Neutron and X-Ray reflectometry on complex biological systems on solid surfaces

Cell adhesion is a fascinating albeit very complex process. It is mediated by the perhaps most abundant self-assembling molecular system in nature: the highly ordered, heterogeneous and immensely diverse cell membrane. Lipids are the main constituents of the cell membrane. The hydrophobic interactions between them and the surrounding are the main driving force for the self-assembly. The cell adhesion process and the structure and functionality of the cell membrane are a prerequisite for life. A deeper understanding is of high biological and pharmaceutical relevance. In the scope of this thesis, the interface between solid substrates and membranes was studied with X-ray and neutron reflectometry. Resolving the lipid structure on graphene is a prerequisite for future applications of lipid decorated solution-gated graphene fieldeffect transistor based biosensors. X-ray reflectometry measurements on lipid coated graphene were performed on a custom-built molybdenum-anode-based in-house reflectometer. It was reconstructed during this thesis to meet the requirements regarding measurement time and signal-to-noise ratio. An around 20 Å thick lipid monolayer formation was revealed. The interpretation of the results from an electrical characterization of the lipid graphene interface and lipid spreading experiments correspond to the results of the reflectometry experiments. Solution-gated graphene field-effect transistors were applied to monitor the formation of lipid layers and to investigate the influence of differently charged lipid layers. Insitu monitoring of the formation of lipid layers in real time was demonstrated. Cell adhesion on a silicon surface has been studied in neutron and X-ray reflectivity experiments. The neutron reflectometry experiments were conducted at FRM II. Sample chambers were developed, which provide the required sterile and temperature-stable sample environment for performing in-vitro reflectometry experiments on cell layers. Furthermore they allow for an exchange of the cell medium hence ensuring the necessary medium exchange for cell culturing and contrast variation. Neutron reflectivity experiments with confluent layers of epithelial cells adhering to silicon in cell medium with different scattering contrasts were performed. This contrast variation reduces the ambiguity of modelling the neutron data. The average thickness and degree of hydration of the interface between the adherent cells and the substrate were determined. A three-layered interfacial organization is suggested, consisting of a very dense 70 120 Å thick protein film bound to the silicon surface interface, followed by a highly hydrated, 180 280 Å thick layer. The third, several hundred Å thick layer is attributed to the less hydrated composite membrane. This layer structure is in agreement with the theory of cellular adhesion and immobile receptors. It is further suggested that the bottom dense protein layer is formed by the cells to generate a bioanalogue tissue film. Additionally, X-ray reflectometry measurements were performed. The measurements did not provide more insight, what is attributed to beam damage in the sample. Nevertheless, the prerequisites for continuative measurements were created.