The ability to measure low concentrations of analytes (e.g, antigens/antibodies) circulating in bodily fluids is important to detecting the onset of fatal diseases like cancers in their early stages. Although miniaturization efforts have helped in significantly cutting down the sample volume requirement of immunoassays, there is a need to reduce the minimum analyte concentration detectable (limit of detection) by these techniques to broaden their utility in biomedical applications. Here, we present a relatively simple method to improve the limit of detection (LOD) of enzyme-linked immunosorbent assays (ELISA) through solution-phase pre-concentration of the enzyme reaction product (resorufin) in a glass microchannel. To accomplish this goal, an ELISA surface for detecting TNF-a was first created on the walls of our fluidic ducts followed by introduction of the enzyme substrate solution prepared in 10mM sodium tetraborate into the entire microfluidic network. Once the enzyme reaction had proceeded for 30min., the resorufin molecules were hydrodynamically transported towards a 100mM sodium tetraborate stream while applying an electric field. The reduction in the electric field across the junction of the two buffer streams then allowed the resorufin species to slow down at this interface leading to their pre-concentration. Our experiments show that this enhanced detectability of resorufin molecules leads to a significant reduction in the LOD of the ELISA method. For human TNF-a samples we have realized a 17-fold improvement in LOD over that realized on conventional microtiter plates (0.22 versus 3.82pg/mL). Moreover, this improvement in ELISA sensitivity was accomplished using only 1�L of the sample as compared to 100�L required in the microtiter plate format. A unique feature of our pre-concentration approach is that it does not rely on the stacking of molecules against a physical barrier (e.g., membrane), thus simplifying both the fabrication and operation of the reported device.

Quantitation of low concentration of TNF-a using solution phase pre-concentration in a microfluidic device

246th National Meeting (2013)

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