Development of an integrated optical interferometric sensor of refractive index changes and evaluation of nickel(II)nitrilotriacetic acid-dextran as a reusable matrix for studies of histidine tagged protein.

摘要

In the first part of this project a refractive index sensor was designed that will ultimately allow the study of ligand-protein and protein-protein interactions. The setup consists of a flow cell that contains an integrated optics Mach-Zehnder interferometer chip, into which laser fight can be introduced by end- or prism-coupling. The optical parameters of the interferometer were chosen to achieve sensitivities as low as 0.07 ng/cm2. Optimized procedures for the interferometer fabrication, dimensions of the individual sensor parts as well as parameters for the optical alignment are described in great detail. While successful light coupling into the waveguiding channel of the interferometer was achieved, improvements in the light conductivity of the titanium-silica waveguides are still necessary to make this setup work.; A second issue of this thesis is the preparation of a polymeric matrix for biosensors, that consists of dextran functionalized with nickel(II)nitrilotriacetic acid (NiNTA-dextran) and the characterization of its binding affinity toward histidine oligopeptides and histidine-tagged proteins by isothermal titration calorimetry (ITC). Enhanced binding affinities of the oligopeptides to NiNTA-dextran relative to NiNTA that are due to polyvalent interactions between the NiNTA-moieties on the modified dextran and neighboring histidine groups in the peptides were found. An increase in the strength of the interaction between NiNTA-dextran and the oligohistidines was observed as the number of histidine residues in the oligopeptide were increased. Proteins with at least four neighboring histidine groups are expected to be particularly suited for the generation of stable affinity matrices out of NiNTA-dextran. A decrease in the stability of the NiNTA-dextran matrices that are loaded with histidine-tagged proteins by buffering reagents was demonstrated, underscoring the importance of choosing an appropriate buffer system in biosensor applications. Finally a model for the structure of complexes between Ni-NTA and oligohistidine residues is proposed based on the stoichiometry of binding observed by ITC.