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We present a fractional sensitivity analysis of a competitive microfluidic heterogeneous immunoassay for a small molecule analyte. A simple two-dimensional finite element model is used to determine the fractional sensitivity of the assay signal with respect to analyte concentration, flow rate, initial surface density of binding sites, and antibody concentration. The fractional sensitivity analysis can be used to identify (1) the system parameters for which it is most crucial to control or quantify the variability between assays and (2) operating ranges for these parameters that improve assay sensitivity (within the constraints of the experimental system). Experimental assay results for the drug phenytoin, obtained using surface plasmon resonance imaging, are shown to be consistent with the predictions of the model.

We describe a novel microfluidic immunoassay method based on the diffusion of a small-molecule analyte into a parallel-flowing stream containing a cognate antibody. This interdiffusion results in a steady-state gradient of antibody binding site occupancy transverse to convective flow. In contrast to the diffusion immunoassay (Hatch, A.; Kamholz, A. E.; Hawkins, K. R.; Munson, M. S.; Schilling, E. A.; Weigl, B. H.; Yager, P. Nat. Bioteehnol. 2001, 19, 461-465.), this antibody occupancy gradient is interrogated by a sensor surface coated with a functional analogue of the analyte. Antibodies with at least one unoccupied binding site may specifically bind to this functionalized surface, leading to a quantifiable change in surface coverage by the antibody. SPR imaging is used to probe the spatial distribution of antibody binding to the surface and, therefore, the outcome of the assay. We show that the pattern of antibody binding to the SPR sensing surface correlates with the concentration of a model analyte (phenytoin) in the sample stream. Using an inexpensive disposable microfluidic device, we demonstrate assays for phenytoin ranging in concentration from 75 to 1000 nM in phosphate buffer. At a total volumetric flow rate of 90 nL/s, the assays are complete within 10 min. Inclusion of an additional flow stream on the side of the antibody stream opposite to that of the sample enables simultaneous calibration of the assay. This assay method is suitable for rapid quantitative detection of low molecular weight analytes for point-of-care diagnostic instrumentation.

A novel microfluidic surface-based competition immunoassay, termed the concentration gradient immunoassay (described in detail in a companion paper (Nelson, K.; Foley, J.; Yager, P. Anal Chem. 2007, 79, 3542-3548.) uses surface plasmon resonance (SPR) imaging to rapidly measure the concentration of small molecules. To conduct this assay, antibody and analyte are introduced into the two inlets of a T-sensor (Weigi, B. H.; Yager, P. Science 1999, 283, 346-347. Kamholz, A. E.; Weigl, B. H.; Finlayson, B. A.; Yager, P. Anal. Chem. 1999, 71, 5340-5347). Several millimeters downstream, antibody molecules with open binding sites can bind to a surface functionalized with immobilized antigen. This space- and time-dependent binding can be sensitively observed using SPR imaging. In this paper, we describe a complex three-dimensional finite element model developed to better understand the dynamic processes occurring with this assay. The model shows strong qualitative agreement with experimental results for small-molecule detection. The model confirms the experimental finding that the position within the microchannel at which the antibody binds to the immobilized analyte may be used to quantify the concentration of analyte in the sample. In addition, the model was used to explore the sensitivity of assay performance to parameters such as antibody and analyte concentrations, thereby giving insight into ways to optimize analysis speed and accuracy. Given the experimental verification of the computational results, this model serves as an efficient method to explore the influence of the flow rate, microchannel dimensions, and antibody concentration on the sensitivity of the assay.