Date of Award
Doctor of Philosophy (PhD)
Kilbey II , S M
Metters , Andrew T
Luzinov , Igor
This dissertation deals with the fundamental studies of peptide adsorption on surfaces modified by surface-confined polymer films, with an application emphasis on membrane bioseparations. In order to understand protein adsorption at a fundamental level, it is important to study the specific residue-level interactions with surfaces. Keeping this idea in view, the present work describes experimental measurements of submolecular-level interaction energies involved in the process of peptide adsorption on polymer films using surface plasmon resonance spectroscopy. Gibbs energy change on adsorption (ΔGad) for tyrosine, phenylalanine, and glycine homopeptides were measured at 25 °C and pH 7 on highly uniform, nanothin polymer films, and the results were used to predict ΔGad for homologous homopeptides with a larger number of residue units. Nanothin poly(2-vinylpyridine), poly(styrene) and poly(1-benzyl-2-vinylpyridinium bromide) films were used for the adsorption studies; they were prepared using a graft polymerization methodology. In-situ swelling experiments were done with ellipsometry to examine the uniformity of the surfaces and to ensure that the graft densities of the different polymer films were similar to facilitate the comparison of adsorption results on these surfaces. To extend this approach to a mixed-residue peptide, measurements were made for glycine, phenylalanine, and tyrosine-leucine subunits found in leucine enkephalin. It was found that combining ΔGads values for adsorption of the individual peptide units in a short- chain peptide allows us to predict its overall ΔGads value with reasonable estimates. Calculations for uncharged surfaces (poly(2-vinylpyridine) and poly(styrene)) gave estimates deviating by no more than |9%| from experiment. Deviations between measured and predicted adsorption energies were larger for the charged poly(1-benzyl-2-vinylpyridinium bromide) surface relative to uncharged surfaces, and, generally speaking, measured uncertainty values were slightly larger for the charged surface. Nevertheless, the adsorption energies were found to be additive within experimental uncertainties for the charged surface as well.
One of the central parts of my dissertation is the fabrication of uniform polymer nanolayers with independent control of the layer thickness and chain grafting density using surface-initiated polymerization. Surface-tethered polymer brushes with independently variable grafting densities and layer thicknesses were fabricated for peptide adsorption and cell-adhesion studies. Surface-initiated atom transfer radical polymerization (ATRP) was used together with thiol self-assembly to generate these nanothin polymer brush layers of poly((polyethylene glycol) methacrylate). A kinetic study was done to measure the layer thickness growth rate at room temperature from flat gold substrates presenting different polymerization initiator molecule surface densities. The polymer brush layers transition from mushroom to brush regimes with increasing grafting density. The results showed that layer properties such as wettability and dry layer thickness depend strongly on initiator surface density. Ultimately, the interaction energy of an RGD-containing synthetic peptide Gly-Arg-Gly-Asp-Ser and the adhesion and spreading of cells were correlated with surface properties, which continues to be a major research theme in biomedical and biomaterials research.
Finally, the work was extended to the surface modification of polymeric membranes to tune the physical and chemical properties of the membranes. I describe a methodology to surface modify commercially available membranes with various functionalities to prepare ion-exchange membranes using graft polymerization from the surfaces of the membranes. ATRP was used to modify the membranes with pyridinium exchange groups and carboxylic acid groups. Polymerization time was used as the independent variable to manipulate the amount of grafted polymer on the membrane surface. ATRP was used to make adsorptive (ion-exchange) membranes with among the highest static and dynamic protein binding capacities, and in a way that allowed us to control the impact on membrane permeability. Confocal laser scanning microscopy was used to visualize membrane pore structure of the unmodified and modified membranes to prove that modification by ATRP did not impact the membrane pore structure detrimentally and also to visualize binding of fluorescently labeled lysozyme.
Singh, Nripen, "Thermodynamic Adsorption Studies of Peptides on Well-defined, Surface-Confined Polymers with Applications in Membrane Bioseparations" (2007). All Dissertations. 120.