In this paper, we present a novel and cost-effective lab-on-paper microfluidics platform for performing ELISA autonomously, with no user intervention beyond adding the sample. The platform utilizes two Bi-Material Cantilever Valves placed in a specially designed housing. The integration of these valves in a specific channel network forms a complete fluidic logic circuit for performing ELISA on paper. The housing also incorporates an innovative reagent storage and release mechanism that minimizes variability in the volume of reagents released into the reagent pads. The platform design was optimized to minimize variance in the time of fluid wicking from the reagent pad, using a randomized design of experiment. The platform adheres to the World Health Organization’s ASSURED principles. The optimized design was used to conduct an ELISA for detecting rabbit immunoglobulin G (IgG) in a buffer, with a limit of detection of 2.27 ng/mL and a limit of quantification of 8.33 ng/mL. This represents a 58% improvement over previous ELISA methods for detecting rabbit IgG in buffer using portable microfluidic technology.

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 research explores the dynamics of a fluidically loaded Bi-Material cantilever (B-MaC), a critical component of μPADs (microfluidic paper-based analytical devices) used in point-of-care diagnostics. Constructed from Scotch Tape and Whatman Grade 41 filter paper strips, the B-MaC’s behavior under fluid imbibition is examined. A capillary fluid flow model is formulated for the B-MaC, adhering to the Lucas–Washburn (LW) equation, and supported by empirical data. This paper further investigates the stress–strain relationship to estimate the modulus of the B-MaC at various saturation levels and to predict the behavior of the fluidically loaded cantilever. The study shows that the Young’s modulus of Whatman Grade 41 filter paper drastically decreases to approximately 20 MPa (about 7% of its dry-state value) upon full saturation. This significant decrease in flexural rigidity, in conjunction with the hygroexpansive strain and coefficient of hygroexpansion (empirically deduced to be 0.008), is essential in determining the B-MaC’s deflection. The proposed moderate deflection formulation effectively predicts the B-MaC’s behavior under fluidic loading, emphasizing the measurement of maximum (tip) deflection using interfacial boundary conditions for the B-MaC’s wet and dry regions. This knowledge of tip deflection will prove instrumental in optimizing the design parameters of B-MaCs.

Microfluidic paper-based analytical devices (μPADs) have witnessed a great extent of innovation over the past decade, developing new components and materials assisting the diagnosis of different diseases and sensing of a wide range of biological, chemical, optical, and electrochemical phenomena. The novel paper-based cantilever (PBC) actuator is one the major components that allows autonomous loading and control of multiple fluid reagents required for the accurate operation of paper-based microfluidic devices. This paper provides an extensive overview of numerical and experimental modeling of fluidically controlled PBC actuators for automation of the paper-based assay. The PBC model undergoing hygro-expansion utilizes quasi-static 2D fluid loaded structure governed by the Euler–Bernoulli beam theory for small and moderately large deflections. The solution for the model can avail the response of paper-based actuators for response deflection θ, within 0° to 10° under the assumption of insignificant cross-sectional deformation. The actuation of PBC obtained using a quasi-static theory shows that our results are consistent with quantitative experiments demonstrating the adequacy of models.

The novel paper-based Bi-Material Cantilever (B-MaC) valve allows the autonomous loading and control of multiple fluid reagents which contributes to the accurate operation of paper-based microfluidic devices utilized for biological and chemical sensing applications. In this paper, an extensive parametric study is presented to evaluate the effects of key geometric parameters of the valve, such as paper direction, cantilever width, paper type, tape type, and sample volume, in addition to the effects of relative humidity and temperature on the functionality of the B-MaC and to provide a better understanding of the rate of fluid flow and resulting deflection of the cantilever. Machine direction, cantilever width, paper type, and tape type were found to be important parameters that affect the B-MAC’s activation time. It was also observed that the rate of fluid imbibition in the B-MaC is considerably affected by change in humidity for high (55 °C) and low (25 °C) temperatures, while humidity levels have no significant effect during imbibition in the B-MaC at an ambient temperature of 45 °C. It was also found that a minimum distance of 4 mm is required between the B-MaC and the stationary component to prevent accidental activation of the B-MaC prior to sample insertion when relative humidity is higher than 90% and temperature is lower than 35 °C. The rate of fluid imbibition that determines the wetted length of the B-MaC and the final deflection of the cantilever are critical in designing and fabricating point-of-care microfluidic paper-based devices. The B-MaC valve can be utilized in a fluidic circuit to sequentially load several reagents, in addition to the sample to the detection area.

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.