AVS2009 Session BI+AS+NS-FrM: Micro and Nanoengineering of Biointerfaces II

Combining nanolithography and biomolecular self-assembly strategies, we report on the fabrication of nanopatterned biomimetic surfaces and their use in a variety of biological studies.

We have fabricated arrays of Au/Pd nano-dots of dimensions down to the sub-10nm regime using electron-beam and nanoimprint lithography. Different chemical strategies at surfaces have been pursued to organize biological relevant nanoarchitectures into hierarchical arrays in which structural parameters, such as the spacing and nature of specific functional groups, could be systematically varied and controlled.

The generation of DNA nano-dot arrays allowed us to follow the activity (at surfaces) of a restriction enzyme in real time and at the nanoscale: fluorescence microscopy enabled the monitoring of the kinetics of such protein-DNA interaction.

Furthermore we will show how our nanopatterned biomimetic surfaces can be used to probe the importance of both the geometric arrangement (i.e. spatial ordering of transmembrane proteins, integrins) as well as the role played by peptide sequences as cell binding domains in the formation of cell focal adhesions.

Finally, we will highlight the broader utility and application of such functional nanopatterned surfaces for nanoscopic control and studies: biochemical specificity can be used to selectively place individual nanocomponents with a high degree of control over both position and orientation, as well as to organize functional nanostructures into dense arrays with very fine pitch.

Two photon photopolymerization (TPP) is a direct laser writing technique, which is known as a powerful tool to make arbitrary 3D structures. Here we demonstrate a method for fabricating high aspect ratio (~10) patterns of varying height by using TPP process in order to study contact guidance of cells. Ridge patterns of various heights and widths were fabricated through single laser scanning steps by low numerical aperture optics, hence at much higher processing throughput. Fibroblast cells were seeded on parallel line patterns of different height (~1.5-μm, ~0.8-μm, and ~0.5-μm) and orthogonal mesh patterns (~8-μm and ~4-μm height, ~5-μm and ~5.5-μm height, and ~5-μm and ~6-μm height). Cells experienced different strength of contact guidance depending on the ridge height. Furthermore, cell morphology and motility on microscale anisotropic cross patterns and parallel line patterns in different aspect ratio (1:2, 1:4, and 1:∞), size of grid (12-, 16-, and 24-µm distance neighboring longer side ridges) was also studied quantitatively. The significant effect of the cross patterns on cell alignment and directionality of migration, and motility was observed on 1:4 cross patterns and parallel line patterns, even though all cross patterns could have an effect on cell attachment and morphology. Overall, it is noted that cell morphology and motility can be influenced by the height of ridges, the aspect ratio of cross pattern and the size of grid.

Monitoring biomolecular recognition events with Surface Plasmon Resonance (SPR) heavily relies on the right surface chemistry. Uniform self assembled monolayers with carboxylic functional groups are widely used but might show steric hindrance, thus limiting the interpretation of the biorecognition kinetics. Furthermore, such negatively charged surface needs to be passivated upon immobilization of the ligand to prevent nonspecific electrostatic-driven binding of components from the analyte matrix.

In the present study we evaluate alternatives based on a 2D and 3D array of carboxylic nanodomains on a chemically contrasting matrix. By means of plasma-based colloidal lithography and e-beam lithography we were able to array ≈ 200 nm wide carboxylic motifs having a hexagonal 2-D crystalline structure on a gold surface. The interstitial gold was further modified with contrasting thiol chemistries or vapour enhanced deposition of nonadhesive material like poly ethylene oxide (PEO). The two 2D nanoarrayed chemical contrasts evaluated were carboxylic nanodomains on either a methyl-based or PEO-based matrix.

In addition, the 3D nanoarray based on a carboxylated dextran hydrogel matrix was evaluated for effects on the mass transport. In these cases, mass transport is one of the major challenges when measuring binding kinetics of biointeractants on a surface using a surface plasmon resonance (SPR) biosensor. The presence of a hydrogel on the surface increases the interactant density improving the sensitivity. Nevertheless, this is done at the expense of aggravating the mass transport phenomena.

The influence of the nanoarrayed chemical contrasts combined with the sensitivity improvement due to the band-gap effect on the kinetics of model biomolecular interactants was evaluated using an imaging SPR system and correlated with surface characterization techniques as atomic force microscopy, ellipsometry, and contact angle measurements.