Preparation and characterization of DNA aptamers against roxithromycin

Abstract

Roxithromycin is a broad-spectrum antibiotic widely used in human and livestock. It is continually released and accumulated in our natural environment. It exhibited an extreme resistance to microbial biodegradation and has a serious impact on ecosystem and human health. It is in urgent need of establishing a rapid and efficient method for the detection of environmental roxithromycin. This study was based on capture-SELEX to select aptamers against roxithromycin from an initial library containing randomized ssDNA sequences. Candidate aptamers were obtained by 16 rounds of capture-SELEX process. Competent clones were prepared for sequencing. Clone Ap01 was chosen for further characterization. SYBR Green I fluorescence assays showed high affinity with roxithromycin. The dissociation constant of Ap01 was 0.46 ± 0.08 μM. Ap01 bound specifically to roxithromycin with capable of distinguish from non-roxithromycin macrolides. There was no cross reaction with the detected non-macrolide compounds. Accordingly, a colorimetric aptasensor has been developed. It has been demonstrated that the detection limit achieved 0.077 μM. To proof the concept, detections of roxithromycin contained in tap water and lake water were evaluated. It laid a foundation for further study on the detection of roxithromycin in actual aquatic environments.

Keywords: Aptamer; AuNPs; Roxithromycin; SELEX; SGI.