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Characterization of curcumin nanocrystals in capillary electrophoresis
Dvořáková, Amálie ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
This Bachelor thesis deals with the characterization of curcumin nanocrystals using capillary electrophoresis with UV-VIS and laser-induced fluorescence (LIF) detection. Curcumin is a natural polyphenolic substance with many biological effects. Its low solubility is one of the main reasons for its low bioavailability, however, it can be increased by preparing curcumin nanocrystals. In the experimental part, the effects of the composition of the background electrolyte (BGE) and the applied voltage on the curcumin nanocrystals were investigated. Background electrolytes were prepared using phosphoric acid, or HEPES and their pH was adjusted to 7.0 by NaOH, or TRIS, to test whether the shape of the peak is influenced by the ionic strength of BGE and not by the increasing concentration of a particular BGE component. Initial experiments did not involve voltage application, but only a pressure of 50 mbar. In following experiments both positive and negative voltages were applied, using a 10mM phosphate buffer, the pH of which was adjusted using TRIS. Most of the experiments were conducted using a 100nm curcumin nanocrystals sample. Curcumin nanocrystals provided an abnormally wide peak in both UV-VIS and LIF detection. It was found that the dispersion of the nanocrystal zone was not caused by diffusion,...
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Modifications of inner walls of electrophoretic capillaries using positively charged anchors
Stávek, Petr ; Křížek, Tomáš (advisor) ; Coufal, Pavel (referee)
Electroosmotic flow plays a crucial role in capillary electrophoresis, as it can impact both the resolution and duration of the separation process. Altering the electroosmotic flow can be achieved through dynamic coating of the inner capillary surface. The primary objective of this work was to investigate the potential of various positively charged anchors in providing a suitable dynamic coating for capillary electrophoresis. At first, experiments were carried out with neopentyl anchors PK 1291, PK 941, PK 1300 and PK 1294 using four different coating methods. Obtained coatings demonstrated the ability to suppress the electroosmotic flow, although the stability of the obtained coatings varied significantly among the individual anchors. PK 941 demonstrated the most promising results within this group. Subsequently, measurements were also performed using the cyclodextrin anchor PK 1281, which yielded the most favorable outcomes. It was observed that depending on the background electrolyte and the chosen coating method, this anchor could either reverse the direction of the electroosmotic flow or nearly completely eliminate it. Key words: capillary electrophoresis, electroosmotic flow, dynamic capilary coating, positively charged anchors.
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Comparison conductive detectors for capillary electrophoresis
Kříček, Zdeněk ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
The aim of this bachelor thesis is comparison of two contactless conductivity detectors in capillary electrophoresis. Two methods were used for comparison: The determination of saccharides, namely glucose, fructose and sucrose, and then the determination of inorganic ions, such as chlorides, nitrates, and sulphates. The theoretical part deals with capillary electrophoresis in general and then the specific use of a contactless conductivity detector in capillary electrophoresis and its optimization. Parameters used for comparison of detectors such as limit of detection or relative standard deviation are described. Afterwards, different methods that can be used for the separation and determination of saccharides and inorganic ions are reviewed. The experimental part contains data that were measured in the optimization part of this study as well as comparison of the two detectors used, Admet and TraceDec. The comparison part starts with the method for the separation of saccharides using short-end injection mode in 40mmol∙l−1 sodium hydroxide with a separation voltage of −15 kV. The internal standard was 0,1mg∙ml−1 lactose. The calibration curve was measured in the concentration range from 0,01 to 0,5 mg∙ml−1 The linearity was measured with values of LOD, LLOQ and RSD determined as well. The values of...
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New procedures for autonomous analyses of dried blood spots by capillary electrophoresis
Profousová, Sylvie ; Glatz, Zdeněk (referee) ; Dvořák, Miloš (advisor)
Collection of capillary blood in the form of dry blood spot (DBS) samples on cellulose sampling cards has been popular in clinical analysis for some time. However, in the diagnosis and monitoring of metabolic disorders and diseases, target analytes (e.g. inorganic and organic ions) are examined from blood plasma or serum. Determination of the target analytes from capillary blood or DBS samples is not the standard and reference values are not available due to the different composition of plasma/serum vs. capillary blood. Moreover, DBS sorbents have been found to release several ions into DBS eluates resulting in their contamination and, together with the effect of blood hematocrit and retention of target analytes, can adversely affect the quantitative analysis of DBS samples. For these reasons, this thesis addresses the design of an alternative concept for DBS collection that would eliminate the use of sampling sorbents and ensure the collection of a defined and precise volume of blood. The experimental part focuses on the determination of selected inorganic and organic ions (NH4+, K+, Na+, Ca2+, Mg2+, Cl, NO3, NO2, SO42, formate, lactate, and acetate) by capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) in various DBS samples. All these ions are present in blood at different concentrations and can be simultaneously released as contaminants from the DBS sorbents. A non-porous polypropylene vial was used for the novel sorbentless DBS collection. A small drop of blood of defined volume (e.g. 5 l) was transferred into this vial and then dried directly therein. The selected vial is suitable for both direct DBS elution and DBS eluate analysis and is fully compatible with autosamplers of commercial CE instruments. The proposed concept of the sorbentless blood collection eliminated unwanted contamination and proved suitable for the determination of the tested cations and anions. The stability of anions was generally lower than that of cations but was still significantly better compared to standard DBS sampling sorbents. Determination of some anions appeared to be acceptable only within 0–48 hours after drying of the sorbentless DBS samples (stored at laboratory temperature), as there was a continuous increase in their content. This was probably due to microbial degradation of the blood matrix. It was confirmed that this increase could be slowed down by storing sorbentless DBS samples at low temperatures. Different blood sampling procedures have been proposed for sorbentless DBS collection that are simple, economical, convenient, and repeatable and are therefore suitable for patient-centric blood sampling as well as for sampling in medical centers. This is a basic pilot study applied only to selected analytes that served as model examples, nevertheless, based on this thesis, more extensive scientific studies will follow up to further develop the new concept of sorbentless DBS collection.
