Electrochemical Researches And Applications On The Monitor Of DNA Damage Induced By Enzyme-catalyzed Fenton Reaction

DNA plays an important role in biological system, which stores the genetic information and controls cell proliferation and differentiation. Many biological processes, such as diseases and aging, have a close relationship with DNA damage. Therefore, it is important to find sensitive, rapid and simple analytical methods to study DNA damage. It is very necessary for investigating the mechanism of biological processes and treatment of dieases.As a novel green non-aqueous electrolyte, room temperature ionic liquids (ILs) have shown many unique electrochemical characteristics in biocatalysis and biosensing with high ionic conductivity, wide electrochemical window, good chemical and thermal stability, less volatile, large viscosity, non-flammable, good solubility and so on. ILs could use as non-aqueous solvents for studying the production of free radical. It establishes a new method to investigate DNA damage and the inhibition of antioxidants on DNA damage. These advantages allow RTILs to be designed for specific reaction system including the generation of radical. Given that·OH is a kind of very unstable and short lifetime specie, it causes great difficulties in the investigation of·OH-induced DNA damage. It is thought the radical travels slow through high viscosity of RTILs. This might reduce the chance of interactive collision among the radicals, which leads to prolong the lifetime of the radical. In other words, the lifetime of the radical in large viscosity of RTILs will distinctly increase.The main contents and results are summarized as follows:(1) ILs were synthesized via the reaction of N, N’-dialkylimidazolium cation and PF6anion, and characterized its structure and properties. It is necessary to choose ILs with good bio-affinity, good solubility of some biomolecular, wide potential window in order to meet the requirements of electrochemical research.(2) A simple procedure for the electrochemical detection of DNA damage and antioxidants protecting DNA from its damage by DNA biosensor is reported. The biosensor was constructed by the co-immobilization of DNA and Glucose oxidase (GOx) on the glassy carbon electrode. Under the aerobic conditions, GOx could catalyze the oxidation of glucose, and the produced H2O2would further react with transition metal ions, generating hydroxyl radical (·OH)(frequently via Fenton-type reactions), which was validated by UV-vis spectroscopy. The produced reactive oxygen species can cause serious oxidative damage to DNA, which could be detected by square wave voltammetry signals of the electroactive indicator Co(bpy)33+. Some conditions, such as pH of phosphate buffer, incubation time and the concentration of glucose and FeSO4were optimized. The effects of antioxidants, such as ascorbic acid and aloe-emodin, on DNA damage were also investigated within the concentration range of5×10-8mol L-1-2×10-4mol L-1in the cleavage agent. This work provides an in vitro model system to mimic the real bioprocess in DNA damage through a simple electrochemical approach.(3) Oxidative DNA damage is the most critical factor implicated in carcinogenesis and other disorders. However, the electrochemical detection of oxidative DNA damage and protection by antioxidants has been reported rarely in room temperature ionic liquids. In this study, a hydrophobic room temperature ionic liquid,1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), was applied as nonaqueous solvent for the generation of hydroxyl radical (OH) through glucose oxidase (GOx)-catalyzed Fenton reaction. The enzyme could catalyze glucose, and the produced H2O2would further react with transition metal ions, generating a reactive oxygen species-hydroxyl radical (·OH).·OH attacked DNA and led to DNA damage. DNA damage could be detected by the square wave voltammetry (SWV) signals of the electroactive indicator Co(bpy)33+. Co(bpy)33+binded more strongly to intact DNA, and the peak currents of the SWV at the potential of0.064V decreased when DNA was damaged. The experiment results testified that the antioxidants, ascorbic acid, aloe-emodin and rutin, could inhibit oxidative DNA damage by hydroxyl radical. The method was promising for rapid, sensitive, and inexpensive detection of DNA damage.(4) The biosensor was constructed by immobilising dsDNA and xanthine oxidase (XOD) on the glassy carbon electrode. The reactive oxygen species (ROSs), hydroxyl radical (OH), generating by the xanthine/FeSO4system on the DNA-XOD/GCE was reported for the first time. The produced reactive oxygen species, which was verified by UV-vis spectroscopy, were very active and could cause serious oxidative damage to DNA. Square wave voltammetry (SWV) signals of the electroactive indicator Co(bpy)33+was selected for the development of this methodology. There was less oxidative damage when reactive antioxidants were added. The antioxidants used in this work were ascorbic acid (AA), aloe-emodin (AE) and rutin. The method was promising for rapid, sensitive, and inexpensive detection of DNA damage.