Surface immobilization of probe molecules in surface based assays is a
key area of research in the continued development of immunoassay microarrays.
Interest continues to grow in microarray based immunoassays given their
potential as a high throughput technique for immunodiagnostics. Therefore, it is
important to thoroughly study and understand the implications of interfacial
chemistry and immobilization conditions on the performance of the assay. This
thesis presents a body of work that examines the impact of probe density,
interfacial chemistry, and enhancement factors for arrays read with surface
plasmon resonance (SPR) imaging.
An array of structurally similar Salmonella disaccharides was immobilized
at varying densities and the interface formed was thoroughly investigated to
determine the properties of the interface. The arrays were then used with SPR
imaging to evaluate the binding of an antibody specific for one disaccharide of the
three stereoisomers on the array. A dilute disaccharide surface was found to
provide optimal antibody binding. Higher densities result in steric hindrance of
antibody binding by not allowing the disaccharide to insert into the antibody
binding pocket.
The role of interfacial chemistry in antibody attachment was studied to
determine optimum conditions. The study examined physical adsorption,
covalent attachment, and affinity capture. It was found that covalent attachment
provided the most stable attachment and resulted in the lowest levels of antigen
detection. Both the physical adsorption and affinity capture provided larger
antigen binding capacity and therefore more sensitive antigen detection. The
covalent attachment was chosen to evaluate an enhanced assay with the
incorporation of gold nanoparticles. These particles provided detection limits that
were an order of magnitude improved over those excluding the nanoparticles.
A novel surface chemistry for antibody immobilization in SPR imaging
studies was evaluated. This involved the electrochemical driven formation of
mono- to multilayers of diazonium benzoic acid films. The studies showed the
ability to control the thickness of the films formed and also the ability of the
antibody chips to capture antigen from solution.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:AEU.10048/499 |
| Date | 11 1900 |
| Creators | Grant, Chris |
| Contributors | McDermott, Mark T. (Chemistry), Harrison, D. Jed (Chemistry), Hall, Dennis G. (Chemistry), Campbell, Robert E. (Chemistry), Gallin, Warren J. (Biological Sciences), MacKenzie, C. Roger (External, Environmental Biology, University of Guelph) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 2848506 bytes, application/pdf |
| Relation | Grant, Chris (2008).http://pubs.acs.org/doi/abs/10.1021/la8026489 |
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