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

Abstract 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.