this study,using Spinosad as the target,the aptamers that recognize Spinosad were screened by magnetic bead-SELEX technology.Then,based on the aptamer,the electrochemical aptamer sensor,the disposable aptamer sensor and the fluorescent aptamer sensor were established,which provides a variety of methods for the rapid detection of Spinosad.(1)Based on the magnetic bead-SELEX method,the aptamers of Spinosad were screened,and the aptamers were sequenced and identified.Taking Spinosad as the aimed object,a random ssDNA library was designed and fixed on streptavidin magnetic beads,and a SELEX screening method based on magnetic beads was constructed.Combined with Q-PCR technology,the enriched library was obtained after six rounds of screening,and more than 7000 sequences were obtained by high-throughput sequencing.According to the enrichment and composition of the sequences,48 candidate sequences were selected.These sequences were analyzed by phylogenetic tree,and then the affinity identification was carried out by isothermal titration calorimetry.The results showed that 06-aptamer and 11-aptamer had thermal reaction,and the equilibrium dissociation constants were 518e-6±27.3e-3 M and 70.1e-6±612e-6 M,respectively.The 11-aptamer with stronger affinity was selected as the optimal aptamer.(2)Construct the electrochemical aptamer sensor based on an ordinary gold electrode.Taking 11-aptamer as the aimed object,the sulfhydryl group was modified at its 5’ end,which was fixed on the gold electrode by gold-sulfur bond.The 6-mercaptohex-l-ol was added dropwise to prevent ssDNA lodging and non-specific adsorption.The working electrode was prepared.Using platinum wire as the counter electrode and Ag/AgCl chloride as the reference electrode,a three-electrode system was formed together with the working electrode,and the electrochemical signal was detected by differential pulse voltammetry.The electrochemical aptamer sensor was successfully constructed by optimizing the concentration of aptamer and self-assembly time.Within the detection concentration range of 10-2～102 nmol/L,the peak current difference and the log value of the Spinosad concentration showed a good linear relationship,and the detection limit was 0.01 nmol/L.(3)Design of double-stranded probes to construct the disposable aptamer sensor base on screen-printed electrodes.The double-stranded probe was designed according to the secondary structure of 11aptamer,which solves the problem of uneven distribution and easy lodging of ssDNA.The sulfhydryl group was modified at the 5’ end of the aptamer and the 3’ end of the paired primer.And the double-stranded probe was formed by complementary pairing,which was immobilized on the nano-gold printed electrode and electrochemically detected by square wave voltammetry.The disposable aptamer sensor was successfully constructed by optimizing the concentration of double-stranded probe and binding temperature.Within the detection concentration range of 10-1～104 nmol/L,the peak current difference and the log value of the Spinosad concentration showed a good linear relationship,and the detection limit was 0.1 nmol/L.(4)Construct the fluorescent aptamer sensor based on graphene oxide(GO)and use nucleases to achieve signal cycle amplification.The FAM fluorophore was modified at the 3’ end of aptamer,and the aptamer was adsorbed by GO after the addition of GO,and the fluorescence was also quenched.After adding Spinosad,the target and aptamer form a complex and dissociate into the solution,and the fluorescence signal was restored.During the process,a microplate reader is used to detect the fluorescence signal.The fluorescent aptamer sensor was successfully constructed by optimizing the concentration of GO,quenching time and response time.Within the detection concentration range of 1～100 μmol/L,the fluorescence intensity signal and the Spinosad concentration showed a good linear relationship,and the detection limit was 1 μmol/L.The fluorescence intensity recovery after the introduction of DNase I for target cyclic signal amplification is more obvious,about twice as high as before.The detection limit was as low as 100 nmol/L,and the sensitivity of the fluorescent aptamer sensor was improved.