Sensitive detection of antibiotic residues in food is of great significance for food safety monitoring. Herein, a novel nucleic acid amplification sensing platform for protein enzyme-free, label-free, and homogeneous fluorescence detection of kanamycin had been established through combining catalytic hairpin assembly (CHA) circuit and double-end G-quadruplex signal amplification. Here, kanamycin initiated the CHA circuit between two assembled probes by specifically binding to aptamer sequence. Each of the two assembled probes encapsulated the G-rich sequence in the stem region, so kanamycin-catalyzed CHA circuit produced the active double-end G-quadruplex assemblies under the action of K+. The double-end G-quadruplex assemblies further were combined with N-methylmesoporphyrin IX to induce a distinctly amplified fluorescence signal. The CHA-based double-end G-quadruplex signal amplification significantly improved the detection sensitivity of kanamycin. The fluorescence signal increment results confirmed that the result of double-end G-quadruplex assemblies was more than twice higher than those of single-end G-quadruplex assemblies. The system exhibited a wide linear range of 1 nM to 500 nM for kanamycin, and the limit of detection (LOD) and the limit of quantitation (LOQ) were 0.27 nM and 0.89 nM respectively. In addition, the sensing system effectively distinguished kanamycin from other antibiotics, exhibiting high selectivity for kanamycin. The method also successfully detected spiked kanamycin in cow milk samples, and the recoveries of spiked kanamycin were in range of 99.00%―100.23% with RSDs in range of 1.71%―2.63%. All results verified the application potential of the system in the field of food detection.

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