Orientation and Conformation of Single Macromolecules on Unmodified and Functionalized Surfaces

Sammanfattning: In this thesis methods for investigation of orientation and conformation of individual macromolecules on surfaces are presented as well as novel methods for functionalization of silicon chips with the possibility to get an ordered immobilization of antibodies. Two novel methods are presented which makes it possible to investigate the orientation of individual macromolecules on different kinds of surfaces with AFM. One is based on threshold patterning where, depending on substrate, side- and end-on adsorbed immunoglobulin molecules could be detected. The other method is using the principle of site-specific ligands where the orientation of proteins adsorbed to various surfaces was evaluated. By measuring the increase in protein volume of the formed protein-ligand complexes with AFM, the amount of protein having an orientation that allows binding can be estimated. The influence of surface chemistry on protein structure was examined using human serum fibronectin adsorbed to hydrophilic and hydrophobic surfaces, where a major difference in structure were seen depending on surface properties.In addition, methods for surface functionalization have been developed which are suitable for immobilization of macromolecules and for basic studies of macromolecule/surface interactions at the nanometer scale. In an effort to immobilize protein in a specific orientation that could be studied with AFM, a new method for preparing reactive disulfides based upon a mixed reaction with 2,2’-dipyridyldisulfide and 2-thiopyridone to a mercapto-silanized silica surface was presented. The possibility to covalently bind proteins to this surface was examined, using beta-galactosidase and Fab’-fragments of IgG.