Paper-Based Vertical Flow Assays for in Vitro Diagnostics and Environmental Monitoring

Abstract

Microfluidic paper-based analytical devices (μPADs) are powerful tools for diagnostic and environmental monitoring. Being affordable and portable, μPADs enable rapid detection of small molecules, heavy metals, and biomolecules, thereby decentralizing diagnostics and expanding biosensor accessibility. However, the reliance on two-dimensional fluid flow restricts the utility of conventional μPADs, presenting challenges for applications that require simultaneous multibiomarker analysis from a single sample. Vertical flow paper-based analytical devices (VF-μPADs) overcome this challenge by allowing axial fluid movement through paper stacks, offering several advantages, including (1) enhanced multiplexing capabilities, (2) reduced hook effect for improved accuracy, and (3) shorter assay times. This review provides an overview of VF-μPADs technologies, exploring structural and functional performance trade-offs between VF-μPADs and conventional lateral flow systems. The sensing performance, fabrication methods, and applications in in vitro diagnostics and environmental monitoring are discussed. Furthermore, critical challenges─such as fabrication complexity, data analysis, and scalability─are addressed, along with proposed strategies for mitigating these barriers to facilitate broader adoption. By examining these strengths and challenges, this review presents the potential of VF-μPADs to advance point-of-care testing, particularly in resource-limited settings.

Keywords: Environmental monitoring; In vitro diagnostics; Multiplexing; Paper-based analytical devices; Point of care testing; Vertical flow assay; Vertical flow paper-based analytical devices.

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Comparison of lateral flow-μPADs (LF-μPADs) and VF-μPADs. (A) LF-μPADs leverage horizontal fluid flow across a porous membrane, making them simple, cost-effective, and scalable for mass production, with easily interpretable results. However, they are constrained by the hook effect at high analyte concentrations and offer limited multiplexing abilities. (B) VF-μPADs harness vertical fluid flow through stacked paper layers, enabling enhanced multiplexing capabilities, reduced hook effect, and the integration of functional layers to accommodate complex samples. Despite these advantages, VF-μPADs face challenges with more intricate fabrication and less intuitive result interpretation.

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Comparison of lateral flow and vertical flow with illustrations of VF-μPADs in POCT. (A) Illustration of the shorter fluid path in VF-μPADs, enabling rapid fluid transfer through vertically stacked layers. This arrangement reduces assay time. Reproduced with permission from ref (). Available under a CC-BY license. Copyright 2021 MDPI. (B) Difference in color signal uniformity between LF-μPADs and VF-μPADs depending on flow direction. Reproduced with permission from ref (). Copyright 2019 American Chemical Society. (C) Effect of flow direction on assay performance. Schematic representation illustrating fluid division into two streams directed into the control and test zones, effectively mitigating the hook effect through vertical flow. Reproduced with permission from ref (). Copyright 2013 Royal Society of Chemistry. (D) Multiplexing in VF-μPADs achieved by folding and unfolding patterned paper with photoresist. This design allows simultaneous detection of multiple analytes within a compact device. Reproduced with permission from ref (). Copyright 2011 American Chemical Society. (E) Blood separation in VF-μPADs using a filtration layer. Reproduced with permission from ref (). Copyright 2012 American Chemical Society. (F) Fluid control in VF-μPADs using multiple functional layers. Reproduced with permission from ref (). Copyright 2017 Elsevier B.V.

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Structural components and configurations of VF-μPADs for enhanced diagnostic performance in POC applications. (A) Basic structure of a VF-μPAD consisting of multiple wax-printed layers, including a NC membrane for detection and an absorbent pad. Reproduced with permission from ref (). Copyright 2023 Elsevier B.V. (B) Schematic showing the inclusion of a conjugation pad in VF-μPADs. Cross-sectional illustration of the conjugation pad with in the layered structure. Reproduced with permission from ref (). Available under a CC-BY license. Copyright 2021 MDPI. (C) Integration of a vertical flow diffusers (VFDs) to ensure uniform fluid distribution across the sensing membrane (Left). Schematic of a VF-μPAD with VFD included, illustrating how fluid flow is directed evenly (Right). Reproduced with permission from ref (). Copyright 2019 Royal Society of Chemistry. (D) Use of a separation membrane for sample filtration in complex biological samples. The separation membrane, placed above the conjugation pad, filters out larger debris, such as cells and proteins, before they reach the sensing layers. Reproduced with permission from ref. (). Available under a CC-BY license. Copyright 2023 Springer Nature. (E) Single-membrane VF-μPAD design for simplified fabrication. A single cellulose membrane functions as both the conjugation pad and sensing membrane, reducing the need for complex layer alignment. Reproduced with permission from ref (). Copyright 2019 Royal Society of Chemistry.

