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Study of Genotoxicity of Organic Compounds Using DNA Biosensors
Augustín, Michal ; Vyskočil, Vlastimil (advisor) ; Trnková, Libuše (referee) ; Labuda, Ján (referee)
Proposed dissertation thesis presents novel hybrid electrochemical DNA biosensors based on distinct forms of pyrolytic graphite ("edge-plane" and "basal-plane") and their potential in terms of applicability in the field of DNA electrochemistry. Testing of the applicability of the presented bioanalytical tools was preceded by thorough optimization process involving selection of the optimal values for the most crucial operational parameters of the electrochemical DNA biosensors fabricated by means of electrostatic adsorption - deposition potential (Edep), deposition time (tdep), optimal DNA concentration for electrostatic adsorption (cg(DNA)), and possible involvement of convection during the adsorption procedure. Initially, applicability of presented bioanalytical tools was investigated in relation to monitoring DNA damage by utilization of triplet of external DNA damaging sources - UV light irradiation, one- electron oxidants, and hydroxy radicals. Outcome of the damaging processes was monitored directly (via monitoring changes in the redox signals of DNA) or indirectly (via monitoring changes in the redox signals of redox indicator) by the use of voltammetric methods - square- wave voltammetry (SWV), cyclic voltammetry (CV), linear-sweep voltammetry (LSV), and electrochemical impedance...
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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...
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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...
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Simple Electrochemical DNA Biosensor for Detection of DNA Damage Caused by UV Radiation
Arustamian, Daria ; Vyskočil, Vlastimil (advisor) ; Dejmková, Hana (referee)
Ultraviolet (UV) radiation is a common DNA damaging agent. Major DNA lesions, such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone (6-4PPs) photoproducts, are carcinogenic and mutagenic. UV induced DNA damage was investigated using a simple electrochemical DNA biosensor based on an ultra-trace graphite electrode (UTGE) and low molecular weight doble-stranded DNA (dsDNA) from salmon sperm. Biosensor was prepared using adsorption of dsDNA on a surface of the UTGE and then used to detect UV-induced DNA damage. Effects of UV radiation were investigated using a combination of several electrochemical technics: square-wave voltammetry (SWV) for direct monitoring of DNA base oxidation and cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), as non-direct methods, using redox-active indicator [Fe(CN)6]4-/3- . CV and EIS, which allow characterization of electrode surface, were used to optimize preparation of the dsDNA/UTGE biosensor. Prepared dsDNA/UTGE biosensor was exposed to UV radiation using UV lamp with two set wavelengths: UVC of 254 nm and UVA of 365 nm. UVC radiation was used to damage DNA. Relative signal decrease was 50% after 20 minutes of exposure to UVC radiation. UVA radiation was used to compare effects of different types of UV radiation. Obtained...
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Electrochemical Investigation of Novel Redox Indicatiors for Detection of DNA Damage
Otépka, Tomáš ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
Cílem této práce bylo zkoumání nových redoxních systémů, které by bylo možné využít pro detekci poškození DNA na rtuťovým meniskem modifikované stříbrné pevné amalgámové elektrodě (m-AgSAE). Jako slibné se nabízely komplexy 1,10-fenantrolinu (phen) a iontů přechodných kovů. Konkrétně byly provedeny pokusy s komplexy kobaltaných iontů a phen, jejichž příprava byla provedena slitím roztoků obou složek v příslušném poměru. Chování komplexů v této směsi bylo porovnáno se syntetizovaným komplexním kationtem [Co(phen)3]3+ , a to jednak s využitím voltametrické techniky, jednak spektrofotometricky pomocí UV/VIS spektrofotometrie. Analýzy na m-AgSAE probíhaly s využitím techniky diferenční pulsní voltamerie (DPV) ve vodném prostředí. Jako základní elektrolyt byl zvolen 0,1 mol.dm-3 KCl, který poskytoval nejširší potenciálové okno v katodické oblasti potenciálů. Nízkomolekulární DNA z lososího spermatu byla využita v následujícím modifikační kroku pro vytvoření modifikované elektrody. Poškození DNA bylo provedeno vystavením takto připraveného biosenzoru UV-C záření o vlnové délce 254 nm. Poškozená DNA naadsorbovaná na m-AgSAE byla testována pomocí DPV v elektrochemické cele obsahující ionty Co2+ nebo komplex Co2+ a phen, obojí o koncentraci 1.10-3 mol.dm-3 . Na získaných voltamogramech byla sledována...
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Simple Electrochemical DNA Biosensor for Detection of DNA Damage Caused by UV Radiation
Arustamian, Daria ; Vyskočil, Vlastimil (advisor) ; Dejmková, Hana (referee)
Ultraviolet (UV) radiation is a common DNA damaging agent. Major DNA lesions, such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone (6-4PPs) photoproducts, are carcinogenic and mutagenic. UV induced DNA damage was investigated using a simple electrochemical DNA biosensor based on an ultra-trace graphite electrode (UTGE) and low molecular weight doble-stranded DNA (dsDNA) from salmon sperm. Biosensor was prepared using adsorption of dsDNA on a surface of the UTGE and then used to detect UV-induced DNA damage. Effects of UV radiation were investigated using a combination of several electrochemical technics: square-wave voltammetry (SWV) for direct monitoring of DNA base oxidation and cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), as non-direct methods, using redox-active indicator [Fe(CN)6]4-/3- . CV and EIS, which allow characterization of electrode surface, were used to optimize preparation of the dsDNA/UTGE biosensor. Prepared dsDNA/UTGE biosensor was exposed to UV radiation using UV lamp with two set wavelengths: UVC of 254 nm and UVA of 365 nm. UVC radiation was used to damage DNA. Relative signal decrease was 50% after 20 minutes of exposure to UVC radiation. UVA radiation was used to compare effects of different types of UV radiation. Obtained...
