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Fluorescence correlation spectroscopy in the study of the properties of colloidal systems
Marková, Kateřina ; Lehocký, Marián (referee) ; Kapusta, Peter (referee) ; Pekař, Miloslav (advisor)
Because of their properties, hydrogels are a highly sought-after matrix for medical purposes. These properties are often conditioned by the structure, therefore the emphasis is put on a precisely defined polymer network. In the presented dissertation, different types of hydrogels were researched using fluorescence correlation spectroscopy (FCS) in conjunction with microrheology. The combination of aforementioned methods is rarely used when determining the properties of hydrogels, and therefore, the method had to be standardized and optimized. For these purposes, fluorescently labeled silicate nanoparticles with a neutral surface charge were selected. In the course of the method optimization, the limits of the device were discovered within which the measured diffusion coefficients could still be considered valid. Furthermore, we have determined the parameters that affect the MSD curve and thus also the correctness of the actual measured data. An aqueous solution of agarose was chosen as a model colloidal system, as it ranges from a viscoelastic liquid to a solid hydrogel depending on the concentration. On this colloidal system, the diffusion properties of the nanoparticles used were tested as well as the structural properties of the system itself. Limit concentrations were found when the diffusion coefficient could no longer be detected using the selected method. The limit was successfully partially extended by adjusting the correlation time, however, the dispersion of the diffusion coefficient values was very high in such case. The values measured in this way were compared with the calibration-free method of bifocal fluorescence correlation spectroscopy (2f–FCS). Furthermore, changes in the diffusion coefficient were determined depending on the type of sample preparation. Alongside these experiments, image analysis was also performed, which provided interesting results in conjunction with FCS. The last experiment, which provided information about the properties of both the embedded nanoparticles and the polymer system, consisted of washing out of the particles from the hydrogel structure. The maximum entropy method with an analysis using log-normal distribution of diffusion coefficients was selected as the last and the most advanced polymer network analysis of the, which defined the system in a new and an alternative way. The results obtained using these advanced analyses were similar to the data calculated using the mathematical model of anomalous diffusion. The description of properties using rheological modules was a completely new approach. The properties were obtained using a series of conversions from the measured autocorrelation curve. Therefore, a rheological module obtained from microrheological data was the output of the approach. The shape of the curve is comparable to classical (macro)rheology, but the numerical values are lower by an entire order of magnitude. The smallest particles in the entire concentration range behaved as if they were in a purely viscous environment while the largest particles defined the behavior of the system depending on the concentration from very viscous to viscoelastic. The last type of measurement was the study of hyaluronan using selected nanoparticles and its fluorescently labeled analogue. All the methods that were used in the study of agarose hydrogel were applied to the viscoelastic system of hyaluronan to verify their applicability.
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