Low-background fluorescent biosensor based on graphene oxide and aptamer biorecognition for sensitive detection of kanamycin

The detection of kanamycin has attracted extensive attention in the field of food safety. A label-free, low-background fluorescent biosensor was successfully constructed based on graphene oxide and aptamer biorecognition in this paper for kanamycin detection in milk samples. In this biosensor, the integrated probe with the aptamer sequence of kanamycin and the G-rich sequence was designed. The aptamer sequence of kanamycin was used as the recognition part, the G-quadruplex/N-methyl mesoporphyrin IX (NMM) complex as the fluorescent probe, and graphene oxide as the fluorescent quencher. Initially, the integrated probe could be adsorbed on the graphene oxide surface through π-π stacking interactions, while the fluorescence of NMM could be efficiently quenched because of the resonance energy transfer between NMM and graphene oxide, which was beneficial to the low background signal of the system. During the detection process, the aptamer sequence preferred to bind to kanamycin, forming aptamer-kanamycin complex. At the same time, the G-rich sequence folded into G-quadruplex structure that specifically bound to NMM under the action of K+, forming G-quadruplex/NMM complex. Under the synergistic effects of the two complexes, the integrated probe with aptamer-kanamycin complex and G-quadruplex/NMM complex was released from the graphene oxide surface due to the steric hindrance effect, restoring the strong fluorescence signal. By combining graphene oxide and G-quadruplex/NMM complex, the fluorescent biosensor could effectively achieve label-free and low-background detection of kanamycin. The linear calibration curve was in the range of 1 ~ 400nM with a detection limit of 0.60nM for kanamycin. It was successfully applied to analyze raw milk samples (spiked kanamycin) with recoveries of 98.33% to 101.25% and also exhibited high selectivity for kanamycin. The experimental data showed that the biosensor was a potentially powerful tool for monitoring food safety.