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Comparison of sensitivity of drop coating deposition Raman spectroscopy and surface enhanced Raman spectroscopy for study of biologically important molecules
Sayedová, Shirin ; Kočišová, Eva (advisor) ; Hofbauerová, Kateřina (referee)
The main objective of this work was to compare the sensitivity of two Raman spectroscopy methods, namely the method of drop coating deposition Raman spectroscopy and surface-enhanced Raman spectroscopy for selected biologically significant molecules: dipicolinic acid, acetylsalicylic acid, methylene blue and riboflavin. For the DCDR experiments three hydrophobic surfaces were used: commercial substrate Tienta, polished CaF2 substrateand surface with copper nanoparticles with a gradient of hydrophobicity. Two commercial surfaces were used for the SERS method. Both techniques are suitable for sample measurement at low concentrations and with small initial volumes of solutions. The DCDR method, in contrast to the SERS method, allowed to work with higher excitation radiation power. The SERS technique was markedly more sensitive for a selection of suitable SERS active surface. The spectra obtained by the SERS method were influenced by the way of adsorption of the molecules to the surface. By studying the surface with a gradient of hydrophobicity, we have revealed an effect on the shape of dried drops. The dependence on Raman intensity was not found in the case of methylene blue. For both methods we determined the limit concentrations of solutions of measured molecules that can be detected. We have shown that the...
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Functional characterization of MDR pump Pdr5p responsible for multiple drug resistance in yeast Saccharomyces cerevisiae
Sayedová, Shirin ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
One of the main reasons for the treatment failure of infections caused by pathogenic microorganisms is the overexpressing of efflux membrane proteins, which actively remove drugs from cells, leading to a phenomenon called multidrug resistance MDR. In this work, we focused on the functional characterization of the MDR pump Pdr5p in the yeast Saccharomyces cerevisiae. We have verified that diS-C3(3) fluorescence method can be used to determine the binding sites where the substrates bind in the binding pocket of the pump ScPdr5p. We focused on the study of the ScPdr5p binding pocket using triazole derivatives: ravuconazole, voriconazole and fluconazole. Using disc diffusion assay, we showed that all three studied triazoles are substrates of the pump ScPdr5p. We have found that these structural analogs have a significantly different effect on the inhibition of the potentiometric fluorescent probe diS-C3(3) transport by the pump ScPdr5p, and also that ravuconazole and voriconazole compete with each other for transport by the pump ScPdr5p. We have used a fluorescent approach to study the binding of azoles to the binding pocket of pump ScPdr5p using benchmark substrates, that bind selectively to only one binding site in the binding pocket of the pump ScPdr5p, and we have supported the hypothesis that ravuconazole...
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Comparison of sensitivity of drop coating deposition Raman spectroscopy and surface enhanced Raman spectroscopy for study of biologically important molecules
Sayedová, Shirin ; Kočišová, Eva (advisor) ; Hofbauerová, Kateřina (referee)
The main objective of this work was to compare the sensitivity of two Raman spectroscopy methods, namely the method of drop coating deposition Raman spectroscopy and surface-enhanced Raman spectroscopy for selected biologically significant molecules: dipicolinic acid, acetylsalicylic acid, methylene blue and riboflavin. For the DCDR experiments three hydrophobic surfaces were used: commercial substrate Tienta, polished CaF2 substrateand surface with copper nanoparticles with a gradient of hydrophobicity. Two commercial surfaces were used for the SERS method. Both techniques are suitable for sample measurement at low concentrations and with small initial volumes of solutions. The DCDR method, in contrast to the SERS method, allowed to work with higher excitation radiation power. The SERS technique was markedly more sensitive for a selection of suitable SERS active surface. The spectra obtained by the SERS method were influenced by the way of adsorption of the molecules to the surface. By studying the surface with a gradient of hydrophobicity, we have revealed an effect on the shape of dried drops. The dependence on Raman intensity was not found in the case of methylene blue. For both methods we determined the limit concentrations of solutions of measured molecules that can be detected. We have shown that the...
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