Magnetic relaxation switching biosensor based on CRISPR-mediated cascade reaction for the amplification-free...

Sensitive and accurate detection of foodborne pathogens is crucial for preventing foodborne outbreaks and ensuring public health safety, but challenged by extremely low infectious doses of many foodborne pathogens and matrix interference effects. In this study, we developed a magnetic relaxation switching biosensor boosted by CRISPR/Cas12a-mediated enzymatic cascade reaction (CMCR-MRS) for amplification-free detection of Salmonella typhimurium (S. typhimurium). CRISPR/Cas12a was designed to precisely target the pathogen-specific DNA and efficiently cleaved single-stranded DNA (ssDNA) immobilized on the magnetic nanoparticle-alkaline phosphatase (MNP-ALP) probes through trans-cleavage activity. Subsequently, the released ALP facilitated the conversion of paramagnetic Mn(VII) to Mn(II), which resulted in the change of transverse relaxation time (T₂), achieving a high signal-to-background ratio with superior biocompatibility and minimal background interference, making them highly advantageous for sensitive detection in complex biological samples. Our assay showed a broad dynamic range from 40 to 10⁷ CFU/mL and a limit of detection (LOD) of 10 CFU/mL for S. typhimurium without amplification. Furthermore, it has been successfully validated in real food samples, demonstrating strong consistency (R² = 0.989) with the quantitative real-time polymerase chain reaction (qPCR) test. CMCR-MRS can serve as a highly effective and reliable strategy for achieving sensitive and accurate pathogen detection.