Electroactive Nanoarrays for the Biospecific-Ligand Mediated Study of Single Cell Adhesion and Polarization.

Cell adhesion, polarization, and migration are vital to numerous biological phenomena. Therefore, a greater understanding of the mechanisms of these processes will have broad impacts in fields ranging from developmental biology to medicine. This work has focused on developing a nanoscale model system that will allow one to study the effect of the spatial presentation of immobilized ligands on the nanoarchitecture of adherent cells. In Chapter 2, the development of electroactive nanoarrays of hydroquinone-terminated alkanethiol, produced by dip-pen nanolithography (DPN) is described. These nanoarrays, in conjunction with an oxime-chemistry based chemoselective immobilization strategy and high-resolution fluorescence microscopy, were used to study biospecific-ligand mediated single cell adhesion. The difference in ligand affinity of linear and cyclic Arg-Gly-Asp (RGD) was shown to have a dramatic affect on the intracellular nanoarchitecture of adherent fibroblasts. The production of asymmetric nanoarrays used to study single cell polarization is described in Chapter 3. Asymmetric nanoarrays presenting linear RGD were found to induce net directional cell polarization in adherent fibroblasts, while linear RGD-presenting symmetric nanoarrays did not induce net polarity. This demonstrates a direct correlation between the spatial distribution of cell adhesive ligand and the establishment and maintenance of directional cell polarization. In addition, there was no net directional cell polarity found on asymmetric nanoarrays presenting a higher affinity ligand cyclic RGD, indicating that ligand affinity also has a profound effect on cell polarization. The relationship between ligand affinity and spatial distribution of immobilized ligand was further explored through double asymmetric nanoarrays presenting cyclic RGD, which were shown to impose directional cell polarization. In order to extend this methodology to examine other aspects of cell adhesion and polarization on electroactive nanoarrays other methods of visualization were considered. There have been conflicting reports regarding the use of total internal reflection fluorescence microscopy (TIRFM) to visualize cells near thin metal layers. In Chapter 4, it was determined that TIRFM is an effective method to examine intercellular structures of cells adhered to patterned SAMs on gold surfaces. This was demonstrated through the use of microcontact printing and DPN patterning methods. Future applications of this research are presented in Chapter 5.