Nanoscale Protein Patterning via Nanoimprint Lithography and Ultrafast Laser Irradiation.

Abstract

The diverse biological roles of proteins include catalysis, force generation, mechanical support, signaling and sensing. Beyond their central importance to biology, proteins are of interest because these nano-machines have potential to be integrated into micro- fabricated devices to create low-cost, robust technologies of unprecedented small scale and high efficiency. Applications include biosensors, actuation of micro-electromechanical systems (MEMS), and tissue engineering, as well as screening tools for proteomics and pharmacology, and basic biological research. However, both the study and application of proteins has been challenged by the inherent difficulties associated with positioning these tiny objects. Thus, a primary enabling technology is the ability to immobilize biomolecules in well-defined patterns while retaining their functionality.

Towards achieving this goal, we have developed two approaches capable of producing high resolution protein patterns. First, we immobilized proteins in patterns defined by nanoimprint lithography, which offers the advantages of high throughput, high reproducibility, and low cost. We demonstrate patterning of bioactive antibodies with sub-100nm feature resolutions.

The second technique uses tightly focused ultrafast laser pulses which, through a non-linear damage mechanism, are known to be capable of ablating features far smaller than the diffraction-limited spot size. We find that proteins can be removed from a glass surface at intensities considerably below the ablation threshold of glass, cleaning the surface without damaging the underlying substrate. AFM and epifluorescent analyses indicate near-total removal of proteins from a glass surface with well-defined nanoscale features. We describe potential mechanisms for the damage and/or removal of proteins from the surface.

Glass surfaces irradiated at these low intensities exhibit marked changes in surface chemistry. We characterize the adsorption of several model proteins as well as small charged fluorophores. Based on the adsorptive behaviors of these molecules, we describe a sub-threshold damage mechanism which alters the long-term chemical state, surface charge, and adsorptivity of irradiated glass surfaces.

Finally, we made use of the laser-based protein removal technique described above to selectively remove fibronectin from the path of motile fibroblasts. We demonstrate that we are able to guide movement by this in situ modification of the cells’ microenvironment.