Nanofabricated Molecular-Scale Devices for the Study of Cytoskeletal Protein Binding Interactions and Their Effect on Cell Motility

This thesis describes a study of the role of the spatial arrangement of extracellular binding ligands for transmembrane receptors (integrin molecules) in the formation of focal adhesion in cells. Biomimeitic surfaces, which contain arrays of such ligands, spatially arranged in a controlled fashion were used for the vitro study of cell adhesion and motility. Those arrays were implemented through nanofabricated patterns of metal dots of the same or smaller size than that of integrin molecule (10 nm), biofunctionalized with the binding ligands. Each biofunctionalized dot works as a single integrin molecule binding site, and controlled variation in the dot arrangement allows the study of protein interactions on single-molecule level. Nanoimprint lithography was used for the fabrication of the arrays. Nanoimprint molds were fabricated of diamond-like carbon (DLC) films grown on silicon substrates, patterned with e-beam lithography and etched by plasma, in combination with a novel anti-adhesion surface coating, based on fluorocarbon plasma. An alternative method for mold fabrication was based on the direct patterning of silicon substrates with HSQ resist, followed by thermal annealing. A process based on an angle evaporated metal mask and further pattern transfer by metallization and liftoff, followed by thermal annealing was developed to obtain arrays of sub-10 nm AuPd on the imprinted substrates. The reduction of the imprinted features, controlled by the thickness of the angle-evaporated mask, allowed fabrication of arrays of sub-5 nm AuPd dots. The fabricated arrays were biofunctionalized with thiolated monolayers, and conjugated to integrin binding ligands through biotin-streptavidin binding, and passivation of unpatterned areas with PEG. Fluorescence microscopy of labeled streptavidin was used to monitor the quality of the functionalization process. Arrays of dots arranged in pairs, trimers, and extended hexagons were examined, with the inter-dot spacing ranging in 50 nm-100 nm. The best cell spreading and adhesion was found for hexagonal arrays with the lower inter-dot spacing, showing the importance of the global density of dots rather than inter-dot spacing for the examined geometries. Clustering effect of the binding sites was studied using arrays of 7 sites with different global densities, that resulted in the same high efficiency of cell adhesion and spreading, emphasizing the importance of clustering compared to other arrangement parameters. Further study of cell spreading on the arrays with the different cluster size showed a significantly improved cell spreading on the arrays of cluster of 4 and more binding sites.