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Fluorescence correlation spectroscopy in colloid systems
Janíček, Tomáš ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This work is focused on studying of stability and interaction in colloidal systems created by micelles of surfactant CTAB in solution of hyaluronic acid and water. As method chose for this work was selected fluorescence correlation spectroscopy. As dye was used Nile red. Measurements were divided firstly by size of polymeric chain and secondly by concentration of hyaluronic acid. Purpose of this work was to find out how will CTAB particles react with different sizes and concentration of hyaluronic acid. For comparison to hyaluronic acid was used Poly(sodium-4-styrenesulfonate).
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Optimalization of techniques of microrheology for characterization of biopolymer hydrogels
Dušenková, Alica ; Smilek, Jiří (referee) ; Sedláček, Petr (advisor)
The main aim of the bachelor thesis is optimalization of microrheological techniques for characterization of biopolymer hydrogels. Hydrogels, based on thermoreversible biopolymer agarose, were selected for these experiments. The influence of incorporated poly(sodium 4-styrenesulfonate) on structure and viscoelastic properties of agarose hydrogels was investigated through diffusion coefficient and MSD curves. Microrheological properties were studied by fluorescence correlation spectroscopy, by using fluorescent beads, which were embedded in hydrogel systems.
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Advanced microrheological techniques in the research of hydrogels
Kábrtová, Petra ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with the use of fluorescence correlation spectroscopy technique for microrheological characterization of hydrogel in a system of hyaluronate-cetyltrimethylammonium bromide. Fluorescently labelled particles were used for microrheological FCS analysis. To optimize the method the most appropriate size of particles was chosen on the basis of Newtonian glycerol solutions analysis. Among other things, the discussion was focused on the influence of refractive index change of analysed solutions on analysis results. After hyaluronate solutions analysis it was possible to assess the biopolymer concentration and molecular weight impact on the FCS microrheology results, which could then be compared with analysis results of model hydrogels of hyaluronate and CTAB. Finally, usability and limitations of FCS microrheology have been discussed.
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Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek ; Hof, Martin (advisor) ; Fidler, Vlastimil (referee) ; Konopásek, Ivo (referee)
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
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Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
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Study on Interconnection between Structure and Rheological Properties of Hydrogels on Macro and Microscopic Level
Lepíková, Jana ; Klučáková, Martina (referee) ; Kalina, Michal (advisor)
Diploma thesis main goal is to obtain new pieces of knowledge about relationship between hydrogel structures and its flow and transport properties. Thesis is mainly focusing on combining pertinent biopolymers into model hydrogels based on agarose. Then perform correlation of results obtained by diffusion methods, and by rheologic measurements on macroscopic and microscopic level. Properties of hydrogels were measured by selected rheologic measurements, dynamic light scattering method, and correlative fluorescence spectroscopy. From these methods various parameters (MSD modules, values of complex viscosity) were obtained. Afterwards transport properties of prepared hydrogels were studied by observing Rhodamine 6G diffusion. Here two different approaches were used. From macroscopic perspective, simple principles of mass diffusion from dye solution to cuvettes filled with hydrogels containing individual biopolymers were used. From microscopic perspective, dye was added during the sample preparation and then the mass diffusion was investigated using FCS. Based on evaluated results it was discovered that added biopolymers don’t influence properties of carrier medium, in this case agarose hydrogels. During the study of prepared hydrogels’ reactivity and barrier properties some differences were observed. Charge of biopolymer and its charge density were discovered as main factors influencing transport of charged solutes into prepared hydrogels.
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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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Transport of Biomedical Active Compounds Through Porous Membranes
Vašíčková, Kateřina ; Smilková, Marcela (referee) ; Smilek, Jiří (advisor)
This bachelor thesis deals with a study of transport of biomedical active compounds through porous membranes. The main goal is to optimize a penetration method of micellar solutions with a solubilized dye and subsequently with solubilized drug. Cetyltrimethylammonium bromide is chosen as a surfactant and a nile red dye is used as a diffusion and fluorescence probe. Selected active compound is a nonsteroidal anti-inflammatory drug ketoprofen with analgesic and antipyretic effects. The diffusion processes are realized using Franz diffusion cells with polycarbonate membrane with a 2 µm size of poruses. The samples are withdrawn in a chosen time intervals and afterwards evaluated by a fluorescence correlation spectroscopy. Using this method, it is possible to screen the diffusion in time even in nanomolar concentrations of a fluorescence probe chosen. This thesis provides an overview of nile red behaviour in the micellar solutions of surfactant during diffusion processes. The experiment can be extended using an active compound, for instance using a ketoprofen as it is mentioned above.
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Fluorescence correlation spectroscopy in hydrophilic colloids
Rýcová, Eva ; Mondek, Jakub (referee) ; Venerová, Tereza (advisor)
This work is focused on studying of hydrophilic colloids using fluorescence correlation spectroscopy (FCS). The gel systems were studied with several fluorescent probes, in 0,15 M sodium chloride (NaCl) consist of hyaluronan and surfactant cetyltrimethylammonium bromide (CTAB). Probe ATTO 655 was chosen for closer examination due to the suppression of triplet state. Every surrounding occuring in the gel systems was examine individually. Results showed that the measurement of gel systems by this method can be realized under certain conditions, the repeatability of the results was loaded with a small standard deviation. Extension of diffusion times caused the presence of micelles, triplet state was caused by a characteristic environment of the hydrogel. The probe also showed free diffusion motion in all environments. This diffusion motion was represented by the shortest diffusion time.
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Using diffusion techniques in the study of hydrogels
Holubová, Anna ; Žitňan, Michal (referee) ; Mravec, Filip (advisor)
Properties of colloidal systems, especially hydrogels, were studied using the method of fluorescence correlation spectroscopy (FCS). Hydrogels were prepared based on hyaluronan and cationic surfactant CTAB interactions. The proposed hydrophilic fluorescent probe, suitable for the study of phase-separated hydrogels in polymer-surfactant systems, was chosen ATTO 488 due to the suppression of triplet state. As a hydrophobic probe was chosen Nile Red. Individually were examined micellar systems of 10 mM CTAB and 0,5% sodium hyaluronate in water and 0,15 M NaCl in different concentrations of selected fluorescent probes. Subsequently the hydrogel systems were studied. The results showed that the measurement of hydrogels by this method is realizable. Short lifetimes were created by a complex probe and CTAB causing quenching and low levels of diffusion coefficients characterize the viscous environment of study system.
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