This study presents an integrated approach to understanding fluid dynamics in Microfluidic Paper-Based Analytical Devices (µPADs), combining empirical investigations with advanced numerical modeling. Paper-based devices are recognized for their low cost, portability, and simplicity and are increasingly applied in health, environmental monitoring, and food quality analysis. However, challenges such as lack of flow control and the need for advanced detection methods have limited their widespread adoption. To address these challenges, our study introduces a novel numerical model that incorporates factors such as pore size, fiber orientation, and porosity, thus providing a comprehensive understanding of fluid dynamics across various saturation levels of paper. Empirical results focused on observing the wetted length in saturated paper substrates. The numerical model, integrating the Highly Simplified Marker and Cell (HSMAC) method and the High Order accuracy scheme Reducing Numerical Error Terms (HORNET) scheme, successfully predicts fluid flow in scenarios challenging for empirical observation, especially at high saturation levels. The model effectively mimicked the Lucas–Washburn relation for dry paper and demonstrated the increasing time requirement for fluid movement with rising saturation levels. It also accurately predicted faster fluid flow in Whatman Grade 4 filter paper compared with Grade 41 due to its larger pore size and forecasted an increased flow rate in the machine direction fiber orientation of Whatman Grade 4. These findings have significant implications for the design and application of µPADs, emphasizing the need for precise control of fluid flow and the consideration of substrate microstructural properties. The study’s combination of empirical data and advanced numerical modeling marks a considerable advancement in paper-based microfluidics, offering robust frameworks for future development and optimization of paper-based assays.

This study presents an innovative lateral flow microfluidic paper-based analytical device (μPAD) designed for conducting quantitative paper-based enzyme-linked immunosorbent assays (p-ELISA), seamlessly executing conventional ELISA steps in a paper-based format. The p-ELISA device utilizes a passive fluidic circuit with functional elements such as a multi-bi-material cantilever (B-MaC) assembly, delay channels, and a buffer zone, all enclosed within housing for autonomous, sequential loading of critical reagents onto the detection zone. This novel approach not only demonstrates a rapid assay completion time of under 30 min, but also boasts reduced reagent requirements, minimal equipment needs, and broad applicability across clinical diagnostics and environmental surveillance. Through detailed descriptions of the design, materials, and fabrication methods for the multi-directional flow assay (MDFA), this manuscript highlights the device’s potential for complex biochemical analyses in a user-friendly and versatile format. Analytical performance evaluation, including a limit of detection (LOD) of 8.4 pM for Rabbit IgG, benchmarks the device’s efficacy compared to existing p-ELISA methodologies. This pioneering work lays the groundwork for future advancements in autonomous diagnostics, aiming to enhance global health outcomes through accessible and reliable testing solutions.