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

Staphylococcus aureus (S. aureus) and Salmonella 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 S. aureus (1.06 × 10¹-1.06 × 10⁷ CFU/mL) and Salmonella (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.