Optical Detection of the Mitochondrial DNA 8-Hydroxy-2''–Deoxyguanosine Mutations

• Main Authors: Chien-Hui Su, 蘇千惠
• Other Authors: Gou-Jen Wang
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/17694570921067450102

碩士 === 國立中興大學 === 機械工程學系所 === 104 === This study utilizes small fluorescent molecules and photosensitizers to detect the production of 8-OHdG arising from oxidative damage. Firstly, the absorption and fluorescence spectra for each of the nucleosides of mitochondrial DNA (Guanosine, Cytidine, Thymidine, Adenosine) and 8-OHdG were obtained. The 8-OHdG absorption spectra showed that there are two significant peaks below the wavelength of 300 nm (at a pH of 7, 248 and 296 nm), and as the pH of the test solution was increased the absorption peaks of each nucleoside becomes indistinguishable from one another, while at a pH of 7 the peaks can be clearly seen. An excitation wavelength of 295 nm was selected for fluorescence spectroscopy of 8-OHdG, and it was found that at a pH of 11 a maximum fluorescence intensity was obtained at a wavelength of 362 nm. Next, small fluorescent molecules (Compound B) in combination with fluorescence resonance energy transfer (FRET) was used to amplify the signal obtained from 8-OHdG. The results show that at a pH of 11, using an excitation wavelength of 295 nm, a fluorescent signal can be detected at a wavelength of 575 nm, indicating successful energy transfer from 8-OHdG to Compound B. It was found that a change in concentration of 8-OHdG affected the signal intensity at 575 nm and the lower detection limit was found to be 3.125 μM. Furthermore, the addition of a photosensitizer (Methylene blue) to generate singlet oxygen, after irradiation, may damage the 8-OHdG and allows detection of changes in the absorption spectra at different concentrations. Using a Methylene blue concentration of 5 μM it was possible to distinguish changes in the 8-OHdG concentration and it was found that as the irradiation time increased the absorption intensity of 8-OHdG decreased, showing that Methylene blue can effectively damage 8-OHdG. The results obtained revealed that 8-OHdG completely reacts with Methylene blue after 20 minutes of exposure, and the minimum detectable concentration of 8-OHdG was found to be 3.125 μM.