Elsevier

Electrochimica Acta

Volume 56, Issue 28, 1 December 2011, Pages 10786-10790
Electrochimica Acta

Electrically conducting particle networks in polymer electrolyte as three-dimensional electrodes for hydrogenase electrocatalysis

https://doi.org/10.1016/j.electacta.2011.01.051Get rights and content

Abstract

Efficient H2 oxidation and production by hydrogenase enzymes has attracted much interest because of the possibilities it raises for clean energy cycling without the need for precious metal catalysts. Although hydrogenases are extremely active electrocatalysts, high surface-area electrode structures will be necessary if the enzymes are to find application in energy technologies. Taking inspiration from fuel cell electrode assemblies, in which metal nanoparticles are commonly mounted on particulate carbon supports encased in polymer electrolyte, we show that high surface-area hydrogenase electrodes can be constructed from enzyme-loaded pyrolytic graphite particles in pH-neutralised Nafion. Pyrolytic graphite is the favoured surface for direct electrochemistry of many redox proteins, and on sanding, yields micron-dimension platelike particles. By modifying graphite platelets with hydrogenase before assembling the particles into a network, we ensure a high, uniform enzyme coverage. Incorporation of hydrogenases into high surface-area conducting network electrodes enhanced electrocatalytic H2 oxidation currents by 30-times compared to values obtained for a planar hydrogenase electrode, while retaining efficient conductivity and H2 mass transport through the network. This approach should make it possible to directly compare enzyme and precious metal electrocatalysis and to benchmark what opportunities are possible with selective enzyme catalysts.

Introduction

Direct electron transfer between immobilised redox enzymes and an electrode has facilitated a wide range of studies in which protein film electrochemistry (PFE) is used to control and probe catalysis and reactivity [1]. This approach has been particularly successful for hydrogenases, enzymes which catalyse the reversible interconversion of H2 and H+ very close to the calculated E(2H+/H2) potential and often with impressive selectivity for H2 over other small gaseous molecules. Both the nickel–iron and iron–iron families of hydrogenases possess a relay chain of iron–sulfur electron transfer clusters that links the buried active site to the protein surface and is conducive to efficient electron exchange with an electrode. PFE has provided fundamental insight into the mechanism and reactions of hydrogenases [2], and has also provided a means of exploiting their catalysis in fuel cells [3], [4], [5] and H2 production [6], [7]. High currents are desirable for these applications, and the electrocatalytic current for enzyme electrodes under efficient mass transport conditions is limited by the surface coverage attainable for large enzyme molecules. The typical footprint for a hydrogenase enzyme is about 20 nm2, meaning that the maximum monolayer coverage supported on a disk electrode is about 8 pmol cm−2; that is, the surface density of active sites is very low. Several approaches have previously been pursued for extending enzyme electrodes into three-dimensional space. If redox mediators are incorporated to shuttle electrons between the protein and the electrode, the enzyme of interest can be indirectly wired to the electrode by trapping it in a hydrogel or polymer electrolyte with the free or tethered mediator [8], [9], [10]. For example, ethanol oxidation by alcohol dehydrogenase in tetrabutylammonium bromide ion-exchanged Nafion was utilized at an enzyme fuel cell anode by Minteer and coworkers with electro-regenerated NAD+ as mediator [9], [11]. However when dealing with highly electroactive enzymes, mediated electron transfer is likely to limit catalysis. Direct electron transfer between fructose dehydrogenase and a high surface area carbon electrode was achieved by Kano and coworkers who used poly(vinylidene difluoride) to link a form of carbon particles known as Ketjen Black onto a glassy carbon surface and then allowed the enzyme to slowly adsorb [12]. This gave rise to fructose oxidation current densities of around 8 mA cm−2 at 500 mV above the onset potential for the catalysis. A nickel–iron hydrogenase covalently attached to multi-walled carbon nanotubes grown on a gold support gave diffusion limited current densities above 2 mA cm−2 in a study by De Lacey and coworkers [13].

