Biocompatible surfaces for specific tethering of individual protein molecules

We have characterized the biocompatibility of glass surfaces that were modified by protein layers or linear poly(ethylene glycol) (PEG) brushes with the aim of determining the optimal treatment for the immobilization of single biomolecules by specific attachment, which was done here using a streptavidin-biotin linkage. We have investigated the surface homogeneity by atomic force microscopy and the resistance to nonspecific binding by single-molecule detection of fluorescently labeled RNase H in a scanning confocal fluorescence microscope. The resistance to nonspecific binding of the surfaces was simply examined by omitting the biotinstreptavidin linkage in the surface preparation. We have also carried out fluorescence resonance energy transfer experiments to examine the ability of the surfaces to preserve the native state of specifically attached proteins via streptavidin-biotin linkage. Chemisorption and crosslinking of BSA or streptavidin to amino-functionalized glass yields excellent surface homogeneity and reproducibility, much better than physisorption onto bare glass. PEG 5000 films form the smoothest films and are very resistant to nonspecific protein adsorption, but PEG 2000 has significantly worse properties. Unlike protein-coated surfaces, PEG 5000 surfaces cause partial denaturation of the RNase H molecules, which we explain by intermingling of the PEG and polypeptide chains or by interaction of the underlying aminosilane layer with the protein. These results highlight the importance of a thorough surface characterization and design in fluorescence studies of immobilized single biomolecules.