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Towards biosensor for phosphatidylinositol
Eisenreichová, Andrea ; Bouřa, Evžen (advisor) ; Petráčková, Denisa (referee)
Phosphatidylinositol is a a minor membrane component of eukaryotic cells, however, it plays a crucial role in cell signaling pathways as a precursor for a number of signaling molecules and second messengers. Among the most significant ones are phosphoinositides created by phosphorylation of the hydroxyl groups of phosphatidylinositol at positions 3,4, and 5 of the inositol ring. Despite its significance, the spatial and temporal distribution and dynamics of phosphatidylinositol remains unclear owing mainly to the lack of a specific optical probe (biosensor) to visualize phosphatidylinositol in living cells. Biosensor for inositol phospholipids are based on lipid-binding domains of their effector proteins with high enough affinity and specificity for a given phosphoinositide - but nosuch domain is known for PI. However, an enzyme - phosphatidylinositol-dependent phospholipase C - that specifically recognizes phosphatidylinositol is known. This enzyme catalyzes the hydrolysis of phosphatidylinositol into diacylglycerol and inositol 1-phosphate and unlike eukaryotic homologs does not act upon the phosphorylated forms of phosphatidylinositol. The main aim of this thesis was to solve the structures of several inactive mutant forms of phospholipase C from Bacillus cereus complexed to myo-inositol which...
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Study of Biomolecular Interactions with Surface Plasmon Resonance Biosensors
Šípová, Hana ; Homola, Jiří (advisor) ; Houska, Milan (referee) ; Skládal, Petr (referee)
Surface plasmon resonance (SPR) biosensors represent one of the most advanced sensing technologies for real-time studies of biomolecular interactions. In this thesis, methods for functionalization of SPR substrates were optimized and studied via spectroscopic methods. Effects related to the SPR sensor microfluidic interface on the measured biomolecular interactions were analyzed, and furthermore, means to decrease mass-transport limitations were proposed. Several SPR-based assays regarding the detection of nucleic acids were developed, which allow for the detection of physiologically relevant concentrations of nucleic acids as well as point mutations in a nucleic acid sequence. Assays for the determination of the enzymatic activity of HIV integrase and ribonuclease H were developed. These assays can be employed for the design and synthesis of molecules that function either as antiviral drugs or as gene-regulating agents.
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Calculation of optical response of photonic structures by FDTD method
Vozda, Vojtěch ; Veis, Martin (advisor) ; Richter, Ivan (referee)
FDTD method is based on Maxwell's equations and this thesis describe how to make these differential equations computer readable for numerical solution known as the Yee algorithm. Time step dependence on spatial step is examined here in order to obtain stable solution. Discrete Fourier trasform is defined to obtain frequency dependent transmission and reflection coefficients. Programmed simulation is tested on analytically solvable structures even on slightly more complex systems whose optical response was computed by other type of simulation. Finally photonic crystals and their application as biosensors are discussed. Particular shape of photonic crystal is examined in details (frequency spectrum dependence upon spatial resolution, inaccuracy in geometry, different compounds in holes, geometry modification).
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Technology of wearable sensors
Javor, Adrián ; Majzlíková, Petra (referee) ; Hubálek, Jaromír (advisor)
This bachelor thesis deals with thick-film and thin-film technology of creating wearable sensors on human body, that sense substances in perspiration. Part is devoted to their classification and making subsequent proposal of flexible three-electrode biosensors using 4 different electrochemical transducers. All of these biosensors sense concentration of lactate in human sweat, and are based on immobilization lactate oxidase enzyme on the working electrode. Technological process of production is based on combination of thick-film and thin-film technology. Conclusion of this bachelor thesis contains amperometric evaluations of the lactate concentration, from which calibration curves were created.
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Electrochemical biosensors with flow-through enzymatic reactors
Josypčuk, Oksana ; Josypčuk, Bohdan
Twelve enzymatic biosensors with various silica powder reactors were fabricated and tested.\nExperiments with six silica materials showed that mesoporous silica SBA-15 was the best one\nin terms of covering by enzymes, sensitivity and lifetime of biosensors. The current response\nof the SBA15-glucose oxidase (GOx) sensor started to decrease only after 200-day testing.\nThe used amalgam tubular detector in a flow system allowed working at highly negative\npotentials. Statistical results of parallel measurements of model solutions with the fabricated\nbiosensors show their high accuracy and sensitivity. The proposed SBA15-GOx biosensor\nwas used for determination of glucose in commercial honey.
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Electrochemical biosensor for the study of DNA methylation
Petrula, Jakub ; Hrdý, Radim (referee) ; Hynek, David (advisor)
This bachelor’s thesis deals with design and optimalisation of custom biosensor for detection of methylated DNA. Teoretical part explains the mechanism and importance of DNA methylation. Next section describes analytical methods used in connection with DNA methylation and some basic direct and indirect methods of detection. Final part is dedicated to experiment itself, which is divided into several sections. Section one deals witch modification of working electrode and optimalisation of detection method. Second section introduces two different ways of DNA methylation detection. First is based on direct detection and second one on detection through the biosensor. Final part shows determination of methylcytosine from sample based on analysing characteristic attributes of signal and numeric algorithm based on curve fitting.
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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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Differentiation of yeast colonies and development of new approaches to monitor oxygen and nutrient availability
Vopálenská, Irena ; Janderová, Blanka (advisor) ; Demnerová, Kateřina (referee) ; Pichová, Iva (referee)
Yeast Saccharomyces cerevisiae as an unicellular organism is one of the best-studied experimental organisms. It is an important model organism for the study of intracellular processes of eukaryotic cells. Yeasts are also social organisms with cell-to-cell communication able to form organized multicellular structures (colonies and biofilms). Yeast and other microorganisms in nature prefer to form colonies on solid substrates rather than to grow as "planktonic" single cells (Palková, 2004; Wimpenny, 2009). The yeast S. cerevisiae typically forms colonies, biofilms were described only rarely. Yeast colonies exhibit an organized morphological pattern characteristic of each particular yeast strain (Kocková-Kratochvílová, 1982). This work is focusing on morphology and differentiation of the S. cerevisiae colonies of common laboratory strains forming less structured colonies, and strains of the Σ1278b genetic background forming highly structured "fluffy" colonies. It shows that polarized budding pattern and especially cell ability to form aggregates enable development of structured morphology. During development of "fluffy" colonies two differently regulated events of dimorphic switch from yeast form to filamentous growth occur. One of these events is dependent on the surface glycoprotein, Flo11p flocculin. This...
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Fabrication of well defined nanoporous structures with application in membrane sensing
Fabianová, Kateřina ; Édes, Zoltán (referee) ; Sadílek, Jakub (advisor)
Theme of this bachelor thesis is focused on preparation of the metal nanomenhir structures situated in highly oriented matrix of silicon nitride nanopores based plasmonic biosensor. Porous structures were prepared by reactive ion etching of silicon nitride film using electron beam lithography prepared temporary mask as a template. Deposition of metals was handled by evaporation and magnetron sputtering and results was compared. Finally, this work assumes reached results including successful approach of sensor preparation without contamination of surrounding surface by heavy metal ions.
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