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 106-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.
Cell-based biohybrid sensors using insect olfactory receptors (ORs) have emerged as promising sensing platforms for the selective and sensitive detection of biomarkers. A single sensor cell provides a weak signal, so the readout of most biohybrid sensors is the accumulated response of a whole population of cells. Response heterogeneity within engineered sensor cell populations can limit sensor sensitivity because low-responding cells dilute the bulk-averaged signal. Selecting highly responsive subclones could improve sensor sensitivity. However, most insect cell lines proliferate poorly at low cell density, making it challenging to obtain expandable colonies from a single insect cell. Here, we present a colony-isolation workflow for insect sensor cells using hollow hydrogel microparticles (PicoShells). Insect sensor cells were encapsulated in PicoShells at limiting dilutions to physically isolate one or a few cells. The encapsulated insect sensor cells were co-cultured with non-encapsulated support cells in the same suspension culture to promote colony formation. Individual colony-containing PicoShells were isolated and expanded into subclonal cell lines. Functional screening using a portable fluorescence detector and a microwell array identified a highly responsive subclone. In a human urine sample spiked with acetophenone, a candidate volatile biomarker of disease, this subclone showed an approximately 2.4-fold higher response sensitivity than the parental cell line, and the limit of detection was reduced from 1.56 ± 0.17 μM to 0.93 ± 0.22 μM for this subclone. These results indicate that colony isolation using PicoShells, combined with functional screening, provides a practical strategy for improving the sensitivity of insect cell biohybrid sensors.
