Ross D. Milton

Hydrogen peroxide produced by glucose oxidase affects the performance of laccase cathodes in glucose/oxygen fuel cells: FAD-dependent glucose dehydrogenase as a replacement

Hydrogen peroxide production by glucose oxidase (GOx) and its negative effect on laccase performance have been studied. Simultaneously, FAD-dependent glucose dehydrogenase (FAD-GDH), an O2-insensitive enzyme, has been evaluated as a substitute. Experiments focused on determining the effect of the side reaction of GOx between its natural electron acceptor O2 (consumed) and hydrogen peroxide (produced) in the electrolyte. Firstly, oxygen consumption was investigated by both GOx and FAD-GDH in the presence of substrate. Relatively high electrocatalytic currents were obtained with both enzymes. O2 consumption was observed with immobilized GOx only, whilst O2 concentration remained stable for the FAD-GDH. Dissolved oxygen depletion effects on laccase electrode performances were investigated with both an oxidizing and a reducing electrode immersed in a single compartment. In the presence of glucose, dramatic decreases in cathodic currents were recorded when laccase electrodes were combined with a GOx-based electrode only. Furthermore, it appeared that the major loss of performance of the cathode was due to the increase of H2O2 concentration in the bulk solution induced laccase inhibition. 24 h stability experiments suggest that the use of O2-insensitive FAD-GDH as to obviate in situ peroxide production by GOx is effective. Open-circuit potentials of 0.66 ± 0.03 V and power densities of 122.2 ± 5.8 μW cm(-2) were observed for FAD-GDH/laccase biofuel cells.

A self-powered amperometric lactate biosensor based on lactate oxidase immobilized in dimethylferrocene-modified LPEI.

Lactate is an important biomarker due to its excessive production by the body during anerobic metabolism. Existing methods for electrochemical lactate detection require the use of an external power source to supply a positive potential to the working electrode of a given device. Herein we describe a self-powered amperometric lactate biosensor that utilizes a dimethylferrocene-modified linear poly(ethylenimine) (FcMe2-LPEI) hydrogel to simultaneously immobilize and mediate electron transfer from lactate oxidase (LOx) at the anode and a previously described enzymatic cathode. Operating as a half-cell, the FcMe2-LPEI electrode material generates a jmax of 1.51 ± 0.13 mAcm(-2) with a KM of 1.6 ± 0.1 mM and a sensitivity of 400 ± 20 μAcm(-2)mM(-1) while operating with an applied potential of 0.3 V vs. SCE. When coupled with an enzymatic biocathode, the self-powered biosensor has a detection range between 0mM and 5mM lactate with a sensitivity of 45 ± 6 μAcm(-2)mM(-1). Additionally, the FcMe2-LPEI/LOx-based self-powered sensor is capable of generating a power density of 122 ± 5 μWcm(-2) with a current density of 657 ± 17 μAcm(-2) and an open circuit potential of 0.57 ± 0.01 V, which is sufficient to act as a supplemental power source for additional small electronic devices.

Employing FAD-dependent glucose dehydrogenase within a glucose/oxygen enzymatic fuel cell operating in human serum

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) is emerging as an oxygen-insensitive alternative to glucose oxidase (GOx) as the biocatalyst for bioelectrodes and bioanodes in glucose sensing and glucose enzymatic fuel cells (EFCs). Glucose EFCs, which utilize oxygen as the oxidant and final electron acceptor, have the added benefit of being able to be implanted within living hosts. These can then produce electrical energy from physiological glucose concentrations and power internal or external devices. EFCs were prepared with FAD-GDH and bilirubin oxidase (BOx) to evaluate the suitability of FAD-GDH within an implantable setting. Maximum current and power densities of 186.6±7.1 μA cm(-2) and 39.5±1.3 μW cm(-2) were observed when operating in human serum at 21 °C, which increased to 285.7±31.3 μA cm(-2) and 57.5±5.4 μW cm(-2) at 37 °C. Although good stability was observed with continual near-optimal operation of the EFCs in human serum at 21 °C for 24 h, device failure was observed between 13-14 h when continually operated at 37 °C.

Tailoring Biointerfaces for Electrocatalysis

Bioelectrocatalysis is an expanding research area due to the use of this type of electrocatalysis in electrochemical biosensors, biofuel cells, bioelectrochemical cells, and biosolar cells. This feature article discusses recent advancements in tailoring the biointerface between electrodes and biocatalysts for facile electrocatalysis. This includes the design of pyrene moieties for directing the orientation of biocatalysts on electrode surfaces and mediation as well as the rational design of redox polymers for self-exchange-based electron transport to/from biocatalysts and the electrode and the use of bioscaffolding techniques for designing the bioelectrode structure. However, recent advances in the past decade have shown the importance of hybrid bioelectrocatalytic systems, and future work will be needed to use these same pyrene, redox polymer, and bioscaffolding techniques for hybrid bioelectrocatalysis.

