| This dissertation focuses on the application of capillary electrophoresis related techniques (CE) and laser induced fluorescence (LIF) to DNA sequencing, chiral separation and on-line immunoassay.; First, the matrix effect on fluorescence lifetime detection of dye-labeled DNA fragments (both dye-labeled M13 primers and PCR products) in CE is presented for application to four-decay detection in DNA sequencing. In four-decay fluorescence detection scheme, fluorescence lifetime is used to identify the four base-specific fluorescent labels for base-calling. Optimization of lifetime detection and improved detection limits were achieved through screening and modifying sieving matrices. Several commercially available sieving matrices and their solvent modulated forms were investigated to enhance on-capillary fluorescence lifetime detection. Of these, a sieving matrix was identified that showed the best combination of peak resolution and accurate lifetime recovery for DNA sequencing products. It was demonstrated that environmental effects, which are easily masked in color or intensity based detection, showed up more directly in lifetime analysis through recovery of unexpected lifetimes, leading to a more robust and accurate detection scheme for DNA sequencing.; Second, studies of enantiomeric separation using capillary electrochromatography (CEC) with aptamer stationary phases are described. Aptamers are short oligonucleotides that selectively bind to a target compound with high binding affinity. Our interest in aptamers lies in the interactions that they show with molecules other than the target species and their potential as stationary phases for chemical separation. This research has involved in two stages of experimentation: attachment of aptamers to capillary inner surfaces and enantiomeric separations using the aptamer-coated capillaries. Four immobilization methods were evaluated in terms of their stability and performance for separation under CEC conditions. Enantiomeric separations of several amino acids and pharmaceutical drugs were achieved.; Third, preliminary studies were performed towards the development of a novel, on-line immunoassay for cytokines involved in wound healing process. Two approaches, immunoaffinity CE and competitive assay, were investigated. Wound healing processes are closely related to many disease states. Better understanding of these processes will lead to more efficient therapy. It has been found that several cytokines in body fluids are important indicators for wound healing processes. It is therefore important to develop a technique for rapid, efficient and economical detection of these indicators. Studies included optimization of protein labeling, effects of pH of run buffer on the fluorescence intensity of the dye-labeled antigen, temperature control and separation of antigen-antibody complex from free antigen. |
