The presence of trace kanamycin (KANA) residues in animal-derived foods from its improper use present a critical public health issue, making the urgent need for the development of sensitive and selective KANA detection methods. In this study, we developed molecularly imprinted polymers (MIPs)-based electrochemical sensor for the detection of KANA, incorporating Co, Mo-doped carbon nanofibres (Co, Mo@CNFs) to modify a glassy carbon electrode (GCE). Then, the MIPs layer was synthesized via electropolymerization using KANA as a template molecule and 3-aminophenylboronic acid (3-APBA) as the functional monomer (as MIPs/Co, Mo@CNFs/GCE), facilitating selective recognition through boric acid affinity. The presence of Co, Mo@CNFs enhanced electron transport across the otherwise insulating MIPs layer, thereby improving the sensor’s selective recognition capabilities. Under optimal conditions, the MIPs/Co, Mo@CNFs/GCE sensor demonstrated precise KANA detection within a concentration range of 1 × 10−2 to 1 × 105 nmol·L−1, achieving a detection limit of 2.56 pmol·L−1. Furthermore, the sensor’s applicability was validated in complex matrices, successfully detecting KANA in serum and milk samples with recovery rates ranging from 95.05 % to 116.43 %. These results confirmed that the proposed sensor is highly effective for detecting KANA in both biological and animal-origin food samples.
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