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Study of Electrochemical Processes on Neuromodulation Electrodes
Vašíček, Čeněk ; Kratochvíl, Matouš (referee) ; Ehlich, Jiří (advisor)
This bachelor’s thesis deals with experiments on a neuromodulation electrode made of a platinum-iridium alloy. The thesis is focused primarily on local changes in concentration of oxygen and hydrogen peroxide based on a potential, that is applied. Experiments take place in two electrolytes – PBS and a mixture of Dulbecco’s modified eagle medium with fetal bovine serum (90:10). The goal is to compare the electrode’s behaviour in these electrolytes, mainly on how the surface of the electrode would be affected, if the electrochemical reactions would happen when a different potential is applied, or if they would be less effective in general etc. Changes in oxygen and hydrogen peroxide concentrations are measured using amperometric sensors, with the added measurement of hydrogen peroxide with a spectrophotometric assay consisting of horseradish peroxidase (HRP) and 3,3‘,5,5‘-tetramethylbenzidine (TMB). The next thing that the thesis deals with is stability of hydrogen peroxide in both electrolytes used. In the last part, suggestions on next steps in this topic are mentioned.
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Study of the Influence of Faradaic Processes on the Efficiency of Yeast Electroporation
Martinů, Dominik ; Krčma, František (referee) ; Ehlich, Jiří (advisor)
Electroporation is a widely used method in biotechnology and healthcare. It involves the application of short, intense electric pulses, which create pores in the cell membrane. These pores allow the exchange of molecules between the electrolyte and the intracellular environment. Although electroporation is a standard technique, its exact mechanism remains unknown and is the subject of current research. It is assumed that the primary mechanism of electroporation is the effect of the electric field itself. Recent studies, however, suggest that one of the accompanying mechanisms may also be the effect of reactive oxygen species (ROS) and generally the products of faradaic processes. These highly oxidizing molecules can influence both the efficiency of electroporation and cell viability. The hypothesis was that by anodizing electroporation electrodes, i.e., creating a thin dielectric layer on their surface, the production of ROS can be prevented. By subsequently comparing the efficiency of electroporation using prepared capacitive electrodes and classic faradaic electrodes, it would be possible to reveal the effect of ROS on electroporation. The production of H2O2, as a representative of ROS, was mapped using standard electroporation buffers, protocols, and various electrode materials. The concentration of H2O2 was determined spectrophotometrically by its reaction with 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by horseradish peroxidase (HRP). Electrodes were selected to achieve low, medium, and high production of H2O2. These selected electrodes were used for in vitro electroporation of Saccharomyces cerevisiae yeast, with the efficiency of permeabilization determined by electrochemical impedance spectroscopy (EIS) and fluorescence measurement of propidium iodide (PI) intake. It was found that the action of ROS is not the primary mechanism of pore formation in the cell membrane during electroporation and does not significantly affect the efficiency of this method. However, the results do not exclude their role in secondary processes that affect the kinetics of pore closure and cell viability after electroporation.
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The influence of electrochemically generated hydrogen peroxide on the stress response of microorganisms for the purpose of increased PHA production
Najbrtová, Johana ; Nováčková, Ivana (referee) ; Ehlich, Jiří (advisor)
The subject of the presented thesis is the effect of electrochemically formed hydrogen peroxide on the production of polyhydroxyalkanoate. In this work, hydrogen peroxide was generated directly in the medium with bacteria by two-electron reduction of oxygen. This thesis is divided into a theoretical part, which is devoted to approaching the topic of polyhydroxyalkanoate, oxidative stress, electrochemical production of H2O2 and the connection of electrochemistry with biotechnology. The experimental part is focused on the application of these findings. Hydrogen peroxide was generated by applying an electrical potential to an electrode system composed of a stainless-steel working electrode, a titanium counter electrode, and a pseudo-reference Ag/AgCl electrode embedded in a polypropylene holder. After initial electrochemical characterization of the prepared system, a potential of 0,8 V and -1 V was selected for application in cultivation experiments. Here, the effect of H2O2 on the stress response of the bacterium Cupriavidus necator H16 from the Czech collection of microorganisms was investigated. From the results of the cultivation experiments, it was evident that the electrochemically produced H2O2 does not have a significant effect on the development of the bacterial culture. However, the electrode system itself positively affects bacterial growth. The influence of H2O2 was not noticeable due to the low production in medium. This was caused by a low concentration of oxygen in the medium. During this work, a new electrode system with higher H2O2 production was designed, which could use atmospheric oxygen for reduction to H2O2. However, even with this system, a desired stress response was not achieved, because increased production of H2O2 was prevented by the formation of biofilm on the electrodes. Furthermore, it was found that the materials used for the electrode system support the formation of biofilm, mainly titanium, stainless-steel mesh and polypropylene.
