Electroactive Nanoarrays for Biospecific Ligand Mediated Studies of Cell Adhesion

Dip-pen nanolithography (DPN), which has revolutionized nanotechnology, is based on a scanning-probe technique in which an atomic force microscopy (AFM) tip is used to pattern molecules onto a surface with precise nanometer-scale features. A wide variety of materials have been patterned by DPN for applications ranging from the fabrication of nanostructures to ultra-high-density DNA arrays. More recently, DPN has been extended to the development of protein nanoarrays for high-throughput genetic and biomarker analysis. Several protein nanoarrays have been generated by using DPN to directly pattern proteins onto a variety of substrates. Other strategies have used bioconjugation methods such as antibody–antigen affinities to further functionalize the nanoACHTUNGTRENNUNGarrays in order to generate more complex protein surfaces. A major area of research in which DPN nanoarray technology will make a significant impact is in cell biology. For example, the spatial presentation of cell-adhesive ligands influences the subcellular nanoarchitecture of adherent cells and affects their behavior. In particular, the number and size of biospecific interactions between extracellular ligands and cell-surface receptors is critical for cell adhesion and cell migration; however, they remain poorly understood and elusive due to the lack of molecularly defined nanopatterned model substrates. While a few nanoarrays for cell adhesion have been reported, most are formed by the unspecific adsorption of protein to nanometersized patterns. Consequently, these substrates do not offer precise control over the orientation and spatial distribution of the immobilized ligand and do not allow for the immobilization of a variety of ligands with precise control of density. In order to study the effect of immobilized ligands on the nanoarchitecture of adherent cells, the model substrate must meet several criteria. First, the feature sizes of the ligands must be in the nanometer range. Next, the spatial distribution of the immobilized ligands must be precisely defined. Also, the interactions between the immobilized ligands and cell-surface receptors must be biospecific. Finally, the substrate must be compatible with cell culture, and amenable to high-resolution fluorescence microscopy. In this communication, we show a chemoselective peptideimmobilized electroactive nanoarray for biospecific cell-adhesion studies. We show that the affinity of peptide ligands and nanoarray spacing have a dramatic effect on focal adhesion number, structure, and size for cell adhesion on nanopatterned surfaces. Our strategy is based on a redox-active hydroquinone-terminated alkanethiol that is patterned by dip-pen nanolithography in nanometer-sized spots on a gold substrate (Figure 1). The remaining bare gold region was then backfilled