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3D Printing techniques in the fabrication of VF-μPADs for POCT. (A) FDM approach for VF-μPAD fabrication using PLA and wax filaments: (i) Illustration of an FDM 3D printer depositing PLA and wax filaments onto Whatman chromatography paper to create hydrophobic barriers; (ii) view of the VF-μPAD layers produced by FDM, including the sample inlet, reaction zones, and absorbent pad; and (iii) VF-μPADs for dengue virus serotype detection using cell-free reactions. Reproduced with permission from ref (). Copyright 2021 Elsevier B.V. (B) DLP 3D printing approach for VF-μPAD fabrication with integrated plasma separation functionality: (i) Schematic of DLP printing setup showing the integration of a parylene C-coated PSM within a photocurable polymer matrix and (ii) illustration of the wetting of the plasma separation membrane (PSM) and the corresponding color generation in the detection zone using 10, 30, and 100 μL of whole blood containing 5 mM glucose. Reproduced with permission from ref (). Copyright 2019, American Chemical Society.

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Applications of VF-μPADs for noninfectious diseases. (A) VF-μPADs for POC measurement of iron from whole blood: (i) Sensor strip consists of four sandwiched membranes and hosts 50 μL of whole blood sample; (ii) GF membrane which acts as a primary filtration layer (top) while asymmetric polysulfone membrane which acts as a secondary filtration layer (bottom); and (iii) calibration curve for blood ion detection exhibited a slope of 0.0004 with a coefficient of determination (R²) of 0.95. Reproduced with permission from ref (). Copyright 2021 Royal Society of Chemistry. (B) VF-μPADs fabricated by origami method for programmable multifold analyte preconcentration: (i) Schematic representation and assembly of the 3D paper-based origami device for programmable analyte preconcentration and (ii) histograms showing a 407% and 406% signal increase for 5 and 10 ppb glucose, respectively, after preconcentration. Reproduced with permission from ref (). Copyright 2024 American Chemical Society.

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Applications of VF-μPADs for infectious diseases. (A) Smartphone-compatible VF-μPADs for rapid SARSCoV-2 antigen detection: (i) Assay workflow illustrating the VF-μPADs setup for SARS-CoV-2 antigen detection and (ii) device validation and calibration data with images of a light-controlled box and smartphone interface for real-time result analysis. Reproduced with permission from ref (). Copyright 2022 Royal Society of Chemistry. (B) Multiplexed VF-μPADs for the early detection of Lyme disease (LD): (i) illustration of the multiplexed immunoreactions that occur on the sensing membrane during the xVFA operation and (ii) photograph of the mobile-phone reader with an opened xVFA cassette and example images of the sensing membrane. Reproduced with permission from ref (). Copyright 2020 American Chemical Society. (C) DNA extraction and damage detection using an origami μPAD: (i) schematic of the assay workflow showing the DNA extraction integrated with TUNEL reaction on paper, creating a streamlined approach to DNA damage analysis and (ii) time-dependent S/B values showing that the paper-based TUNEL exhibited a significantly faster reaction rate constant (k ^paper = 1.12 min^–1) compared to the solution-based TUNEL (k ^solution = 0.25 min^–1). To demonstrate its practical application, genomic DNA with and without H₂O₂ treatment was mixed with TUNEL reagents and applied to the paper chip. Reproduced with permission from ref (). Copyright 2021 Royal Society of Chemistry. (D) Slidingstrip loop-mediated isothermal amplification (LAMP) device for rapid and sensitive molecular diagnostics: Schematic of the LAMP device showing the sequential addition of sample, buffer, and reagents and sensitivity results. Reproduced with permission from ref (). Copyright 2015 American Chemical Society.

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Application of VF-μPADs for environmental monitoring and agriculture. (A) VF-μPAD design with a functional waste reservoir layer, enabling increased sample volume capacity for colorimetric assay of heavy metal ions. Reproduced with permission from ref (). Copyright 2020 Elsevier B.V. (B) Enclosed VF-μPADs for the simultaneous detection of heavy metal ions and a radioactive isotope in water environment. (i) Schematic illustration of enclosed VF-μPADs with four channels for multiplex detection. (ii) (a, b) Multiplex colorimetric response for detection of water samples contaminated with nickel (Ni²⁺), copper (Cu²⁺), mercury (Hg²⁺), and cesium (Cs⁺) ions. Reproduced with permission from ref (). Available under a CC-BY license. Copyright 2023 MDPI. (C) Sensor integrated VF-μPADs for on-site and prompt evaluation of chloride contamination in concrete structures. (i) Design of the vertical flow paper-based sensor. The device is loaded with a few microliters of two solutions having different concentrations of chlorides, drop-cast on the top layer and on the bottom layer. (ii) Photos of the experimental setup applied to concrete. (iii) Chloride evaluation: a portion of the building fundament, a wall from the basement, and the monumental stairs. Reproduced with permission from ref (). Copyright 2021 American Chemical Society. (D) VF-μPADs combined with metal–organic frameworks in a single device for phenolic content assessment in fruits. Reproduced with permission from ref (). Available under a CC-BY license. Copyright 2023 Springer Nature. (E) VF-μPADs for the visual determination of alcohol content in whiskey samples. (i) Layout of the foldable VF-μPADs and the test spots for before and end point of detection (ii) Calibration curve for ethanol and alcoholic content determined for seized samples using a foldable VF-μPADs. Reproduced with permission from ref (). Copyright 2018 Elsevier B.V.