Magnetic Nanoparticle-Assisted Cleavage-Based Detection of Foodborne Pathogens Using Nuclease-Responsive DNA Probes

We report a nuclease-responsive DNA probe platform integrated with magnetic separation for ultrarapid and sensitive bacterial detection. The sensing strategy employs dual-labeled DNA probes featuring a 3'-biotin and a 5'-signal reporter (fluorophore, enzyme, or chromophore), which undergo cleavage by nucleases released from viable bacteria. Streptavidin-conjugated magnetic nanoparticles enable efficient separation of cleaved signal fragments from biotin bound strands, thereby minimizing background noise and maximizing assay specificity. The entire workflow-including bacterial lysis, enzymatic cleavage, magnetic separation, and signal readout-can be completed within 3 min. Using Escherichia coli as a model, the platform achieved detection limits below 10 CFU per reaction with fluorescence or chemiluminescence methods, and as few as 10² CFU per reaction with a colorimetric method. This platform selectively detected viable bacteria and demonstrates real-world applicability through a custom-designed point-of-care testing (POCT) device, which successfully identified simulated bacterial contamination in various food samples. Overall, this technology offers a practical and rapid solution for frontline microbial surveillance in food and environmental safety.