The authors describe the deposition of single biomolecules on substrates at defined spacing by pure self-assembly. The substrate is equipped with an array of 8 nm large gold particles which form the template for biomolecule binding. The authors verified the successful binding of single biomolecules via specific antibody labeling and imaging by fluorescence microscopy. Scanning force microscopy provided evidence that every gold nanoparticle of the pattern is occupied by at least one biomolecule. Furthermore, gold conjugated secondary antibodies in combination with scanning electron microscopy proved that at least 75% of the nanoparticles carried only one active biomolecule. The precision given by such surface densities is molecularly defined and such considerably higher than in any other case reported so far.

We report a study on the interaction of the extracellular domain of trans-membrane proteins N-cadherin and L1 with nitrilotriacetic acid (NTA)-terminated self-assembled monolayers (SAMs) grown on silver and gold surfaces. Quartz crystal microbalance (QCM) and reflection absorption infrared spectroscopy (RAIRS) measurements reveal that upon addition of protein to an NTA-SAM there is a subsequent change in the mass and average chemical structure inside the films formed on the metal substrates. By using vibrational sum frequency generation (VSFG) spectroscopy and making a comparison to SAMs prepared with n-alkanethiols, we find that the formed NTA-SAMs are terminated by ethanol molecules from solution. The ethanol signature vanishes after the addition of L1, which indicates that the L1 proteins can interact specifically with the NTA complex. Although the RAIRS spectra display signatures in the amide and fingerprint regions, the VSFG spectra display only a weak feature at 866 cm(-1), which possibly indicates that some of the abundant phenyl rings in the complex are ordered. Although cell biology experiments suggest the directional complexation of L1, the VSFG experiments suggest that the alpha-helices and beta-sheets of L1 lack any preferential ordering.

There is currently great interest in material science to mimic cellular microenvironments with controlled bioactive features and biomolecule presentation at the nanometer length scale. Target cells exposed to those 2- or 3D-substrates are stimulated to manifest a variety of cellular functions like cell adhesion, migration and differentiation.Based on micellar nanolithography, we developed an experimental setup to present single biomolecules on a 2D-nanoarray over large areas on cell-adhesive or cell-repellent substrates. The spacing between biomolecules can be adjusted to 30-250 nm. To mimic a more complex cellular micro-environment with cell-cell contacts as well as cell-extracellular matrix (ECM) contacts, we used biomolecules from different cell adhesion molecule families, like cadherins, immunglobulin-superfamily, integrins and laminins. Via a Ni2+- nitrilo triacetic acid (NTA) system, the amount and the orientation of proteins is chemically controlled such that it resembles the native in vivo settings in the cellular microenvironment. By using poly-l-lysine-grafted-polyethylene glycol (PLL-g-PEG) molecules, we generated 2D-protein-nanoarrays with a cell repellent background matrix. After modification of this matrix we incorporated small bioactive peptides to create a microenvironment which allows cell adhesion. These approaches resulted in substrates with specific presentation of biomolecules to mimic cell-cell contacts and cell-ECM interactions simultaneously.With these substrates we investigated primary fibroblast and neuronal cell adhesion mediated by different biomolecules and with different nanometer spacing. We quantified neurite outgrowth of dorsal root ganglion (DRG) neurons as a differentiation paradigm on nanostructured substrates. Gold nanoparticles were functionalized with peptides, containing the arginine-glycine-aspartic acid (RGD) motif, an integrin activation sequence. Cell adhesion as well as differentiation showed a dependency on the nanometer length scale and on the presented biomolecules.