Direct enzymatic bioelectrocatalysis: differentiating between myth and reality

Enzymatic bioelectrocatalysis is being increasingly exploited to better understand oxidoreductase enzymes, to develop minimalistic yet specific biosensor platforms, and to develop alternative energy conversion devices and bioelectrosynthetic devices for the production of energy and/or important chemical commodities. In some cases, these enzymes are able to electronically communicate with an appropriately designed electrode surface without the requirement of an electron mediator to shuttle electrons between the enzyme and electrode. This phenomenon has been termed direct electron transfer or direct bioelectrocatalysis. While many thorough studies have extensively investigated this fascinating feat, it is sometimes difficult to differentiate desirable enzymatic bioelectrocatalysis from electrocatalysis deriving from inactivated enzyme that may have also released its catalytic cofactor. This article will review direct bioelectrocatalysis of several oxidoreductases, with an emphasis on experiments that provide support for direct bioelectrocatalysis versus denatured enzyme or dissociated cofactor. Finally, this review will conclude with a series of proposed control experiments that could be adopted to discern successful direct electronic communication of an enzyme from its denatured counterpart.

NAD-dependent dehydrogenase bioelectrocatalysis: the ability of a naphthoquinone redox polymer to regenerate NAD

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.

FAD-Dependent Glucose Dehydrogenase Immobilization and Mediation Within a Naphthoquinone Redox Polymer

Electrochemically-active polymers (redox polymers) are useful tools for simultaneous immobilization and electron transfer of enzymes at electrode surfaces, which also serve to increase the localized concentration of the biocatalyst. The properties of the employed redox couple must be compatible with the target biocatalyst from both an electrochemical (potential) and biochemical standpoint. This chapter details the synthesis of a naphthoquinone-functionalized redox polymer (NQ-LPEI) that is used to immobilize and electronically communicate with flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH), yielding an enzymatic bioanode that is able to deliver large catalytic current densities for glucose oxidation at a relatively low associated potential.

Mechanism of Nitrogenase H2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects

Nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H2. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H2. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to the MoFe protein allowing examination of the mechanism of H2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, was found to change linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate-limiting step of H2 formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein, either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H2 formation where the S-H+ bond breaks and H+ attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.

Mediator-free enzymatic electrosynthesis of formate by the Methanococcus maripaludis heterodisulfide reductase supercomplex

Electrosynthesis of formate is a promising technology to convert CO2 and electricity from renewable sources into a biocompatible, soluble, non-flammable, and easily storable compound. In the model methanogen Methanococcus maripaludis, uptake of cathodic electrons was shown to proceed indirectly via formation of formate or H2 by undefined, cell-derived enzymes. Here, we identified that the multi-enzyme heterodisulfide reductase supercomplex (Hdr-SC) of M. maripaludis is capable of direct electron uptake and catalyzes rapid H2 and formate formation in electrochemical reactors (-800 mV vs Ag/AgCl) and in Fe(0) corrosion assays. In Fe(0) corrosion assays and electrochemical reactors, purified Hdr-SC primarily catalyzed CO2 reduction to formate with a coulombic efficiency of 90% in the electrochemical cells for 5 days. Thus, this report identified the first enzyme that stably catalyzes the mediator-free electrochemical reduction of CO2 to formate, which can serve as the basis of an enzyme electrode for sustained electrochemical production of formate.

Catalysts for nitrogen reduction to ammonia

AbstractThe production of synthetic ammonia remains dependent on the energy- and capital-intensive Haber–Bosch process. Extensive research in molecular catalysis has demonstrated ammonia production from dinitrogen, albeit at low production rates. Mechanistic understanding of dinitrogen reduction to ammonia continues to be delineated through study of molecular catalyst structure, as well as through understanding the naturally occurring nitrogenase enzyme. The transition to Haber–Bosch alternatives through robust, heterogeneous catalyst surfaces remains an unsolved research challenge. Catalysts for electrochemical reduction of dinitrogen to ammonia are a specific focus of research, due to the potential to compete with the Haber–Bosch process and reduce associated carbon dioxide emissions. However, limited progress has been made to date, as most electrocatalyst surfaces lack specificity towards nitrogen fixation. In this Review, we discuss the progress of the field in developing a mechanistic understanding of nitrogenase-promoted and molecular catalyst-promoted ammonia synthesis and provide a review of the state of the art and scientific needs for heterogeneous electrocatalysts. The artificial synthesis of ammonia remains one of the most important catalytic processes worldwide, over 100 years after its development. In this Review, recent developments in enzymatic, homogeneous and heterogeneous catalysis towards the conversion of nitrogen to ammonia are discussed, with a particular focus on how mechanistic understanding informs catalyst design.