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Redox značení DNA - od jednoduché detekce DNA pomocí modifikace komplexy oxidu osmičelého k ratiometrické analýze sekvencí
Havran, Luděk ; Balintová, Jana ; Vidláková, Pavlína ; Macíčková-Cahová, Hana ; Pivoňková, Hana ; Hocek, Michal ; Fojta, Miroslav
Elektrochemické metody našly široké uplatnění jak v analýze DNA a studiu jejích interakcí s různými látkami, tak i při vývoji senzorů pro detekci hybridizace DNA. DNA je přirozeně elektroaktivní molekula, která poskytuje na různých typech pracovních elektrod řadu analyticky využitelných elektrochemických signálů. Pro některé typy analýz, zvláště pak při vývoji DNA hybridizačních senzorů, se osvědčilo užití redox aktivních značek. Jedním ze způsobů jak zavést do DNA elektroaktivní značku je její modifikace pomocí komplex oxidu osmičelého s dusíkatými ligandy (Os,L). Vznikající adukty poskytují na rtuťových i uhlíkových elektrodách elektrochemické signály v důsledku redukce/oxidace atomu osmia v molekule aduktu. Tato metoda značení DNA našla uplatnění ve vysoce citlivé analýze DNA a také v analýze nukleotidových sekvencí. Další z metod pro přípravu redox značené DNA je enzymatická inkorporace značených deoxyribonukleotid trifosfátů (dNTP) pomocí DNA polymeráz. Značené dNTP lze připravit jednoduchou cross-coupling reakcí ve vodném prostředí. V tomto příspěvku bude demonstrována aplikace různých redox aktivních značek v elektrochemické analýze DNA.
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Electrochemical Reduction of 1,3-Alt-tetranitrothiacalix[4]arenes
Liška, Alan ; Ludvík, Jiří
Calixarenes are organic molecules which contain several aromatic units (in this work four) connected by methylene („calixarenes“) or sulfur („thiacalixarenes“) bridges. Calix[4]arenes can exist in four defined conformations: cone-, partial cone-, 1,2- and 1,3-alternating (Fig. 1). The conformational stability of a (thia)calix[4]arene is controlled by the size of the substituents on the upper or the lower rim, or by other interactions (e.g. hydrogen bonds). These compounds are very attractive for supramolecular chemists, since they have the shape of a cavity, being able to exhibit host-guest interaction according to the substitution 1,2. The calixarene skeleton itself is not reducible. Therefore it´s necessary to introduce a suitable redox probe in the calixarene frame in order to use electrochemical methods for investigations of calixarene properties. For the purpose of this study the nitro group was chosen because its electrochemical reduction proceeds easily in a well-defined way.
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Elektrochemická studie triazaborinových chromoforů
Mikysek, T. ; Josefík, F. ; Ludvík, Jiří
Nové organické sloučeniny jsou, v současné dob , v popředí zájmu materiálových chemik , kteří se snaží najít vhodné materiály pro moderní technologie 1. Žádané jsou především „charge-transfer“ chromofory vykazující fluorescenci v pevném stavu, dobrou propustnost sv tla, rozpustnost, tepelnou stabilitu nebo jsou biologicky aktivní 2,3.
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Electrochemistry of Flavonolignans and their Interactions with DNA and Proteins
Pyszková, M. ; Zatloukalová, M. ; Biedermann, David ; Křen, Vladimír ; Ulrichová, J. ; Ramešová, Šárka ; Sokolová, Romana ; Vacek, J.
Electrochemical oxidation of flavonolignans silybin, silydianin, silychristin and their 2,3-dehydroderivatives, was studied using ex situ and in situ cyclic and square wave voltammetry at pyrolytic graphite electrode. The pilot results presented here could be used for further investigation of mechanism of oxidation and reactivity of flavonolignans to DNA and proteins.
