Advancements in Methodologies and Theories Regarding Model Membrane Environments by Total Internal Reflection with Fluorescence Correlation Spectroscopy Public Deposited

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  • March 19, 2019
  • Pero, Jamie Kay
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • Total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) was utilized to determine the diffusion coefficient of nine fluorescently labeled antibodies, antibody fragments, and antibody complexes approximately 85 nm from a planar membrane. The diffusion coefficient decreased with increasing molecular size over what would be expected from the Stokes-Einstein Equation. Theory was derived specific to use with TIRFCS to describe spatially dependent diffusion near membranes. The decreased diffusion is likely due to increased frictional coefficients when molecules are in close proximity to membranes. This described spatially dependent diffusion could be one contributor to the nonideality observed in ligand-receptor kinetics at membranes. A stacked phospholipid bilayer system was developed by utilizing the interaction between NeutrAvidin and biotin. A biotinylated bilayer was deposited on a substrate and then treated with NeutrAvidin. Finally, a second biotinylated bilayer was allowed to adsorb and fuse atop the NeutrAvidin. This stacked bilayer system was characterized using epifluorescence, fluorescence pattern photobleaching recovery (FPPR), order parameter measurements, and atomic force microscopy (AFM). These techniques indicated that the stacked bilayer was relatively continuous but did exhibit some gaps where bilayer was missing. Fluorescence experiments indicated that the second bilayer was less fluorescent than the primary bilayer. The stacked bilayer system will have application in multilayer systems and as a cushioning system. Other applications are likely forthcoming. High refractive index substrates would be beneficial for application in TIR-FCS because they produce much shallower evanescent wave depths (18-43 nm) than fused silica (63-104 nm). It was verified that phospholipid bilayers can be formed upon TiO2 and SrTiO3. A second verification was required because conflicting reports were present in the literature. TIR-FCS was attempted upon these substrates with some success. The data were fit to a simplified version of the appropriate autocorrelation function. The data did autocorrelate, but the fit was extremely noisy and did not fit well at fast times (< 0.1ms). TiO2 and SrTiO3 were determined to be natively luminescent in the visible region with SrTiO3 being about 6x more luminescent than TiO2. Further investigations are required to fully amend the use of TiO2 to TIR based techniques.
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  • Thompson, Nancy
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