An enzyme-free and label-free fluorescent aptasensor for sensitive detection of kanamycin in milk samples based on hybridization chain reaction

Kanamycin residues accumulate in the human body and endanger human health, so the sensitive detection of kanamycin has important research significance. Herein, we constructed an enzyme-free and label-free fluorescent aptasensor for sensitive detection of kanamycin in milk samples based on hybridization chain reaction. We first designed a kanamycin recognition probe with a blocked aptamer and a blocked trigger sequence. Kanamycin specifically recognized the aptamer and then initiated the conformational transition process of the recognition probe to generate an aptamer-kanamycin complex with an activated trigger sequence. The trigger sequence initiated alternate hybridization of two partially complementary assembled probes to generate a long double -stranded DNA with many intact G-quadruplex structures for amplified, enzyme-free detection of kanamycin. The fluorescent dye N-methylmesoporphyrin IX was inserted into the intact G-quadruplex structures, and then it emitted a strong fluorescent signal for amplified, label-free detection of kanamycin. The linear range was from 2.0 to 200 nM for kanamycin, and the limit of detection (LOD), limit of quantification (LOQ) were 0.562 nM and 1.855 nM respectively. Moreover, the fluorescent aptasensor presented high selectivity for kanamycin compared with other antibiotics. Furthermore, the recoveries of spiked kanamycin in milk samples ranged from 98.66% to 100.7%, indicating that the fluorescent aptasensor had a great application prospect in food safety detection.