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Vertical flow immunoassays (VFIAs) are considered potential point-of-care testing (POCT) devices compared to lateral flow assays due to their ability to analyze a comparatively large sample volume and ease of multiplexing. However, VFIA devices are limited by low analytical sensitivity when coupled with a visual colorimetric signal. Herein, we carefully analyzed key parameters that accounted for the proper functionality of VFIA that can be modified to enhance the overall sensitivity of VFIA. In particular, we focused on improving the stability of conjugate pads impregnated with capture antibodies, maintaining a controlled flow rate to ensure higher analyte reactivity with capture antibodies, and enhancing the absorption efficiency. The results showed that air-drying of conjugate pads in the presence of 5% (w/v) lactose significantly improved the stability of antibodies during long-term storage. Integration of dissolvable polyvinyl alcohol (PVA) membrane of optimal concentration as a time-barrier film into the sensor delayed the flow of samples, thereby increasing the biorecognition interaction time between immunoreagents for the formation of immuno-complexes, which in turn led to higher sensitivity of the assay. Furthermore, the employment of an absorbent pad with higher water holding capacity significantly reduced the non-specific binding of immunocomplexes, thereby reducing the possibility of false-negative results.

Optimizing Effective Parameters to Enhance the Sensitivity of Vertical Flow Assay for Detection of Escherichia coli