An Amplification-Free Digital Assay Based on Primer Exchange Reaction-Mediated Botryoidal-Like Fluorescent Polystyrene Dots to Detect Multiple Pathogenic Bacteria

Multiple and ultrasensitive detection of pathogenic bacteria is critical but remains a challenge. Here, we introduce a digital assay for multiplexed and target DNA amplification-free detection of pathogenic bacteria using botryoidal-like fluorescent polystyrene dots (PS-dots), which were first prepared through the hybridization reaction between primer exchange reaction chains and polystyrene nanospheres that encapsulated polymer dots for signal preamplification. The pathogenic bacteria's DNA was cleavaged by the argonaute (Ago) protein-mediated multiple and precise cleavage reactions, where the obtained target sequences bridged the magnetic beads (MBs) and botryoidal-like PS-dots via a hybridization reaction, and the fluorescent MB-botryoidal PS-dot complexes were utilized as digital probes based on colors and sizes for digital encoding. An artificial-intelligence-fluorescent microsphere counting algorithm was applied to identify and count the fluorescent MBs for digital readout. This digital assay combined the ultrabright botryoidal-like PS-dots with