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Optimizing the determination of distribution of surfactant at phase separation in polymer-surfactant system
Sátorová, Kateřina ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
This diploma thesis is focused on the determination of distribution a surfactant at phase separation in polymer-surfactant system. Sodium hyaluronate of three molecular weights was chosen as a polymer, CTAB was used as a surfactant. The experiments were performed in an environment of 0,15 M NaCl. The measurement of the CTAB concentration in samples was based on the formation of coloured complexes of CTAB and picric acid in chloroform. UV-VIS spectroscopy was used for the detection of these complexes. The content of CTAB was determined in four gels of different composition (2% Hya + 200 mM CTAB, 2 % Hya + 50 mM CTAB, 0,5 % Hya + 200 mM CTAB, 0,5 % Hya + 50 mM CTAB). All gels were gradually washed five times with 0,15 M NaCl. The content of CTAB was determined for newly prepared gels and for gels after 2 and 4 months since the preparation. Initial concentrations of CTAB and hyaluronan have greater influence on the distribution of CTAB after phase separation, than the molecular weight of hyaluronan. The content of CTAB in newly prepared gels and after 4 months since preparation is very similar and demonstrates the stability of the system.
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Preparation of model membranes to study their interactions with biopolymers using fluorescence correlation spectroscopy
Adamcová, Zuzana ; Márová, Ivana (referee) ; Mravec, Filip (advisor)
This diploma thesis is focused on preparation and characterization of supported lipid bilayers as simplified models of cell membranes. The bilayers were prepared from source system of lecithin liposomes in phosphate buffer using the vesicle fusion method on a cover glass sufrace hydrophilized by plasma. Three fluorescent probes – Nile red, Oregon Green DHPE and DiO – were utilized to characterize diffusion within the bilayer using fluorescence correlation spectroscopy. For this purpose Z-scan FCS, which is a method developed specially for planar samples, was used. After the process of preparation and characterization of supported lipid bilayer was optimalized, interaction between this artificial membrane and solution of hyaluronic acid in phosphate buffer was studied. It was found out, that addition of this biopolymer causes slowing the diffusion of the fluorescent probe within the bilayer.
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Dual-focus FCS in colloidal research
Chovancová, Romana ; Kapusta, Peter (referee) ; Mravec, Filip (advisor)
Tato práce se zabývá studiem fluorescenčně značeného hyaluronanu, konkrétně rhodaminylamino hyaluronátu sodného (Hya-Rh, 40 kDa), pomocí dvouohniskové fluorescenční korelační spektroskopie (2f-FCS). Nejdříve byla prostudována literatura týkající se využití FCS techniky v koloidní chemii a při studiu polymerů, přičemž následně byly shrnuty veškeré poznatky o využití 2f-FCS metody. Na základě prvotních měření byl zjištěn vhodný postup přípravy a způsob uchovávání vzorku Hya-Rh používaném pro následující experimenty. Záhy byly prostudovány možné vlivy koncentrace Hya-Rh na jeho difúzní charakteristiky jak ve vodě, tak ve fyziologickém roztoku. Následně bylo studováno chování Hya-Rh a vliv koncentrace solí alkalických kovů ve vodných roztocích těchto solí a fluorescenčně značeného hyaluronanu. Poté bylo sledováno chování Hya-Rh v závislosti na koncentraci velmi nízkomolekulárního hyaluronanu (VLMW HA, 404 kDa) v čisté vodě i ve fyziologickém roztoku, přičemž získané výsledky byly mezi sebou porovnány. Nakonec bylo využití 2f-FCS metody celkově zhodnoceno a popsáno z hlediska studia chování fluorescenčně značeného hyaluronanu v roztocích.
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Passive microrheology of colloidal systems based on biopolymers.
Bjalončíková, Petra ; Burgert, Ladislav (referee) ; Mravec, Filip (advisor)
Diploma thesis was aimed to deal with evaluation of microrheology method in the research of biopolymer-protein. Used biopolymer was sodium hyaluronate and proteins were trypsin and chymotrypsin. For measuring of microrheology were used particles with different radius (0,5 m and 1 m). It was found, that both substances have viscous charakter. Passive microrheology is suitable for measuring the viscoelastic properties of biopolymers.
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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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Time-resolved fluorescence study of liquid and condensed systems based on biopolymer-surfactant interactions.
Černá, Ladislava ; Žitňan, Michal (referee) ; Mravec, Filip (advisor)
This thesis studies properties of hydrogel, which arises on the basis of electrostatic and hydrophobic interactions between hyaluronan chain and micelles of cationic surfactant. A native sodium hyaluronan at molecular weight 750–1 000 kDa and a cationic surfactant CTAB (cetyltrimethylammonium bromide) were used. This hydrogel was assessed as a material for drug delivery systems. The hydrogels were made by mixing 200mM CTAB with 0.5% hyaluronan, both dissolved in 0.15M aqueous solution of NaCl simulating physiological solution. Methods used in this study were steady-state and time-resolved fluorescence spectroscopy, more accurately time-resolved emission spectra (TRES) and deconvolution of steady-state emission spectra of a whole sample by means of parameters gained from fluorescence intensity decays at a set of wavelenghts. Selected systems were investigated by three fluorescent probes, prodan, laurdan and rhodamine 6G. The first two mentioned probes were in hydrogel localized only within micelles in three different microenvironments. Rhodamine 6G pointed out that in hydrogel the aqueous environment is significantly restricted in comparison to purely micellar solution. In addition, rhodamine informed about less available micelle surfaces, caused by hyaluronan chains occupation. There were no interactions between the probes and hyaluronan chains. Freshly made hydrogels showed almost the same results as after a week of maturation under its supernatant.
