Protein engineering provides the tools to develop novel biomaterials endowed with multiple functionalities. The focus of much of the work in this area has been the development of bioactive hydrogels for drug delivery and tissue engineering applications. The fields of biosensing and bioelectrocatalysis will benefit from the advantages of protein-based hydrogels provided that electron transport and catalytic functionalities can be incorporated into the protein structures. Here we present two bifunctional protein building blocks that co-assemble into a bioelectrocatalytic hydrogel. One building block, a metallo-polypeptide, supports electron transfer, and a second, an engineered chimeric poly-phenol oxidase, provides enzymatic activity towards the reduction of dioxygen to water. Both proteins are bifunctional in that, in addition to electron transport and catalytic functionalities, they contain physical cross-linking domains that promote the self-assembly of a supramolecular hydrogel. The metallo-polypeptide is based on a previously designed tri-block polypeptide comprised of a soluble region flanked by two α-helical leucine zipper domains. The helical domains self-assemble into physically cross-linked junctions through coiled-coil formation. Compatible helices genetically fused to the poly-phenol oxidase allow mixtures of the two building blocks to co-assemble into bioelectrocatalytic hydrogel.

Electron transport functionality of the metallo-polypeptide is derived from the divalent attachment of an osmium redox moiety to the tri-block polypeptide. Attachment of the osmium moiety is demonstrated by mass spectroscopy (MS-MALDI-TOF) and cyclic voltammetry. Physical cross-linking functionality of α-helical domains in both building blocks is confirmed by circular dichroism spectroscopy and by rheological measurements. Catalytic activity of the chimeric poly-phenol oxidase and electron conduction of the metallo-polypeptide are evaluated by dilute solution kinetic assays and cyclic voltammetry, respectively. Mixtures of the two building blocks co-assemble into a bioelectrocatalytic hydrogel that generates a catalytic current in the presence of oxygen.

This bioelectrocatalytic system is the first example of an all protein-based electrode surface modification. With further optimization, the system could readily be used as the cathode to a biofuel cell or as an oxygen sensor. The design is also an example of a general method of multi-functional materials design, the co-assembly of multi-functional protein building blocks. In addition, the modularity of the system allows for the independent tuning of the physical properties of the hydrogel and its bulk function, an aspect of hydrogel design that will prove beneficial to a wide range of applications.