Programmable Electrochemical Cefapirin Antibiotic Aptasensor Powered by Highly Conductive Bimetallic NiCu(HITP) MOF and Dendritic DNA Amplifications

Abstract

Residues of veterinary antibiotics in animal-derived foods motivate the development of analytical methods that combine high sensitivity with straightforward operation. Cefapirin, a β-lactam used extensively for the treatment of bovine mastitis, may persist in milk and other commodities; therefore, sensitive detection of trace-level cefapirin residues is essential for food-safety compliance. Herein, we report an electrochemical aptasensor for cefapirin that integrates a bimetallic conductive NiCu(HITP) metal–organic framework (MOF) interface with a two-stage nucleic acid amplification cascade comprising self-priming strand displacement amplification (SPSDA) and nonlinear hybridization chain reaction (nHCR). The NiCu(HITP) MOF provides a highly conductive electrode coating and exhibits enhanced electrocatalytic activity toward hexaammineruthenium(III) chloride (RuHex). Upon target binding, aptamer recognition releases an initiator strand that triggers SPSDA, generating abundant single-stranded DNA products from a single recognition event. These products then initiate surface-confined nHCR to form dendritic DNA assemblies on the electrode. The polyanionic nanostructures further enrich RuHex via electrostatic accumulation, while the NiCu(HITP) MOF accelerates its electrochemical turnover, collectively yielding a significantly amplified catalytic current. The resulting aptasensor achieves a limit of detection of 31 pM for cefapirin and enables quantification in milk matrices. Because the recognition is programmed by the aptamer and the amplification modules are sequence-addressable, such sensor architecture can be readily reconfigured for other small molecule residues by substituting the aptamer and reoptimizing the associated DNA sequences to maintain the thermodynamic balance of the cascade reactions, offering a general route to trace-level surveillance of antibiotic contaminants in food.