Label-free characterization of aptamer-oxytetracycline binding and detection of oxytetracycline with fluorescently labeled aptamer based on fluorescence anisotropy analysis

Oxytetracycline (OTC) is one commonly used antibiotic, and its overuse has led to contamination in environment and food, posing substantial risks to both human health and ecology systems. Development of sensitive and rapid methods for OTC detection is in demands. High-affinity DNA aptamers (OTC5) for OTC recently have been selected, providing new tools for OTC detection. In this work, based on aptamer-binding induced increase of fluorescence anisotropy (FA) of OTC, we utilized FA analysis to systematically characterize affinity of aptamers and mutant sequences. We identified the key regions of OTC5 aptamer for OTC recognition and conserved bases of aptamer sequence by fully testing single base mutation. In addition, we found the OTC5 aptamer with mutation of C10 base to deoxyuridine (dU) (OTC5-C10dU) showed enhanced affinity. We further developed a direct FA sensor for OTC detection by using this mutated aptamer with internal tetramethylrhodamine (TMR) label. The OTC binding caused significant changes in FA signals of TMR due to the conformational changes of the TMR-labeled aptamers. We screened out the aptamer with TMR label at the 18th T base, which produced the most sensitive FA response to OTC. Using this TMR-labeled aptamer, we achieved sensitive and selective FA detection of OTC within a short analysis time of 10 min, with a detection limit as low as 0.6 nM (0.28 µg L −1 ). This FA aptamer sensor allowed the detection of spiked OTC in diluted skim milk and lake water samples, showing potential for application in real food and environmental samples.