Cascaded and nonlinear DNA assembly amplification for sensitive and aptamer-based detection of kanamycin

Simple, selective and sensitive monitoring of antibiotic residues in food is essential for food safety and human health because of its side effects upon inappropriate usage. Here, with a new label- and enzyme-free significant signal enhancement approach by the coupling of catalytic hairpin assembly (CHA) with nonlinear hybridization chain reaction (nHCR), we developed a fluorescence aptamer sensor for the detection of trace kanamycin in milk samples with high selectivity and sensitivity. The binding of the target kanamycin to the aptamer probe could initiate the CHA between two hairpins for the formation of partial DNA duplexes, which further triggered the nHCR of other three hairpins to yield branched DNA complexes with a multitude of active G-quadruplex structures. The subsequent intercalation of the organic dye, thioflavin T, into G-quadruplex structures resulted in significantly enhanced fluorescence responses for realizing sensitive sensing of kanamycin in the dynamic range of 0.1-300 nM with a detection limit of 46.1 pM. Besides, this strategy could also achieve the monitoring of kanamycin selectively in spiked milk samples. With features of high sensitivity and simplicity in a non-enzyme/label fashion, our signal amplification strategy has high potentials for establishing sensitive and convenient means to monitor various antibiotics.