A hydrodynamic microfluidic system for the analysis of glycoproteins

Elizabeth Marie Miller, Purdue University

Abstract

An integrated device has been described to perform a variety of enzymatic and immunological assays for applications to proteomics, disease diagnosis, and disease state monitoring. The work presented here focused on the use of this system to perform solution enzyme assays and lectin immunoassays. ^ In typical microfluidic devices, fluid manipulation is conducted via microfabricated pumps or electroosmotic flow. On such a small scale, pumps and valves are extremely difficult to fabricate and have a high failure rate. Electroosmotic flow requires expensive power supplies, incurs a sampling bias, and can result in joule heating within plastic devices. A plastic device was proposed that employed only hydrodynamic flow for fluid transport. This device was used to carry out enzymatic assays for two small molecule analytes. Several coating strategies were considered to prevent fouling of the device as a result of adsorption of both large and small molecules to the hydrophobic walls. ^ Post-translational modifications such as glycosylation and phosphorylation are of great interest in proteomics, and high-throughput methods are needed for the identification of trends in these modifications. An immunoassay was developed for the characterization of glycoproteins. Antibodies were used to capture target glycoproteins, and lectins used for qualitative and quantitative analysis of the glycans present on the protein, The assay was carried out in a traditional format in microtitre wells and in a microscale format with microcontact printing of assay arrays. The assay successfully distinguished between types of glycosylation on two standard proteins, and quantitation was achieved by two independent detection methods. It was also determined that the hydrodynamic flow system was an effective means of concentration of low-abundance proteins on the assay. ^ The lectin immunoassay was adapted to investigate two known protein markers for rheumatoid arthritis. A variety of lectins were used to characterize the types of glycosylation on both standard proteins, and a comparative study was performed to identify differences in glycosylation between samples of serum from a normal pool and a rheumatoid arthritis patient. This assay successfully identified trends in glycosylation that are consistent with those reported in the literature.^

Degree

Ph.D.

Advisors

Fred E. Regnier, Purdue University.

Subject Area

Chemistry, Analytical

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