Fe₃O₄@Au Nanoparticle-Enabled Magnetic Separation Coupled with CRISPR/Cas12a for Ultrasensitive Detection of Foodborne Pathogens

The rapid detection of foodborne pathogens, such as <i>Staphylococcus aureus</i> and <i>Salmonella</i>, is critical for ensuring food safety. Herein, we present a magnetically controlled electrochemical biosensor integrating CRISPR/Cas12a with Fe₃O₄@Au nanoparticles designed to achieve ultrasensitive and multiplexed detection. By utilization of the magnetic separation of CRISPR-cleaved ssDNA from Fe₃O₄@Au nanoparticles, the sensor circumvents intricate electrode modifications, enabling direct signal readout. This approach expedites the workflow to 65 min while achieving a detection limit of 2 CFU/mL. Additionally, the sensor exhibits signal stability over 45 days and demonstrates its versatility by enabling the separate detection of both Gram-positive (<i>S. aureus</i>) and Gram-negative (<i>Salmonella</i>) pathogens. With validation in milk samples with high interference resistance, this platform bridges CRISPR programmability with practical deployability, offering a robust solution for on-site monitoring. The innovation lies in its simplified design, enhanced stability, and clinical versatility, setting a new benchmark for rapid, low-cost pathogen detection in resource-limited environments.