Selection And Application Of Aminoglycoside Antibiotics-specific Single-stranded DNA Aptamers

Both gentamicin and tobramycin belong to aminoglycoside antibiotics,which have antibacterial effect against a variety of Gram-negative bacteria.However,the excessive use of aminoglycoside antibiotics leads to their residue in animal-derived foods,which can finally accumulate in human through the food chain and cause risks to human health.The detection of residual aminoglycoside antibiotics using aptamers as recognition elements is of great importance in food and environment safety and other related areas.In this thesis,single-stranded DNA（ssDNA）aptamers specific for gentamicin were firstly screened,and their structures and properties were characterized.Then the obtained optimal aptamer as well as the reported tobramycin-specific aptamer were utilized to construct analytical method for these antibiotics.The selection of the aptamers was carried out using gentamicin-immobilized Sepharose affinity chromatography-based systematic evolution of ligands by exponential enrichment（SELEX）technique from an ssDN A initial library（full length of 79 nucleotides containing 35 random nucleotides in the center）.The affinity of the selected pool reached saturated after nine rounds of selection.The screened products of the last round were amplified by PC R.After transformation,positive clones were picked and sequenced.The nucleotide sequences of these 34 aptamers were analyzed by DNAMAN and divided into six families according to their homology.The secondary structure and stability of these aptamers were analyzed by Mfold online software.The values of dissociation constants（K_d）of four of the most stable aptamers（Ap-5,-8,-22 and-26）were determined by fluorescent method,which were74.50±9.77 nmol·L^-1,80.25±9.04 nmol·L^-1,96.34±18.96 nmol·L^-1 and 14.00±3.34nmol·L^-1,respectively.Among them,Ap-26 showed the highest affinity for gentamicin.The aptamer showed poor binding ability to other antibiotics,indicating satisfactory specificity.Then the interaction between Ap-26 and gentamicin was simulated by Autodock 4.0 software.The results showed that nucleotides No.17-20and 54-56 are the binding sites.Using the reported tobramycin-specific aptamer and the obtained gentamicin-specific aptamer in this thesis,a colorimetric method for the determination of tobramycin and gentamicin based on the changes of the color of gold nanoparticles（AuNPs）was established.In the concentration range of 100¹400nmol·L^-11 and 50⁷00 nmol·L^-1,respectively,the absorbance of AuNPs solution at 520nm was gradually decreased with the increased concentration of tobramycin and gentamicin,where the linear relationships were derived,with the low detection limits of 37.9 nmol·L^-1and 11.50 nmol·L^-1 for tobramycin and gentamycin,respectively.The specificity of this method was also verified.Then the method was further applied to detect gentamicin in milk samples,and the results showed that the matrix of milk hardly influences the determination of gentamicin.Therefore,the screened aptamer and the established assay can be well applied to detect antibiotic residues in actual samples.