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Detection of Oxidative Stress Using Electrochemical DNA Biosensors
Jurečková, Zuzana ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
Presented Diploma Thesis is focused on the development, characterization, and utilization of simple and inexpensive electrochemical DNA biosensor for the detection of DNA damage caused by oxidative stress. The initial part of the work is devoted to preparation and characterization of a large-surface carbon film electrode (ls-CFE) modified with carbon nanotubes (CNT/ls-CFE). Carbon nanotubes improve electrochemical properties of the transducer and increase the amount of adsorbed DNA on the electrode surface. Testing of the electrode surface modified with multiwalled carbon nanotubes (MWCNT) was performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using a redox system [Fe(CN)6]4-/3- and using square wave voltammetry without any redox indicator. Carbon nanotubes have proved to be unsuitable material for our type of biosensor, but it can be used inanalytical chemistry for the determination of electroactive substances. The second part of this Thesis deals with the application of the prepared DNA biosensor for the detection of DNA damage by oxidative stress. The biosensor based on the ls-CFE was chosen for this purpose, having several advantages, such as its fast preparation, a simple mechanical renewal of the electrode surface, a good reproducibility of measurements,...
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The Use of an Electrochemical DNA Biosensor in Detection of DNA Damage Caused by Genotoxic 2-Nitrofluorene
Stávková, Klára ; Vyskočil, Vlastimil (advisor) ; Zima, Jiří (referee)
2-Nitrofluorene is a model representative of nitrated polycyclic aromatic hydrocarbons (NPAH) which belongs to a group of mutagens and carcinogens. Interaction of DNA with genotoxic 2-nitrofluorene was monitored by an electrochemical DNA biosensor made of a glassy carbon electrode (GCE) and low molecular weight DNA from salmon sperm. Techniques used are electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square-wave voltammetry (SWV). Using the EIS technique, no damage to DNA, which would cause strand breaks in DNA, was observed, whereas using the CV technique, the intercalation of NF to the structure of DNA was observed, leading to the formation of a NF-DNA complex. The intercalation results in a reduction of electroactive sites which can be oxidized. It was verified using the SWV technique, by which a decrease of the peak heights of adenosine and guanosine was observed. Because of the dangerous effect of NF on the structure of DNA, an electroanalytical method for its determination was developed. An applicability of the method was successfully tested on a model sample of sand. For the development of the technique, differential pulse voltammetry (DPV) was used in a mixture of the Britton-Robinson buffer of pH 7.0 and ethanol in a ratio of 7:3 (v/v) and with a periodic...
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The Use of a Simple Electrochemical DNA Biosensor for the Determination of Environmental Pollutants and Investigation of Their Interaction with DNA
Blašková, Marta ; Vyskočil, Vlastimil (advisor) ; Zima, Jiří (referee)
The interaction between three selected representatives of environmental pollutants - naphthalene, anthracene, and 2-aminoanthracene - and DNA was investigated using an electrochemical DNA biosensor based on a glassy carbon electrode (GCE) and low molecular weight DNA from salmon sperm (DNA/GCE). The interactions with DNA were monitored using square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS). For naphthalene, there was no DNA damaging interaction observed. In the case of anthracene, the formation of an intercalation complex [DNA-anthracene] was observed. However, its formation does not cause DNA strand breaks. The formation of similar intercalation complex was observed for 2-aminoanthracene [DNA-2-aminoanthracene], where we suppose on the basis of the results obtained that the intercalation of 2-aminoanthracene into the DNA double helix induces a tension and subsequent formation of single-strand breaks, which cause that the fragments of DNA fall away from the electrode surface. The intercalative interaction of DNA with anthracene a 2-aminoanthracene was used in the development of electrochemical methods for determination of these compounds at the GCE and DNA/GCE. At the development of the methods, DC voltammetry (DCV) and differential pulse voltammetry (DPV) were used....
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Novel Voltammetric DNA Biosensor for the Detection of the DNA Damage Caused by Oncological Drugs
Hrochová, Zuzana ; Vyskočil, Vlastimil (advisor) ; Zima, Jiří (referee)
This Bachelor Thesis is focused on the optimization of conditions for the use of a simple voltammetric DNA biosensor, based on a large-surface carbon film electrode (ls-CFE), for the detection of the DNA damage caused by oncological drugs. ls-CFE was used for its advantageous properties, such as its fast preparation, a simple mechanical renewal of the electrode surface, a good reproducibility of results, a simple chemical modification, and, last but not least, low preparation costs. A content of ethanol in the solution, in which the biosensor was incubated together with the damaging agent, was the main optimization parameter investigated in this Thesis. The contents of ethanol in the range from 0 to 50 % (v/v) of ethanol in 0.1 mol/l phosphate buffer of pH 7.15 (PBS) were tested. After the incubation of the biosensor, the measurements were performed using cyclic voltammetry (CV) in the presence of the redox indicator [Fe(CN)6]4-/3- in PBS. The optimum content of ethanol was found as 5 %. Afterwards, the DNA biosensor, which preparation was optimized in a previous Bachelor Thesis, was used for the detection of the DNA damage caused by model cytostatic agent - Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazol). Using the CV technique and the redox indicator [Fe(CN)6]4-/3- , the response of the biosensor to...
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