Date of this Version
Vargas Badilla, J.A. Applications of High Performance Affinity Chromatography with High Capacity Stationary Phases Made by Entrapment, Ph.D. dissertation, University of Nebraska-Lincoln, Lincoln, NE, December 2015
High performance affinity chromatography (HPAC) is a technique that uses a biologically-related agent such as a transport protein or an antibody as the stationary phase in an HPLC system. In recent years, HPAC has been shown to be a valuable bioanalytical tool for studying solute-protein interactions. Human serum albumin (HSA), the most abundant protein in blood (with concentrations of 35 to 50 mg/mL in serum), has been shown to interact with many drugs, affecting their transport, excretion and metabolism. A physical method for immobilizing proteins in HPAC supports has been optimized in this dissertation by using HSA as a model protein.
This method involved the encapsulation of this protein inside the pores or near the surface of hydrazide-activated silica by using mildly oxidized glycogen as a capping agent for the hydrazide groups. Previous work has shown that this approach is able to retain the activity of the entrapped protein and to produce binding that is comparable to what is seen for the protein in its soluble form.
The entrapment process was optimized in a slurry based-format by altering the purification method for the oxidized glycogen, as well as increasing the concentration of protein and decreasing the total reaction volume that were used in this format. Through these changes, it was possible to obtain HPAC stationary phases with a nine-fold increase in their retention factor for warfarin. An on-column entrapment approach was also examined, in which HSA and oxidized glycogen were reacted with hydrazide-activated silica in a flow-based format. This second approach provided retention factors for warfarin that were up to three-fold higher than those obtained by the slurry method. The ability of the on-column method to produce higher capacity stationary phases was then exploited for making chromatographic supports capable of separating sugars by using immobilized lectins. This on-column approach was also used for studying the effects of glycation of HSA on its binding properties with various sulfonylurea drugs that are used to treat diabetes. It was found that normal HSA and HSA with various levels of glycation, as immobilized by entrapment, did show changes in their drug binding parameters that depended on the level of glycation.
Finally, organic monoliths containing silver nanoparticles (AgNPs) were placed on glass slides and within microchannels and used for detecting a near infrared fluorescent dye. Up to ten-fold enhancement in the fluorescence signal of the dye was obtained for monoliths containing AgNPs when compared to control monoliths. This higher sensitivity can be exploited in making stationary phases or detection elements in microfluidic devices that can be utilized in the analysis of small samples and in applications such as flow-based biointeraction studies.
Advisor: David S. Hage