Dense, ultrathin networks of isocyanate terminated star-shaped poly(ethylene oxide) (PEO) molecules, cross-linked at their chain ends via urea groups, were shown to be extremely resistant to unspecific adsorption of proteins while at the same time suitable for easy biocompatible modification. Application by spin coating offers a simple procedure for the preparation of minimally interacting surfaces that are functionalized by suitable linker groups to immobilize proteins in their native conformations. These coatings form a versatile basis for biofunctional and biomimetic surfaces. We have demonstrated their advantageous properties by using single-molecule fluorescence microscopy to study immobilized proteins under destabilizing conditions. Biotinylated ribonuclease H (RNase H) was labeled with a fluorescence resonance energy transfer (FRET) pair of fluorescent dyes and attached to the surface by a biotin-streptavidin linkage. FRET analysis demonstrated completely reversible denaturation/renaturation behavior upon exposure of the surface-immobilized proteins to 6 M guanidinium chloride (GdmCl) followed by washing in buffer. A comparison with bovine serum albumin (BSA) coated surfaces and linear PEO brush surfaces yielded superior performance in terms of chemical stability, inertness and noninteracting nature of the star-polymer derived films.

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][5][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][9][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]...

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.