Label-free biomolecular interaction sensing on microarrays using surface plasmon resonance imaging

This thesis, “Biomolecular interaction sensing on microarrays using surface plasmon resonance imaging��?, gives a brief introduction to biomolecular interactions in general. And includes interactions of peptides and proteins, antibodies and autoimmune diseases. Furhtermore, the concept of label-free biosensing is introduced, with a special emphasis on SPR imaging. The principle and role of microarrays and data analysis are also described. Chapter 3 explains the soft lithography process to develop various types of PDMS spotting devices for the immobilization of proteins in confined surface areas in a microarray format. PDMS was explored to provide a disposable alternative for the TopSpot, however, in combination with our experimental restrictions the PDMS based method did not succeed and we continued using the TopSpot. In chapter 4 and 5 the use of a scanning-angle surface plasmon resonance (SPR) imaging instrument for monitoring the binding of biomolecules on user-defined regions of interest of a microarray is described. Peptides and proteins were both spotted on the same sensor chip to illustrate that both, low and high molecular weight ligands with initial large differences in off-set SPR angles, can be applied within the same experiment. The effectiveness of this system is demonstrated by automatically monitoring the interactions between citrullinated peptides and serum autoantibodies of 50 rheumatoid arthritis (RA) patients and 29 controls in a single step. Chapter 6 deals with a new assay protocol based on injecting a single analyte concentration, and exposing it to spots with various ligand densities, rather than multiple analyte injections of varying analyte concentrations exposed to a single ligand surface, as is done in the conventional procedure is proposed. The new method uses controlled dilution of ligands with background molecules which, facilitates immobilization of a precise ligand density on the surface, which is a prerequisite for direct kinetic analysis. This alternative to the conventional multi-analyte overlay plot for calculating the rate- and affinity constants of a biomolecular interaction, has the advantage of requiring only a single analyte injection, fewer surface regeneration steps, and a reduction of the overall assay time. As a model system, various biotin specific interactions have been tested by means of SPR imaging for their affinity toward surface immobilized biotinylated peptides.