| Description |
The focus of this dissertation is on the investigation of the physical parameters impacting phospholipid membrane structure and dynamics. Sum-frequency spectroscopy (SFVS) was used to investigate the structural organization of planar supported lipid membranes. Specifically, the structural characteristics of cholesterol influencing the kinetic and thermodynamics of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) flip-flop was examined by incorporating cholestanol or cholestene into a DSPC membrane. Cholestanol lacks the double bond in the sterol ring and cholestene is without the hydroxyl group on carbon 3. These studies found that the structural modifications to cholesterol altered the thermodynamics of lipid flip-flop by changing the physical compressibility of the membranes. The impact of membrane physical parameters was further explored by developing a biologically compatible thin conductive oxide material. Conductive metal oxide materials are attractive substrates for the analysis of membrane electrochemical properties as model lipid membranes are more easily created on oxides compared to metal surface electrodes. The widely used material of indium tin oxide has a limited functional pH range and is easily removed from a surface. A new, robust thin conductive oxide was created out of ruthenium dioxide (RuO2) using simplistic colloidal solution-gelation (sol-gel) chemistry that has widely been established for synthesizing amorphous silica glass films. These electrodes demonstrated unique stability under harsh physical and chemical conditions making them suitable for long term use in studying planar membrane models. The structure of membranes formed by vesicle fusion on planar substrates was investigated using SFVS, fluorescence microscopy and electrochemical impedance spectroscopy (EIS). This methodology is widely used in model membranes to study the interactions plasma membrane components. Previously implemented techniques studying the mechanism of vesicle fusion are incapable of investigating possible population differences between leaflets that may arise during bilayer formation. SFVS is capable of distinguishing the interleaflet structure of vesicle fused membranes resulting in increased comprehension of vesicle fusion. These experiments were coupled with epifluorescence microscopy and EIS of membranes on RuO2 electrodes to examine the integrity of vesicle fused membranes. This dissertation demonstrates the capabilities of SFVS for use in the investigation of the chemical interactions between lipid components within planar supported model membranes. |
