Electrochemical Detection of Kanamycin Utilizing Analyte‐Induced Homogeneous Biorecognition and Atom Transfer Radical Polymerization

The rapid and accurate detection of antibiotic residues remains a critical challenge in agricultural food safety and veterinary drug analysis. Herein, we report a novel electrochemical biosensor for kanamycin (KAN) detection, which integrates analyte‐induced homogeneous biorecognition with atom transfer radical polymerization for signal amplification. In the presence of KAN, a DNA complex containing a KAN‐specific aptamer and a hairpin DNA (HP) undergoes Exonuclease I (Exo I)‐assisted cyclic digestion, releasing abundant HP strands. These strands hybridize with DNA probes on the electrode to form Y‐shaped nanostructures with terminal azide groups. Subsequently, a conductive polymer film is grown on the electrode via Cu(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) reaction and electrochemically mediated atom transfer radical polymerization (eATRP) reaction. This process culminates in a cascade signal amplification, significantly enhancing the sensor’s sensitivity. The successful stepwise formation of the polymer layer and the resulting surface morphology were further confirmed by scanning electron microscopy. Under optimal conditions, the biosensor exhibits excellent analytical performance, with a linear detection range from 0.001 to 100 µg/mL and a low detection limit of 0.97 ng/mL (S/N = 3), as well as strong anti‐interference capability against potentially coexisting substances. This strategy not only meets the requirements for specific and accurate KAN residue detection but also holds great potential for ensuring product quality and safeguarding human health.