A Colorimetric Biosensor Integrating Rotifer-Mimicking Magnetic Separation with RAA/CRISPR-Cas12a for Rapid and Sensitive Detection of Salmonella

Efficient detection of foodborne bacteria is crucial for ensuring food safety, yet current methods often fall short in balancing speed, accuracy, sensitivity, and cost. This study presents an integrated biosensing platform for the rapid and sensitive detection of Salmonella in large-volume food samples. The platform incorporates a Rotifer-Mimicking Magnetic Separator (RMMS) that enhances the sample pretreatment by effectively mixing and isolating the bacteria from the sample. Coupled with this, the colorimetric biosensor utilizes a streamlined one-pot system that combines Recombinase Aided Amplification (RAA), betaine, and CRISPR-Cas12a to enable efficient pathogen detection. Initially, phenylboronic acid-modified magnetic beads (PBA-MBs) capture Salmonella, forming bacteria-PBA-MB complexes, which are then isolated using the RMMS. Target DNA amplicons activate ribonucleoprotein complexes, and Au@PtNPs-MBs with linker single DNAs are cleaved to release Au@PtNPs. The Au@PtNPs catalyze the H 2 O 2 -3,3′,5,5′-tetramethylbenzidine, producing a visible blue color that indicates Salmonella concentration. This biosensor successfully detects Salmonella in 40 mL spiked milk samples within 75 min, achieving a detection limit of 89 CFU/mL. This work offers a simple, sensitive, low-cost detection method with potential applications in on-site testing, significantly enhancing food safety monitoring.