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 biotin-streptavidin 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 homogenei...