Rapid multiplex detection of bloodstream pathogens using microsphere suspension arrays functionalized with RNA-cleaving fluorogenic DNAzymes

Bloodstream infections (BSIs) are a major cause of morbidity and mortality worldwide, and delays in pathogen identification significantly increase mortality risk. Here, we developed a rapid, sensitive, and multiplex suspension array platform integrating RNA-cleaving fluorogenic DNAzymes (RFDs) with flow cytometry (FCM) for the direct identification of BSI pathogens from positive blood culture bottles. Our platform utilized fluorescently barcoded microspheres functionalized with pathogen-specific RFDs targeting Staphylococcus aureus (SA), Escherichia coli (EC), Klebsiella pneumoniae (KP), and Clostridioides difficile (CD). Under optimized conditions, the assay achieved a detection limit of ~10 3 CFU/mL, exhibited excellent specificity without cross-reactivity, and enabled simultaneous detection of four pathogens in a single measurement. Clinical validation using 135 patient samples demonstrated 100% concordance with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) results for SA and EC, and 90% concordance for KP, with accurate identification of dual infections. By eliminating the need for subculture and leveraging widely available FCM instrumentation, this platform substantially shortens the time to pathogen identification and holds promise as a scalable tool for accelerating targeted antimicrobial therapy in BSI patients.