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Preparation of organic electrochemical tranzistors for biosensorics
Ehlich, Jiří ; Vala, Martin (referee) ; Salyk, Ota (advisor)
The goal of this thesis is to describe the process used in preparation of organic electrochemical transistors thus biosensors for testing potential drugs aimed for cardiomyocytes. Then explore some physical properties of developed sensors such as adhesion of materials used, conductivity of developed structures and stability of developed structures in various water environments. As primary technology used for creating our OECTs was used inkjet printing of special inks with electrical properties on PET and PEN substrate cured with special treatment before and after printing. This thesis results in complete view on technology and development of biosensors we had created.
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Optimalization of printing methods of organic semiconducting layers preparation
Ehlich, Jiří ; Vala, Martin (referee) ; Salyk, Ota (advisor)
Electrophysiological biosensors enables a novel way to measure electrical activity of biological structures both in-vitro and in-vivo and represents valuable alternative to current cellular activity measuring methods. Within this work we will be focusing on development of organic semiconductor (PEDOT:PSS) based Organic Electrochemical Transistors (OECTs) and optimization of material printing methods used in their development. These transistors are meant to be able to transfer electrochemical signals within the cell membrane to electrical signal. Such sensors should be used for cytotoxicity testing of chemicals and potential drugs on cardiomyocytes. Main benefits of OECTs are in their higher sensitivity thanks to their ability to locally amplify electric signals, better noise-signal ratio and outstanding biocompatibility. Their development is undemanding and inexpensive due material printing methods and materials processable at room temperatures.
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Development and characterization of micro-reference electrodes for bioelectronics applications
Florian, Vojtěch ; Glowacki, Eric Daniel (referee) ; Ehlich, Jiří (advisor)
The subject of this work was the construction and long term stability measurement of micro-reference electrodes based on anodized silver wire and titanium wire with Ag/AgCl paste. A measuring apparatus, with which the long term stability was measured, was constructed as a part of this work. These days, commercial reference electrodes are available on the market. These reference electrodes are characterized by their excellent long term stability, however, they are too large and too expensive to be used in bioelectronic systems. Because of this, the science community has been trying to miniaturize reference electrodes in the past years. Without reference electrodes, it is impossible to do most electrochemical measurements. Based on the long term stability of measured samples it was determined which of the samples meet the criteria for bioelectronic measurements. It was also determined which of the studied samples were most suitable. The last result of this work is a measuring apparatus that can measure the long term stability of up to thirty-two electrodes.
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Electrochemical characterization of bioelectronic systems
Runštuková, Nikola ; Salyk, Ota (referee) ; Ehlich, Jiří (advisor)
The aim of this thesis was electrochemical characterization of 24 microbial fuel cells platform for high-throughput screening of electrogenic microorganisms and comparison of measured values across all the fuel cells. In the theoretical part, there is a description of electrogenic bacteria, which are essential for current genereation in microbial fuel cells, description of microbial fuel cells and methods used for their characterization. In experimental part, there is a description of assembly process of the platform and measurement process. The result of this thesis is characterization of the platform before and after inoculation using open-circuit potential measurement, continuous current measurement, cyclic voltametry, linear sweep voltametry and electrochemical impedance spectroscopy. Thanks to this methods the values of open-circuit potencial, maximum power and internal resistance were obtained and their distribution was compared. These performed experiments verified the correct function of presented fuel cell platform.
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Electrochemical evolution of hydrogen peroxide for biotechnological applications
Runštuková, Nikola ; Kratochvíl, Matouš (referee) ; Ehlich, Jiří (advisor)
This master‘s thesis deals with the connection of two distinct fields, electrochemistry and biotechnology, where electrochemically generated peroxide was subsequently used in bacterial cultivation. The peroxide was synthesized in an electrolytic cell by reducing oxygen at the cathode to which suitable electrical potential was applied. The aim of this thesis was to investigate the dependence of the amount of hydrogen peroxide produced on a few selected parameters, such as cathode potential, electrode material, the geometry of the measuring cell, or the stirring speed, and then to apply these findings in the continuous stressing of the bacterium Cupriavidus necator H16, in order to increase the production of polyhydroxyalkanoates. The selected parameters were first tested in small-scale measuring cells and then experiments were carried out on a larger scale, in Erlenmeyer flasks. During the experiments, the amount of electric current produced was measured and also the peroxide concentration was examined spectrophotometrically along with the oxygen concentration measured by using an optical sensor. Based on the obtained results, bacteria were cultivated in flasks with a built-in system of steel electrodes, due to which oxidative stress in the form of in situ generated hydrogen peroxide was continuously applied. The bacterial cultures were evaluated by measuring the change in optical density, gravimetric analysis and GC-FID analysis. It was found that even small amounts of hydrogen peroxide (~25 M) can induce a stress response in the organism in the form of increased polyhydroxybutyrate (PHB) synthesis. A percentage increase in the PHB content of the biomass was achieved, however, the overall polymer yield was lower due to lower biomass growth.
