Universität Stuttgart
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Item Open Access Surfaces for functional and patterned immobilization of proteins(2007) Stegmaier, Petra; Brunner, Herwig (Prof. Dr. )Functional and patterned immobilization of proteins onto solid substrates is an important issue in biotechnological processes involving protein purification or detection, or in the study of protein-protein interactions. This thesis presents a new strategy to functional immobilize proteins in selected microregions of a substrate based on photosensitive surface layers containing caged ligands and protein repellent EG chains. Special effort has been paid to the development of surface compositions and architectures that retain protein function in the surface mobilized state. In the first part of the thesis, branched silanes containing protein repellent OEG arms terminated with one inert -OMe group and one photocleavable, caged amino functionality were synthesized. Silica surfaces were modified with these molecules and the properties of the surface layers were characterized. According to the ellipsometric data, submonolayers with amino surface densities lower than in SAMs of thiols were obtained after optimization of the conditions for the surface reaction. The photolytic properties of the surfaces were analyzed. Irradiation of the surface at 355 nm cleaved the photoremovable cage and liberated the amino functionalities. These were then used to immobilize protein targets from solution after appropriate biofunctionalization. Reflectance Interference Spectroscopy (RIFS) was used to monitor and quantify protein binding. Low non-specific adsorption of proteins onto the surfaces as a consequence of the presence of EG chains was proven. Binding efficiencies were shown to be better than binding onto surface layers containing linear and shorter EG chains. For surface layers with similar architectures (either linear or branched) binding results correlate with the surface density of ligands. In the second part, silanes possessing different photocleavable groups able to be cleaved individually by using light of different wavelengths (NVoc and DEACM) were synthesized. UV measurements revealed that DEACM can be easily cleaved off upon UV irradiation at 412 nm, without damaging the NVoc group. The NVoc group can be removed at 345 nm. Surface layers containing a mixture of both groups were prepared and used for coupling two different fluorescent dyes to selected microregions of a surface. The expected fluorescence pattern was observed, confirming the possibility of generating complex chemical patterns by using different cages that can be independently addressed. In a last part of the thesis, magnetite nanoparticles were modified with mixed silane layers containing amino and OH/ OMe terminal groups in different molar ratios and connected to the surface by EG spacers with different lengths. The influence of the amine density and accessibility on the protein loading capacity of the nanoparticles has been analyzed. The optimum silane surface composition for maximum protein loading was identified.