Target-to-Signal Conversion and Spatial Enrichment Cascade Boosts CRISPR/Cas12a Biosensing for Trace-Level Pathogen Detection

Abstract

typhimurium is a widely distributed and highly pathogenic Gram-negative bacterium capable of causing outbreaks of foodborne illness through the ingestion of contaminated food. Consequently, the development of a rapid and accurate detection strategy for S. Typhimurium is of paramount importance. This study constructed a CRISPR/Cas12a-activated DNA nanowire biosensor based on DNAzyme-catalysed amplification for the sensitive detection of Salmonella Typhimurium. Specific cleavage by DNAzymes generates single-stranded DNA to activate HCR, enabling the serial HCR signal enrichment of three DNA hairpin structures, whose branched ends bear PAM sequences. Subsequent introduction of CRISPR/Cas12a cleaves the DNA NW, restoring quenched fluorescence signals to achieve ultra-sensitive detection of Salmonella Typhimurium. Under optimal conditions, this biosensor exhibits a linear detection range of 10 ¹ -10 ⁵ cfu/mL, with an estimated limit of detection (LOD) of 6.26 cfu/mL. The method offers advantages of straightforward operation, rapid response, and high sensitivity. Furthermore, by modifying the recognition module and primer sequences, this biosensor holds promise for highly sensitive detection of multiple foodborne pathogens.