Multiplexed Pathogenic Bacteria Detection via a Two-Dimensional Encoded Fluorescent Microsphere System

We developed an advanced microscopy imaging platform enabling amplification-free, multiplex detection of pathogenic bacteria in food and clinical samples, eliminating the need for DNA extraction. This platform leverages two-dimensional encoded polystyrene (PS) microspheres and an Argonaute-based decoding system to create multiplexed signal libraries. Each PS microsphere probe, encoded with spectrally distinct fluorophores and differing particle sizes, achieves high fluorescence through a tetrahedral DNA-enhanced hybridization chain reaction (TDNA-HCR), significantly enhancing signal intensity and reducing reaction time by 67%. Pathogenic bacteria identification relies on aptamer-specific recognition, which transduces pathogenic bacteria presence into guide DNA (gDNA) signals that activate Clostridium butyricum Argonaute (CbAgo) for precise DNA cleavage, encoding pathogenic bacteria type and concentration in the color, size, and count of fluorescent PS probes. A custom computer vision-powered algorithm processes these signals, offering sensitive detection at 10 2 CFU/mL within 1.5 h, demonstrating significant potential for food safety and clinical diagnostic applications.