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Study of physical gels with hydrophobic domains
Kovářová, Lenka ; Márová, Ivana (referee) ; Mravec, Filip (advisor)
The thesis is focused on physical hyaluronan gels. The object of study is the interaction of hyaluronan (HyA) with oppositely charged surfactants in physiologic solution (0.15 M NaCl), leading to the formation of gel. In the first part of work have been determined the solids´ contents (X) in gels and their supernatants in percentage and their correlation with molecular weight concentration of original HyA solution and the ratio of binding sites on hyaluronan chain and surfactant CTAB. To conclude, decrease in HyA concentration results in higher values of X and vice versa. On the other hand, increase in the value of X with increasing molecular weight of HyA is not so significant. Analogous conclusions have been made for supernatants and the amount of solids in gel. Drying process has been recorded by drying curves. Swelling process has been used for the characterization of gels. The percentage of water that can be absorbed by dried gel, was determined. The results are in agreement with the measurements of solids´ content in gels. In the next part, the correlation between rheological properties of gels and HyA concentration, HyA molecular weight and concentration of CTAB have been studied by the oscillation and flow tests. The samples with the highest molecular weight and concentration have the most viscoelastic character. The flow test confirmed the assumed pseudoplastic behavior of gels. A very interesting trend arose while comparing HyA concentrations and viscosity in stock solutions and gels. Whereas in stock solution viscosity (at low shear rate) is lower with increasing of HyA concentration, the situation was exactly the opposite in gels. The results are in agreement with frequency tests and observed character of gels.
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Colloidal particles marked with biopolymer
Pihíková, Dominika ; Víteček,, Jan (referee) ; Mravec, Filip (advisor)
The effect of hydrophobically modified hyaluronan on surfactants aggregation has been studied in this master’s thesis. The value of critical micelle concentration of anionic surfactant SDBS (sodium dodecylbenzensulfonate), cationic surfactant CTAB (cetyltrimethylamonnium bromide) and nonionic surfactant Triton X-100 (octylphenol ethoxylate) was determined by fluorescence spectroscopy using pyrene probe. Aggregation behavior of surfactants was performed with addition of hydrophobically modified hyaluronan of two molecular weights (17 kDa, 206 kDa) in aqueous solution. The greatest influence of hydrophobized hyaluronan on aggregation behavior was observed in system with cationic surfactant CTAB. Stability of system containing cationic surfactant and hydrophobically modified hyaluronan was established through zeta potential. Last part of thesis deals with size determination using dynamic light scattering.
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Use of fluorescent techniques in study of aerosol deposition
Lippay, Josef ; Jedelský, Jan (referee) ; Mravec, Filip (advisor)
Several experiments were designed for utilization of fluorescence spectroscopy as a method of aerosol particle detection in a model of lungs. One of the experiments was to arranged use luminescent properties of DEHS (bis(2-ethylhexyl)decandioate) for calculating aerosol deposition. The outcome of this experiment was confirmation of clusters existence, which causes luminescence of DEHS. But the luminescence is not enough dependent on concentration and as such is not suitable for calculation of aerosol deposition. As the next experiment DEHS-fluorescein particles were generated by condensation monodisperse aerosol generator (CMAG), where water was used instead of isopropyl alcohol as a solvent. By this alteration the negative influence of DEHS was eliminated, which caused results refinements of aerosol deposition. Generation of fluorescein sodium salt particles by small-scale powder disperser (SSPD) was designed as a last experiment. The lower deposition efficiency measured by this method was caused by particles polydispersion. Photo records were used for documentation of Hot-spots. Outcomes of this study are new knowledge of fluorescence spectroscopy utilization for study of aerosol deposition and possibilities of fluorescent aerosol particles generation. Acquired data can serve for knowledge extension of possible detection methods for aerosol particles in the model of lung and can serve for validation of numerical simulations.
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Ionogenic fluorescence probes in research of colloidal system
Střondalová, Hana ; Burgert,, Ladislav (referee) ; Mravec, Filip (advisor)
This diploma thesis studied the interaction of ionic and amphiphilic fluorescent probes with polyelectrolytes. The research of such colloidal system could be helpful in deeper understanding of interactions between polyelectrolytes and surfactants. The aim of this thesis was to determine whether ionic or amphiphilic probes are suitable for this research. Two polyelectrolytes, sodium polystyrene sulfonate and hyaluronan were used in this study. Interactions of polyelectrolytes with fluorescent probes (4-Di-2-Asp, methylene blue, dodecyl acridine orange, DiO and DiA) were studied in this work. Then cationic surfactant cetyltrimethylamonium bromide was added to this system. Samples were studied using fluorescence and UV/VIS spectroscopy. Emission and absorption spectra of these probes were recorded. This experiment was based on hypothesis that due to positive charge these probes will bind to the negatively charged polyelectrolyte. Formation of dimers or other aggegates was presumed for fluorescent probes dodecyl acridine orange and methylene blue. When 4-Di-2-Asp was used,changes in fluorescence intensity and absorption spectra in connection with possible formation of aggregates was supposed to be observed. It should be noted that formation of dimers for 4-Di-2-Asp is not described in literature. The formation of dimers wasn't clearly demonstrated during the measurement. Formation of dimers was observed only with dodecyl acridine orange. Probes DiO and DiA were used only in the study of the interaction with polyelectrolytes. These fluorescent probes showed up as inappropriate, because they adsorbed on tube walls. When cationic surfactant was added to hyaluronan-fluorescent probe system, surfactant pushed away fluorescent probe and bonded electrostatically to negatively charged groups of polyelectrolyte.
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