In the context of this doctoral thesis, methods for affinity investigations of a reprocessed collagen sample and proteins were developed using capillary electrophoresis, microscale thermophoresis and spectral shift analysis. The collagen source material was provided by botiss biomaterials GmbH and represents a prototype for use in dental wound healing. Due to collagen being insoluble under physiological conditions, the raw fleece material was ground, resuspended and finally freeze-dried, to achieve defined mass concentrations in later experiments. Subsequently, the resulting suspension was analyzed by raster electron microscopy and particle size distribution. The particles were of various shapes and sizes (approximately 3 - 1000 μm in diameter), which tend to aggregate depending on their concentration and pre-treatment. Furthermore, the collagen was examined using CE-SDS. However, the method was not optimal because electrokinetic injection and denaturation of large collagen particles proved problematic. In contrast, the separation of commercial rat tail collagen dissolved in acetic acid was more successful and in accordance with literature values. The majority of this study focuses on method development using mobility shift affinity capillary electrophoresis (msACE). The principle of this technique is based on changes in electrophoretic mobility depending on ligand concentration. Hence, binding constants serve as parameters to characterize the strength of interactions, which are determined by non-linear least squares fitting of experimental data to a model function. In this work, an msACE method was successfully developed, which investigated the affinity of reprocessed collagen to epidermal growth factor (EGF) as model protein. The specialties were the use of an LEDIF detector, the application of constant power instead of constant voltage and the use of suspensions instead of solutions. In addition, the influence of viscosity and dielectricity has been studied and included in the form of correction factors. The influence of the EOF when using a coated capillary and the effect of buffer depletion were further considered. As a result, an affinity between collagen and EGF was found, although molar binding constants could not be determined due to the lack of molecular weight of collagen. In addition, two modern capillary based methods for measuring interactions were presented by microscale thermophoresis (MST) and spectral shift (SpS) analytic, which are combined into one device. MST is based on a change in fluorescence signal upon heating, while SpS is founded on a shift in emission spectra, both of which depend on the binding behavior of a ligand. Consequently, a binding to the reprocessed collagen was indicated for EGF, vascular endothelial growth factor (VEGF) and human serum albumine (HSA).
Sophie Hartung (Fri,) studied this question.