Preparation of DNA aptamer and development of lateral flow aptasensor combining recombinase polymerase amplification for detection of erythromycin

Abstract

Erythromycin has polluted our aquatic environment for decades, leading to the risk of bacterial resistance and harmful effects on human beings, wildlife and ecosystem. There is an urgent demand of developing a portable tool capable of detecting erythromycin on site. In this study, ten aptamer candidates against erythromycin were prepared through Capture-SELEX (systematic evolution of ligands by exponential enrichment) process in 20 rounds. Aptamer candidate Ery_06 with the highest enrichment was chosen for further study, whose affinity was characterized by gold nanoparticles colorimetric assay, quartz crystal microbalance with dissipation and agarose chasing diffusion assay. It was determined by SYBR Green I fluorimetric assay that the characterized aptamer binds to erythromycin with high affinity (Kd: 20 ± 9 nM). Its specificity was also characterized by distinguishing erythromycin from different antibiotics tested. A novel lateral flow aptasensor was constructed by using the newly identified aptamer combined with recombinase polymerase amplification (RPA) and lateral flow strip (LFS). Aptamer acted as a sensing element anchoring on the surface of solid phase could be eluted by erythromycin. RPA functioned to amplify and convert the signal to be visible on LFS. The lateral flow was completed in 15 min, achieving a detection limit of 3 pM. The application feasibility of the aptasensor was proved by the detection of tap water samples spiked with erythromycin.