A Portable Colorimetric Device for Rapid Bacterial Detection with Cleavable Functional Nucleic Acid Probes for A Common Bacterial Endoribonuclease

ABSTRACT Rapid bacterial detection, including multiplex formats, is critical for clinical diagnostics, particularly when distinct pathogens cause similar symptoms and complicate differential diagnosis. Here we report a portable gold‐coated filter tip‐based assay (GFTA) for rapid, colorimetric, and two‐plex bacterial detection. Cleavable functional nucleic acid (FNA) probes were obtained through in vitro selection to target RNase H2, a conserved bacterial endoribonuclease. In GFTA, horseradish peroxidase (HRP) serves as the reporter, while a bacterium‐specific FNA probe functions both as the recognition element and as a molecular bridge linking HRP to a gold‐coated filter tip. When a target bacterium is present, its RNase H2 cleaves the FNA probe and releases HRP. The released enzyme is then captured by a second gold‐coated filter tip, where it catalyzes tetramethylbenzidine oxidation to generate a visible color change. The assay is completed within 30 min. GFTA enabled specific detection of Clostridioides difficile and Salmonella typhimurium at concentrations as low as as low as 1.3 × 10 3 CFU/mL in fecal samples. Clinical validation using 60 human fecal samples achieved 83.3% sensitivity and 100% specificity for C. difficile detection. Its simplicity, portability, and instrument‐free colorimetric readout make GFTA attractive for resource‐limited settings.