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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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HPLC-ED/UV with Solid Phase Extraction for the Determination of 5-Hydroxyindole-3-acetic Acid
Němečková-Makrlíková, Anna ; Dejmková, H. ; Navrátil, Tomáš ; Barek, J. ; Vyskočil, V.
Determination of 5-HIAA in human urine was successfully performed using HPLC-ED/UV with SPE. HPLC-ED is a very sensitive technique, presented method does not need any sample pre-treatment apart from SPE procedure, which replaced filtration of samples of human urine to avoid problems with clogging a HPLC column. The found native\nconcentrations of 5-HIAA in human urine using HPLC-ED with SPE fully corresponds with published normal concentrations in urine (17.8 - 58.3 micromol l-1). The found native concentrations of 5-HIAA were 18.4 micromol L-1 (calculated from peak height). The presented method is fast and could be used for simple\nscreening of human urine, because HPLC-ED/UV allows simultaneous determination of 5-HIAA, HVA, and VMA as important tumour biomarkers.
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Application of a Micro-Volume Voltammetric Cell for Determination of Doxorubicin Hydrochloride
Skalová, Štěpánka ; Navrátil, Tomáš ; Barek, J. ; Vyskočil, V.
Doxorubicin is an anticancer drug, which is used for treatment of various cancer types. It contains a quinone structure, similarly as some other drugs belonging to the same category (e.g., daunorubicin and epirubicin). Its monitoring can lead to optimization of individual patient dosages and increase chances for recovery of patients. A micro-volume voltammetric cell and differential pulse cathodic stripping voltammetry have been used for determination of doxorubicin hydrochloride. A volume of used sample was 50 microL. Limit of detection was 0.39 micromol L-1. An applicability of this method was verified on samples of drinking water and of human urine.
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Voltammetric Determination of Tumor Biomarkers using Flow Injection Analysis with Amperometric Detection
Makrlíková, Anna ; Matysik, F.-M. ; Barek, J. ; Vyskočil, V.
Three tumor biomarkers (homovanillic acid, vanillylmandelic acid,\nand 5-hydroxyindole-3-acetic acid) have been determined by flow\ninjection analysis with amperometric detection at screen-printed\ncarbon electrodes in optimum medium of Britton-Robinson buffer\n(0.04 mol l-1, pH = 2.0). Dependences of the peaks current on the\nconcentration of biomarkers were linear in the tested concentration\nregion from 0.05 to 100 mu mol l-1, with the limits of detection of\n0.065 mu mol l-1 for homovanillic acid, 0.053 mu mol l-1 for vanillylmandelic\nacid, and 0.033 mu mol l-1 for 5-hydroxyindole-3-acetic acid (calculated\nfrom heights), and 0.024 mu mol 1-1 for homovanillic acid,\n0.020 mu mol l-1 forvanillylmandelic acid, and 0.012 mu mol l-1 for 5-hydroxyindole-3-acetic acid (calculated from areas), respectively.
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Pilot Experiments With a Micro-Volume Voltammetric Cell for the Determination of Electrochemically Reducible Organic Compounds
Skalová, Štěpánka ; Barek, J. ; Rodrigues, J. A. ; Goncalves, L.M. ; Navrátil, Tomáš ; Vyskočil, V.
A new micro-volume voltammetric cell for analysis of small volumes\nof electrochemically reducible organic compounds was developed\nand tested. The sample (20-100 mu L) is placed in a narrow glass tube\nwith an agar membrane at the bottom and the working electrode is\nimmersed into the sample. The agar membrane, as anion permeable\nlayer, electrically connects the workin g e lectrode immersed in the\nanalysed sample in the glass tube with a large-volume compartment\n(20 mL) filled with Britton-Robinson buffer, where conventional nonminiaturized\nreference and auxiliary electrodes are placed. The system\nwas tested using a polished silver solid amalgam electrode\n(p-AgSAE) as a working electrode. Sodium anthraquinone-2-sulfonate\nwas used as a model compound because it is intended to use this\nsystem to monitor electrochemically reducible organic compounds.
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Mercury Electrodes as Tools for Voltammetric Determination of Biologically Active Organic Compounds and for Detection of Their Interaction with DNA
Horáková, Eva ; Vyskočil, Vlastimil (advisor) ; Ludvík, Jiří (referee) ; Vytřas, Karel (referee)
The main aim of this work was to use traditional mercury electrodes for the development of voltammetric methods of determination of organic xenobiotics and for the electrochemical study of the interaction between double-stranded deoxyribonucleic acid (DNA) and these compounds. In relation to my previous research work (conducted in the framework of my diploma thesis), firstly, 4-nitrobiphenyl (4-NBP), the suspected carcinogen, was studied. Interaction of DNA with 4-NBP was studied using differential pulse voltammetry (DPV), cyclic voltammetry (CV), and chronocoulometry at a hanging mercury drop electrode (HMDE), and using CV and alternating current voltammetry at a DNA modified HMDE. Using CV, the reduction mechanism was investigated. The interaction of DNA with 4-aminobiphenyl (4-ABP), a metabolite of 4-NBP, and 4-NBP reduction intermediates was studied. It was found that the interaction of DNA with 4-NBP or 4-ABP results in a formation of a DNA aggregate with these analytes. The second studied analyte was methyl violet 2B (MV). For determination of MV in a buffered solution were used: direct current tast polarography and differential pulse polarography at a dropping mercury electrode, and direct current voltammetry, DPV, and differential pulse adsorptive stripping voltammetry (DPAdSV) at HMDE. The...
