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Synthesis of core/shell quantum dots for diagnostics
Mihajlović, Ana ; Vaculovičová, Markéta (referee) ; Pekárková, Jana (advisor)
This thesis deals with biosensors based on modified semiconductor core/shell quantum dots (QDs) for diagnosis. The work is divided into four main parts. The first one discusses the theory required for the use of QDs in bioaplications, there are described methods of synthesis, modification, application and bioconjugation of QDs. In the experimental part, CdTe/ZnS QDs with core/shell structure were prepared, in which the core was modified by MPA, GSH and TGA. In the next step, these QDs were further modified using CDI, EDC and NHS as mediators in order to increase affinity to BSA (bovine serum albumine) and IgG (imunoglobuline G). Prepared conjugates were characterized by fluorescence spectroscopy (Infinite M200Pro, Tecan) and capillary electrophoresis (Agilent 7100).
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Study of UV-generated fluorescent zinc complexes by fluorescence spectroscopy
Havlíková, Martina ; Vaculovičová, Markéta (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of UV light-generated zinc complexes with cadmium and organic molecules SAM, SAH, CYS, HCYS and GSSG, specifically at 375 nm. Furthemore, the aim of the work is to characterize the precursors spectrally and temporally before and after irradiation in the transilluminator at 250 nm. Study of genesis these complexes was performed by FLIM. Thanks to this method, it was found that the formation of complexes occurs only with Zn:SAH, Zn:GSSG and Zn:Cd. The formation of complexes is influenced by the method of preparation. The spectral characteristic was performed on a fluorimeter where the increase in fluorescence intensity of the irradiated solution with the precursors was expected. These were turbid solutions where sedimentation of the particles was observed and the intensity of fluorescence was changed. In the Zn:SAM and Zn:CYS sample, the sedimentation increased in intensity, while in Zn:SAH and Zn:HCYS decreased. The Zn:Cd precursor solution was clear and there was no change in intensity. Zn:Cd showed the best spectral properties, while the Zn:SAM sample, whose excitation and emission maxima are very close to each other, appeared to be the worst. A sample with Zn:CYS and Zn:HCYS showed almost the same spectra and respective peak results. Based on lifetime characteristics by TCSPC, the sample with Zn:CYS, Zn:HCYS and Zn:GSSG, which showed 3 lifetimes, was best treated. Lifetime could not be unambiguously determined for SAM and SAH samples. Zn:Cd had 4 lifetimes
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Study of metallothionein conjugates by electromigration methods
Johaníková, Klára ; Zemánková, Kristýna (referee) ; Vaculovičová, Markéta (advisor)
The bachelor thesis deals with the study of conjugates of metallothionein (MT) with fluorescent nanoparticles using electromigration methods. Particular attention is paid to the preparation and characterization of quantum dots (QDs) and their bioconjugates with the metallothionein protein, whose separation conditions (concentration and suitable electrolyte pH) have been carefully optimized. For the preparation of QDs, the action of UV radiation for one type of quantum dots and the thermal action of high temperature for the second type of species were used. Conjugation of QDs to the protein was performed via a carbodiimide-zero-length crosslinker (EDC / sulfo-NHS), which activates carboxyl groups and allows bioconjugation of the ligand by covalent bonding. The resulting bioconjugates were studied by capillary electrophoresis with absorption detection ( 214).
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Optimization of a method for the determination of inorganic ions using capillary electrophoresis
Špačková, Simona ; Bezděková, Jaroslava (referee) ; Vaculovičová, Markéta (advisor)
Inorganic ions are one of the most important group chemicals. Their importance is crucial for all living organisms on the planet and monitoring of their content is essential for this purpose in clinical or environmental analysis but also in the food industry, agriculture or many other industries. Many analytical methods for ion detection have been designed and used, depending on the area of their use. Recently, numerous methods have been adapted to areas where not primarily intended. One such methods may be, for example, laser ablation with inductively coupled plasma mass spectrometry, which has recently begun to affect the analysis of biological samples. As a complementary method, able to detect also non-metallic ions is capillary electrophoresis. In this master thesis, the possibility of applying capillary electrophoresis with indirect photometric detection for the separation of inorganic ions in cell lysates of HeLa cells by capillary electrophoresis was verified. Main attention was paid to chloride ions. The ability to separate inorganic compounds in cellular material using salicylic acid as an absorbing anion in the base electrolyte was tested.
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Origin of biomolecules over clays under conditions of early terrestrial planets
Hrnčířová, Jana ; Ferus, Martin (advisor) ; Vaculovičová, Markéta (referee)
The mankind has always been interested in the origin of life on Earth or in the universe. The Oparin's theory1 replaced creationist metaphysical theories (of gods' actions on Earth to create life) in the beginning of the twentieth century. This theory explained production of basic building blocks of life like amino acids, nucleobases and sugars from abiogenic mixture of gases and subsequent formation of so-called coacervates. The synthesis of amino acids was then confirmed by experiments of Miller and Urey2 . Especially the theory of simple formation of nucleobases became an essential contradiction between evolutionary and molecular biologists and chemists. From biological point of view it seems very probable that primal life structures were based on the self-replicating molecule of RNA. From chemical point of view the synthesis of nucleobases seemed very problematic, therefore the models of life structures based on amino acids were favorited. This opposes the basic dogma of molecular biology, which postulates one-way transcriptions of nucleobases' sequence into amino acids' sequence exclusively. In our experiments we follow the idea of simple production of nucleobases from formamide molecule in possible primordial conditions. The mixture of formamide with different types of clays was exposed to...
