Highly amine-reactive graphene-oxide EM grids for biochemical surface modification in aqueous buffer

Abstract

AbstractGraphene oxide (GO), an oxidized derivative of graphene, has found application in cryo-electron microscopy (cryo-EM) as a hydrophilic and transparent solid support on which to adsorb biological macromolecules, providing an alternative to traditional aqueous films. Current applications generally adsorb the macromolecule directly onto unmodified GO or modify the GO surface with polyethylene glycol-amine reagents. This nucleophilic amine reaction must be performed in an aprotic organic solvent and therefore precludes the use of biological samples such as nucleic acids and peptides. The utility of GO could be expanded by the ability to covalently modify its surface with biochemical affinity reagents such as small- molecule metabolites, peptides, or nucleic acids, in aqueous buffer at neutral pH. Presented here is a chemical procedure that converts all oxygen functionalities of GO to highly amine- reactive glycidyl epoxide groups, achieved without the need of specialized laboratory equipment. We show that single sheets of glycidyl epoxide-modified GO react on the EM grid with primary amines at micromolar concentrations in minutes at room temperature in aqueous buffer. Given the ease of derivatizing biochemical reagents with amines, the chemistry described here will enable imaging of macromolecules immobilized on GO through specific biochemical and biologically relevant binding interactions.