Electrochemical RNA-based aptasensor for neomycin detection in milk samples

Mastitis is one of the most common diseases in dairy cattle, leading to significant financial losses. Veterinary drugs containing neomycin (Neo) are frequently used to treat this condition. However, the overuse of neomycin results in antibiotic residues in milk, increased microorganism resistance, and reduced therapeutic efficacy. Therefore, there is a growing need for simple and cost-effective methods of neomycin detection. This study presents the development of an electrochemical aptasensor for neomycin detection in cow's milk. A 2′-O-methylated ssRNA aptamer (APT_Neo) was used as the receptor element and was covalently immobilized on the surface of the gold electrode alongside an anti-adhesive compound, 4-mercaptophenol (4-MCP). The interaction between neomycin and the 2′-O-methylated ssRNA aptamer was monitored through changes in the electron transfer resistance (ΔR) of the electrode, recorded using electrochemical impedance spectroscopy (EIS) in the presence of an electroactive label redox couple, Fe(CN)₆^3−/4- in the supporting solution. The developed electrochemical aptasensor exhibits high sensitivity and a low detection limit (36.3 nM in a 10-fold-diluted cow's milk sample), and selectivity for neomycin over structurally similar compounds (kanamycin and streptomycin) as well as antibiotics from tetracycline group (tetracycline and oxytetracycline). The proposed aptasensor is user-friendly and scalable, paving the way for the development of innovative miniaturized electrochemical sensor systems for practical applications in neomycin detection in cow's milk samples and food safety monitoring.