A Cu₂O/hemin-GO nanozyme with aptamer-enhanced peroxidase-mimic activity for colorimetric detection of kanamycin in milk

Kanamycin (KAN) is an aminoglycoside antibiotic employed for the treatment of bacterial infections in livestock and poultry and is widely used as a veterinary drug in animal husbandry. Its overuse poses serious threats to public health and ecosystems; while traditional detection methods cannot meet the requirements of widespread application and rapid detection, developing rapid and reliable technology for KAN detection is essential. In this study, a colorimetric aptasensor was developed to rapidly detect KAN residues, leveraging the enhanced catalytic effect of aptamers on the peroxidase-like activity of the Cu₂O/hemin-graphene oxide (Cu₂O/hemin-GO) nanozyme. In the presence of hydrogen peroxide, the Cu₂O/hemin-GO nanozyme demonstrated excellent peroxidase-like catalytic activity, oxidizing 3,3',5,5'-tetramethylbenzidine (TMB). The adsorption of aptamers on the nanozyme surface increased the negative charge density and significantly enhanced the affinity of the nanozyme for the positively charged chromogenic substrate TMB, boosting the catalytic activity nearly threefold. Upon exposure to KAN, the specific binding of aptamers to KAN reduced their adsorption on the Cu₂O/hemin-GO nanozyme, leading to a decrease in the peroxidase-like activity of the Cu₂O/hemin-GO nanozyme and a corresponding reduction in color change. The aptasensor exhibited excellent sensitivity with a linear detection range from 0.1 pM to 1 μM and a detection limit of 16 fM. The recovery rates for KAN in milk samples ranged from 92.05% to 110.71%. Furthermore, the colorimetric aptasensor demonstrated high selectivity and reproducibility. Overall, the colorimetric approach achieved the economical, simple and sensitive detection of KAN residues, indicating its promising potential in real-world applications. In addition, this sensing method could be applied to other targets by replacing the aptamer, providing a strategy for developing sensors facilitating rapid detection.