A CRISPR/Cas12a-hyperbranched rolling circle amplification sensor for ultrasensitive visual bacteria detection

The swift spread of pathogenic bacteria via food, air, and water poses severe risks to human health. Conventional detection methods often suffer from time-consuming operations, bulky instruments, and insufficient sensitivity for on-site screening, highlighting an urgent demand for sensitive and visual platforms. Herein, a detection platform (Cas12a-HRCA) was constructed for sensitive pathogen quantification by integrating hyperbranched rolling circle amplification (HRCA), CRISPR/Cas12a system, copper fluorescence nanoparticles (CuNPs) and smartphone-based signal readout. In this strategy, pathogen target-activated CRISPR/Cas12a precisely regulates HRCA initiation, which exponentially generates AT-TA-rich sequences. These products serve as templates for the self-assembly of the fluorescence CuNPs, achieving integrated rapid signal amplification without requiring sample preprocessing steps. Combined with smartphone-based RGB analysis, the platform enables direct, on-site quantitative readout. Cas12a-HRCA demonstrated exceptional performance for S. aureus, with a detection limit of 1 CFU/mL, high specificity, and 91%-106% reliable recovery in spiked milk samples without pre-enrichment or purification, showcasing great potential for point-of-need food safety monitoring. the detection of various pathogenic bacteria in food, clinical or environmental settings.