Screening And Application Of Metronidazole Specific Aptamers

Metronidazole,one of nitroimidazole derivative drugs well-known for its outstanding antimicrobial properties,is effective against trichomonas,amoeba and anaerobic bacteria.However,excessive use of metronidazole can be teratogenic,carcinogenic,and neurotoxic.Hence,it is important to develop an alternative method for metronidazole determination with high sensitivity and selectivity.Aptamers are single-stranded oligonucleotides that possess unique properties folding into a defined 3D structure,by which they specifically bind to various targets.Aptamers are manufactured by chemical synthesis methods,so they do not induce immunogenicity,and can be chemically modified to improve stability or achieve labeling,which enables reproducible,inexpensive and rapid production.Aptamers are selected from a large library of oligonucleotides that contains approximately 10¹⁵ different sequences by systematic evolution of ligands by exponential enrichment,a process termed SELEX.This process consists of repetitive cycles of selection and amplification.During each cycle,oligonucleotides with a specific affinity for a desired target are retained,recovered,and amplified.After 10-15 cycles,this procedure leads to the enrichment of a library,predominantly consisting of putative target binding species,which can be ultimately called the aptamers.Truncation of aptamers is an effective strategy to improve affinity and reduce detection costs.The sequence of the original aptamers is too long when applied,so further optimization is needed to obtain more efficient aptamers.Metronidazole-specific aptamers were screened from a random ssDNA library with the full length of 79 nt based on DNA library-immobilized magnetic beads SELEX technology.Four representative aptamers with high enrichment and rich conserved sequences were selected for structural simulation and affinity test.Among them,AP32 with high affinity and good specificity was selected as the best aptamer for further optimization.Through the secondary structure analysis,molecular docking and affinity evaluation of the structural properties of truncated aptamers,truncation experiments of various schemes were carried out to study the changes in affinity and structural characteristics of aptamers before and after truncation.With little change in affinity,the redundant nucleotides in AP32 are gradually removed.According to this strategy,the truncated aptamer AP32-4 with a length of only 15 nt was finally screened,and its dissociation constant was 77.22±11.27 nM,which was almost equal to the original aptamer AP32,and verified its specificity for metronidazole.Metronidazole fluorescent biosensor based on aptamer AP32-4 was successfully applied to the actual detection of metronidazole in solution with the help of exonuclease-assisted target cycle amplification.Graphene oxide?GO?is widely used in the SELEX process and the construction of aptamer-based biosensors due to its excellent ability to adsorb ssDNA and quench fluorescence.The biosensor uses graphene oxide as a carrier to adsorb the modified aptamer of the fluorescent group.In the presence of metronidazole,it binds to the aptamer AP32-4 and falls off the surface of graphene oxide.Then the exonuclease RecJf completely hydrolyzes the aptamer,which releases metronidazole to bind other aptamers and start a new cycle to achieve target cycle.The sensitivity of metronidazole was improved through the optimization of detection conditions.Finally,the biosensor was used to detect metronidazole in honey samples.The linear detection range of biosensor detection was 25-800 nM,and the detection limit was 10.50 nM.The recovery rate is from 98.25%to 102.1%,and the relative standard deviation?RSD?is from 3.92%to 5.08%.The parameters indicate that the proposed method is reliable in analyzing samples containing metronidazole.Therefore,the method designed in this paper can be used to quantify metronidazole in actual samples.