Preparation And Characterization Of Dna Aptamers Against Three Small-molecule Pollutants In Water Sources

Nowadays,due to the development of industry and agriculture and the improvement of people's living standard,the pollution of small molecules in water sources such as persistent organic pollutants and endocrine disrupting chemicals is becoming increasingly serious.These pollutants have adverse impacts on people's health.Therefore,it is particularly important to quickly and accurately detect them.Generally,these substances are weak in immunogenicity and difficult to detect them by traditional antibody-based assays.Aptamers,new type of recognition molecules,are single-stranded DNA?ssDNA?or RNA,which are obtained through in vitro selection methods.Aptamers have not only antibody-equivalent affinity and specificity,but also a wide range of targets including small molecules.Aptamers are also characterized by good stability,easy modification,in vitro selection and easy synthesis.In recent years,selections and applications of aptamers in detection of various small molecules have been fast developed,and they just emerged in the analysis and detection of small-molecule pollutants in water recently.In this paper,three small-molecule pollutants in water sources were studied.Their DNA aptamers were selected by improved immobilized and/or nonimmobilized target systematic evolution of ligands by exponential enrichment?SELEX?technology.In additional,by using magnetic bead separation and personal glucose meter?PGM?,a portable aptasensor?aptamer-based biosensor?was developed.Methods,which developed in this study for detecting smallmolecule pollutants,provide new ideas and solutions for detecting and monitoring more small-molecule pollutants in water sources.Firstly,we invented a novel gel-based diffusion method for monitoring aptamer-target binding.We used 3D printing technology to prepare the gel models.Chasing diffusion was successfully developed for monitoring the small molecules and their aptamers.This experiment is convenient for library monitoring during aptamer selection process,especially for small-molecule targets.Secondly,two magnetic beads?MB?-based SELEX methods were applied to select aptamers against DDA,one of DDT metabolites.In the MB-based target coupling method,an enriched library was obtained after 9 rounds of selection.The library was cloned and sequenced.The sequences from individual clones were synthesized and taken into further characterization.Results showed that binding affinities of candidate aptamers against DDA were not significant improved.Thus,we turned to use the Capture-SELEX.In the MB-based Capture-SELEX,DNA aptamers of DDA were successfully obtained after 13 rounds of selection.The candidate aptamers were identified by gold nanoparticles?AuNPs?assay and SYBR Green I?SGI?method,respectively.Results showed that one of the candidate aptamers,designated DDA₀8,had high affinity?dissociation constant,Kd,3.9 nM?and specificity with DDA,which indicated DDA₀8 had the potential for DDA detection in the future.Thirdly,two different types of SELEX were applied for selection of aptamers against perfluorooctanoic acid?PFOA?,one of the perfluorinated compounds?PFCs?.In the nitrocellulose?NC?membrane-based target immobilization SELEX?NC-SELEX?,BSA-PFOA complex was synthesized to immobilize PFOA onto the NC membrane.After 13 rounds of selection,an enriched library was obtained and sequenced.Results showed that candidate aptamers were bound to both BSA and BSA-PFOA.We obtained aptamers bound to BSA,which was the carrier protein in NC-SELEX.Therefore,we found that NC-SELEX is not suitable for aptamer selection of small-molecule targets,such as PFOA molecule.In the MB-based Capture-SELEX,in first 5 rounds of selection,we found that the libraries were not enriched effectively,which monitored by real time PCR.Then we introduced mutagenesis PCR to increase the diversity of PFOA-derived library.With the continues 11 rounds of selection,DNA aptamers against PFOA were obtained and identified.The potential affinities of the candidate aptamers were analyzed by SGI assay and chase diffusion,respectively.Fourth,selection of aptamers against o,p'-DDT was conducted by using MB-based Capture-SELEX.Three of seventeen candidate sequences were selected for preliminary characterization by SGI assay.One sequence with highest fluorescence response with o,p'-DDT,designated DDT₁3,was chosen for further characterization.Its Kd was determined to be 412.3±124.6 nM.DDT₁3 exhibited low cross-binding activities on other tested small molecules.Fine bioactivities of DDT₁3 were demonstrated for analysis of spiked lake water and tap water samples.This study provides a novel o,p'-DDT-specific probe for its future applications.Fifth,a portable aptasensor by using a PGM and MBs was developed.In this study,o,p'-DDT aptamer was firstly coupled with the invertase.Then the complementary DNA?cDNA?was immobilized onto the MBs.The two elements described above were assembled together to construct the testing system.The detection principle is that conformational changes can be induced by the target,leading the release the invertase-aptamer from the MBs surfaces.The portable aptasensor was developed by detecting glucose signal,which was triggered by catalyzing the sucrose by target-induced invertase-aptamers.A good linear relationship of o,p'-DDT concentrations ranging from 0 to 0.5 ?M was obtained.In summary,a gel-based diffusion method was invented for monitoring the aptamer-target binding.Then selection of aptamers against DDA,PFOA,and o,p'-DDT was conducted by optimizing SELEX method coupling with immobilization and/or non-immobilization target.The aptamers selected in this study had high affinity and specificity.Meanwhile,we also concluded that Capture-SELEX was the most suitable method for selection of aptamers against small-molecule targets.A portable aptasensor by using a PGM and MBs was developed.This study provides suitable technical supports for the development of new technologies for the detection of small-molecule pollutants,but no limited to,in water sources.