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Permanent link (DOI): https://doi.org/10.7939/R3136G

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Development and Characterization of Interfacial Chemistry for Biomolecule Immobilization in Surface Plasmon Resonance (SPR) Imaging Studies Open Access

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Other title
Subject/Keyword
Biomolecule Immobilization, Surface Plasmon Resonance (SPR) Imaging, Interfacial Chemistry, Protein Adsorption, Immunoassay
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Grant, Chris
Supervisor and department
McDermott, Mark T. (Chemistry)
Examining committee member and department
MacKenzie, C. Roger (External, Environmental Biology, University of Guelph)
Gallin, Warren J. (Biological Sciences)
Campbell, Robert E. (Chemistry)
Hall, Dennis G. (Chemistry)
Harrison, D. Jed (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2009-08-05T21:05:18Z
Graduation date
2009-11
Degree
Doctor of Philisophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R3136G
Rights
License granted by Chris Grant (cfgrant@ualberta.ca) on 2009-08-01T22:27:56Z (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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File size: 2848506
Last modified: 2015:10:12 12:21:24-06:00
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File title: Prefatory pages ALL1.pdf
File title: Microsoft Word - Prefatory pages ALL.doc
File author: Chris Grant
Page count: 189
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