University of Missouri - Saint Louis

The Graduate School

Announcement

An oral examination in defense of the dissertation for the degree

Doctor of Philosophy in Chemistry

Binod P. Pandey
MS, Chemistry, University of Missouri-St. Louis. May 2009
MSc., Physical Chemistry, Tribhuvan University, Kathmandu, Nepal, 2004
B.Sc., Chemistry, Tribhuvan University, Kathmandu, Nepal, 2001


Nanoporous gold as a support for immobilization of biomolecules for the development of immunoassay, and for biomolecular interaction studies

 

Abstract

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 limits of these assays were 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 were also studied. Pure and mixed SAMs of 8-mercaptooctyl α-D-mannopyranoside (αMan-C8-SH) and α-D-Gal-(1→4)-β-D-Gal-(1→4)-D-Glc1-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 were also sensitive to the chemical composition of the modified surfaces.

 

Date: November 5, 2012

Time: 1:00 pm – 3:00 pm

Place: 451 Benton Hall

 

Defense of Dissertation Committee

 

Dr. Keith J. Stine (Advisor)

Dr. Alexei V. Demchenko
  Dr. James Chickos Dr. Michael Nichols