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New technique on a chip for rapid detection of biological materials
Pejović Simeunović, Jelena ; Foret,, František (referee) ; Táborský,, Petr (referee) ; Hubálek, Jaromír (advisor)
Tato práce navrhuje techniku separace a detekce na čipu pro kvantové tečky (QD, „quantum dots“) konjugované s různými proteiny, za účelem sledování vlivu vazebného činidla na potlačení intenzity uorescence QD způsobené konjugací s proteinem a za účelem provedení multianalytické imunoanalýzy k identifikaci malých množství daného proteinu. Za optimálních podmínek byly biokonjugované QD úspěšně odděleny od těch nezkonjugovaných během 10 minut. Částice a cílové roztoky byly smíchány a detekce na čipu byla provedena za pomoci zařízení vyvinutého v naší laboratoři. Byl použit pouze jeden zdroj excitačního světla v kombinaci s několika filtry pro různé emisní vlnové délky. Fluorescence emitovaná dvěma typy konjugovaných QD mohla být poté zaznamenána současně, protože QD emitovaly světlo na různých vlnových délkách, ačkoliv byly excitovány při stejné vlnové délce. Smícháním dvou typů QD biokonjugovaných se dvěma druhy proteinů a protilátek jsme dokázali detekovat imunokomplexní píky s různými plochami. Plocha pod píkem závisela na koncentraci QD a antigenů, na postupu reakcí protilátka–antigen a ukázalo se, že je lineárně korelována s koncentrací antigenu. Ukázali jsme, že kapilární elektroforéza QD na čipu může být použita jako citlivá technika pro detekci biologických molekul. Hlavními výhodami této metody jsou jednoduchost, malé požadavky na objem vzorku i činidla a také vysoká účinnost separace.
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Determination of hyaluronan by electromigration methods
Bártová, Adéla ; Mravcová, Ludmila (referee) ; Řezáčová, Veronika (advisor)
This thesis is focused on description hyaluronic acid, characterization of its behaviour in water solutions, its chemical-physical properties and its using in different industrial sectors. The next part deals with possibilities of its analysis. The following section of the thesis is focused on electromigration techniques with special attention dedicated to capillary electrophoretic methods, especially capillary zone electrophoresis. The aim of this thesis is optimization of the method of determination of hyaluronic acid in water solutions by capillary zone electrophoresis.
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Use of fluorescence methods for the study of protein interactions
Johaníková, Klára ; Bezděková,, Jaroslava (referee) ; Pavelicová, Kristýna (advisor)
The diploma thesis "Use of fluorescence methods for the study of protein interactions" is focused on the use of fluorescence methods for the study of protein interactions using electromigration methods and Förster resonance energy transfer (FRET). The aim of this work was to create a bioconjugate of metallothionein (MT) protein with quantum dots (QDs) and commercial dyes. FRET was subsequently studied between these conjugates. QDs were synthesized under UV light and conjugation with MT was performed via a carbodiimide zerolengthcrooss-linker (EDC / sulfo-NHS), which serves to activate carboxyl groups and allows bioconjugation of the ligand by covalent bonding. Due to the high proportion of cysteines in MT, this protein has a very high affinity for metals. It is also involved in scavenging free radicals and there are studies that show that MT is overexpressed in cancer cells. Attention was also paid to the study of MT dimerization, which leads to an understanding of oxidative dimerization of MT and thus can contribute to understanding the formation of free radicals in the body and to deepen the knowledge about neurodegenerative disorders such as Parkinson's or Alzheimer's disease or amyotrophic lateral sclerosis. The formation of the MT dimer was confirmed by energy transfer between the donor (QDs) and the acceptor (commercial dye-cyanine) through the physical phenomenon of FRET and MALDI-TOF-MS.
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Analysis of mycotoxins from biological matrices using biomembranes and capillary electrophoresis
Kubová, Natália ; Šesták,, Jozef (referee) ; Duša,, Filip (advisor)
This thesis summarizes knowledge about mycotoxins, with focus to ochratoxin A. It also summarizes its tolerable levels of food intake, detoxification and analytical methods for mycotoxins. The work also includes a chapter describing liposomes that were used for the analysis of ochratoxin A by liposomal electrokinetic capillary electrophoresis (LECK). The practical part includes the analysis of ochratoxin A from Aspergillus ochraceus Wilhelm and Aspergillus melleus Yukawa fungi cultivated on a rye and optimization of the method for analysis of ochratoxin A based on liposomes of different compositions. By capillary zone electrophoresis, ochratoxin A is not sufficiently separated and detected in the extracted mixture; conversely, when liposome solutions are used, different migration behavior can be achieved while stabilizing ochratoxin A in solution due to amphiphilic interactions between mycotoxins and liposomes. Therefore, the LEKC method was used for this work. The most suitable liposome composition has been shown to be molar ratios of 25% cholesterol (membrane stabilization) / 50% 2-oleoyl-1-palmitoyl-sn-glycerol 3-phosphocholine (main zwitterionic lipid) / (25% 1,2-diacyl-sn-glycerol)-3-phospho-L-serine (introduction of negative charge).
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