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

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 × 10³ 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.