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Microfluidic Frameworks for Immunoanalysis of Multiple Proteins

Abstract

Immunoassays are workhorse laboratory tools for detecting protein targets based on highly specific antibody-antigen binding interactions. Greater confidence can be ascribed to an immunoassay when the specific binding interaction is coupled with a separation process based upon some characteristic physicochemical property of the analyte. This dissertation describes the maturation and optimization of microfluidic immunoassay technology to enable the simultaneous detection of multiple targets.

A multi-analytical immunoblotting technique is reported which integrates electrophoretic protein separation with antibody-mediated target capture in a two dimensional microgel. This immunoassay overcomes challenges to traditional multi-analytical protein blotting, which involves repeated cycles of antibody stripping and reprobing, exacerbating the laborious nature of conventional Western Blots. In contrast, an automated and integrated blotting workflow is made possible through a novel multi-stage photolithography process to pattern antigen capture gels at adjacent regions with zero dead volume. The blotting assay completes within 5 minutes, and exhibits a linear dynamic range from 8-800 nM. A label free detection strategy is also introduced to enable simultaneous and quantitative multiplexed measurements without the need for sample prelabeling.

The challenge of separating a post-translationally modified protein target from its unmodified counterpart motivated the development of an immunoprobed isoelectric focusing (IEF) separation within a polyacrylamide gel. Charge separated proteins are immobilized via covalent attachment to polyacrylamide gel, followed by multispectral antibody-based detection, allowing for correlation of probed post-translational modifications to matching protein targets as well as characteristic pI shifts. Device performance was characterized through analysis of phosphorylated and acetylated proteins forms of heat shock protein 27 and superoxide dismutase 2, respectively. The assay reported protein isoforms in immune purified sample and raw cell lysate in two hours with sample volume requirements of 2 microliters.

An analytical model based upon Langmuir immunoaffinity interactions is described to inform the rational design and operation of both immunoassays. The non-dimensional Damkohler number serves as an instructive measure of device performance for each application. 2D immunoblotting requires operation within a mass transport limited regime for effective target capture, whereas the design of the IEF immunoprobe system is specified to be reaction-limited to enable rapid and sensitive fluorescence probe measurements.

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