Isolation And High Throughput Characterization Of Aptamers Against Antibiotics

Antibiotics are essential for the treatment of bacterial infectious diseases,but when overused and released into the environment,they affect the balance of the entire ecosystem and lead to the generation of drug-resistant bacteria,which has a significant impact on human health.At present,antibiotic residues in aqueous samples are mostly detected using high performance liquid chromatography,gas chromatography-mass spectrometry,and other methods that require complicated instruments and professional technicians.Aptamer-based biosensors have received a lot of interest because of their good selectivity,specificity,and sensitivity.At present,a variety of aptamer-based biosensors have been used to detect different antibiotics.Aptamers are convenient to produce and label,making them ideal recognition elements in biosensors.Various types of biosensors made of aptamers against a variety of antibiotics have been reported.There is still a great demand for the screening of aptamers with high affinity and specificity for more kinds of antibiotics and the development of biosensors.The conventional SELEX of aptamers for small molecule targets is tedious,and the failure rate is high.Moreover,it is difficult to identify the affinity sequences from enormous candidate sequences of the enriched library,and there is no simple,economical and high-throughput method to measure the affinity between sequence and small molecule.In published articles,sequences from the library were amplified onto the surface of the beads using a method called particle display,which greatly reduced the number of screening cycles.However,there are still some issues with this strategy such as the droplets are vary in size.Based on the principle of particle display,this paper uses the characteristics of droplet digital PCR that can perform uniform large-scale parallel amplification of a single template,combined with the high-speed analysis and single particle sorting ability of flow cytometry,aptamers that specifically recognize ciprofloxacin and vancomycin were directly selected from the pre-enriched library in one round.Thus,the long process and low success rate of aptamer screening and identification of small molecule targets can be solved.Firstly,the possibility of using droplet digital PCR and flow cytometry for single round high-throughput identification was investigated by literature review.After further optimizing each specific experiment,including beads coupled with primers,droplet digital PCR conditions,and parameter setting of single bead collection with FACS,the system was successfully used to screen and identify four DNA aptamers binding ciprofloxacin from the pre-enrichment library in one round,and one of the aptamers was truncated to 29 bases to obtain the core sequence,which maintained good binding with the target ciprofloxacin(Kd=84 μM).The fluorescence property of ciprofloxacin was used in the sorting process,therefore,we further verified the feasibility and accuracy of the system by using the dye Thioflavin T(ThT)as a model target,and screened the aptamers with different fluorescence enhancement effects with ThT.The fluorescence intensity can be well correlated with the fluorescence collection unit set during sorting,which realized rapid matching of aptamer sequence acquisition and the binding ability.Then,for small molecules without fluorescent properties,we choose vancomycin as the screening target.By introducing the fluorescent modified oligo as the reporter sequence,the beads will display fluorescence signal on and off changes before and after binding to the target thus achieving the specific collection of target beads.This scheme breaks the restriction that the target lacks fluorescence throughout the screening process.After a round of screening,we successfully obtained several aptamers binding vancomycin,and the truncated aptamer with 47 bases have good specificity and high affinity(58 μM)with the target.Based on the aptamer of vancomycin,a fluorescent biosensor was designed for sensitive detection of vancomycin with a linear range of 1 to 20 μM,the limit of detection was 0.7 μM.Moreover,the sensor can detect vancomycin in 90%river water and 90%human serum,and the linear range of detection is 1 μM～10 μM and 4 μM～60 μM.There are few reports on the generation of uniform aptamer particles based on droplet digital PCR,combined with high-throughput detection and single particle sorting of FACS to obtain DNA aptamers in one round.The high-throughput identification method in this paper helps to lessen the cycles of traditional small molecule screening,greatly simplifies the process of identifying candidate sequences in enriched library,and improves the success rate of SELEX.In this paper,the aptamers specifically binding to ciprofloxacin and vancomycin were successfully screened by this method,and based on the truncated aptamers of vancomycin,a fluorescent biosensor for the specific detection of vancomycin was developed by using the molecular beacon method.