The rough ‘edge’ surface of pyrolytic graphite (PG) [14] has been found to be an excellent electrode material for direct electrochemical studies of hydrogenases [2], [15] and many other redox enzymes including carbon monoxide dehydrogenase [16], nitric oxide reductase [17], formate dehydrogenase [18], fumarate reductase [19], and NAD+/NADH cycling domains of repiratory complex I [20]. Direct adsorption of proteins on PG probably benefits from the range of functional groups available on a freshly abraded surface of this material. We have therefore developed a method for assembling high surface-area electrodes for direct electron transfer to adsorbed enzymes, taking advantage of PG as the electrode material. Particles of PG can be prepared readily by abrasion of a piece of PG with emery paper (Fig. 1A) [21]. Particles are platelike, with a planar basal surface, and a jagged edge surface that should be ideal for enzyme adsorption. We show here that incorporation of a dense paste of hydrogenase-modified particles into the polymer electrolyte Nafion, titrated to neutral pH with phosphate [22], provides a straightforward route to assembling a three-dimensional hydrogenase electrode, as shown schematically in Fig. 1B. Hydrogenase electrodes assembled in this way were found to give substantially higher current relative to hydrogenase on a rotating disk electrode (RDE) of corresponding geometrical surface area. The integrity of hydrogenase in the film is confirmed by the electrocatalytic wave shape which closely resembles that at a conventional RDE.

Our test enzymes are the relatively robust NiFe hydrogenases 1 and 2 (Hyd-1 and Hyd-2) from Escherichia (E.) coli. These enzymes have already been shown to absorb on PG to give electroactive films that are stable over several hours [23]. Hyd-1 is strongly biased in the direction of H2 oxidation, with H+ reduction being strongly inhibited by its product, H2. In the H2 oxidation direction, a reversible oxidative inactivation/reductive reactivation process gives rise to a characteristic switch off/switch on in the electrocatalytic current that serves as a useful fingerprint for effective potential control of the enzyme [23], [24]. Hyd-1 belongs to a class of O2-tolerant hydrogenases that are able to sustain H2 oxidation in air and have been demonstrated as viable anode electrocatalysts in mixed-feed H2/O2 fuel cells [4]. Incorporation of Hyd-1 into high surface area network electrodes should open up new possibilities for enzyme fuel cell construction and testing. Hyd-2 is active for both H+ reduction (particularly at low H2 partial pressures) and H2 oxidation, particularly at H2 partial pressures below 1 bar. The approach in which particles are first modified with protein before assembly of the particle network should also be useful for exploitation and study of other redox proteins that engage in direct electron transfer at PG.

Section snippets

Experimental

All experiments were conducted in a N2-filled anaerobic glove box (Mbraun <1 ppm O2). Electrochemical experiments involving a rotating disk electrode (RDE) were carried out in a glass electrochemical cell, water-jacketed for temperature control, and sealed with an o-ring onto an electrode rotator (EG&G model 636). The cell was equipped with gas inlet and outlet fittings to allow exchange of gases in the headspace. Gases used were: H2 (Premier Plus, Air Products), and 5% H2 in N2 (Protec 5, Air

Results and discussion

A voltammogram for an unmodified electrode in Fig. 2A (i) confirms that no electrocatalytic current is observed under H2 in the absence of hydrogenase. Trace (II) in Fig. 2A (enlarged in the inset) shows a typical voltammogram at 1 bar H2 for a film of E. coli hydrogenase 1 (Hyd-1), prepared by adsorbing enzyme directly onto a pyrolytic graphite (PG) ‘edge’ RDE. The aerobically isolated enzyme requires a slow reductive activation step before full activity is reached [1], [2] so the electrode was

Conclusions

The method we describe for assembling high surface-area enzyme electrodes is likely to be useful in applications of hydrogenase electrocatalysis in fuel cells or H2 production where high electrocatalytic currents are required. It remains unclear whether the long-term stability of hydrogenase electrodes will be suitable for their application in energy devices, but this approach should make it possible to test hydrogenases alongside more conventional electrocatalysts and assess the possibilities

Acknowledgements

This work was supported by research grants from the Royal Society and the John Fell Fund. K.A.V. is a Royal Society Research Fellow. A.J.H. is supported by a scholarship from the John Fell Fund. H.R. is grateful for support from Christ Church, Oxford and A.P. is supported by a Junior Research Fellowship from Merton College, Oxford.

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