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Enzymatic bioelectrocatalysis often requires an artificial redox mediator to observe significant electron transfer rates. The use of such mediators can add a substantial overpotential and obfuscate the protein's native kinetics, which limits the voltage of a biofuel cell and alters the analytical performance of biosensors. Herein, we describe a material for facilitating direct electrochemical communication with redox proteins based on a novel pyrene-modified linear poly(ethyleneimine). This method was applied for promoting direct bioelectrocatalytic reduction of O 2 by laccase and, by immobilizing the catalytic subunit of nitrogenase (MoFe protein), to demonstrate the ATP-independent direct electroenzymatic reduction of N 2 to NH 3 .

The development of a hybrid, tri-catalytic architecture is demonstrated by immobilizing MWCNTs, TEMPO-modified linear poly(ethylenimine) and oxalate decarboxylase on an electrode to enable enhanced electrochemical oxidation of glycerol. This immobilized, hybrid catalytic motif results in a synergistic 3.3-fold enhancement of glycerol oxidation and collects up to 14 electrons per molecule of glycerol.

Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD + . We demonstrate how bioelectrocatalytic NAD + /NADH inter-conversion can transform a glucose/O 2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

Glycerol is a common fuel considered for bioenergy applications. Computational docking studies were performed on formate dehydrogenase from Candida boidinii (cbFDH) that showed that mesoxalate can bind to the buried active site of the holo form predicting that mesoxalate, a byproduct of glycerol oxidation, may act as its substrate. Spectroscopic assays and characterization by HPLC and GC/TCD have shown for the first time that cbFDH can act as a decarboxylase with mesoxalate. From this assessment, cbFDH was combined with NH 2 -TEMPO to form a novel hybrid anode to oxidize glycerol to carbon dioxide at near-neutral pH.

Electron mediation between NAD-dependent enzymes using quinone moieties typically requires the use of a diaphorase as an intermediary enzyme. The ability for a naphthoquinone redox polymer to independently oxidize enzymatically-generated NADH is demonstrated for application to glucose/O2 enzymatic fuel cells.

The complete electro-oxidation of glycerol to CO2 is performed through an oxidation cascade using a hybrid catalytic system combining a recombinant enzyme, oxalate decarboxylase from Bacillus subtilis, and an organic oxidation catalyst, 4-amino-TEMPO. This system is capable of electrochemically oxidizing glycerol at a carbon electrode collecting all 14 electrons per molecule.

Enzymatic fuel cells (EFCs) are devices that can produce electrical energy by enzymatic oxidation of energy-dense fuels (such as glucose). When considering bioanode construction for EFCs, it is desirable to use a system with a low onset potential and high catalytic current density. While these two properties are typically mutually exclusive, merging these two properties will significantly enhance EFC performance. We present the rational design and preparation of an alternative naphthoquinone-based redox polymer hydrogel that is able to facilitate enzymatic glucose oxidation at low oxidation potentials while simultaneously producing high catalytic current densities. When coupled with an enzymatic biocathode, the resulting glucose/O 2 EFC possessed an open-circuit potential of 0.864 ± 0.006 V, with an associated maximum current density of 5.4 ± 0.5 mA cm -2 . Moreover, the EFC delivered its maximum power density (2.3 ± 0.2 mW cm -2 ) at a high operational potential of 0.55 V.

The electrochemical detection of dehydrogenase activity in crude cell lysates is performed simultaneously using 96 carbon electrodes modified with electrografted phenazines. The method is applied to the screening of a library of formate dehydrogenase mutants obtained by directed evolution.