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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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Application of Antimony Film Electrodes for Determination of Pesticide Trifluralin
Gajdár, Július ; Fischer, Jan (advisor) ; Vyskočil, Vlastimil (referee)
Antimony film electrode was studied for the use in a voltammetric analysis of organic compounds. The substance chosen as an analyte was trifluralin, which is used as a pesticide. The comparison of different substrate electrodes was carried out between five electrodes, which were gold, silver, copper, polished amalgam and glassy carbon electrode (GCE). Best performance was observed on antimony film glassy carbon electrode (SbFGCE). It provided higher sensitivity and lower limit of quantification in comparison with bare GCE. The antimony film was stable and it provided good reproducibility (RSD = 5.2 %). Parameters of an electrochemical preparation of SbFGCE were optimized. Conditions for determination of concentration of trifluralin were optimized on newly prepared SbFGCE. The best conditions were in a solution of methanol and 0.1 M hydrochloric acid in 1:1 ratio measured by differential pulse voltammetry. The limit of quantification was determined as 1.2·10-6 mol·l-1 . A direct voltammetric measurement on SbFGCE was carried out in a model river sample. Lower limits of quantification were achieved with solid phase extraction (SPE). Recovery values were 86 ± 8 % in deionized water with a preconcentration factor of 125. The limit of quantification was lowered to value 1.1·10-8 mol·l-1 . The extraction...
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Voltammetric determination of 2-amino-3-nitrotoluene on silver solid amalgam electrode
Bergerová, Michaela ; Fischer, Jan (advisor) ; Vyskočil, Vlastimil (referee)
This thesis is focused on development of electrochemical method for determination of 2-amino-3-nitrotoluene. The technique of DC voltammetry (DCV) and differential pulse voltammetry (DPV) were used on a silver solid amalgam electrode modified by mercury meniscus (m-AgSAE) with the Britton-Robinson (BR) buffer supporting electrolyte. In the next step the suitability of developed method was tested for drinking and river water model samples. BR buffer of pH 12.0 was used for determination of 2-amino-3-nitrotoluene by DCV method and BR buffer of pH 7.0 was used for DPV method. Both methods were used for concentration monitoring of studied compounds in the range from 1·10-6 to 1·10-4 mol·l-1 . Limit of quantification of DCV method in BR buffer with deionized water was 2,7·10- 6 mol·l-1 whereas 1,2·10-6 mol·l-1 in drinking water and 1,5·10-6 mol·l-1 in river water. Limit of quantification of DPV method was 1,5·10-6 mol·l-1 in BR buffer, 7,3·10-7 mol·l-1 in drinking water and 1,4·10-6 mol·l-1 in river water. Stability of stock solution of the studied compound of 1·10-3 mol·l-1 concentration in water was measured by spectrometry at 3 wavelengths 222 nm, 293 nm, 416 nm. After one month there was no significant decrease in absorbance so the sample was considered to be stable and it was kept in the dark at...
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Determination of benzophenone-3 on boron-doped diamond film electrode
Procházková, Kateřina ; Dejmková, Hana (advisor) ; Vyskočil, Vlastimil (referee)
Voltammetric behaviour of benzophenone-3 on a diamond film electrode doped by boron was studied by difference pulse voltammetry in an anodic area in the Britton-Robinson buffer medium. The influence of supporting electrolyte pH on the determination was monitored as well as the influence of methanol and acetonitrile content in the measured sample. pH 12 was selected as the optimum value for the determination. Methanol did not have any influence on the determination up to the content of 20 % (v/v), acetonitrile up to content of 70 % (v/v). A linear calibration dependence was measured in the medium of Britton-Robinson buffer pH 12 within the benzophenone-3 concentration range from 1·10-6 mol·L-1 to 1·10-4 mol·L-1 . Detection limit 1.5·10-6 mol·L-1 and determination limit 5.0·10-6 mol·L-1 have been reached. Key words Benzophenone-3 Boron-doped diamond film electrode Differential pulse voltammetry
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Determination of Lead Using FIA Technique with Spectrophotometric and Electrochemical Detection
Hála, Petr ; Vyskočil, Vlastimil (advisor) ; Rychlovský, Petr (referee)
The aim of this thesis was the determination of lead in water samples using complexating reaction with 4-(2-pyridylazo)-resorcinol in medium of 2-amino-2- hydroxymethyl-propan-1,3-diol hydrochloride buffer (Tris·HCl). Firstly, for the determination was chosen UV/VIS molecular spectrometry in a static arrangement. Subsequently, the method was performed in flow injection arrangement. The calibration was performed under optimal experimental conditions. The limits of detection for static and flow injection arrangements were 0.097 mol dm-3 and 0.27 mol dm-3 , respectively. Secondly, the determination of lead by differential pulse voltammetry was performed. The electrochemical properties of lead complex were investigated. During these experiments it was found out that small amount of this complex was adsorbed on the surface of silver solid amalgam electrode. The calibration was performed, the limit of detection was 0.020 mol dm-3 . Keywords Lead, 4-(2-pyridylazo)-resorcinol, flow injection analysis, UV/VIS molecular spectrometry, differential pulse voltammetry, silver solid amalgam electrode.