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Optimization of a method for the determination of inorganic ions using capillary electrophoresis
Špačková, Simona ; Bezděková, Jaroslava (referee) ; Vaculovičová, Markéta (advisor)
Inorganic ions are one of the most important group chemicals. Their importance is crucial for all living organisms on the planet and monitoring of their content is essential for this purpose in clinical or environmental analysis but also in the food industry, agriculture or many other industries. Many analytical methods for ion detection have been designed and used, depending on the area of their use. Recently, numerous methods have been adapted to areas where not primarily intended. One such methods may be, for example, laser ablation with inductively coupled plasma mass spectrometry, which has recently begun to affect the analysis of biological samples. As a complementary method, able to detect also non-metallic ions is capillary electrophoresis. In this master thesis, the possibility of applying capillary electrophoresis with indirect photometric detection for the separation of inorganic ions in cell lysates of HeLa cells by capillary electrophoresis was verified. Main attention was paid to chloride ions. The ability to separate inorganic compounds in cellular material using salicylic acid as an absorbing anion in the base electrolyte was tested.
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Study of metallothionein conjugates by electromigration methods
Johaníková, Klára ; Zemánková, Kristýna (referee) ; Vaculovičová, Markéta (advisor)
The bachelor thesis deals with the study of conjugates of metallothionein (MT) with fluorescent nanoparticles using electromigration methods. Particular attention is paid to the preparation and characterization of quantum dots (QDs) and their bioconjugates with the metallothionein protein, whose separation conditions (concentration and suitable electrolyte pH) have been carefully optimized. For the preparation of QDs, the action of UV radiation for one type of quantum dots and the thermal action of high temperature for the second type of species were used. Conjugation of QDs to the protein was performed via a carbodiimide-zero-length crosslinker (EDC / sulfo-NHS), which activates carboxyl groups and allows bioconjugation of the ligand by covalent bonding. The resulting bioconjugates were studied by capillary electrophoresis with absorption detection ( 214).
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Study of UV-generated fluorescent zinc complexes by fluorescence spectroscopy
Havlíková, Martina ; Vaculovičová, Markéta (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of UV light-generated zinc complexes with cadmium and organic molecules SAM, SAH, CYS, HCYS and GSSG, specifically at 375 nm. Furthemore, the aim of the work is to characterize the precursors spectrally and temporally before and after irradiation in the transilluminator at 250 nm. Study of genesis these complexes was performed by FLIM. Thanks to this method, it was found that the formation of complexes occurs only with Zn:SAH, Zn:GSSG and Zn:Cd. The formation of complexes is influenced by the method of preparation. The spectral characteristic was performed on a fluorimeter where the increase in fluorescence intensity of the irradiated solution with the precursors was expected. These were turbid solutions where sedimentation of the particles was observed and the intensity of fluorescence was changed. In the Zn:SAM and Zn:CYS sample, the sedimentation increased in intensity, while in Zn:SAH and Zn:HCYS decreased. The Zn:Cd precursor solution was clear and there was no change in intensity. Zn:Cd showed the best spectral properties, while the Zn:SAM sample, whose excitation and emission maxima are very close to each other, appeared to be the worst. A sample with Zn:CYS and Zn:HCYS showed almost the same spectra and respective peak results. Based on lifetime characteristics by TCSPC, the sample with Zn:CYS, Zn:HCYS and Zn:GSSG, which showed 3 lifetimes, was best treated. Lifetime could not be unambiguously determined for SAM and SAH samples. Zn:Cd had 4 lifetimes
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Origin of biomolecules over clays under conditions of early terrestrial planets
Hrnčířová, Jana ; Ferus, Martin (advisor) ; Vaculovičová, Markéta (referee)
The mankind has always been interested in the origin of life on Earth or in the universe. The Oparin's theory1 replaced creationist metaphysical theories (of gods' actions on Earth to create life) in the beginning of the twentieth century. This theory explained production of basic building blocks of life like amino acids, nucleobases and sugars from abiogenic mixture of gases and subsequent formation of so-called coacervates. The synthesis of amino acids was then confirmed by experiments of Miller and Urey2 . Especially the theory of simple formation of nucleobases became an essential contradiction between evolutionary and molecular biologists and chemists. From biological point of view it seems very probable that primal life structures were based on the self-replicating molecule of RNA. From chemical point of view the synthesis of nucleobases seemed very problematic, therefore the models of life structures based on amino acids were favorited. This opposes the basic dogma of molecular biology, which postulates one-way transcriptions of nucleobases' sequence into amino acids' sequence exclusively. In our experiments we follow the idea of simple production of nucleobases from formamide molecule in possible primordial conditions. The mixture of formamide with different types of clays was exposed to...
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Synthesis of core/shell quantum dots for diagnostics
Mihajlović, Ana ; Vaculovičová, Markéta (referee) ; Pekárková, Jana (advisor)
This thesis deals with biosensors based on modified semiconductor core/shell quantum dots (QDs) for diagnosis. The work is divided into four main parts. The first one discusses the theory required for the use of QDs in bioaplications, there are described methods of synthesis, modification, application and bioconjugation of QDs. In the experimental part, CdTe/ZnS QDs with core/shell structure were prepared, in which the core was modified by MPA, GSH and TGA. In the next step, these QDs were further modified using CDI, EDC and NHS as mediators in order to increase affinity to BSA (bovine serum albumine) and IgG (imunoglobuline G). Prepared conjugates were characterized by fluorescence spectroscopy (Infinite M200Pro, Tecan) and capillary electrophoresis (Agilent 7100).
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