INTEGRATED BIOPHOTONICS APPROACH FOR COMPREHENSIVE SPATIO-TEMPORAL MEASUREMENTS OF THE BIOPHYSICAL PROPERTIES OF BIOMOLECULAR SYSTEMS AND A CHARACTERIZATION OF BIOFUNCTIONALIZED NANOFIBRILLOUS POLYTETRAFLUOROETHYLENE

Open Access
Author:
Proia, Michael Anthony
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 05, 2010
Committee Members:
  • John V Badding, Dissertation Advisor
  • John V Badding, Committee Chair
  • Harry R Allcock, Committee Member
  • David Lawrence Allara, Committee Member
  • Dr Paul Brown, Committee Member
Keywords:
  • Biophotonics
  • Polytetrafluoroethylene
  • fluorescence correlation spectroscopy
  • jet blowing
  • endothelialization
  • biocompatibility
Abstract:
Various biophotonic techniques can be integrated to provide a comprehensive understanding of a system of interest, such as the use of Rh123 to probe the heterogeneity of mitochondrial environments within living cells. This multimodal system can be applied to other biological and biochemical studies at single molecule and macro scale levels without compromising the detection efficiency and spatio-temporal resolution. Here, fluorescence (confocal and two-photon) and DIC microscopy were used to provide cell morphology and mitochondrial distribution. Excited-state fluorescence lifetime imaging microscopy (FLIM) was used to identify the effects of mitochondrial environments on the quantum yield of Rh123. FLIM was also used to convert fluorescence intensity images into concentration images. Fluorescence polarization anisotropy imaging and rotational diffusion was used to probe information concerning structural conformation, hydrodynamic volume, and the surrounding environment of Rh123 in mitochondria. FCS was used to extend the observation time to include translational diffusion with single molecule sensitivity. Several of these integrated biophotonic techniques were applied in an attempt obtain information pertaining to the binding stoichiometry and molecular dynamics of T4-bacteriophage replisome protein gp59 binding to forked DNA. Although a definitive stoichiometry of gp59 on fDNA was not obtained, several other insights into gp59 and fDNA interactions were. Decreasing fluorescence lifetime of Hex-labeled fDNA as gp59 concentration increases suggests a spatial proximity that initiates non-radiative pathways. Fluorescence polarization anisotropy measurements show Hex-labeled fDNA undergoes ultrafast conformational changes (segmental mobility), which are sensitive to the presence of bound gp59. Single-molecule FCS measurements suggest that [fDNA(gp59)n]m aggregates are formed at high gp59 concentrations. Jet Blowing processing of PTFE has been shown to provide control over the nanoscale morphological properties of processed PTFE fibers by adjusting processing parameters such as temperature, pressure, and gas selection. Three distinct morphologies were identified under various different processing conditions which include primarily starting material nodules, highly fibrillous PTFE, and sintered material. BET surface area measurements are convoluted with the surface areas of individual components such as starting material nodules, high aspect ratio fibers, nodule agglomerates, and varied fiber diameters which all compete with increasing and decreasing effects to provide the total measurable BET surface area. Jet Blowing PTFE heated to 270 C with nitrogen at a pressure of 70 MPa was found to be the optimum processing conditions to produce highly fibrillous products. Endothelialization of vascular grafts is critical for the production of artificial organs with enhanced hemocompatibility. Jet Blown nPTFE surfaces were functionalized with hydroxyl groups through the use of RF generated Ar/N2 atmospheric plasma. These hydroxyl groups were activated with tresyl chloride to allow for the covalent binding of the adhesion peptide GYIGSR via the N-terminus. Cell proliferation was measured on native, plasma functionalized, and GYIGSR modified surfaces of nPTFE and ePTFE. All nPTFE surfaces outperformed, with respect to endothelialization, equivalently treated ePTFE surface. This enhancement to cell proliferation is not an effect of differences in cell accessible surface area and therefore, there must be a relationship between the nanoscale morphology of nPTFE and its enhanced endothelial cell proliferation rates.