Autocatalytic MNAzyme-integrated surface plasmon resonance biosensor for simultaneous detection of bacteria from nosocomial bloodstream infection specimens

Abstract Early accurate profiling of pathogenic bacteria in patients with nosocomial bloodstream infections (nBSIs) is crucial to optimize antibiotic therapy and patient survival. We present a novel autocatalytic Multi-component DNAzyme (MNAzyme)-integrated surface plasmon resonance (SPR) biosensor assay that rapidly and sensitively detects the DNA of three nBSI pathogenic bacteria. This strategy combines autocatalytic MNAzyme cleavage on magnetic beads, as the molecular recognition and signal amplification element, with hybridization chain reaction (HCR)-mediated nanowire assembly on SPR sensing film, as the signal readout and further amplification. Once the MNAzyme complex was activated as a biocatalyst by co-recognizing the bacterial DNA and initial probes, the MNAzyme substrate was catalytically cleaved into two DNA segments. One hybridized with immobilized probes triggering folded hairpin probes to self-assemble into massive nanowire structures via HCR on the sensing interface, and the other co-recognized the initial probes trigging another cycle of multi-turnover cleavage. As a result, the amount of cleaved DNA segments was increasing and greatly enhancing the SPR signal from the analyte. This assay system can provide the fast and quantitative analysis at low concentration (67 cfu/mL of S. aureus, 57 cfu/mL of K. pneumonia and 61 cfu/mL of E. coli, respectively) in a nonenzymatic manner. More importantly, the biosensor was successfully applied to DNA extracted from clinical blood specimens of patients with nBSIs, showing good consistence and practicability. The developed autocatalytic MNAzyme-integrated SPR biosensor provides a sensitive, rapid, reliable and culture-independent approach to efficiently determine multiple pathogenic bacteria for the early diagnosis of nBSIs.