Retaining the Activity of Enzymes and Fluorophores Attached to Graphene Oxide

Abstract

Graphene oxide (GO) has drawn interest for impermeable coatings, water purification, drug delivery vectors, and other applications that involve functionalizing its surface with molecular, polymeric, or biomolecular species. Existing GO functionalization strategies rely on covalent attachment to polar functionalities at its oxidized regions or weak nonspecific absorption to the graphitic regions. These modifications risk disrupting GO’s aqueous dispersibility and leave its hydrophobic patches available to disrupt protein structure and quench the emission of fluorophores. Here, we demonstrate a general strategy to functionalize GO noncovalently using tripodal binding motifs, which present three pyrene moieties that bind to the hydrophobic regions. Tripods immobilize the serine protease enzyme chymotrypsin (ChT) onto GO and preserve its native structure and activity. In contrast, unmodified GO is one of the strongest known ChT inhibitors, which we show to arise from interactions with both GO’s hydrophilic regions and hydrophobic regions. Furthermore, GO quenches the photoemission of many fluorescent probes, and its weak inherent photoemission is inconvenient for imaging via fluorescence microscopy. When presented on the GO surface through a tripod, the fluorescent dye Alexa Fluor 488 retains its fluorescence and allows the GO sheets to be imaged using a standard fluorescence microscope. As such, tripod-binding groups represent a useful strategy to functionalize GO with biomolecules and study its interactions with cells and living organisms.