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Isolation and Characterization of Protoplasts and their Utilization for Model Membrane
Nováková, Kateřina ; Navrátil, Tomáš ; Šestáková, Ivana ; Langmaier, Jan ; Heyrovský, Michael ; Zámečníková, B. ; Vodičková, H.
Protoplasts are prepared form plant cells by removing their cell walls, which are usually destroyed mechanically or by enzymes. Protoplasts exhibit the spherical shape after removing of their cell walls. They can be isolated from a range of plant tissues: leaves, stems, roots, anthers, and even pollen. Plasmatic membranes surrounding protoplasts can be used as a source of model membranes. Therefore, some parts of plasmatic membranes or subcellular organelle membranes, which contain specific transport systems, can be isolated and integrated to model phospholipid membranes. State of such model membranes can be characterized by electrochemical impedance spectrometry (EIS) and the transported species can be determined with the use of voltammetric methods.
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Use of the Silver Solid Amalgam Electrode for Determination of 5-Nitroindazole
Nováková, Kateřina ; Navrátil, Tomáš ; Hrdlička, V. ; Vyskočil, V. ; Barek, J. ; Chýlková, J.
The voltammetric behavior of 5- nitroindazole (5-NI) has been investigated using mercury meniscus modified (m-AgSAE) silver solid amalgam electrode (inner diameter 0.5 mm). Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were used for study of electrochemical behavior of 5-NI. Britton-Robinson buffer was found to be the best available supporting electrolyte for 5-NI determination. The reaction mechanism was investigated by CV and by elimination voltammetry with linear scan (EVLS). DPV with optimized working parameters was applied for analysis of model solutions containing 5-NI. The limit of detection was calculated as 0.14 mol L for m-AgSAE. Proposed method was successfully tested for analysis of real water samples spiked by 5-NI.
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Methanol Outbreak in the Czech Republic in the year 2012 – Almost Two Years Later
Navrátil, Tomáš ; Zakharov, S. ; Pelclová, D. ; Mrázová, K.
Almost two years ago, in autumn 2012, a large methanol outbreak was registered in the Czech Republic (CR). The first case was registered in September 2012 in Moravian city Havířov. The source was connected with illegal production and sale of adulterated spirits. About 50 dead and more than 150 cases of methanol intoxications have been reported since September 2012. Some cases of intoxications, connected with old methanol sources distributed in 2012, have been registered up to now. Not all of total amount of prepared methanolic drinks has been found and hundreds liters of toxic methanol have remained in stores or in households. n the other hand, many people intoxicated during this outbreak and classified as “fully recovered” by their hospital discharge, have suffered by sequels of their intoxication at present (visual and nervous system disturbances) and their number has been increasing.
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Electrochemical Biosensors Based on Enzymatic Reactor with Amalgam Powder
Josypčuk, Bohdan ; Barek, J. ; Josypčuk, Oksana
Enzymatic reactor based on the powder of silver solid amalgam was suggested as the main part of biosensors in flow systems for the first time. 4-aminothiophenol, glutaraldehyde and enzyme were gradually bonded to the amalgam surface. Large surface of the fine amalgam particles maintains a big quantity of enzyme molecules. Biosensors were tested with enzymes ascorbate oxidase, glucose oxidase, catalase, tyrosinase and laccase. Electrochemical detection of the oxygen concentration change (for the first 3 enzymes) or the quinones concentration (tyrosinase and laccase) in the measured solution was done amperometrically by means of the tubular detector of silver solid amalgam. The current response of each biosensor was optimized with respect to the detection potential, the flow rate of the mobile phase, the injection volume and the enzymatic reactor volume. Under the found optimum conditions, concentration dependence and some statistical parameters of repeated measurements (relative standard deviation (RSD) for the studied enzymes was in the range 0.81 – 2.1 %) were measured. Biosensor with the ascorbate oxidase reactor was used for determination of ascorbic acid in the vitamin tablet Celaskon®. Results of the analysis were in good agreement with the contents of ascorbic acid declared by manufacturer and the RSD of these analyses was 2.0 %.
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