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A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 1996 / The electric field strength and pH gradients generated in isotachophoretic systems may be used for the separation of biomolecules. Poly (2 acrylamido 2-methyl-1-propanesulfonic acid) polymers of a uniform distribution of molecular mass were synthesized and used as spacers in isotachophoresis in order to generate linear electric field strength gradients in which biomolecules could be focused. These novel spacers are designed to operate within sieving media, and hence are useful for the separation of nucleic acids. [Abbreviated Abstract. Open document to view full version]

Microfluidics is the science and technology of manipulating fluids at small scales ranging from microlitres (10$^{-6}$) to picolitres (10$^{-12}$). The fundamental physics is distinct from fluid behaviour on bulk scales and laminar flow is the key characteristic on this scale. Microfluidic systems have a wide range of applications in many disciplines from engineering to physics, chemistry and biotechnology. In this thesis, I explore different strategies exploiting the capabilities of microfluidic devices for manipulating and analysing biomolecules. A particular focus of the work is on the study of amyloid fibrils. These species are protein aggregates related to a wide range of human diseases and functional materials. In chapter 3 I demonstrate an efficient way to separate particles in different sizes based on a microfluidic diffusion method. This method enables us to explore the properties of amyloid fibrils, such as their growth kinetics and interaction with small molecules. Rapid binding information could be obtained with only microlitres of sample in tens of seconds time scale. A further manipulation method for charged particles is introduced in chapter 4, based on the integration of microfluidics and free flow electrophoresis. I present a very effective and simple way to overcome one of the most critical problem in this situation. High electric filed can be applied through two streams of conductive solutions, with all the electrolysis by products, e.g., gas bubbles and other deposits, removed simultaneously without interfering with the system. In addition to microfluidic devices made by soft lithography in PDMS, I also set up a hot embossing fabrication process with the Teflon material (chapter 5). Teflon has many advantages compared with PDMS, such as lower protein adsorption, higher mechanical strength and better chemical compatibility. With different materials and structures, microfluidic devices can be expanded to more applications.

Analysis of complex protein samples is demanding due to the wide dynamic range of expression levels and the limited detection range of technology. Proteomics relies heavily on the development of new fractionation strategies to help reduce complexity, and overcome the technological and biological challenges associated with proteome analysis. Here, the development of a prototype instrument named ??Microflow MF10?? was explored to enrich for particular classes of proteins. The MF10 was found to have a number of advantages over commercially available fractionation systems. Due to the reduced separation electrode distance, fractionation was rapid, occuring within ~0.125 kVH over 2-6 fractions under native conditions but longer under denaturing conditions. As low as 2 ng peptide could be fractionated with recovery for downstream analysis achievable. The ability to alter protein charge by changing the pH (acidic (pI 3.6) to basic environments (pI 10.4)) allows selection of proteins based on charge/mobility, size, shape, buffer ionic strength, pH and field strength. Proteins <10 kDa are also not routinely analysed because current technology is unable to cater for this region of the proteome. Peptide enrichment using the MF10 was achieved using a 7-protein/peptide standard mix (1-25 kDa), to the 1-5 kDa fraction with simultaneous fractionation of the higher mass protein standards. Plasma was also used to enrich for the peptidome (< 5 kDa) in the presence of the proteome. Enrichment of 73 proteins inclusive of 22 proteins in the 1-25 kDa fraction was achieved compared to a total equivalent of 42 proteins from unfractionated plasma. Rare samples (≤ 106 cells) from stem cell populations or derived clinically are challenging due to the absolute limits in protein copy number and abundance. CD34+ haematopoietic stem cells and CD4+/CD8+ T-cells were used to develop fractionation methods and elucidate the cell differentiation process. MF10 fractionation and analysis by SDS-PAGE and LC-MS/MS revealed 24 differentially expressed proteins between the 3 cell populations, which may be involved in cell differentiation. To quantify these expression differences, iTRAQ with 2-D LC-MS/MS was applied. This study has highlighted the challenges associated with samples of limited quantity. It has been successful in understanding the effects of various conditions on the electrophoretic mobility of proteins, which in proteomics, has remained largely unexplored.