DNA-modified surfaces have been widely studied for microarray and biosensor applications, in particular sequence-specific detection of DNA, for which electrochemical and optical signs can be produced. Variations in the organization and surface density of adsorbed DNA are known to affect the sensitivity and reliability of assays performed using such surfaces, however most measurements of such surfaces to date have little to no spatial resolution, limiting the information that can be gathered regarding the heterogeneity of the organization of adsorbed DNA molecules. We have applied in situ epi-fluorescence microscopic imaging in conjunction with electrochemical measurements to fluorescently labelled thiolate DNA, adsorbed on polycrystalline gold electrodes with a mercaptohexanol (MCH) passive layer. Spatially resolved information on the organization of adsorbed DNA on the surface is gathered within an area measuring 520by 730micrometres with a 0.96 micrometre resolution. The technique has enabled us to investigate "hotspots" (regions of anomalously bright fluorescence) and regional variation in fluorescence; since molecular fluorescence is quenched as a function of distance from the metal substrate, potential modulation with consequent DNA reorientation or layer specificity of the adsorption. Furthermore, an alternative means to the conventional preparation of thiolate-DNA / MCH monolayers has been developed. In this new method, a gold substrate passivated with MCH is subsequently immersed in an aqueous solution of 5'hexylthiol modified DNA. Through a ligand exchange process, DNA is immobilized forming a mixed MCH / DNA monolayer. Samples prepared via the new method display fewer hotspots and improved fluorescence switching of the DNA during electromodulation for samples made with single stranded (ss) DNA and with double stranded (ds) DNA. Measurement of the DNA surface concentration using ruthenium (III) hexaammine chloride with cyclic voltammetry for self assembled monolayers (SAMs) prepared via the new method are on the order of 1% of the maximum grafting density obtainable for both ssDNA and dsDNA by conventional methods.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:BVAU.2429/2784 |
| Date | 11 1900 |
| Creators | Murphy, Jeffrey N. |
| Publisher | University of British Columbia |
| 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 | Electronic Thesis or Dissertation |
Page generated in 0.0036 seconds