UBC Theses and Dissertations
Fluorescent detection of DNA single nucleotide polymorphism by electric field assisted hybridization/melting of surface-immobilized oligonucleotides Verhaven, Alexandra Gaia
Deoxyribonucleic acid (DNA) self-assembled monolayers (SAMs) immobilized on gold electrodes are the basis of many electrochemical biosensors. Control of the interfacial behavior of DNA by means of an electric field is of interest for sensing applications such as the detection of single nucleotide polymorphisms (SNPs). Moreover, the in situ characterization of immobilized DNA monolayers at a molecular level is important for the fabrication of robust, reliable and sensitive sensors. The thesis aims at studying the discrimination between DNA strands containing SNPs on the basis of electric-field assisted hybridization/denaturation of DNA. In situ electrochemical fluorescence microscopy is used as a detection methodology and characterization tool for DNA interfaces. For this purpose, fluorescently labeled DNA sequences are immobilized at gold electrodes as thiol SAMs. First, the SAMs under investigation were composed of perfect match or SNP-containing target sequences. The relationship between the applied potential and the denaturation of DNA duplexes was investigated. Electrochemical melting was observed at -0.25 V vs. Ag|AgCl and attributed to an electrostatics-based melting mechanism. A model based on electrical double layer theories was proposed to explain the observed partial electrochemical melting. The influence of various parameters was systematically investigated such as the assembly of the SAM and the measurement conditions. The observed trends were attributed to a destabilization of the duplex. The most influential variables were the DNA sequence (e.g. mismatch near the electrode surface), ionic strength and temperature. Next, a FRET methodology was investigated by studying a model DNA SAM system labeled with a FRET pair. The aim of the study was to gain information regarding the local molecular scale environment of a DNA SAM under measurement conditions. The influence of surface crystallography on the SAM organization was studied by wide-field FRET microscopy. FRET measurements were used in a semi quantitative way to characterize the uniformity of the DNA monolayer. A departure from the ideal uniform DNA distribution was observed for the (111) and (110) surface regions. The study provided a proof of concept for the use of electrochemical FRET microscopy as an in situ characterization tool for DNA modified electrode surfaces.
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