Ceres, Donato Marino (2006) Electron transfer at DNA-modified electrodes. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06202006-113417
The DNA pi stack provides an efficient pathway for transport of electron and electron holes. Ground-state electron transport is furthermore extremely sensitive to subtle DNA structural perturbations, such as a single base mismatch, that alter pi-stacking. As a result, DNA-modified electrodes have allowed the development of highly sensitive diagnostic devices for the detection of base mismatches, lesions, and mutations. We have been able to apply DNA-mediated charge transduction, using a methylene blue/ferricyanide electrocatalytic cycle, in a DNA chip format for the detection of a single base mismatches at a microelectrode. Electrocatalysis is detected at DNA-modified electrodes down to 40 um electrode in diameter, where 108 DNA molecules are responsible for the electron transduction. This exquisite sensitivity both for mismatch detection irrespective of sequence context and to a small number of molecules is an important requisite for the development of a device able to detect multiple genetic variations in the absence of DNA amplification.
We have also investigated in detail the electrochemical properties of DNA films. DNA is a highly charged molecule and, when self-assembled on a gold surface in a dense array, its properties are similar to those of polyelectrolyte films. We have found that the structure of the DNA film is sensitive to ion concentration and identity. Variations of the electrostatic potential across the film can sensitively affect both thermodynamics and kinetics of redox reporters incorporated in the film. Methylene blue reduction in the DNA film occurs via a two electron, one proton process. The Pourbaix diagram is linear in the case of a monovalent anionic buffer, while it is curved in phosphate buffer. Electron transfer kinetics are also affected by the relative concentration of divalent anions: at low pH the film is compressed in the linker portion and the rate of electron transfer is faster. Based on this understanding of the electrostatic balance inside the DNA film, a new analytical tool for monitoring hybridization events on gold surfaces has been developed using electrochemical impedance spectroscopy of ferricyanide.
In order to explore the electron transport properties of DNA films mechanistically scanning tunneling microscopy (STM) has also been employed. These experiments provide a first opportunity to examine DNA conductivity under physiological conditions. These STM experiments on DNA films show that DNA, when perpendicularly oriented with respect to the surface, is coupled to the STM tip and the local density of states contribute to the measured tunneling current. At positive biases, when the surface is positive, the DNA is tilted towards the surface and as a result decoupled from the tip; the DNA appears "transparent" and the underlying surface instead is imaged. Also important is the integrity of the base stack. When the percentage of DNA duplexes containing a single base mismatch in the film is increased, the conductivity of the film decreases. The STM tip, being held at a constant current, approaches the DNA film until, at a critical mismatch content, the tip must penetrate the film and image resolution is lost. The current versus voltage characteristic of the DNA film has furthermore been determined through a new scanning tunneling spectroscopic technique that provides highly stable and reproducible measurements. We find that DNA duplex films under physiological conditions exhibit negative differential resistance, which is a feature that is typical of resonant electron tunneling via energetically localized molecular orbitals. This observation provides an experimental evidence for the existence of localized states within the DNA HOMO-LUMO gap that can be responsible for the ground state electron transport observed in electrochemical experiments.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||conductivity; DNA chip; electrochemistry; electron tunneling; in situ STM; intercalator; methylene blue; mutation detection; resonant states|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||18 May 2006|
|Non-Caltech Author Email:||ceres (AT) caltech.edu|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||22 Jun 2006|
|Last Modified:||26 Dec 2012 02:53|
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