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RESEARCH PROFILES Concentrating on small molecules
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Antibody Sample
Control
Flow
Most medical diagnostic assays are still being done on blood samples that are collected from patients and delivered to a lab. Test results can take hours to days this way. In a pair of papers in the May 15 issue of Analytical Chemistry (pp 3542–3548; 3549–3553), Paul Yager and colleagues at the University of Washington Seattle introduce a new microfluidic immunoassay intended to make the quantitative analysis of lowmolecular-weight compounds rapid, simple, and routine. The focus, Yager says, is on “moving testing technology from the centralized lab to a small, portable, and even handheld instrument, no matter how complex the test protocols.” Dubbed the concentration gradient immunoassay (CGIA), the new approach takes advantage of the diffusion of a small-molecule analyte between microfluidic streams that flow parallel to one another. In a prototype device, three fluid streams converge into a single 3.6-mm-wide channel. The first, a sample stream, contains the analyte of interest. The adjacent central stream contains an antibody against that analyte, and the third stream serves as a reference, or control. The upper portion of the main channel is a “prebinding zone” of defined length—generally >22 mm, but the dimensions can be varied to suit a particular assay. This section of the channel is treated to reduce nonspecific surface adsorption. The downstream “sensing surface” is patterned with a band of immobilized analyte (or suitable analog). Device geometry ensures laminar, parallel flow so that no bulk mixing of the three streams occurs. Instead, a stable diffusion gradient of analyte is established, extending outward from the sample stream and into the adjacent antibody stream. Some of the streaming antibody binds the diffusing analyte molecules, whereas excess antibody is left free to bind to the analyte band that is immobilized downstream.
Prebinding zone
Sensing surface
Outlet
Schematic showing CGIA geometry (not to scale). Analyte diffuses from the sample stream (left) to bind with antibody in the center stream. The degree of diffusion controls further binding to immobilized analyte on the sensing surface.
The assay is read by surface plasmon resonance (SPR), which requires neither labeled reagents nor expensive components. The authors note that SPR’s potential in miniaturized diagnostics has not been widely exploited to date. In this case, says Kjell Nelson, first author of the experimental paper, SPR detects antibody binding to the sensing surface. The pattern of this binding is determined by the extent of analyte diffusion into the antibody stream, and therefore it is directly proportional to the concentration of analyte in the original sample. The surface-binding pattern is dynamic in space and time. It depends on flow rate, microchannel dimensions, antibody concentration, and analyte concentration. A complex 3D finite-element model developed by Jennifer Foley, lead author of the accompanying
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theoretical paper, was an invaluable guide to both optimization and interpretation of the CGIA method, says Nelson. “I don’t think we really had a good appreciation for the subtleties and complexities of the processes occurring in this assay method until we got started,” he says. “Jenn’s models really helped us understand how we might interpret the information obtained from the method and what parameters controlled the outcomes.” Tests were conducted in a model system with phenytoin (a drug used to control seizures that is sold as Dilantin in the U.S.) as the analyte and a bovine serum albumin–phenytoin conjugate patterned on the sensing surface. With 400 nM analyte, 75 nM antiphenytoin antibody, and a flow rate of 90 nL/s, a stable antibody binding pattern was established in
