Catalytic hairpin assembly-based double-end G-quadruplex signal amplification for sensitive fluorescence detection of kanamycin

Abstract

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 was established by combining a 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 an aptamer sequence. Each of the two assembled probes encapsulated a G-rich sequence in the stem region such that the kanamycin-catalyzed CHA circuit produced active double-end G-quadruplex assemblies under the action of K⁺. The double-end G-quadruplex assemblies were further 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 confirmed that the result of double-end G-quadruplex assemblies was more than twice that of single-end G-quadruplex assemblies. The system exhibited a wide linear range of 1–500 nM for kanamycin, with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.27 nM and 0.89 nM, respectively. In addition, the sensing system effectively distinguished kanamycin from other antibiotics, thus exhibiting high selectivity for kanamycin. Moreover, the method successfully detected spiked kanamycin in cow milk samples, and the recoveries of spiked kanamycin were in the range of 99.00%–100.23% with RSDs in the range of 1.71–2.63%. All results confirm the application potential of this system in the field of food monitoring.