A dual-mode vertical flow assay for simultaneous bacteria identification and load assessment in urine

Rapid urine-based bacteria screening requires not only pathogen identification but also estimation of total bacteria burden. However, most paper-based assays provide only one of these outputs. Here, we present a dual-mode vertical flow assay (VFA) designed to simultaneously perform species-specific bacteria identification and total bacteria load assessment from a single urine sample.
The platform integrates two complementary colorimetric modules within one vertical flow device: an HRP–antibody/TMB immunodetection module for species-specific recognition and a WST-8–mPMS metabolic module for quantifying viable bacteria load. These reactions are arranged in spatially separated detection spots to minimize chemical and optical interference while enabling parallel readouts under a simple vertical-flow workflow. A graded porous membrane stack was used to regulate capillary-driven transport, improve residence time at the detection interface, and support consistent color development.
Using Escherichia coli as the target bacteria, the immunodetection module produced concentration-dependent signals in both tryptic soy broth and human urine, achieving stronger quantitative fitting in urine with Hill and linear regression coefficients of R² = 0.8643 and R² = 0.8764, respectively. The WST-8–mPMS metabolic module further enabled total bacteria load assessment across different bacterial species, including E. coli and Staphylococcus aureus, with linear fitting coefficients ranging from R² = 0.72 to 0.84 across broth and urine conditions. In the integrated device, both modules generated concentration-dependent responses from a single urine sample, demonstrating threshold-oriented detection over 5 × 10⁴ to 10⁸ CFU/mL and covering the clinically relevant bacteriuria level of 10⁵ CFU/mL.
These results demonstrate that dual-mode VFA can provide complementary information on bacteria identity and overall bacteria burden within a single assay format. This strategy offers a practical framework for rapid urine bacteria screening and may support future development of integrated paper-based diagnostic platforms.