Dean Y. Stevens

Microfluidics without pumps: reinventing the T-sensor and H-filter in paper networks.

Conventional microfluidic devices typically require highly precise pumps or pneumatic control systems, which add considerable cost and the requirement for power. These restrictions have limited the adoption of microfluidic technologies for point-of-care applications. Paper networks provide an extremely low-cost and pumpless alternative to conventional microfluidic devices by generating fluid transport through capillarity. We revisit well-known microfluidic devices for hydrodynamic focusing, sized-based extraction of molecules from complex mixtures, micromixing, and dilution, and demonstrate that paper-based devices can replace their expensive conventional microfluidic counterparts.

Enabling a microfluidic immunoassay for the developing world by integration of on-card dry reagent storage

As part of an effort to create a point-of-care diagnostic system for the developing world, we present a microfluidic flow-through membrane immunoassay with on-card dry reagent storage. By preserving reagent function, the storage and reconstitution of anhydrous reagents enables the devices to remain viable in challenging, unregulated environmental conditions. The assay takes place on a disposable laminate card containing both a porous membrane patterned with capture molecules and a fibrous pad containing an anhydrous analyte label. To conduct the assay, the card is placed in an external pumping and imaging instrument capable of delivering sample and rehydrated reagent to the assay membrane at controlled flow rates to generate quantitative results. Using the malarial antigen Plasmodium falciparum histidine-rich protein II (PfHRP2) as a model, we demonstrate selection of dry storage conditions, characterization of reagent rehydration, and execution of an automated on-card assay. Gold-antibody conjugates dried in a variety of sugar matrices were shown to retain 80-96% of their activity after 60 days of storage at elevated temperatures, and the release profile of the reconstituted reagent was characterized under flow in microfluidic channels. The system gave a detection limit in the sub-nanomolar range in under nine minutes, showing the potential to expand into quantitative, multi-analyte analysis of human blood samples.

Progress toward multiplexed sample-to-result detection in low resource settings using microfluidic immunoassay cards

In many low resource settings multiple diseases are endemic. There is a need for appropriate multi-analyte diagnostics capable of differentiating between diseases that cause similar clinical symptoms. The work presented here was part of a larger effort to develop a microfluidic point-of-care system, the DxBox, for sample-to-result differential diagnosis of infections that present with high rapid-onset fever. Here we describe a platform that detects disease-specific antigens and IgM antibodies. The disposable microfluidic cards are based on a flow-through membrane immunoassay carried out on porous nitrocellulose, which provides rapid diffusion for short assay times and a high surface area for visual detection of colored assay spots. Fluid motion and on-card valves were driven by a pneumatic system and we present designs for using pneumatic control to carry out assay functions. Pneumatic actuation, while having the potential advantage of inexpensive and robust hardware, introduced bubbles that interfered with fluidic control and affected assay results. The cards performed all sample preparation steps including plasma filtration from whole blood, sample and reagent aliquoting for the two parallel assays, sample dilution, and IgG removal for the IgM assays. We demonstrated the system for detection of the malarial pfHRPII antigen (spiked) and IgM antibodies to Salmonella Typhi LPS (patient plasma samples). All reagents were stored on card in dry form; only the sample and buffer were required to run the tests. Here we detail the development of this platform and discuss its strengths and weaknesses.

Towards a point-of-care diagnostic system: automated analysis of immunoassay test data on a cell phone

Many of the diagnostic tests administered in well-funded clinical laboratories are inappropriate for point-of-care testing in low-resource environments. As a result, inexpensive, portable immunoassay tests have been developed to facilitate the rapid diagnosis of many diseases common to developing countries. However, manually analyzing the test results at the point of care may be complex and error-prone for untrained users reading test results by eye, and providing methods for automatically processing these tests could significantly increase their utility. In this paper, we present a mobile application that automatically quantifies immunoassay test data on a smart phone. The speed and accuracy demonstrated by the application suggest that cell-phone based analysis could aid disease diagnosis at the point of care.

Laboratory-scale protein striping system for patterning biomolecules onto paper-based immunochromatographic test strips

A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.