Biomolecules exhibit remarkable sensitivity, selectivity and efficiency in their response to specific stimuli, which makes them extremely attractive targets for sensor applications.[1] Biosensor designs are often based on hybrid (biotic–abiotic) nanoscale interface technologies, in which biomolecules are immobilized on solid substrates.[2, 3] The delicate structure of proteins requires coating of these surfaces with carefully designed films that interact only weakly with the protein, except through a strong tether for protein attachment, and thus preserve the functionally competent, properly folded conformation. The surface must not only resist interaction with the hydrophilic surface of the native protein structure, rather, for stability over long periods of times and/or under destabilizing conditions (extreme temperatures, cosolvents), the surface must also be inert towards hydrophobic protein moieties that become transiently or permanently exposed upon unfolding. Here we present a versatile surface preparation that matches exactly these requirements. It was specifically developed for single-molecule studies of protein folding, the intriguing process by which the linear polypeptide chain assumes its specific, functionally competent three-dimensional architecture.[4–6] Protein folding is an intrinsically heterogeneous process because a huge number of folding pathways on the free energy surface connect the myriad of unfolded conformations with the much smaller set of conformations belonging to the native state.[7] Förster resonance energy transfer (FRET) between a donor and acceptor fluorophore has been used extensively in equilibrium and time-resolved investigations of protein folding on large protein ensembles.[8–10] With the advent of single-molecule fluorescence spectroscopy, a technique has become available that enables us to examine protein folding pathways of individual protein molecules in real time, and to detect intermediate states and trajectories leading to misfolded structures, which have been implicated in diseases such as the spongiform encephalopathies (BSE, CJD, Alzheimer’s disease).[11] The strong distance dependence of FRET can be exploited to observe the reconfiguration of a single polypeptide chain in real time.[12] To this end, a donor–acceptor pair of fluorescent dye molecules is attached to specific locations along the polypeptide chain so that the two dyes are in close proximity in the folded structure and further apart in the unfolded chain. Such experiments, performed on proteins diffusing freely in solution,[13, 14] have already yielded insightful results. Free diffusion, however, limits the observation time to the transit time of the protein through the sensitive volume of the microscope, which is typically less than 1 ms. Longer observation time can be realized by fixing the molecules in space to within a volume smaller than the extent of the point-spread function of the microscope. Immobilization of proteins inside surface-bound lipid vesicles is an elegant approach,[15] but this does not allow easy solvent exchange. More versatile is the direct tethering of the protein to the surface. This strategy has been pursued to observe conformational dynamics of a small peptide on an amino-silylated glass surface.[16] In close proximity to a nanostructured surface, a biomolecule experiences an environment that is distinctly different from that in solution. Surface-protein interactions can easily be in the range of the overall stabilization energy of the native fold and thus can profoundly change the energy landscape of the protein. Therefore, experiments with surface-immobilized proteins reveal properties of the protein-surface assembly and not of the protein …