Chemical Tagging of Surface Composition Gradients With Polystyrene Nanospheres and the Extracellular Matrix Protein Fibronectin for the Study of Cellular Migration
Oxley, Susan Plummer
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https://hdl.handle.net/2142/84119
Description
Title
Chemical Tagging of Surface Composition Gradients With Polystyrene Nanospheres and the Extracellular Matrix Protein Fibronectin for the Study of Cellular Migration
Author(s)
Oxley, Susan Plummer
Issue Date
2003
Doctoral Committee Chair(s)
Bohn, Paul W.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Language
eng
Abstract
Surface composition gradients show great promise as tools in understanding cellular adhesion and motility. Gradients in surface electrochemical potential were prepared by poising two ends of a thin Au film at different potentials relative to a common solution Ag/AgCl reference couple, resulting in a linear surface electrochemical potential gradient. A pre-assembled thiol monolayer was stripped in regions with local potentials cathodic of the reductive desorption potential, while regions sufficiently anodic of that potential maintained a full thiol monolayer. A transition region bridged the bare areas and those retaining full monolayer coverage. This thesis describes the use of water-soluble carbodiimide coupling chemistry with a succinimide stabilizer to covalently bind polystyrene nanospheres and the protein fibronectin (FN) to thiol gradient surfaces. Fluorescently doped, carboxylic acid-modified nanospheres were reacted with gradients of an amine-terminated thiol. Surface nanosphere distribution was probed with fluorescence microscopy, from which information on the gradient transition region was obtained. The gradient position and width in potential space were found to depend on the offset and magnitude of the applied potential window. The extracellular matrix protein fibronectin (FN) was covalently bound to a counterpropagating two-component gradient formed from thiols with carboxylic acid and hydroxyl terminal functional groups. The acid/alcohol gradients were characterized by tagging with fluorescently doped, amine-modified nanospheres and imaged with fluorescence microscopy. Areas of the surface with initial acid termination reacted with amine groups on the FN, while protein adsorption was resisted on the alcohol regions. FN coverage was determined from surface plasmon resonance reflectometry measurements. FN gradients were mapped with FTIR external reflection spectroscopy and imaged with fluorescence microscopy by tagging with anti-FN and FITC-IgG. The gradients exhibited a sigmoid shape, with the width of the gradient region occupying ∼10% of the total film length. When denatured bovine serum albumin (BSA) was used to block non-specific cellular adhesion, the adhesion of the 3T3 fibroblasts to these gradients followed the spatial composition profile of FN.
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