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Assembly and characterization of protein multi-layer systems


Christensen,  Danica
MPI for Polymer Research, Max Planck Society;

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Christensen, D. (2004). Assembly and characterization of protein multi-layer systems. Master Thesis, Johannes Gutenberg-Universität, Mainz.

Abstract: Biocompatible multi-layer architectures are of great interest for the construction of biosensor applications. We have established a system that is a multi-layer protein architecture with alternating layers of streptavidin (SA) and biotinylated immunoglobulin G (B-IgG). The layer building properties of this protein multi-layer architecture has been studied in situ using the surface plasmon resonance spectroscopy (SPR). Studies were also done using neutron reflectivity to investigate the surface architecture of the multi-layers. The multi-layer architecture has been shown to assemble with a linear growth of the multi-layer thickness with respect to the number of layers deposited. Experiments conducted using SPR have also shown that the multi-layers are stable against free biotin attack and the assembly of the multi-layers is very reproducible. The effects of using SA versus avidin to build the multi-layer system were studied using SPR. Two immobilization techniques for the attachment of the first SA layer to the metal surface were compared with SPR; the first using covalent bonding between the SA and a thiol derivative self-assembled to the surface, the second technique used the binding affinity of SA and biotin to bind the SA directly to a biotinylated thiol on the gold surface. The fluorescence intensity of Alexa fluoro labeled streptavidin (AFSA) was investigated using Surface Plasmon Fluorescence Spectroscopy (SPFS). The effect of adding additional non-labeled protein layers to a layer of AFSA that was placed close to the gold surface, about 3 nm, and a second layer a bit further away from the metal surface, at 15 nm, on the fluorescence intensity was also investigated using SPFS.