Aqueous phospholipid preparations comprised of 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) and 1,2‐dihexanoyl‐sn‐glycero‐3‐phosphocholine (DHPC) are prevalent materials for biological characterization and become gel‐like near physiological temperature, but have a low viscosity below 24°C. The rheology of 20% phospholipid preparations of [DMPC]/[DHPC] = 2.5 reveals that, under conditions utilized for fluid steering, the materials are shear‐thinning power‐law fluids with a power‐law index ranging from 0.30 through 0.90. Phospholipid preparations are utilized to steer fluids in microfluidic chips and support hydrodynamic delivery of sample across a double T injection region in a chip. The fact that the phospholipids are fully integrated as a valving material as well as a separation medium is demonstrated through the separation of linear oligosaccharides labeled with 1‐aminopyrene‐3,6,8‐trisulfonic acid.

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.

For the first time, a semi-permanent phospholipid coating is utilized in capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). Although phospholipids in free solution are generally avoided in ESI, they did not interfere with the detection of linear and branched oligosaccharides using ESI operated in negative mode. The CE and ESI were coupled using a coaxial sheath flow interface. The separation was operated in reversed polarity and the electroosmotic flow was effectively suppressed by the phospholipid coating. The method was characterized with linear oligosaccharides and used to monitor the enzymatic hydrolysis of maltooligosaccharides with α-amyloglucosidase. Branched oligosaccharides were separated and detected with the system. The enzyme β1-4 galactosidase was used to distinguish branched isomeric oligosaccharides derived from asialofetuin.

Capillary Electrophoresis for Measuring Biological Interactions: Binding Affinity and Enzyme Kinetics By Ted Langan This dissertation is based upon research in the use of capillary electrophoresis for the measurement of biological interactions. The studies presented here research two classes of analytes, steroids and oligosaccharides, both of which have profound importance to biological systems. Capillary electrophoresis separation methods were utilized to determine binding affinity and enzyme kinetic constants. The second chapter describes the first use of a semi-permanent phospholipid coating that is utilized in capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) for the purpose of monitoring enzymatic hydrolysis and the detection of maltooligosaccharides. Although phospholipids in free solution are generally unused in ESI, they did not interfere with the detection of linear and branched oligosaccharides using ESI operated in negative mode. The CE and ESI were coupled using a coaxial sheath flow interface. The separation was operated in reversed polarity, and the electroosmotic flow was effectively suppressed by the phospholipid coating. The method was characterized with linear oligosaccharides and used to monitor the enzymatic hydrolysis of maltooligosaccharides with α-amyloglucosidase. Branched oligosaccharides were separated and detected with the system. The enzyme β1-4 galactosidase was used to distinguish branched isomeric oligosaccharides derived from asialofetuin. The third chapter of this dissertation presents the measurement of the binding affinity of 17β-estradiol with an immobilized DNA aptamer by using capillary electrophoresis. Estradiol captured by the immobilized aptamer was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 µM. The method was also used to screen binding affinity of the aptamer for other steroids. Chapter three demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers. iv Dedication This research is dedicated to my grandmother, Corlista (Pat) Murphy Crowe, who instilled the need to do things in the best possible way, to be honest, to persevere, and to advance my education. She has dedicated her life to learning, working hard, and teaching others. My work has been completed with the constant support and love of my family: James,