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Advanced Fluorescence Techniques in Research on Micellar Systems and Their Interactions with Biopolymers
Holínková, Petra ; Burgert, Ladislav (referee) ; Táborský, Petr (referee) ; Pekař, Miloslav (advisor)
The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.
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Interaction of phospholipids with polyelectrolytes in aqueous medium
Maivaldová, Iva ; RNDr.Miroslav Gál, Ph.D. (referee) ; Klučáková, Martina (advisor)
This diploma thesis is focused on determination of aggregation behavior of selected phospholipids (lecithin; 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) in water and on the effect of native hyaluronan addition of various molecular weights and concentrations on this behavior. The behavior has been investigated with fluorescence spectroscopy using pyrene and perylene as fluorescence probes being able to penetrate into hydrophobic cavities of formed aggregates. Critical aggregation concentration and the concentration at which lecithin begins to aggregate have been determined. Regarding 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, it was possible to determine only the beginning of aggregation value. The values of this parameter for lecithin and for 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine correspond in the order. It has been investigated, that the addition of native hyaluronan has only in some systems slight effect on the aggregate behavior of selected phospholipids.
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Time-resolved fluorescence in investigation of colloid system
Černá, Ladislava ; Ouzzane, Imad (referee) ; Mravec, Filip (advisor)
This thesis is focused on determination of the basic spectral characteristics, such as fluorescence maximum and lifetime, of fluorescent probes prodan [6-propionyl-2-(dimethylamino)naphthalene] and perylene by steady-state and time-resolved fluorescence spectroscopy. Time-resolved measurements were realized through TCSPC method (time-correlated single photon counting). The characteristics of prodan were found in water, acetone, n-heptane and in mixtures of these, so that a polar scale of environments would arise. Both of the mentioned probes were further investigated in systems of aqueous solutions of hyaluronic acid (300 kDa) at the concentration of 10 mg•l-1 together with cationic surfactant CTAB (cetyltrimethylammonium bromide) at the concentration range from 0.01 to 0.20 mmol•l-1 (before CMC). The aim was to confirm the reach and the range of critical aggregation concentration of the surfactant (CAC), to uncover the basic spectral characteristics of the mentioned probes in this concentration range and to verify the stability of this system in the presence of NaCl at the concentration of 0.15 mol•l-1. Furthermore, by TCSPC method were obtained time-resolved emission spectra of prodan from the system of hyaluronic acid and CTAB at one concentration of CTAB in CAC interval both without salt and with salt at the concentration of 0.15 mol•l-1. The obtained time-resolved emission spectra correlated with the results of the CAC concentration range measurement. After addition of salt the system of CAC proved to be unstable.
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Study of aggregation in a biopolymer-surfactant system at low surfactant concentration.
Stiborský, Filip ; Vojtová, Lucy (referee) ; Mravec, Filip (advisor)
In this bachelor thesis, the interaction between hyaluronate and surfactant using fluorescent spectroscopy and pyrene as fluorescent probe was studied. A cationic surfactant cetyltrimethylammonium bromide and a native hyaluronate with different molecular size was used. Before a detected critical micelle concentracion, there were no interactions observed in phosphate buffer. However, we could observe electrostatical interactions in the water systém measuring. At low concentration of surfactant (between critical aggregation concentration and critical micelle concentration), the production of gel phase appeared.
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Hyaluronan ionocomplexes for cosmetic and farmaceutical applications
Cimalová, Jana ; Obruča, Stanislav (referee) ; Pekař, Miloslav (advisor)
This bachelor thesis is focused on the study of a system of cationic surfactant – hyaluronan. Using fluorescent spectroscopy the critical micellar concentration of Carbethopendecinium bromide (Septonex) was determined in three environments – water, 0,15 M NaCl, and buffer PBS, with three fluorescent probes – pyrene, perylene, and nile red. It was found that the physiological environments (buffer and NaCl) reduce the value of CMC about one order. During the experiments with native hyaluronan, precipitate and gel formation were observed. The following part dealt with aggregate behaviour of the new biologically acceptable amphoteric surfactant tetradecylfosfocholine (TPC) in the environment of water and NaCl according to the increasing molar molecular weight of hyaluronan. As a fluorescent probe perylene was used. These measurements showed that the molar molecular weight or a change of ionic force has no effect on the value of CMC and therefore there are no interactions with hyaluronan. The last surfactant to study was 1,2-dihexadecanoyl-3-dimetylammonium-propan (DPTAP), which is water insoluble. DPTAP is soluble in chloroform, but after evaporation of chloroform and after addition of hyaluronan, it precipitated. The results so far, have confirmed that in terms of research of aggregation by fluorescence spectroscopy the new cationic surfactants TPC and DPTAP are not suitable for complexation with hyaluronan.
