|
|
Novel Electrochemical Biosensor for the Detection of DNA Damage Based on a Large-Surface Carbon Film Electrode
Jurečková, Zuzana ; Vyskočil, Vlastimil (advisor) ; Zima, Jiří (referee)
Presented Bachelor Thesis is focused on the development and utilization of a simple and inexpensive electrochemical DNA biosensor for the detection of DNA damage caused by chemical carcinogens. A large-surface carbon film electrode (ls-CFE), having several advantages, such as its fast preparation, a simple mechanical renewal of the electrode surface, a good reproducibility of measurements, an absence of problems connected with "electrode history", and simple chemical modification, was used for its preparation. The initial part of the work is devoted to the investigation of the composition of a carbon ink suspension, which was optimized and tested using a [Fe(CN)6]4-/3- redox system by cyclic voltammetry (CV), further to the optimization of the DNA biosensor (DNA/ls-CFE) preparation and to its characterization, which was performed using two electrochemical techniques - CV and electrochemical impedance spectroscopy (EIS). The second part of this Thesis deals with the application of the newly prepared DNA biosensor for the detection of DNA damage by model chemical carcinogens. The direct interaction of DNA with fluorene and 2-aminofluorene (2-AF) was investigated using CV and EIS at the DNA/ls-CFE. The obtained results confirmed that the interaction of DNA with fluorene and 2-AF causes DNA damage,...
|
|
|
Novel Electrochemical Biosensor for the Detection of DNA Damage Caused by Chemical Carcinogens
Blašková, Marta ; Vyskočil, Vlastimil (advisor) ; Zima, Jiří (referee)
Presented Bachelor Thesis is focused on the development and utilization of a simple and inexpensive electrochemical DNA biosensor for the detection of DNA damage caused by chemical carcinogens. A glassy carbon electrode (GCE), having several advantages such as broad potential window and well-renewable surface, was used for its preparation. A low- molecular-weight DNA isolated from salmon sperm was used. The initial part of the work is devoted to the optimization of the biosensor preparation and to its characterization, which was performed using several electrochemical techniques - cyclic voltammetry (CV), square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS). The influence of the DNA immobilization type on the quality of the biosensor response was also investigated. Two approaches were tested: (i) spontaneous adsorption of DNA by leaving the DNA stock solution to dry on the GCE surface and (ii) adsorptive accumulation of DNA on the GCE surface from the solution (selected as the optimum one). The second part of this Thesis deals with the detection of DNA damage by various chemical carcinogens. Four model substances were tested: flutamide (an antiandrogen antitumor drug), 4-nitro-3-(trifluoromethyl)aniline (NTMA; a metabolite of flutamide), 2-aminoanthracene (a genotoxic...
|
|
|
Effect of Genotoxic 2-Nitrofluorene and Its Metabolites on DNA In Vivo and Possibilities of Investigation of this Effect Using Electrochemical DNA Biosensors In Vitro
Stávková, Klára ; Vyskočil, Vlastimil (advisor) ; Fišer, Radovan (referee)
Presented Bachelor Thesis is targeted on the topic of hazardous 2-nitrofluorene (NF), which is known as one of the markers of the presence of nitrated polycyclic aromatic hydrocarbons. This Thesis summarizes the current knowledge about the formation of this xenobiotic, its occurrence and transport in living and working environment, and about its negative effects on living organisms, especially on their DNA. These findings obtained upon the in vivo investigations are compared in this Thesis with information obtained using electrochemical DNA biosensors, which represent very promising in vitro alternative to the study of processes proceeding in living organisms upon the interaction of their DNA with the xenobiotic. NF is mainly released to the atmosphere through the exhaust gases of diesel and gasoline engines. It is adsorbed onto solid particulate matters, through which it is spread over long distances and can enter the air passages of animals. Through an atmospheric fall-out, NF contaminates waters and soils, where it is transported together with other nutrients and food to plants and animals; by consummation of such organisms, it can finally enter the body of higher animals and the human. It has been shown that NF is a chemically stable substance. On the other hand, it is metabolized intensively...
|
|
|
Voltammetric Determination of DNA Damage Caused by Genotoxic 2-Aminofluorene at a Carbon Film Electrode
Faťarová, Mária ; Vyskočil, Vlastimil (advisor) ; Zima, Jiří (referee)
A large-surface carbon film electrode (ls-CFE) represents a suitable alternative to the commercially available disposable screen-printed carbon working electrodes. Its simple, fast and inexpensive preparation, simple mechanical renewal of the electrode surface (by wiping off the old film with filter paper and forming a new one), good repro- ducibility of measurements, elimination of problems connected with "electrode history" and simple chemical modification are its main advantages. In this Bachelor Thesis, the utilization of ls-CFE for the preparation of a simple elec- trochemical DNA biosensor for the detection of carcinogenic environmental pollutant, 2-aminofluorene (2-AF), is presented. The composition of carbon ink suspension was optimized and tested using a Fe(II)/Fe(III) redox system by cyclic voltammetry (CV). A large-surface solid electrode, covered by a carbon film of the optimum composition, was then used as a transducer for the preparation of double stranded DNA (dsDNA) based electrochemical DNA biosensor (dsDNA/ls-CFE). The electrochemical behavior of dsDNA (i) present in the measured supporting electrolyte (0.1 mol.L−1 acetate buffer of pH 4.7) or (ii) attached at the ls-CFE surface (by covering with dsDNA stock solution and leaving to dry) was investigated using square wave voltammetry...
|
|
|
Voltammetric Study of the Interaction of Genotoxic 2-Nitrofluorene with DNA at a Hanging Mercury Drop Electrode
Krejčová, Zuzana ; Vyskočil, Vlastimil (advisor) ; Nesměrák, Karel (referee)
In this Diploma Thesis, an interaction of genotoxic environmental pollutant 2-nitrofluorene with a double-stranded calf thymus DNA has been studied using a hanging mercury drop electrode (HMDE) as an electrochemical sensor. Two types of DNA damage were investigated and electrochemically detected (using cyclic voltammetry and differential pulse voltammetry): (i) The DNA damage caused by the direct interaction with 2-nitrofluorene and (ii) the DNA damage caused by short-lived radicals generated by the electrochemical reduction of the nitro group in 2-nitrofluorene. For the study of direct interaction, HMDE was modified by DNA and the interaction of DNA with 2-nitrofluorene was studied, after their incubation, right at the HMDE surface (adsorptive transfer stripping technique) or the DNA was preincubated with 2-nitrofluorene and, subsequently, the interaction was studied voltammetrically (DNA titration technique). Using both detection techniques, the formation of DNA - 2-nitrofluorene complex was observed and the mutual interaction was interpreted as an intercalation between the DNA base pairs, although such interaction was not clearly confirmed by UV-visible absorption spectroscopy. An electrostatic binding of 2-nitrofluorene on DNA sugar-phosphate backbone was partially formed at low concentrations of...
|
guest ::
