Paolo Spicar-Mihalic

Two-dimensional paper network format that enables simple multistep assays for use in low-resource settings in the context of malaria antigen detection.

The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PfHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card closed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold particle label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.

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.

Rapid prototyping of glass microchannels

This paper describes two methods by which we rapidly and economically fabricate microfluidic systems in glass. The first strategy relies on transferring patterns of microchannels in poly(dimethylsiloxane) (PDMS) onto glass by using PDMS molds that are conformally sealed to glass for confining the etching solution, which then defines the etched pattern. The second strategy uses patterned deposition of surface activators and sensitizers for the electroless and electrolytic plating of nickel, which can then be used as a mask for either wet etching or dry reactive ion etching. We also characterize and compare the morphologies and surface roughness of the glass microchannels fabricated using these two methods.

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.

CO2 laser cutting and ablative etching for the fabrication of paper-based devices

We describe a method for fabricating paper-based microfluidic devices using a commercially available CO2 laser system. The method is versatile and allows for controlled through-cutting and ablative etching of nitrocellulose substrates. In addition, the laser system can cut a variety of components that are useful in the fabrication of paper-based devices, including cellulose wicking pads, glass fiber source pads and Mylar-based substrates for the device housing.