A Microfluidic Chip-Based Electrochemical Biosensor Coupled with CRISPR/Cas12a for Simultaneous Detection of Foodborne Pathogens

<i>Staphylococcus aureus</i> (<i>S. aureus</i>) and <i>Salmonella</i> frequently cocontaminate food products, posing significant health threats. We developed a microfluidic electrochemical biosensor that enables simultaneous detection of both pathogens within 65 min. The chip integrates sample loading, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based recognition into the upper layer. Reaction products enter a detection chamber with a single-walled carbon nanohorns-polypyrrole-gold nanoparticles (SWCNHs-ppy-AuNPs)-modified three-electrode system, where ssDNA probes act as signal reporters. Upon target recognition, Cas12a cleaves the probes, releasing electroactive molecules and reducing the current. The sensor exhibits linear responses for <i>S. aureus</i> (1.06 × 10¹-1.06 × 10⁷ CFU/mL) and <i>Salmonella</i> (1.04 × 10¹-1.04 × 10⁷ CFU/mL), with detection limits of 3 CFU/mL. This platform offers a rapid, sensitive, and accurate tool for on-site detection of foodborne pathogens in food products.