Automated liquid handling robot for rapid lateral flow assay development.

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.

Threshold-Based Quantification in a Multiline Lateral Flow Assay via Computationally Designed Capture Efficiency.

Lateral flow assays (LFAs) are widely used for yes/no detection of analytes, but they are not well-suited for quantification. We show that the sensitivity of the test line in a lateral flow assay can be tuned to appear at a specific sample concentration by varying the density of capture molecules at the test line and that when test lines tuned for different responses are combined into a single test strip, lines appear at specific thresholds of sample concentration. We also developed a model based on mass-action kinetics that accurately described test line signal and shape over a wide matrix of capture molecules and sample concentrations in single-line strips. The model was used to design a three-line test strip with lines designed to appear at logarithmically spaced sample concentrations, and the experiments showed a remarkable match to predictions. The response of this "graded ladder bar" format is due to the effect of test line concentration on capture efficiency at each test line, not on sample depletion effects, and the effect is maintained whether a system is under kinetic or equilibrium control. These features enable design of nonlinear responses (logarithmic here) and suggest robustness for different systems. Thus, the graded ladder bar format could be a useful tool for applications requiring quantification of sample concentrations over a wide dynamic range.

Sensitivity enhancement in lateral flow assays: a systems perspective.

Lateral flow assays (LFAs) are rapid, inexpensive, easy-to-manufacture and -use tests widely employed in medical and environmental applications, particularly in low resource settings. Historically, LFAs have been stigmatized as having limited sensitivity. However, as their global usage expands, extensive research has demonstrated that it is possible to substantially improve LFA sensitivity without sacrificing their advantages. In this critical review, we have compiled state-of-the-art approaches to LFA sensitivity enhancement. Moreover, we have organized and evaluated these approaches from a system-level perspective, as we have observed that the advantages and disadvantages of each approach have arisen from the integrated and tightly interconnected chemical, physical, and optical properties of LFAs.

Improving Lateral Flow Assay Performance Using Computational Modeling.

The performance, field utility, and low cost of lateral flow assays (LFAs) have driven a tremendous shift in global health care practices by enabling diagnostic testing in previously unserved settings. This success has motivated the continued improvement of LFAs through increasingly sophisticated materials and reagents. However, our mechanistic understanding of the underlying processes that drive the informed design of these systems has not received commensurate attention. Here, we review the principles underpinning LFAs and the historical evolution of theory to predict their performance. As this theory is integrated into computational models and becomes testable, the criteria for quantifying performance and validating predictive power are critical. The integration of computational design with LFA development offers a promising and coherent framework to choose from an increasing number of novel materials, techniques, and reagents to deliver the low-cost, high-fidelity assays of the future.

Application of mass transfer theory to biomarker capture by surface functionalized magnetic beads in microcentrifuge tubes.

In many diagnostic assays, specific biomarker extraction and purification from a patient sample is performed in microcentrifuge tubes using surface-functionalized magnetic beads. Although assay binding times are known to be highly dependent on sample viscosity, sample volume, capture reagent, and fluid mixing, the theoretical mass transport framework that has been developed and validated in engineering has yet to be applied in this context. In this work, we adapt this existing framework for simultaneous mass transfer and surface reaction and apply it to the binding of biomarkers in clinical samples to surface-functionalized magnetic beads. We discuss the fundamental fluid dynamics of vortex mixing within microcentrifuge tubes as well as describe how particles and biomolecules interact with the fluid. The model is solved over a wide range of parameters, and we present scenarios when a simplified analytical expression would be most accurate. Next, we review of some relevant techniques for model parameter estimation. Finally, we apply the mass transfer theory to practical use-case scenarios of immediate use to clinicians and assay developers. Throughout, we highlight where further characterization is necessary to bridge the gap between theory and practical application.

Rapid concentration and elution of malarial antigen histidine-rich protein II using solid phase Zn(II) resin in a simple flow-through pipette tip format.

Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/µl. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more Plasmodium falciparum histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with Plasmodium falciparum-specific RDTs. A systematic investigation of immobilized metal affinity chromatography divalent metal species and solid phase supports established the optimal design parameters necessary to create a flow-through column incorporated into a standard pipette tip. The bidirectional flow inherent to this format maximizes mixing efficiency so that in less than 5 min of sample processing, the test band signal intensity was increased up to a factor of twelve from HRPII concentrations as low as 25 pM. In addition, the limit of detection per sample was decreased by a factor of five when compared to the RDT manufacturer's suggested protocol. Both the development process and commercial viability of this application are explored, serving as a potential model for future applications.

Mass transfer limitations at crystallizing interfaces in an atomic force microscopy fluid cell: a finite element analysis.

Although atomic force microscopy (AFM) has emerged as the preeminent experimental tool for real-time in situ measurements of crystal growth processes in solution, relatively little is known about the mass transfer limitations that may impact these measurements. We present a continuum analysis of flow and mass transfer in an atomic force microscope fluid cell during crystal growth, using data acquired from calcium oxalate monohydrate (COM) crystal growth measurements as a comparison. Steady-state flows and solute concentration fields are computed using a three-dimensional, finite element method implemented on a parallel supercomputer. Steady-state flow results are compared with flow visualization experiments to validate the model. Computations of the flow field demonstrate how nonlinear momentum transport alters the spatial structure of the flow with increasing flow volume, altering mass transport conditions near the AFM cantilever and tip. The simulations demonstrate that the combination of solute depletion from crystal growth and mass transfer resistance lowers the solute concentration in the region between the tip and the crystal compared with the solute concentration at the inlet of the AFM cell. For example, using experimentally measured growth rates for COM, the solute concentration in this region is 3.1% lower than the inlet value because the solute consumed by crystal growth beneath the AFM tip cannot be replenished fully due to mass transport limitations. The simulations also reveal that increasing the flow rate through the cell does not affect this difference significantly because of the inherent shielding by the AFM tip in proximity with the crystal surface. Models such as the one presented here, used in conjunction with AFM measurements, promise more precise interpretations of measurement data.