Detection of DNA Hybridization and Dehybridization With Surface Enhanced Raman Spectroscopy (SERS) by Using Gold Nanovoids Electrodes

Abstract

Approximately every 30 base pairs may contain an SNP. Thus rapid and sensitive detection of SNPs is essential. Therefore, this thesis was focused on the development of a DNA sensor with the surface enhanced Raman spectroscopy based detection of single nucleotide polymorphism (SNP). The study was accomplished in three steps. The first step was the preparation of the SERS substrates and their optimization in order to obtain high sensitivity. Smooth Au wafers were covered with polystyrene beads using the Langmuir-Blodgett method. Subsequently, electrochemical Au deposition was performed to form the nanostructures. In order to determine the optimal Au thickness the bipolar electrochemistry was employed to form an Au thickness gradient. The bipolar electrodes were modified with 4-nitrothiophenol (4-NTP), and the signal intensities along the bipolar electrodes were detected using SERS. The polystyrene spheres with 200 nm diameter provided the highest signal enhancement with 177.8 nm Au thickness. The second step was the formation of a sensing platform by means of the potential-assisted immobilization method on optimized SERS substrates. The prepared sensors were then hybridized with fully-matched target DNA and the electrochemical DNA melting kinetics was monitored via in-situ Raman measurements. Furthermore, the ssDNA desorption kinetics was also analysed via in-situ Raman measurements. Since the electrochemical melting starts at potential of -1100 mV vs. Ag/AgCl (3 M KCl) and the ssDNA starts to desorb at -1300 mV vs. Ag/AgCl (3 M KCl), -1250 mV vs. Ag/AgCl (3 M KCl) was chosen for further dsDNA dehybridization. In the last step, SNP detection was accomplished by monitoring the signal decrease with time due to the applied potential. Modified SERS substrates were hybridized with single mismatched target DNA and fully-matched target DNA separately. Potential-assisted DNA melting was monitored via in-situ Raman measurements. The results showed that 60% of the signal from SNP-containing target DNA was lost after 60 s of melting, while only 10% of fully matched target DNA was dehybridized in that time. Furthermore, the reproducibility of the designed sensor was satisfying.