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.

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.

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.

Selective electroless and electrolytic deposition of metal for applications in microfluidics: fabrication of a microthermocouple.

This paper describes a general strategy for the fabrication of a microthermocouple based on the spatially defined electroless deposition of metal, followed by annealing and electroplating. We present scanning electron microscopy and atomic force microscopy characterizations of the deposition and annealing process, as well as the performance of the microfabricated Ni-Ag thermocouple. The temperature-voltage curve for this Ni-Ag microthermocouple is linear over the range 0-50 degrees C with a slope of 61.9 degrees C mV(-1). The sensitivity of our temperature measurement, which is limited by the uncertainty of our calibration curve, is approximately 1 degrees C. The optimum figure of merit (Z(opt)) is 1.0 x 10(-5) for this type of Ag-Ni thermocouple. We have fabricated microthermocouples ranging in size from 50 to 300 microm. The microthermocouple was integrated into microchannels and used to measure the in-channel temperature rise caused by the following: (1) a simple acid-base reaction, HCl + NaOH --> H2O + NaCl, and (2) an enzyme-catalyzed biochemical reaction, H2O2 + catalase --> H2O + 1/2 O2. We have also profiled the temperature increase in the presence of electroosmotic flow for a 100-, 200-, and 300-microm channel.