Toehold-mediated enzyme-free cascade signal amplification for ratiometric fluorescent detection of kanamycin

Abstract

• An enzyme-free cascade signal amplification for the detection of kanamycin has been constructed. • The out-put ratiometric signal was calculated by two reversible fluorescence signal changes. • The reversible signal change was recorded by synchronous fluorescence spectrum. Ratiometric measurements provide built-in self-calibration for signal correction that enabling eliminate interferences arise from target-independent factors. Herein, we developed a ratiometric fluorescence platform for the enzyme-free signal amplify determination of kanamycin. In our design, catalytic hairpin assembly (CHA) and DNAzyme-powered walking machine were coupled via toehold mediated strand displacement to construct cascade reaction. To construct ratiometric platform, two fluorophores were inserted in the upstream and downstream reactions, respectively. Once the cascade reaction is activated, through DNA hybridization and cleavage of DNAzyme, two fluorophores present reversible signal change, which can be recorded by synchronous spectrum simultaneously. Under the optimum condition, this cascade signal amplification exhibited sensitive determination of kanamycin as low as 21.71 pM. Furthermore, in the spiked milk and honey samples assay, this method also exhibited satisfied recovery results. This enzyme-free cascade signal amplification method with ratiometric signal out-put manifests potential application in antibiotics detection.