Nanoscale orthogonal biofunctionalization imprint lithography and its applications for studying nanoscale cell surfaces interactions
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Surfaces with nanopatterned biological functionality are important prerequisites for many applications including developing biosensors, tissue engineering scaffolds and Bio-MEMS devices. This work presents a versatile technique, termed nanoscale orthogonal biofunctionalization imprint lithography, which allows "top-down" highprecision nanopatterning of proteins that can meet the demands of various applications. To show applicability of this technique, it was used to create disposable large scale arrays of nanopatterned cell adhesion proteins for cell culture for the purpose of investigating the influence of nanoscale geometrical parameters on cell-surface interactions. These cell culture arrays were used to systematically vary the size, spacing and density of fibronectin adhesion clusters, which are expected to modulate the signaling induced by the cell adhesion, the clustering of adhesion molecules and the force generated in the cytoskeleton. As a result, it was first determined that the nanopatterned adhesion sites provided an upper limit to the size of a corresponding cell focal adhesion. Cell morphology, actin stress fibers, vinculin distribution, proliferation and motility were all influenced by nanoscale fibronectin island size, and in some cases, the distance between patterns. Several parameters depended biphasically on the pattern size, indicating a very fine regulation of the associated cell signaling. Adhesion area and local stress on the adhesion are modulated by the adhesion size, and the cell response on the nanopattern shows strong parallels to the response on elastic adhesion substrates. In addition, chemical signaling may be influenced directly by changing the activity of associated enzymes. The results of this work build a basis for an understanding of adhesion on the nanoscale level and offer design criteria for the engineering of biomaterials and tissue scaffolds.