Nanoporous Gold as a Solid Support for Protein Immobilization for the Development of Immunoassays, and for Biomolecular Interaction Studies.

摘要

Nanoporous gold (NPG) is a versatile material of high surface area to volume ratio that can be readily modified with self-assembled monolayers of alkanethiols to which biomolecules can be linked. NPG presents new opportunities for the development of immunoassays, and for the development of carbohydrate based assays. This thesis explores the use of NPG as a support for self-assembled monolayers, their linkage to antibody-enzyme conjugates for immunoassay development, and for the study and application of carbohydrate-protein interactions.;Direct kinetic electrochemical immunoassays were developed on NPG for prostate specific antigen (PSA) and carcinoembryonic antigen (CEA). The decrease in enzymatic conversion of p-aminophenylphosphate to p-aminophenol, by alkaline phosphatase conjugated to an antibody, due to steric hindrance caused by the presence of antigen on antibody, was observed as a drop in peak current in square-wave voltammetry. Detection limit of these assays was 0.075 ng mL -1 and 0.015 ng mL-1 for PSA and CEA, respectively. Similarly, the linear range of determination of these biomarkers extended up to 30 ng mL-1 and 10 ng mL-1 for PSA and CEA, respectively. Minimal interference was observed using newborn calf serum as a substitute for the human serum matrix. A rapid and sensitive enzyme linked lectinsorbant assay was also developed for the study of glycoprotein-lectin interactions on the NPG surface.;Self-assembled monolayers of alkanethiols on NPG were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Similarly, the applicability of this surface for the formation of carbohydrate monolayers and its application for lectin carbohydrate interactions was also studied. Pure and mixed SAMs of 8-mercaptooctyl β-D-mannopyranoside (αMan-C8-SH) and α-D-Gal-(1→4)-β-D-Gal-(1α)-D-Glc-1-O-mercaptooctane (Gb3-C8-SH) with alkanethiols having varying tail groups were prepared. Binding affinity and binding kinetics of concanavalin A to mannoside and soybean agglutinin to galactose in these SAMs were found to be different on NPG than on flat polycrystalline gold, and was also sensitive to the chemical composition of the modified surfaces.