AVS 49th International Symposium
    Biomaterials Monday Sessions
       Session BI+VT-MoA

Paper BI+VT-MoA10
Study of the Adsorption Kinetics and Conformational Changes of Human Serum Albumin and Human Plasma Fibronectin using PM-RAIRS, Radiolabelling and Atomic Force Microscopy

Monday, November 4, 2002, 5:00 pm, Room C-201

Session: Protein Surface Interactions
Presenter: R.J. Manning, University of Manchester Institute of Science and Technology, UK
Authors: R.J. Manning, University of Manchester Institute of Science and Technology, UK
C.M.J. Fauroux, University of Manchester Institute of Science and Technology, UK
M.J. Pilling, University of Manchester Institute of Science and Technology, UK
P. Gardner, University of Manchester Institute of Science and Technology, UK
G.J. Leggett, University of Manchester Institute of Science and Technology, UK
Correspondent: Click to Email
The kinetics of adsorption of proteins has been studied on self assembled monolayers (SAMs) on gold, formed by the adsorption of alkanethiolates with differing functional groups and varying alkyl chain lengths. The adsorption of human serum albumin (HSA) and human plasma fibronectin (HPF) has been studied using three complementary techniques: post modulation fourier transform reflection adsorption infrared spectroscopy (PM-RAIRS), radiolabelling, and atomic force microscopy (AFM). Initial adsorption kinetics of HSA and HPF were established using FTIR. It was found that monolayer coverage was reached faster on methyl terminated SAMs than on hydroxyl and carboxylic acid terminated hydrophilic monolayers. Tritium radiolabelling of HSA and HPF confirmed the trends observed with FTIR. The conformations of the adsorbed proteins were followed using PM-RAIRS, enabling quantitative monitoring of the percentage of @alpha@-helix, @beta@-sheet, @beta@-turn and random coils, indicating the degree of denaturation on differing surfaces over time. Finally, AFM was used to generate direct observations of layers of adsorbed proteins, providing useful insights into the distribution of proteins across the differing surfaces and enabling individual molecules to be observed. HSA was found to form a fibrillar network on methyl terminated SAMs at low concentrations and short adsorption times, whilst individual molecules were observed on hydroxyl and carboxylic acid terminated monolayers. This study demonstrates the complimentarity of FTIR, radiolabelling and AFM in understanding the adsorption of proteins on well-ordered SAMs.