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Voltammetric Determination of Diacetyl and Pyruvate by Derivatization with o-Phenylenediamine
Kotasová, Marcela ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
The aim of this Diploma Thesis was to develop sensitive, inexpensive, and less time- consuming voltammetric methods for the determination of diacetyle and pyruvate, when both analytes are derivatized by ofenylendiamine (OPDA) and the resulting reaction products are determined. For this purpose, techniques DC voltammetry (DCV) and differential pulse voltammetry (DPV) at a mercury meniscus modified silver solid amalgam electrode (m- AgSAE) were used. The optimization consisted of finding a suitable medium and regeneration of the electrode surface for the determination 2,3dimethylquinoxaline (2,3DMQ) and 2hydroxymethylquinoxaline (2OH3MQ), products of derivatization reactions. The optimal medium for the 2,3DMQ determination was a solution of the Britton-Robinson buffer (BRbuffer) of pH 6.0 for DCV (optimal regeneration potentials Ein = 400 mV and Efin = 900 mV, with limit of quantification LQ ≈ 1.2·10-6 mol·L-1 ) and pH 7.0 for DPV (Ein = 200 mV and Efin = -900 mV, LQ ≈ 1.1·10-7 mol·L-1 ). For 2OH3MQ, the optimal medium was BRbuffer of pH 4.0 and regeneration potentials Ein = 500 mV and Efin = 900 mV for both techniques (LQ ≈ 4.8·10-7 mol·L1 for DCV and 3,4·10-7 mol·L-1 for DPV). Optimization of derivatization reactions for the determination of diacetyle and pyruvate consisted of...
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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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Voltammetric Determination of Quinoxaline at a Mercury Meniscus Modified Silver Solid Amalgam Electrode
Kotasová, Marcela ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
The aim of this Bachelor Thesis was to develop sensitive, inexpensive, and less time-consuming voltammetric methods for the determination of quinoxaline. For this purpose, DC voltammetry (DCV) and differential pulse voltammetry (DPV) at a mercury meniscus modified silver solid amalgam electrode (m-AgSAE) were used. The optimization consisted of finding a suitable medium and a suitable regeneration of the electrode surface. The optimal medium for the determination of quinoxaline was a solution of the Britton-Robinson buffer of pH 13,0, and it was also found that for both techniques, it is advisable to use regeneration potentials Ein = -100 mV a Efin = -1800 mV for the cleaning of the electrode surface. Concentration dependences were measured in the concentration range of 1.10-7 - 1.10-4 mol.L-1 of quinoxaline. Over all concentration ranges, the dependences were non-linear, however, it was possible to fit them with a high correlation using a polynomial function of the second order. The limits of quantification (LQ) reached were 8.2×10-7 mol.L-1 (DCV - evaluated the peak height), 3.9×10-7 mol.L-1 (DPV - evaluated the peak height), and 2.8×10-7 mol.L-1 (DPV - evaluated the peak area). The spectrophotometric determination of quinoxaline, which was performed for a comparison, provided two peaks with the...
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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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Voltammetric Determination of the Explosive Penthrite
Vyvadil, Jan ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
This Bachelor Thesis is focused on the investigation of voltammetric behavior of the explosive penthrite (pentaerythritol tetranitrate) (PETN) and on searching for optimum conditions for its determination using differential pulse voltammetry (DPV) at a hanging mercury drop electrode (HMDE). Mixtures of Britton-Robinson buffer (BR-buffer) and methanol in a volume ratio of 9:1 were used as supporting electrolytes in the investigation of voltammetric behavior of PETN in aqueous-methanolic media, and methanolic solutions of tetramethylammonium bromide (TMAB) and tetrabutylammonium chloride (TBAC) were used for investigations in non-aqueous (methanolic) media. Initially, the study of the influence of pH on voltammetric behavior of PETN was performed. Three pH values of the BR-buffer were chosen as suitable: 2.0, 6.0, and 9.0. At these pH values, the influence of the content of methanol in a supporting electrolyte on the voltammetric response of PETN was measured. At all three pH values, the best responses were obtained at a volume ratio of the BR-buffer and methanol of 9:1 or at a 100% content of methanol (here, TMAB and TBAC methanolic supporting electrolytes were tested and TMAB was selected as the optimum one). Concentration dependences were measured in five different media (in the concentration...
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