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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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Low-cost microextraction and preconcentration procedures for biomedical applications
Vašátko, Jan ; Diviš, Pavel (referee) ; Dvořák, Miloš (advisor)
This thesis focuses on low-cost microextraction techniques and their application for purification and preconcentration of biological samples, specifically on the experimental study of supported liquid membrane (SLM) extraction. The described microextraction technique uses commercially available filtration plates as the extraction units and allows the extraction of basic drugs from biological samples of urine and blood (in the form of dried blood spots). The experimental part includes the optimization of microextraction conditions of basic drugs from real samples through a SLM coupled in-line to lab-made capillary electrophoresis. The basic optimization of microextraction conditions involved selecting the appropriate organic phase for membrane impregnation (1:1 mixture of ENB and DHE), appropriate agitation speed for sample convection during extraction (1000 rpm), and optimal ratio of donor to acceptor volumes for high preconcentration of the analytes (400:15 µL). After basic optimization, the effect of donor alkalization with NaOH on extraction recovery (ER) was investigated. For all matrices used (saline solution, undiluted human urine samples, human capillary blood eluted from dry blood spots with deionized water), the highest ER values were achieved using a neutral donor and an acidic acceptor. The extraction time (60 minutes) was optimized based on the time profile of the microextraction for 120 minutes. This optimized microextraction method is suitable for the determination of basic drugs in real matrices with sufficient sample clean-up, preconcentration and ER values.
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Determination of organic tin compounds in the environment
Fojt, Jakub ; Mravcová, Ludmila (referee) ; Řezáčová, Veronika (advisor)
Organotin compounds are one of the most produced and most used organometallic compounds. Some of these substances are endocrine disruptors, persistent organic polutants and their high toxic effects are observed. That’s why their presence in the environment caused by human activity could endanger many organisms. The aim of this thesis is summarize their properties and their occurrence in the environment. Then the quick, easy and relatively cheap method for determination of trialkyltin compounds in heavily poluted aquatic sediments using capillary zone electrophoresis is developed.
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Application of capillary electrophoresis for determination of surfactants
Langová, Lenka ; Čáslavský, Josef (referee) ; Vávrová, Milada (advisor)
Tensides rank among the surfactants with ability to reduce surface tension. This ability is used to remove impurities. Surfactants can be divided into four basic groups: anionic, cationic, nonionic and zwitterionic. Anionic surfactants belong to the most widespread surfactants in the Czech Republic today. They get into sewage waters from various cleansing and washing agents which are used both in households and industry. So this diploma thesis deals with their assessment by application of capillary electrophoresis.
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Molecularly imprinted polymers as a tool for the isolation of key biologically active molecules
Vodová, Milada ; Jaroslava, Bezděková (referee) ; Nejdl, Lukáš (advisor)
This bachelor thesis is focused on preparation and optimization of molecularly imprinted polymers that are selective for the selected nucleobase(uracil). Molecularly imprinted polymers, which wereused for selective uracil isolation, have been prepared non-covalent imprinting technique. As a function monomer was used in this case dopamine. The detection ofisolated uracil was made by capillary electrophoresis with absorption detection (–260 nm). Conditions for preparation of molecularly imprinted polymers,their binding properties, adsorption kinetics, and selectivity wereinvestigatedin detail. Mentioned polymer materials were as well used for uracil selective isolation and detection from complexsamples. Tomato products were used as complex samples. Uracil in those samples was usedasanindicator ofbacterial contamination.
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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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