PCR-free real-time antibiotic-resistant gene detection in wastewater through Impedimetric DNA sensor based on gold-modified NiO nanostructures

Antimicrobial resistance (AMR) poses a significant global health threat, necessitating robust and widespread detection strategies to combat its diffusion. This study addresses the urgent need for sensitive and specific detection of antibiotic-resistant genes (ARGs) by developing an electrochemical DNA sensor based on Au-decorated NiO nanowalls for efficient detection of blaCTX-M (β-lactamase CTX-M gene), an ARG conferring resistance against critical antibiotics like cephalosporin. The sensor fabrication involves Ni(OH) 2 nanowall growth via a simple chemical method, followed by thermal conversion to NiO and Au layer sputtering, enabling precise immobilization of a thiolated probe DNA for blaCTX-M. Morphological characterizations of the electrode confirm the nanowalls' high surface area and uniform probe DNA distribution, which are crucial factors for obtaining high-performance DNA detection. Indeed, after incubation with different concentrations of synthetic target DNA and real DNA samples collected from wastewater plants, electrochemical impedance spectroscopy (EIS) analysis reveals the high sensitivity of the electrode, capable of detecting blaCTX-M from a high concentration of 5 μM for the synthetic oligonucleotide down to 2.7 fM for the real DNA, and an excellent selectivity against non-complementary ARGs oligonucleotides. These results, combined with the scalable fabrication of Au/NiO nanowalls, make this electrode a promising candidate for real-time monitoring of ARGs in contaminated water sources, as well as other pathogens upon appropriate selection of the probe DNA. • Development of a PCR-free sensor for antibiotic-resistant gene (ARG) detection in wastewater. • Au/NiO nanowalls provide high sensitivity down to 2.7 fM blaCTX-M DNA. • Selective DNA recognition via probe-target hybridization with no false positives. • Impedance-based detection allows real-time, label-free ARGs monitoring. • Sensor fabrication is low-cost, scalable, and compatible with portable devices.