Silane-Coupled Photo-Oxidized Graphene Grids for Controlling Protein-Surface Interactions in Cryo-EM Sample Preparation.

Cryo-electron microscopy (cryo-EM) has become a powerful tool for the high-resolution structural analysis of biological macromolecules; however, protein behavior at support-film and air-water interfaces often leads to preferred orientation and particle loss during sample preparation. Here, we report a versatile strategy for engineering graphene (GP)-based cryo-EM grids via silane coupling agent (SCA) modification of photo-oxidized GP. Photo-oxidation introduces reactive oxygen-containing functionalities onto the GP surface, enabling rapid SCA modification with silane precursors bearing distinct molecular architectures. Surface analyses, including water contact angle measurements, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and fluorescence microscopy, supported the presence of SCA-derived components on the GP surface. Cryo-EM observations using GroEL revealed that the resulting interfacial molecular layers influenced particle orientation distributions, indicating the modulation of protein-surface interactions. In addition, negative control experiments using unoxidized GP suggested that photo-oxidation pretreatment is important for establishing a stable functionalized interface. The practical utility of the functionalized grids was further demonstrated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein frequently affected by preferred orientation. Epoxy-functionalized and polyethylene glycol/N-hydroxysuccinimide (PEG/NHS) ester-functionalized grids enabled high-resolution three-dimensional reconstructions at 2.26 and 2.58 Å, respectively, using a 200 kV microscope. Notably, the epoxy-functionalized grid achieved efficient particle capture even at low protein concentration. Because interfacial properties can be varied by the choice of SCA, this approach provides a versatile platform for modulating protein-surface interactions and expanding the molecular design space of graphene-based interfacial layers for cryo-EM sample preparation.