Identifier

etd-11112013-104001

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples like serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to cytosolic proteins, have various functions and can provide insight into the state of disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques and two-dimensional protein profile patterns have been used as potential diagnostic tools for biomarker discovery and the profiles from protein content of body fluids or cells are available in databases. However, gel electrophoretic methods are not always suitable for particular protein samples. Microfluidics offers the potential as a fully automated platform for the efficient analysis of complex samples, such as membrane proteins and do so with performance metrics that exceed their bench top counterparts. In recent years, there have been various applications and improvements to microfluidics and their use for proteomic analysis reported in the literature. In addition, microfluidics offers the potential of a disposable, low cost, and easily fabricated method to perform analysis on complex samples. In this work through the use of microfluidic devices, we demonstrate the ability to effectively extract and purify biotinylated cell surface membrane proteins from the cell lysate of MCF-7 human breast carcinoma. In addition, we also attempt to separate membrane proteins from MCF-7 cells. Our on-chip assay (µ-solid-phase extraction, µSPE) allows us to extract membrane proteins and rid the sample of contaminating cytosolic proteins (purification) in order to do further analysis on the membrane proteins. We also attempted to separate a complex biological sample using a microchip that is suitable for multidimensional techniques that employed sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS µ-CGE) in the 1st dimension and micro-micellar electrokinetic capillary chromatography (µ-MEKC) in the 2nd dimension. Proteins were detected by laser-induced fluorescence following their labeling with dyes. Because our overall goal of this work is the development of a completely integrated system for the analysis of complex protein samples, we also discuss the integration of the extraction module with the separation module along with fabrication steps toward the integration of modules for the digestion of proteins on chip and interfacing the device with MALDI-MS.

Date

2013

Document Availability at the Time of Submission

Release the entire work immediately for access worldwide.

Committee Chair

Soper, Steven A.

Included in

Chemistry Commons

Share

COinS