Precise Fabrication of Graphite-Like Material Directly on a Biological Membrane Enabled by Ethanol Ice Resist

Ice lithography holds the potential to bridge cryogenic electron microscopy and electron-beam lithography and achieve direct high-precision functionalization of fragile biomaterials. Here we demonstrate that 5 keV electron irradiation of ethanol ice creates a material, patterned with <100 nm resolution, that is stable in the solid phase under ambient conditions. Employing the purple membrane from <i>Halobacterium salinarum</i> as a test target, we additionally show that the fabrication process results in minimal biomaterial mass loss. Ketene, an unstable intermediate, was identified in the irradiated ice via Fourier transform infrared spectroscopy and is likely an important factor triggering formation of the ethanol-based material. Surface-enhanced Raman spectroscopy and additional characterization methodologies revealed that the material contains disordered graphite similar to carbon fiber and is mechanically stiff and electrically insulating. This work demonstrates a novel material for additive manufacturing in general and for the precise functionalization of biological membranes in particular.