Multiparametrická fluorescenční spektroskopie
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Táto diplomová práca sa zaoberá možnosťami multiparametrickej fluorescenčnej spektroskopie, keďže hlavným cieľom tohto experimentu bolo vyhodnotiť možnosti multiparametrického merania v Laboratóriu fluorescenčnej spektroskopie. Na základe literárnej rešerše bola navrhnutá vhodná fluorescenčná sonda pre tento experiment, SNARF-4F AM, ktorá vykazuje vysokú citlivosť na zmeny pH prostredia. Fluorofor bol rozpustený v roztokoch s rozdielnym pH a tento systém bol skúmaný pomocou časovo rozlíšeného spektrofluorimetra. Metódou TRES (časovo rozlíšené emisné spektrá) boli preskúmané emisné spektrá fluorescenčnej sondy a nájdené emisné maximá. Pomocou parametra poklesu intenzity fluorescencie pri daných vlnových dĺžkach bola vytvorená dekonvolúcia emisného spektra, ktorá poskytovala informácie o dobe života a relatívnom zastúpení jednotlivých stavov fluorescenčnej sondy v roztoku. Neskôr, sonda bola rozpustená v roztokoch s odlišnou hustotou a pH – tento systém slúžil na anizotropické meranie, počas ktorého boli získané jednotlivé korelačno-rotačné časy fluoroforu. Získané výsledky potom slúžili ako podklad pre multiparametrickú analýzu, ktorá bola vykonaná pomocou fluorescenčného korelačného mikroskopu a spektrografu. Táto kombinácia umožňuje namerať potrebné fluorescenčné parametre v jednom kroku. Bol vytvorený štandardný operačný postup na ovládanie spektrografu. Na základe získaných informácií bola posúdená vhodnosť, presnosť a citlivosť multiparametrickej analýzy.
This diploma thesis deals with the possibilities of multiparametric fluorescence spectroscopy, since the main objective of this experiment was to evaluate the possibilities of multiparametric measurements in the fluorescence spectroscopy laboratory. A suitable fluorescence probe was proposed for this type of experiment that shows high sensitivity for pH changes in the environment, SNARF-4F AM, based on a literature research. The fluorophore was dissolved in solutions of different pH and this system was examined using a time-resolved spectrofluorimeter. The method named TRES (time-resolved emission spectra) was used to obtain the emission spectra of the probe and to find the emission maximum. Fluorescence intensity decay measurements as a function of wavelengths allowed to create deconvolution of the emission spectra, which provided information about the fluorescent lifetime and the relative representation of the states of probes in the solution. Later, the probe was dissolved in solutions of different density and pH - this system served for anisotropic measurements, during which the individual correlation-rotational times of the fluorophore were obtained. The obtained results were then used as the basis for multiparametric analysis, which was performed by using a fluorescence correlation microscope and a spectrograph. This combination allows to measure the necessary fluorescence parameters in one step. A standard operating procedure was created for the spectrograph’s control. On the basis of the obtained information the suitability, accuracy and sensitivity of the multiparametric analysis were qualified.
This diploma thesis deals with the possibilities of multiparametric fluorescence spectroscopy, since the main objective of this experiment was to evaluate the possibilities of multiparametric measurements in the fluorescence spectroscopy laboratory. A suitable fluorescence probe was proposed for this type of experiment that shows high sensitivity for pH changes in the environment, SNARF-4F AM, based on a literature research. The fluorophore was dissolved in solutions of different pH and this system was examined using a time-resolved spectrofluorimeter. The method named TRES (time-resolved emission spectra) was used to obtain the emission spectra of the probe and to find the emission maximum. Fluorescence intensity decay measurements as a function of wavelengths allowed to create deconvolution of the emission spectra, which provided information about the fluorescent lifetime and the relative representation of the states of probes in the solution. Later, the probe was dissolved in solutions of different density and pH - this system served for anisotropic measurements, during which the individual correlation-rotational times of the fluorophore were obtained. The obtained results were then used as the basis for multiparametric analysis, which was performed by using a fluorescence correlation microscope and a spectrograph. This combination allows to measure the necessary fluorescence parameters in one step. A standard operating procedure was created for the spectrograph’s control. On the basis of the obtained information the suitability, accuracy and sensitivity of the multiparametric analysis were qualified.
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Citation
LACKO, K. Multiparametrická fluorescenční spektroskopie [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2017.
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Document version
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cs
Study field
Chemie pro medicínské aplikace
Comittee
prof. Ing. Miloslav Pekař, CSc. (předseda)
prof. RNDr. Ivana Márová, CSc. (místopředseda)
doc. Ing. Pavel Diviš, Ph.D. (člen)
doc. Ing. Petr Dzik, Ph.D. (člen)
doc. Ing. Stanislav Obruča, Ph.D. (člen)
prof. RNDr. Zbyněk Zdráhal, Dr. (člen)
Date of acceptance
2017-06-06
Defence
Pekař: Na základě jakých úvah byla volena použitá fluorescenční sonda?
Jaké bude následné využití vytvořeného standardního operačního postupu?
Do jaké míry je vypracovaný postup limitovaný omezenou dostupností excitačních vlnových délek?
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení