Abstract
Colorimetric detection of intact bacteria is attractive for simple screening, but its sensitivity is often limited by inefficient surface labeling and insufficient signal generation. Herein, an aptamer-replaceable bifunctional DNA nanowire was developed for colorimetric detection of bacterial pathogens by integrating multivalent aptamer recognition and G-quadruplex/hemin catalysis on a hybridization chain reaction (HCR) scaffold. The HCR product served as a programmable nanowire scaffold, on which target-specific aptamer branches enabled bacterial labeling and repeated G-quadruplex/hemin DNAzyme branches generated an amplified ABTS colorimetric readout. By replacing only the aptamer branch, the same scaffold design was separately configured for Salmonella Typhimurium ( S. Typhimurium ) and Staphylococcus aureus ( S. aureus ) as representative Gram-negative and Gram-positive pathogens. Compared with the corresponding monovalent aptamer-G-quadruplex probes under matched total aptamer and G-quadruplex inputs, the nanowire produced 2.08- and 2.24-fold higher absorbance responses for S. Typhimurium and S. aureus , respectively. Under optimized conditions, the corresponding target-specific assays showed linear calibration ranges of 10 2 –10 7 CFU/mL, with limits of detection (LODs) of 16 and 14 CFU/mL for S. Typhimurium and S. aureus , respectively. The assay discriminated the targets from the tested non-target bacteria and retained analytical responses in spiked milk and serum samples, with LODs of 31–46 CFU/mL within 80min. Preliminary blind-sample analysis showed qualitative agreement with qPCR. This aptamer-replaceable DNA-scaffolded design provides a simple strategy for coupling multivalent bacterial labeling with catalytic colorimetric amplification.