Applications of capillary electrophoresis in biological and clinical research

High sensitivity and high efficiency analysis of biomolecules has become increasingly important in biological and clinical research. In this thesis, development and application of techniques such as capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), polymerase chain reaction (PCR), cell synchronization and slab gel electrophoresis are presented in an effort to address a few of the challenging goals in this field.; A CE-LIF based method for accurate and reliable PCR quantification was developed. Using duck hepatitis B virus (DHBV) as a model, the developed technique was validated by evaluating its linearity, sensitivity, accuracy and precision. The method is more convenient and is applicable to a wider variety of PCR amplifications than other popular assays.; Understanding nucleoside anti-cancer drug resistance mechanisms at the single-cell level is crucial in improving clinical treatment. The human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) are two of the molecular determinants of nucleoside drug resistance. The first use of CE-LIF in hENT1 quantification in the plasma membrane is presented and applied successfully to five cultured human cell lines. Theoretical sensitivity of this method suggests that single cell hENT1 quantification is possible. The potential of measuring dCK activity using a fluorescent substrate was studied as the initial step in developing a single cell enzyme assay. Aspects of separation, substrate preparation, enzyme kinetics, substrate uptake and substrate distribution were studied. Preliminary results indicated a more suitable substrate is required to ensure successful future development. In parallel, studies such as assisting the substrate uptake, optimizing the enzyme assay conditions and localizing the substrate in the cytoplasm are also required.; Until analysis at the single-cell level becomes routine, cell synchronization is necessary in understanding cell cycle-dependent biological events. Two examples are presented in this thesis using HeLa cells. The first study combined cell synchronization techniques with my hENT1-CE-LIF assay. Results showed that hENT1 expression levels changed during the cell cycle and may be correlated with cell proliferation. In the second study, slab gel electrophoresis and capillary gel electrophoresis (CGE) were applied to probe the general protein expression differences at different cycle phases.