A ratiometric fluorescent biosensor based on cascaded amplification strategy for ultrasensitive detection of kanamycin

Antibiotic residues in food products significantly threaten public health. In the present work, a fluorescence resonance energy transfer (FRET)-based ratiometric fluorescent biosensor was developed for high sensitive and selective detection of kanamycin by combining aptamer-recognition assay and signal amplification strategy. To improve detection sensitivity, the optimum FRET efficiency of donor-acceptor was explored by modulating the separation distance between the donor (Cy3) and the acceptor (Cy5). In the developed biosensing system, kanamycin could be trapped by its aptamer, which triggered a polymerase-catalyzed amplification (PCA) process to autonomously generate a single-strand DNA. In subsequence, the generated DNA strand initiated the second amplification process of catalyzed hairpin assembly (CHA) to cause fluorescence decreasing of Cy3 and increasing of Cy5. The signal change caused by FRET of donor (Cy3)-acceptor (Cy5) was then augmented by the PCA and CHA processes. The constructed biosensors results in high sensitive detection of kanamycin as low as 0.29 nM within a linear range from 1.0 nM to 80.0 nM. The potential application of the developed biosensing system was evaluated with milk as real sample. The provided strategy could serve as a prototype for developing FRET-based ratiometric bioassays.