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Novel Devices and Materials for Bioelectronics
Ehlich, Jiří ; Achilleas Savva, Ph.D (referee) ; Cifra,, Michal (referee) ; Salyk, Ota (advisor)
Tato dizertace je syntézou hlavních projektů, kterým jsem se věnoval v průběhu mého doktorského studia. Projekty se rozvíjejí kolem širokého spektra témat, z toho důvodu byl zvolen široký a obecný název. Projekty popsané v této práci se zabývají vývojem elektronických zařízení s využitím v řadě bioelektronických aplikací. První část práce se věnuje vývoji, testování a aplikací platforem pro studium mikroorganismů schopných přímé výměny elektronů s elektronickými zařízeními. Druhá část práce se věnuje elektrické stimulaci kmenových buněk pro jejich řízenou diferenciaci. Dále se zabývá fundamentálním studiem potenciálně škodlivých faradaických reakcí, ke kterým dochází v průběhu stimulačních protokolů používaných v klinické praxi. Všechny popsané projekty sdílí společné teoretické a praktické základy vycházející z chemického inženýrství, elektrochemie a materiálových věd. Sjednocující faktor hrající zásadní roli a objevující se napříč všemi projekty je skupina reakcí souvisejících s redukcí kyslíku. Redukce kyslíku je nezbytná reakce odehrávající se v katodovém poločlánku zařízení pro stadium elektroaktivních mikroorganismů. Redukce kyslíku a následná produkce reaktivních forem kyslíku může mít diskutabilně větší vliv na diferenciaci kmenových buněk pomocí elektrické stimulace než samotná elektrická stimulace. V neposlední řadě reakce související s redukcí kyslíku byly hlavními reakcemi, které jsme pozorovali při aplikaci standardních stimulačních protokolů. Disertace prezentuje stručný teoretický úvod nezbytný pro pochopení prezentovaných projektů. Definuje cíle a prezentuje výsledky formou komentovaného seznamu vědeckých publikací. Rovněž nastiňuje budoucí směřování práce. Hlavní dosažené výsledky mohou být shrnuty následujícím způsobem: Prvním cílem bylo vyvinout platformu založenou na poli mikrobiálních palivových článků pro elektrochemickou charakterizaci elektroaktivních mikroorganismů. Po sérii testovacích prototypů byla vyvinuta platforma splňující všechny předem určené parametry. Platforma se osvědčila jako dostatečně přesná a spolehlivá pro zamýšlené biologické experimenty. Dalším cílem bylo vyrobit platformu pro řízenou diferenciaci kmenových buněk pomocí elektrické stimulace. Bohužel připravená platforma se ukázala jako nespolehlivá a nevhodná pro daný účel. To vedlo k neúspěšné snaze o diferenci kmenových buněk požadovaným směrem. Nakonec jsme se zabývali reakcemi souvisejícími s redukcí kyslíku na elektrodách připravených z nejčastěji používaných materiálů pro neurální stimulaci. Kyslík může být redukovaný na vodu a na peroxid vodíku v závislosti na katalytické aktivitě daného materiálu a charakteru stimulačního protokolu. Změřili jsme množství redukovaného kyslíku a produkovaného peroxidu v blízkosti stimulačních elektrod při aplikaci standardních stimulačních protokolů. Následně jsme porovnali katalytickou aktivitu všech materiálů vzhledem k množství redukovaného kyslíku a produkovaného peroxidu vodíku. Hlavním zjištěním bylo, že všechny elektrodové materiály a všechny testované stimulační protokoly vedou k redukci kyslíku, v některých případech až k lokální hypoxii a všechny materiály generují peroxid vodíku, některé až v řádech jednotek mM. Tyto zjištění považujeme za velmi významné, jelikož ovlivnění koncentrace kyslíku a generace peroxidu vodíku mohou mít výrazný vliv na tkáň v okolí elektrod.
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Development and characterization of micro-reference electrodes for bioelectronics applications
Florian, Vojtěch ; Glowacki, Eric Daniel (referee) ; Ehlich, Jiří (advisor)
The subject of this work was the construction and long term stability measurement of micro-reference electrodes based on anodized silver wire and titanium wire with Ag/AgCl paste. A measuring apparatus, with which the long term stability was measured, was constructed as a part of this work. These days, commercial reference electrodes are available on the market. These reference electrodes are characterized by their excellent long term stability, however, they are too large and too expensive to be used in bioelectronic systems. Because of this, the science community has been trying to miniaturize reference electrodes in the past years. Without reference electrodes, it is impossible to do most electrochemical measurements. Based on the long term stability of measured samples it was determined which of the samples meet the criteria for bioelectronic measurements. It was also determined which of the studied samples were most suitable. The last result of this work is a measuring apparatus that can measure the long term stability of up to thirty-two electrodes.
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