Homogeneous electrochemical analysis of β-glycosidase and pathogenic bacteria in clinical sample assisted by click chemistry and enzyme-free catalytic hairpin assembly

Rapid, simple, and highly sensitive detection of pathogenic bacteria is essential for clinical disease diagnosis and early screening. Here, we sought to construct a homogeneous electrochemical analysis system for the detection of Escherichia coli (E. coli) based on Cu+ catalyzed alkyne-azido cycloaddition reaction (CuAAC) and catalytic hairpin assembly (CHA) amplification, with β-glycosidase (β-Gal) as a marker and G-quadruplex as a regulator of the signal change of methylene blue (MB). β-Gal hydrolyzes the 4-Aminophenyl β-D-galactopyranoside (PAPG), generating p-aminophenol (PAP), which reduces Cu2+ to Cu+. Cu+ initiates the CuAAC reaction to generate long single-stranded DNA and triggers the CHA reaction simultaneously. This strategy realizes the quantitative analysis of enzyme and E. coli as low as 0.01 U/L and 0.2 CFU/mL, respectively, demonstrating good target selectivity. The quantitative results of E. coli in 40 clinical urine samples (2 healthy, 25 E. coli positive infections, and 13 non-E. coli infections) demonstrated high consistency with clinical culture and colony counting results. This approach allows for the distinction of E. coli from other bacterial infections and accurate quantification of E. coli concentration in urine with various infection degrees in clinical reports.