A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns

In situ, cells are highly sensitive to geometrical and mechanical constraints from their microenvironment. These parameters are, however, uncontrolled under classic culture conditions, which are thus highly artefactual. Micro-engineering techniques provide tools to modify the chemical properties of cell culture substrates at sub-cellular scales. These can be used to restrict the location and shape of the substrate regions, in which cells can attach, so-called micropatterns. Recent progress in micropatterning techniques has enabled the control of most of the crucial parameters of the cell microenvironment. Engineered micropatterns can provide a micrometer-scale, soft, 3-dimensional, complex and dynamic microenvironment for individual cells or for multi-cellular arrangements. Although artificial, micropatterned substrates allow the reconstitution of physiological in situ conditions for controlled in vitro cell culture and have been used to reveal fundamental cell morphogenetic processes as highlighted in this review. By manipulating micropattern shapes, cells were shown to precisely adapt their cytoskeleton architecture to the geometry of their microenvironment. Remodelling of actin and microtubule networks participates in the adaptation of the entire cell polarity with respect to external constraints. These modifications further impact cell migration, growth and differentiation.

Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

Dip-pen nanolithography was used to construct arrays of proteins with 100- to 350-nanometer features. These nanoarrays exhibit almost no detectable nonspecific binding of proteins to their passivated portions even in complex mixtures of proteins, and therefore provide the opportunity to study a variety of surface-mediated biological recognition processes. For example, reactions involving the protein features and antigens in complex solutions can be screened easily by atomic force microscopy. As further proof-of-concept, these arrays were used to study cellular adhesion at the submicrometer scale.