Nanostructured substrates for multi-cue Investigations of Single Cells

Cellular adhesion and migration are crucial aspects of many physiological functions which depend on the integration of numerous signaling inputs generated by chemical and physical properties of the substrate. Typically, these chemical/physical parameters are investigated individually, making it difficult to predict how their interdependencies modify the adhesion and migratory process. Furthermore, these cues rarely exist in orderd isotropic patterns that fabrication techniques produce, but rather are present in stochastic gradients in vivo. To address this, we have employed a combination of nano/micro-lithography in conjunction with etching techniques to fabricate substrates which expose individual cells to tightly controlled topographical and chemical cues. Nanolithography is employed to pattern gold nanodots atop a quartz substrate, while selective etching creates nanopillars. Ligand density is tuned by varying the nanodot/pillar pitch and using thiol chemistry techniques for biofunctionalization. Furthermore this experimental platform design enables incorporating nanostructure density gradients to better mimic in vivo multi-cue environments. The approach was validated by imaging individual A549 cells as they simultaneously adhered to distinct biofunctionalized topographies.