Paper-Based Vertical Flow Immunoassays (VFIs): A Short Review on Principles, Advances, and Point-of-Care Applications

Abstract Point-of-care detection tests are becoming increasingly important for the rapid identification of analytes across clinical diagnostics, environmental monitoring, and food safety including diseases, contaminants, and microorganisms. To be effective, these tests must meet specific criteria, including high sensitivity, reliability, rapid response, and affordability. While lateral flow assays (LFAs) are currently the most widely used due to their favorable attributes, they encounter limitations related to sensitivity, restricted multiplexing, incubation time, and result accuracy. To overcome these limitations, vertical flow assays (VFAs) have emerged as a versatile alternative, enabling improved mass transport, enhanced sensitivity under appropriate assay designs, reduced assay time, and the ability to efficiently process larger sample volumes when required, without compromising the assay footprint or time-to-result. This review provides a systematic overview of paper-based VFAs, covering operating principles, device architectures, flow-control strategies, and signal transduction mechanisms. Immunological and nonimmunological formats are included, such as enzymatic inhibition, impedance-based detection, and nanomaterial-enhanced assays, with representative applications in clinical diagnostics, contaminant analysis, and microorganism detection.