A dual-mode vertical flow assay for species-specific identification and total bacteria load assessment from a single...

Rapid urine screening would benefit from simultaneous determination of pathogen identity and clinically relevant bacteria burden, yet most paper-based assays provide only one of these outputs. Here, we report a dual-mode vertical flow assay that combines species-specific HRP-antibody/TMB immunodetection with WST-8-mPMS metabolic quantification in spatially separated detection spots within an integrated vertical flow assay device. The device employs a vertically layered membrane stack with graded pore sizes to regulate capillary-driven flow and residence time, enabling efficient analyte transport and signal development. Quantitative analysis is achieved using smartphone-based RGB imaging converted to grayscale intensity. This platform enables simultaneous bacteria identification and quantification from a single urine sample, demonstrating threshold-oriented detection around the clinically relevant bacteriuria level of 10⁵ CFU mL^-1 in both tryptic soy broth and urine samples. This work demonstrates a dual-mode vertical flow assay capable of simultaneously identifying bacteria species and estimating bacteria burden, providing a practical strategy for rapid threshold-oriented urine screening at the point-of-care.