Selection and identification of a DNA aptamer for fluorescent detection of netilmicin

Abstract

Netilmicin (NET) is an antibiotic widely used in healthcare and agriculture, but it can accumulate in the environment to threat human health. Netilmicin (NET) is an antibiotic used for veterinary purposes, for human therapy and for agricultural purposes. Therefore, there is a need to develop high-sensitive measuring methods to detect NET. Aptamer-based detecting methods are highly sensitive, inexpensive, and portable. In this study, we developed an aptamer-based fluorescence method to detect and quantify NET. NET was first conjugated to magnetic beads by amidation reaction and then NET-coated beads were used as the stationary phase to isolate aptamers by systematic evolution of ligands by exponential enrichment (SELEX) screening method. After ten rounds of SELEX screening, 32 aptamers with NET-binding affinity were obtained and the candidate aptamer APT-21 was finally chosen by comprehensively comparing their secondary structure characters and NET-binding affinity. APT-21 bound to NET with high affinity (Kd = 194.1 nmol/L) and high specificity that it displayed low cross-binding activities on 7 different structural analogs. We also developed a fluorometric assay using SYBR Green I (SG-I) and the APT-21. Key experimental parameters were optimized, including buffer system, SG-I and APT-21 reaction time, SG-I concentration, and aptamer concentration, to improve the detecting sensitivity. Our results suggest that the low limit of detection (LOD) of this method reached a low level of 1.95 nM and it also exhibited a good linear range up to 200 nM. Moreover, we successfully applied our method to detect the NET spiked in tap water and river water with good recoveries in the range from 97% to 111%. In conclusion, our current study isolated a NET-specific aptamer and developed an aptamer-based quantification method, which is promising to apply to detect NET in environmental samples.