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

Abstract

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.

Serodiagnosis of Acute Typhoid Fever in Nigerian Pediatric Cases by Detection of Serum IgA and IgG Against Hemolysin E and Lipopolysaccharide

“…The use of LPS as a serodiagnostic marker of salmonelloses is not new; several studies have reported its utility for detection of acute typhoid in serum and saliva. [19][20][21][22] However, uptake and wide clinical evaluation has been slow, possibly because of perceived problem with a lack of specificity. More detailed studies are underway in this laboratory to evaluate the issue of LPS specificity and cross-reactivity, and to evaluate the diagnostic properties of LPS from several different bacterial species to detect specifically the antibodies of the corresponding infection.…”

Towards nano-diagnostics for rapid diagnosis of infectious diseases – current technological state

“…Using pressure sensitive adhesive, different materials can be bonded to each other. An example is the multilayered microfluidic immunoassay card presented by Lafleur and coworkers (44). Proof of principle was provided with the detection of the IgM antibodies against Salmonella typhi LPS and malarial antigen HRPII from Plasmodium falciparum.…”

Automatic sequential fluid handling with multilayer microfluidic sample isolated pumping

“…The existing sequential flow devices either introduce valving mechanisms into pumping systems, such as paper-based microfluidic circuits, 39,40 or build integrated micropumps based on the flow control structures, such as the Quake systems. 33 In our work, pneumatic diaphragm microvalves are integrated into an mlSIP device since both of the fluidic actuation and the diaphragm microvalves are built with multi-layer structures and driven by the vacuum pressure, 49 which is described as unified pumping and control.…”

“…Microfluidic sequential flow devices have been developed for bioassays, such as pneumatic microvalve-based systems, [30][31][32][33] capillary-based systems, [34][35][36][37][38][39][40] the SlipChip systems, [41][42][43] and manual valving systems. 44 For most of these systems, liquid pumping process and flow control functions are separately driven by different accessory equipment, which ultimately increases the system complexity and the overall cost.…”