The Detection Of DNA Damage Induced By Pharmaceuticals And Their Metabolites Using Electrochemical DNA Biosensor

The thesis includes four parts. The central idea of the thesis is the fabrication of electrochemical DNA biosensor and its application for understanding the mechanism of DNA damage induced by harmful substances and its metabolins. The combination of electrochemical techniques and mimic enzyme are exploited for the development of DNA biosensors to simultaneously detect DNA damage induced by toxic chemicals and its metabolins. This research will extend the application of DNA biosensors in the field of clinical diagnosis, medical verification and environment monitoring.In part one, we introduced modes of DNA damage, summarized various techniques for detection of DNA damage, and prospect the future trend of electrochemical DNA biosensor.1. Electrochemical behavior of nitrofurazone (NFZ) was investigated with the use of cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. The pH-dependence of NFZ was studied at a glass carbon electrode (GCE) in ethanol/Britton-Robinson buffer (30:70), and short-lived nitro-radicals were generated by the reduction of NFZ at high pHs (>7.0). In the presence of DNA, the DPV peak current of NFZ decreased and the peak potential negatively shifted, which indicated that there was an electrostatic interaction between NFZ and DNA. An electrochemical dsDNA/GCE biosensor was prepared to study the DNA damage, and NFZ facilitates this damage, when Co(phen)32+was present as an electroactive probe. Also, the oxidation peaks of guanosine (750mV) and adenosine (980mV) indicated that DNA damage was related directly to the short-lived nitro-radicals. The experiments demonstrated that DNA damage occurred via two different steps while NFZ was metabolized, nitro-radicals were produced. The results suggested that in vivo, the nitro-radicals were more cytotoxic than the NFZ molecules. A linear DPV calibration plot was obtained for NFZ analysis at a modified dsDNA/GCE (concentration range:2.50x10-6-3.75x10-5mol L-1, and the limit of detection:8.0x10-7mol L-1), and NFZ was determined successfully in a pharmaceutical sample.2. A novel biosensor consisting of a glassy carbon electrode (GCE) modified by a polydiphenylamine-4-sulfonic acid (PDPASA, conjugated polymer) film and double-stranded DNA (dsDNA), i.e. dsDNA/PDPASA/GCE, was researched and developed for the analysis of catechol-a potentially toxic substance for humans and the environment. Surface properties of the PDPASA film, particularly after dsDNA was immobilized on it, were characterized with the use of atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The surfaces of the novel DNA/PDPASA/GCE biosensor changed during the fabrication process and displayed high sensitivity for catechol. Oxidation potential of catechol decreased significantly and the corresponding current increased substantially as compared with the values obtained at the GCE alone and at the dsDNA/GCE. Also, with the addition of hydroquinone, two well discriminated CV peaks were obtained, and it was demonstrated that hydroquinone did not interfere with catechol. DPV analysis produced a linear catechol calibration (range:0.750-8.25x10-6mol L-1; detection limit:6.48x10-7mol L-1), and thus, various water samples were analysed successfully by this novel method. In addition, the DNA/PDPASA/GCE was used to study DNA damage in the presence of catechol with the use of the Co(phen)33+electroactive probe. Results indicated that the potentially toxic catechol and its metabolites were all responsible for DNA damage.3. In the present work a new biosensor, which was constructed via a glassy carbon electrode (GCE) modified with DNA, hemin and the mixture of nafion-graphene nanoparticless, was researched and developed for the determination of benzo(a)pyrene and DNA damage induced by benzo(a)pyrene enzyme-catalytic product. The assembled process of this biosensor was performed stepwise and each step was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The results indicated that each step of modified was success and the Hemin/Nafion-graphene/GCE represents a viable platform for the immobilization of DNA. When differential pulse voltammetric (DPV) method was carried out at the range of0.2-1.4V after the prepared DNA biosensor incubated in the benzo(a)pyrene, hydrogen peroxide (H2O2) and mixed solution respectively, it was studied that there is no oxidation peaks was found after the incubation of benzo(a)pyrene solution, but the oxidation peak of guanosine was occurrence after the immersion of H2O2and mixed solution. This phenomenon not only stated that hemin in the present of H2O2could be mimic cytochrome P450to metabolize the benzo(a)pyrene and its metabolite could induce DNA damage, but also proved that benzo(a)pyrene was procarcinogen. This work provides an new timesaving, low-cost in-vitro model system to mimic DNA damage induced by most chemical pollutants and their metabolites. This consequence was validated by EIS and ultraviolet (UV) spectroscopy. In addition, it was found that the current of guanosine was proportional to the concentration of benzo(a)pyrene. Hence, this indirect method could be used to determine benzo(a)pyrene in the concentration range of20.0-220.0×10-9mol L-1, with a detection limit of11.2×10-9mol L-1.