Target Enzyme-Triggered Click Chemistry and Hybridization Chain Reaction for Fluorescence Nonculture Homogeneous Analysis of E. coli in Bloodstream Infections

Escherichia coli is the major pathogen that causes bloodstream infections (BSI). It is critical to develop nonculture identification methods which can meet the urgent need of clinical diagnosis and treatment. In this study, we reported a homogeneous fluorescence E. coli analysis system using β-galactosidase (β-Gal) as the biomarker and double-stranded DNA-templated copper nanoparticles (dsDNA-Cu NPs) as the signal output. The product of the enzymatic hydrolysis reaction, p-aminophenol (PAP), could reduce Cu2+ to Cu+, triggering the alkyne-azido cycloaddition reaction (CuAAC). Subsequently, the hybrid chain reaction (HCR) was initiated, producing the dsDNA template used to generate Cu NPs in situ. The system achieved a wide linear range for β-Gal and E. coli 1–104 mU/L and 10–2–10 colony-forming unit (CFU)/mL, and a detection limit of 0.3 mU/L and 0.003 CFU/mL, respectively. 65 samples (45 blood and 20 urine) were collected to evaluate the clinical practicality. The results demonstrated remarkable area under the curve (AUC) values of 0.95 and 0.916 from uncultured urine and blood, respectively. It had 100% specificity and 83.3% sensitivity. The whole duration of the strategy was 3.5 h, which significantly reduced the turnaround time (TAT) and facilitated early BSI diagnosis to improve patients’ prognosis. Our work had the potential to be an alternative to culture-based methods in clinics.