Characterization and Functionalization of Substrates and Nanoparticles for Detection of Surface Binding Events of Biomolecules

microarrays;DNA hybridization;nanoparticles;photoelectrochemistry

Moses, Selina ; Dr. Stefan Franzen, Committee Chair,Moses, Selina ; Dr. Stefan Franzen ; Committee Chair

University:North Carolina State University

关键词: microarrays;    DNA hybridization;    nanoparticles;    photoelectrochemistry;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/5176/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

This dissertation mainly focuses on detection of biomolecules on metal and metal-oxide surfaces by photoelectrochemistry. Characterization of DNA monolayers on the surface of gold was studied in detail by X-ray photoelectron spectroscopy (XPS), Polarization Modulated Infrared reflectance absorbance Spectroscopy (PMIRRAS) and FTIR. An electrochemical technique Chronocoulometry (CC) quantified the surface coverage of ssDNA on gold using a redox label. The issues that affected the hybridization efficiency of DNA on the gold surface were analyzed and parameters for optimum hybridization were established by the same techniques used to detect ssDNA on the surface. Though gold is a well-known surface, the thiol chemistry, which is the basis of attaching DNA on the surface, is not robust and reproducible due to the desorption of probes from the surface during hybridization, which involves elevating the temperature. Hybridization yield on gold surfaces could not be determined accurately as the desorption of probe from the surface was a competing phenomenon during hybridization event. As a result, new substrates were analyzed and indium-doped tin oxide (ITO) was found to be a very useful substrate because of the ease with which it can be modified with COOH, Phosphates, Phosphonates and silanes. In addition, the physical properties of ITO make it an attractive candidate to use in creating bioarrays. It is highly conductive and tansparent in the visible region and reflective in the IR. Compared to the thiol chemistry on gold the phosphates are much stronger and resistant to elevated temperatures used for hybridization. ITO electrodes can sustain relatively high potentials required to oxidize gold nanoparticles used as labels to detect hybridization. All these properties can be utilized to characterize ssDNA modified ITO surfaces and surface DNA hybridization either by optical or electrical methods. 12-Phosphonododecanoic acid (12-PDA) formed a well-ordered monolayer on the ITO surface. By activating the COOH group by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), an amine terminated DNA probe can be attached to the surface with 12-PDA as the linker. Studies on monolayer of DNA on ITO by XPS and IR show presence of DNA on the ITO surface and electrochemistry quantified the surface coverage. The detection strategies employed in this study to detect hybridization of DNA was by probing the gold nanoparticle labeled target DNA by XPS, AFM, electrochemical stripping and thermography. To further improve the design, TiO2 nanoparticles sensitized with a Ruthenium polypyridyl complex was used as a label to detect DNA hybridization photoelectrochemically. This idea was generalized further to detect viruses - Red Clover Necrotic Mosaic Virus (RCNMV) and HIV from Gag protein immobilized on the ITO surface.