The loss of the native structure of proteins or peptides due to changes in various physical and chemical factors as well as misfolding can lead to the formation of insoluble amyloid fibrils. The accumulation of these fibrils in organs and tissues can lead to the development and progression of various neurodegenerative, metabolic, and functional disorders, such as Alzheimer's disease. Understanding the aggregation mechanism, sensitive detection and knowledge of the structure and stability of amyloid fibrils are essential for early detection of protein aggregation, diagnosis, and the discovery of new therapeutic approaches. Due to its numerous advantages, fluorescence spectroscopy using fluorescent probes is one of the most commonly used methods in the study of amyloidogenic protein or peptide aggregation and the detection of amyloid fibrils. In the experimental part of the master's thesis, we first followed the course of aggregation in vitro and confirmed the formation of insulin fibrils by measuring the relative emission intensity of thioflavin T fluorescence. The formation of amyloid β1–42 fibrils was confirmed by microscopic examination and by measuring the emission intensity of thioflavin T fluorescence. Next, we measured the emission spectra of the fluorescent probes, from which we determined the maximum emission wavelength and the increase in fluorescence emission intensity of the fluorescent probe studied upon binding to amyloid fibrils. Probes with a maximum emission wavelength above 600 nm, which show a significant difference or a much higher relative fluorescence emission when bound to amyloid fibrils compared to the fluorescence emission of the free, unbound probe, are the most suitable for further research. For individual probes, we also determined the binding dissociation constant, which shows the binding affinity of the probe for amyloid fibrils. The values of the binding dissociation constants are in the low micromolar or nanomolar range. To address the binding specificity and selectivity of the probes for amyloid fibrils, we also measured the emission spectra of the fluorescent probes in the presence and absence of bovine and human serum albumin. The results show that the difference in the relative fluorescence emission intensity of the fluorescent probes in the presence and absence of bovine or human serum albumin is very small, demonstrating specificity and selectivity of the investigated probes for amyloid fibrils.
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