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Novel Approaches in Electrochemical Determination of Xenobiotic Compounds and in Study of Their Interaction with DNA
Hájková, Andrea ; Vyskočil, Vlastimil (advisor) ; Trnková, Libuše (referee) ; Labuda, Ján (referee)
Presented Ph.D. Thesis is focused on the development of analytical methods applicable for determination of selected xenobiotic compounds and for monitoring DNA damage they can induce. The main attention has been paid to the development and testing of non-toxic electrode materials for preparation of miniaturized electrochemical devices and novel electrochemical DNA biosensors. 2-Aminofluoren-9-one (2-AFN) was selected as a model environmental pollutant, which belongs to the group of hazardous genotoxic substances. Its carcinogenic and mutagenic effects may represent a risk to living and working environment. 2-AFN has one oxo group, where the cathodic reduction occurs, and one amino group, where the anodic oxidation occurs. The voltammetric behavior of 2-AFN in the negative potential region was investigated at a mercury meniscus modified silver solid amalgam electrode (m-AgSAE) representing a non-toxic and more mechanically robust alternative to mercury electrodes. This working electrode was subsequently used for the development of a newly designed miniaturized electrode system (MES), which has many benefits as the possibility of simple field measurements, easy portability, and the measurement in sample volume 100 µL. Moreover, a glassy carbon electrode (GCE) was used for further investigation of...
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Factors influencing electrochemical oxidation of m-cresol at boron-doped diamond electrode
Procházková, Kateřina ; Schwarzová, Karolina (advisor) ; Vyskočil, Vlastimil (referee)
This study investigates electrochemical oxidation of m-cresol on boron-doped diamond electrode using direct current voltammetry (DCV), differential pulse voltametry (DPV) and cyclic voltammetry (CV). In aqueous media in pH range 2.0 - 12.0 m-cresol provides one oxidation peak. The electrode reaction is diffusion-controled. Because of electrode passivation two types of pretreatment were applied for reactivation of electrode surface., i.e. alumina polishing and anodic activation using potential of +2400 mV. Peak heights and potentials are strongly dipending on the type of pretreatment for DCV and DPV - the difference in peak potentials can reach 430 mV. Using optimal conditions for alumina polishing in 0.01 μmol·L-1 NaOH the linear dynamic range is 1.0 - 75 μmol·L-1 for DCV and 0.75 - 75 μmol·L-1 for DPV. And for anodic activation in BR buffer pH 2.0 the linear dynamic range is 0.75 - 75 μmol·L-1 for DCV and DPV. The influence of boron-doping level was investigated using a semiconductive and mettalic-type BDD film. For the latter the sensitivity in DP voltammetry is two times higer and for both types the linear dynamic range is ca 1 - 25 μmol·L-1 . The voltammetric response of m-cresol was further investigated in the presence of cationic surfactants. In the presence of CTAB and CPB the peak current...
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Electrochemical reduction of dehydrocholic and chenodeoxycholic acid at stationary mercury and amalgam electrodes
Patáková, Adéla ; Schwarzová, Karolina (advisor) ; Vyskočil, Vlastimil (referee)
This thesis deals with the study of electrochemical behaviour of dehydrocholic and chenodeoxycholic acid at stationary mercury-based electrodes - hanging mercury dropping electrode and silver solid amalgam electrode modified by a mercury meniscus. This is the first study of electrochemical behaviour of dehydrocholic acid which offers tree peaks with potentials around -1270 mV, -1450 mV and -1800 mV. The last one with the potential -1800 mV is probably the main peak given by reduction of carboxylic group of side chain of steroid skeleton. By cyclic voltammetry was determined that the process is quasireversible and is strongly influenced by adsorption of DHCA on the electrode surface and also by the pH which determines dissociation degree of carboxylic group. In the environment of BR buffer (pH 7.0) - methanol (9:1) was measured concentration dependence by DC, DP and "square-wave" voltammetry. By neither one of these methods have been reached satisfying limits of detection and wide linear dynamic ranges. However, the repeatable signals in wide range of pH can be used for electrochemical characterization of DHCA. CDCA offers in environment 0,04 mol·l-1 borat buffer (pH 9.1) - methanol (9:1) one signal with potential around -1350 mV. Position of this peak on the potential axe and also its height are...
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Voltammetric Determination of Penthrite Using Mercury and Silver Amalgam Electrodes
Vyvadil, Jan ; Vyskočil, Vlastimil (advisor) ; Dejmková, Hana (referee)
This Diploma Thesis is focused on investigation of the 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) (this part of the Diploma Thesis follows the topic investigated in the author's Bachelor Thesis; in this Diploma Thesis, the results of the Bachelor thesis are applied to the determination of PETN in environmental matrices) and at a mercury meniscus modified silver solid amalgam electrode (m-AgSAE) and on investigation of voltammetric behavior in real samples (deionized, drinking, and river water). For investigating the behavior of PETN in aqueous-methanolic media on the m-AgSAE, mixtures of Britton-Robinson buffers (BR-buffers) and methanol in various volume ratios were used. In non-aqueous media, methanolic solutions of tetramethylammonium bromide (TMAB), tetrabutylammonium iodide (TBAI), and tetrabutylammonium chloride (TBAC) were used as supporting electrolytes. Firstly, the influence of pH (in the range of 2 - 13) and methanol content (10 - 90 vol. %) on voltammetric responses of PETN was studied. The best response was obtained at each of those pH values at the volume ratio of BR-buffer - methanol of 1:9 or...
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