Grafting polymers onto surfaces at high density to yield polymer brush coatings is a widely employed strategy to reduce biofouling and interfacial friction. These brushes almost universally feature synthetic polymers, which are often heterogeneous and do not readily allow incorporation of chemical functionalities at precise sites along the constituent chains. To complement these synthetic systems, we introduce a biomimetic, recombinant intrinsically disordered protein that can assemble into an environment-sensitive brush. This macromolecule adopts an extended conformation and can be grafted to solid supports to form oriented protein brushes that swell and collapse dramatically with changes in solution pH and ionic strength. We illustrate the value of sequence specificity by using proteases with mutually orthogonal recognition sites to modulate brush height in situ to predictable values. This study demonstrates that stimuli-responsive brushes can be fabricated from proteins and introduces them as a new class of smart biomaterial building blocks.

We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level.

We demonstrate the possibility of tuning the degree of functionalization of a surface using photoactivatable chemistries and controlled light exposure. A photosensitive organosilane with a protected amine terminal group and a tetraethyleneglycol spacer was synthesized. A o-nitrobenzyl cage was used as the photoremovable group to cage the amine functionality. Surfaces with phototunable amine densities were generated by controlled irradiation of silica substrates modified with the photosensitive anchor. Protein layers with different densities could be obtained by successive coupling and assembly steps. Protein surface concentrations were quantified by reflectance interference. Our results demonstrate that the protein density correlates with the photogenerated ligand density. The density control was proved over four coupling steps (biotin, SAv, (BT)tris-NTA, MBP, or GFP), indicating that the interactions between underlying layer and soluble targets are highly specific and the immobilized targets at the four levels maintain their full functionality. Protein micropatterns with a gradient of protein density were also obtained.

Functional immobilization and lateral organization of proteins into micro- and nanopatterns is an important prerequisite for miniaturizing bioanalytical and biotechnological devices. Here, we report an approach for efficient site-specific protein immobilization based on enzymatic phosphopantetheinyl transfer (PPT) from coenzyme A (CoA)-functionalized glass-type surfaces to specific peptide tags. We devised a bottom-up surface modification approach for coupling CoA densely to a molecular poly(ethylene glycol) polymer brush. Site-specific enzymatic immobilization of proteins fused to different target peptides for the PPTase Sfp was confirmed by real-time label-free detection. Quantitative protein-protein interaction experiments confirmed that significantly more than 50% of the immobilized protein was fully active. The method was successfully applied with different proteins. However, different immobilization efficiencies of PPT-based immobilization were observed for different peptide tags being fused to the N- and C-termini of proteins. On the basis of this immobilization method, we established photolithographic patterning of proteins into functional binary microstructures.

Protein immobilization into micro and nanoscaled patterns opens exciting possibilities in fundamental and applied research. Developing efficient capturing techniques while preserving the structural and functional integrity of the proteins on surfaces is a key challenge for surface scientists. In this paper, current techniques for site-specific protein immobilization into engineered surface architectures are reviewed. Fundamental principles for functional protein immobilization on solid supports are discussed and popular affinity-based recognition pairs and their application for capturing proteins into nano and microstructures are presented.