Applications and Advancements of Dynamic Isoelectric Focusing

Author

Shannon Courtney Wilson, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Tolley, Luke

Abstract

The work in the dissertation expands the applications of DIEF and describes the development of incorporating DIEF in a microfluidic chip to create a comprehensive proteomics tool. Proof-of-concept DIEF experiments have been done previously, so the focus of this work is to explore the capabilities of DIEF. Dynamic isoelectric focusing (DIEF) is a separation technique invented by Dr. Luke Tolley. It is similar to capillary isoelectric focusing except it uses four high voltage electrodes to form a pH gradient instead of only two. The additional two electrodes are able to manipulate the pH gradient resulting in selection of the region and of the range of pH within a pre-defined sampling or extraction point. One of the first applications described for DIEF was to isolate a single protein from a complex mixture. The protein isolated was a cellulase enzyme capable of degrading multiple cellulose materials over a wide range of environmental conditions. DIEF did isolate the protein in a pH span of 0.005 which is equivalent to 0.075% of the total pH range. Fractions were collected for sequencing analysis, but the fractions were contaminated with keratin both times. DIEF was also successfully performed in an open air channel. Though other electromigration techniques have been successfully done in open air channels, these techniques were severely time and pH limited. In contrast, DIEF in an open air channel is capable of using the entire 3-10 pH range and can perform isolations until the proteins are completely separated. The device developed was also an improvement on increasing sample capacity. The channel was significantly bigger than the traditional glass capillaries used. Since the channel was open, fraction collection was made simpler by collecting using a pipette. This work also demonstrated that DIEF can be made through the use of silicone molding compounds and polyurethane. The amount of milling needed is reduced, the pieces are produced quickly, and a single mold can produce several pieces. Machining pieces with fragile bits is not needed to be done as much since only one acrylic piece is required produce a mold. The mold can produce several polyurethane pieces. This fabrication method has proven useful for making DIEF holders. The next step was to make DIEF a truly comprehensive proteomic tool by incorporating it into a microfluidic chip. Multiple sample fractions are rapidly generated on chip through the use of multiple bubbles simultaneously injected into the separation channel. This stops the separation and, since each droplet is isolated from others by a bubble on each side, the protein peaks are not able to broaden. This novel use of digital microfluidics is still a work in progress, but the fundamentals have been demonstrated. The fabrication protocol for making molds and PDMS casts was developed using materials and procedures that can be done in a common laboratory environment. DIEF is a separation technique still in its infancy, with a wide variety of available applications. DIEF will continue to be tested in other areas and developed into a comprehensive proteomic tool.