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Quality of hydrophobic domains in polyelectrolyte-surfactant system
Holínková, Petra ; Vala, Martin (referee) ; Mravec, Filip (advisor)
In this bachelor thesis were studied the photophysical properties of four fluorescent probes depending on the viscosity of their surroundings by the fluorescence spectroscopy. For probes DPH, DPB a perylene was determinated dependence of fluorescence anisotropy and for P3P dependence of ratio excimer/monomer on the viscosity of the sample. Anisotropy probes DPH and perylene were also used to study interaction between hyaluronan and cationic surfactant CTAB (concentration below CMC) in aqueous solution and 0,15 M NaCl. Information about forming hydrophobic domains were obtained by measurement of fluorescence intensity and anisotropy depending on the concentration of CTAB in the sample. It was found that in an aqueuos solution, after the initial addition of CTAB to hyaluronan formed small hydrophobic domains linked to chain of hyaluronan. Increasing concentration of CTAB leads to the formation of gel and phase separation. After addition of NaCl leads to reorganization of this system and probably the formation of free micelles in the solution with higher concentration of CTAB.
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Anisotropy and time-resolved anisotropy techniques in colloidal systems research
Holínková, Petra ; Táborský,, Petr (referee) ; Mravec, Filip (advisor)
In this diploma thesis were investigated in terms of microviscosity liquid and condensed systems composed of hyaluronan (Hya) and cationic surfactant cetyltrimethylammonium bromide (CTAB). The excitation and emission spectra, lifetime, steady-state fluorescence anisotropy and time-resolved fluorescence anisotropy of the samples were measured. First, was studied the formation of hydrophobic domains in the system Hya-CTAB at concentration of CTAB lower than its critical micelle concentration in an aqueous solution and 0.15M NaCl. It was found that in an aqueous solution small hydrophobic domains linked to chains Hya are formed. Then an increasing concentration of CTAB leads to phase separation and formation of gel. Due to the addition of NaCl then leads to the reorganization of this system and probably the formation of free micelles in the solution. Were also studied condensed phase of system Hya-CTAB-NaCl at high concentrations of surfactant during fourteen days of ageing. It was found that the microviscosity of hydrophobic domains is constant, but the microviscosity of hydrophilic parts gradually decreases.
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Resonance energy transfer in the environment of the hydrogel matrix
Janča, David ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
The diploma thesis deals with resonance energy transfer in hydrogel matrix enviroment. In the theoretical part, all the important features of these systems and their components are summarized and all the principles for explaining the method used are described. The work also includes a short literary background research. In the experimental part, the resonance energy transfer was measured in micellar solutions and hydrogels in 0.15 M NaCl medium. The hydrogels were formed on the basis of interaction of the surfactant with the oppositely charged polyelectrolyte. As a positively charged surfactant, carbethopendecinium bromide (Septonex) was chosen and hyaluronan as the negatively charged polyelectrolyte. Perylene with fluorescein and perylene were selected as fluorescent pairs for RET. It has been found that increasing concentration of the acceptor molecule increases the intensity of RET, in other word the rate of acceptor binding to the donor. In the study of fluorescent pair of perylene with DiO was observed, that it was not appropriate to use fluorescence probe DiO at higher concentrations. Experiments were carried out to determine whether RET changes in the time from the point of mixing the donor and acceptor fluorescence probes together. This phenomenon has not been confirmed. In hydrogels, the effect of increasing surfactant concentration and molecular weight of hyaluronan was observed. The resulting analysis showed that the molecular weight of hyaluronan has a significant effect on RET efficiency in hydrogels.
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Advanced Fluorescence Techniques in Research on Micellar Systems and Their Interactions with Biopolymers
Holínková, Petra ; Burgert, Ladislav (referee) ; Táborský, Petr (referee) ; Pekař, Miloslav (advisor)
The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.
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Resonance energy transfer in the environment of the hydrogel matrix
Janča, David ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
The diploma thesis deals with resonance energy transfer in hydrogel matrix enviroment. In the theoretical part, all the important features of these systems and their components are summarized and all the principles for explaining the method used are described. The work also includes a short literary background research. In the experimental part, the resonance energy transfer was measured in micellar solutions and hydrogels in 0.15 M NaCl medium. The hydrogels were formed on the basis of interaction of the surfactant with the oppositely charged polyelectrolyte. As a positively charged surfactant, carbethopendecinium bromide (Septonex) was chosen and hyaluronan as the negatively charged polyelectrolyte. Perylene with fluorescein and perylene were selected as fluorescent pairs for RET. It has been found that increasing concentration of the acceptor molecule increases the intensity of RET, in other word the rate of acceptor binding to the donor. In the study of fluorescent pair of perylene with DiO was observed, that it was not appropriate to use fluorescence probe DiO at higher concentrations. Experiments were carried out to determine whether RET changes in the time from the point of mixing the donor and acceptor fluorescence probes together. This phenomenon has not been confirmed. In hydrogels, the effect of increasing surfactant concentration and molecular weight of hyaluronan was observed. The resulting analysis showed that the molecular weight of hyaluronan has a significant effect on RET efficiency in hydrogels.
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