Designing the diffusion immunoassay (DIA): how properties of the analyte affect DIA performance

Abstract

The diffusion immunoassay (DIA) is a novel assay technique based on the difference in diffusivity of an analyte molecule when free in solution, versus when it is bound to a cognate antibody. It is uniquely enabled by the laminar flow conditions found in microfluidic devices, making it an exciting new candidate for a miniaturized clinical assay platform---the kind of platform that is envisioned as enabling technology for delivery of disease diagnosis at the point-of-care.The DIA principle was originally evinced with the assay of a small molecule--- phenytoin.1 Extension of the principle to larger analytes is not trivial, yet larger analytes represent an important class of analytes in the clinic. Larger analytes diffuse more slowly, both in translation (which affects the transport of analyte mass between adjacent flow streams) and rotationally (which affects the association rate of the analyte with its cognate antibody). It seemed obvious that both transport and reaction differences would change the behavior of the DIA, but it was not obvious how, or by how much. A quantitative understanding of the effects of transport and reaction (as well as other important design variables) on the DIA enables intelligent design of a DIA for any analyte through the formulation of power law relationships and design rules. This dissertation identifies the key parameters in the DIA design space, characterizes improvements (leading to a second generation DIA), quantifies the power law relationships between design parameters and important metrics of performance, and illustrates the conclusions with a second generation DIA for a clinically significant analyte, C-reactive protein.