Target-triggered exponential amplification-enhanced CRISPR/Cas12a system with a fluorescence-quenching lateral flow assay for ultrasensitive detection of Salmonella typhimurium

A target DNA preamplification-free CRISPR/Cas12a sensing platform was established via TEA system. • TEA system achieves a 10 6 -fold signal enhancement compared with conventional CRISPR/Cas12a system. • TEA-FQLFA system achieves attomolar-level detection of target dsDNA (0.081 aM). • TEA-FQLFA system enables sensitive detection of Salmonella typhimurium with a LOD of 6.94 CFU/mL. The use of the CRISPR/Cas12a system combined with lateral flow assays shows significant promise for point-of-care testing in resource-limited regions. However, the use of isothermal amplification technologies to preenrich target DNA fragments inherently increases the risk of aerosol contamination during strip testing. Here, we introduce a target-triggered exponential amplification-enhanced CRISPR/Cas12a system combined with a fluorescence-quenching lateral flow assay (TEA-FQLFA) for ultrasensitive detection of Salmonella typhimurium . The TEA system converts a single Cas12a recognition event into sequential dual Cas12a activation, initiating a cleavage cascade amplification that achieves a 10 6 -fold signal enhancement without the need for a target DNA preamplification operation. Moreover, hyperbranched gold-coated polymer microspheres (HGPMs), characterized by ultrahigh extinction coefficients and a broad absorption band in the 300–800 nm range, are utilized as fluorescence quenching reporters of the FQLFA for simultaneous colorimetric visualization and fluorescence-based quantitative detection of target DNA concentration. By leveraging the synergistic integration of TEA-mediated signal amplification and HGPM-enabled optical signal enhancement, the TEA-FQLFA system achieves attomolar-level detection of target DNA (0.081 aM) and enables sensitive detection of Salmonella typhimurium in complex food matrices, with detection limits of 7.95 CFU/mL in colorimetric mode and 6.94 CFU/mL in fluorescence mode. In summary, this work establishes a versatile platform that integrates cascade signal amplification with dual-mode lateral-flow for ultrasensitive food safety detection.