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

• Author / Creator

  Grant, Chris

• 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.

• Subjects / Keywords

  • Biomolecule Immobilization
  • Surface Plasmon Resonance (SPR) Imaging
  • Interfacial Chemistry
  • Protein Adsorption
  • Immunoassay

• Graduation date

  Fall 2009

• Type of Item

  Thesis

• Degree

  Doctor of Philisophy

• DOI

  https://doi.org/10.7939/R3136G

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.