ABSTRACT

Bacterial infections are highly prevalent globally, and the health issues they induce often lead to numerous severe problems for human well-being, demanding timely and accurate detection strategies. Herein, We developed a universal electrochemical biosensor for pathogen screening, offering high sensitivity, specificity, rapidity, and multiplex detection. The platform integrates interdigitated electrodes for low-voltage pathogen lysis and nucleic acid release with asymmetric recombinase polymerase amplification (aRPA) to produce single-stranded DNA, simplifying extraction and reducing detection time. Screen-printed electrodes were carboxylated using diazonium salts to immobilize Fc-labeled hairpin DNA via amide bonds. Upon applying a positive voltage, amplified DNA hybridizes with the hairpin probes, distancing Fc molecules from the electrode surface and diminishing electrochemical signals, effectively eliminating false positives. Optimized conditions enabled detection sensitivities of 10 CFU/mL for Staphylococcus aureus and 5 CFU/mL for Acinetobacter baumannii. Additionally, Spiked testing in tap water, milk, and lake water demonstrated consistency with plate counting, validating the rapid system's accuracy and applicability. Remarkably, the assay time was reduced from 6 to 8 h to 25 min while maintaining pathogen specificity. This biosensor shows promise for foodborne pathogen surveillance, environmental monitoring, and point-of-care diagnostics, offering a streamlined platform for rapid, accurate pathogen identification.