One of the most powerful and versatile biomedical diagnostic tools, the immunoassay, is used to monitor the levels of drugs and hormones in body fluids, to diagnose infectious and autoimmune diseases, and to both diagnose and monitor treatment of cancer. The performance of immunoassays is today largely restricted to centralized laboratories because of the need for long assay times, complex and expensive equipment, and highly trained technicians. If a wider range of the 700 million immunoassays performed annually in the US alone could be run more inexpensively, more frequently, and at the point of care, the health of millions of patients could be improved. Recent developments in microfluidics suggest that instruments could soon be developed that would allow immunoassays to be performed as easily as is blood glucose testing today. A microfluidic diffusion immunoassay (DIA) has just been demonstrated that may provide a new set of biochemical processes and a common analytical platform that are well suited to such miniaturized and simplified instrumentation. In this assay, the transport of molecules perpendicular to flow in a microchannel is affected by binding between antigens and antibodies. By imaging the steady-state position of labeled components in a flowing stream, the concentration of very dilute analytes can be measured in a few microliters of sample in seconds. This assay has been demonstrated in the format of a small molecule analyte competition immunoassay using fluorescence imaging detection. The DIA could, then, be used for monitoring drugs, hormones, and other small analytes. Modeling suggests that the current assay can easily detect molecules below the 1-10 nM range in which it has been demonstrated, and could monitor concentrations of analytes as large as proteins, at the cost of increased assay times. Proposed is a 4-year plan to determine, by a series of in vitro tests on increasingly complex samples, the clinical potential of the DIA. To be investigated are 1) the full range of sensitivity and selectivity of the small molecule DIA in the presence of blood and derived biological fluids, and 2) whether the DIA can be extended to detection of large molecule analytes, particularly proteins.
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