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

Abstract

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 <i>Clostridium butyricum</i> 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 10² CFU/mL within 1.5 h, demonstrating significant potential for food safety and clinical diagnostic applications.