Two-Dimensional and High-Throughput Electrophoretic Separation of Proteins Using Polymeric Microchips

Abstract

A major task in proteomics is to identify proteins from a biological sample using two-dimensional (2-D) separation prior to mass spectrometry of peptides generated via proteolytic digestion of the proteins. For 2-D separations, microfluidic devices are superior to bench top and capillary-based systems since they potentially provide higher separation efficiencies due to the minimal dead volumes produced during peak transfer between the two separation dimensions. In addition, fast separations can be envisioned because the column lengths are typically shorter in microfluidic platforms without scarifying peak capacity. High-throughput capabilities are extremely desirable for many types of bio-analytical analyses, such as understanding molecular interactions and the role they play in cellular functioning and drug discovery. Polymeric microchips possess a variety of physiochemical properties to match the intended application and their ease of fabrication increases the accessibility of technology to a large research base. In this dissertation, a comprehensive 2-D separation platform for proteins using a polymeric microchip with the ability to perform high performance separations within a few minutes was established. The system combined sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS µ-CGE) with micellar electrokinetic chromatography (MEKC) in a poly(methyl methacrylate), PMMA, microchip and was reported with a programmed pulse injection/separation protocol with laser-induced fluorescence for detection. A novel sixteen-channel polycarbonate (PC) microfluidic device for high-throughput separations of proteins was also presented using a process to pattern gold features as microelectrode array for sixteen parallel channels on microchips. The system was able to simultaneously analyze sixteen different samples in parallel consisting of native proteins, amino acids, peptides, and oligonucleotides with conductivity. Finally, due to the diverse nature of polymer properties and the large number of potential applications for microfluidic chips, the physiochemical properties of various polymers were investigated to guide researchers in selecting the best material for a given application including protein analysis