A enzyme-free fluorescence quenching sensor for amplified detection of kanamycin in milk based on competitive triggering strategies

In this work, we constructed a FAM fluorescence quenching biosensor based on an aptamer competition recognition and enzyme-free amplification strategy. We design a competing unit consisting of an aptamer chain and a complementary chain, and a catalytic hairpin self-assembly (CHA) unit consisting of two hairpins in which the complementary chain can trigger the catalytic hairpin self-assembly. In the initial state, the aptamer chain is combined with the complementary chain, the catalytic hairpin self-assembly unit is inhibited, the FAM fluorescence group was far away from the BHQ1 quenching group, and the fluorescence is turn-on. In the presence of kanamycin, the aptamer chain recognizes kanamycin and doesn't form double chains, resulting in the free complementary chain triggering hairpin 1 (H1), and then H1 triggering hairpin 2 (H2), FAM fluorophore is close to the BHQ1 quenching group, and the fluorescence is off–on. When H1 and H2 form a cyclic reaction, enzyme-free amplification is achieved and there is significant output of the fluorescence signal. Therefore, the biosensor has good performance in detecting kanamycin, the detection line is 54 nM, the linear range is 54 nM–0.9 μM, and it can achieve highly selective detection of kanamycin. Kanamycin residue may cause serious harm to human health. The high sensitivity detection of kanamycin is urgent, so this project has a great application potential for food detection.