Electrochemistry Communications 10 (2008) 970–972

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Electrochemistry Communications

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Electroreduction of O2 at a mediated Melanocarpus albomyces laccase cathodein a physiological buffer

Paul Kavanagh, Peter Jenkins, Dónal Leech *

Department of Chemistry, National University of Ireland, Galway, Ireland

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 April 2008Accepted 16 April 2008Available online 22 April 2008

Keywords:Biofuel cellBiocathodeLaccaseOsmium complexesOxygen reduction

1388-2481/$ - see front matter � 2008 Elsevier B.V. Adoi:10.1016/j.elecom.2008.04.025

* Corresponding author. Tel.: +353 91 493563; fax:E-mail address: donal.leech@nuigalway.ie (D. Leec

We report on oxygen reduction in a physiological buffer solution (0.05 M phosphate buffer containingdissolved O2, 0.1 M NaCl, pH 7.4, 37 �C) by Melanocarpus albomyces laccase, co-immobilized with[Os(2,2’-bipyridine)2(polyvinylimidazole)10Cl]+/2+ as a mediator, on glassy carbon electrodes. Such oxy-gen cathodes yielded current densities of 3.8 mA cm�2 at 0.2 V vs. Ag/AgCl, the largest current densityreported to date for a mediated laccase cathode in physiological buffer solutions, showing promise fordevelopment of biocatalytic fuel cell prototypes.

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1. Introduction and ionic strength. Our experiments result in the highest current

Laccases are ‘blue’ copper oxygenases that oxidize a broad rangeof substrates, including phenols, aromatic amines, and organic andinorganic mediators, coupled to reduction of O2 [1–3]. Electrodesincorporating laccase are attracting considerable attention due topotential applications in biocatalytic fuel cells [4–6]. It is feasiblethat a laccase cathode coupled to a biocatalytic anode could providethe basis for an implantable fuel cell [5,6]. A major drawback is thatoptimum activity for the vast majority of laccases is observed be-tween pH 3.0–5.0 [1–3,7]. However, a number of laccases have beenproduced that display optimal activity closer to neutral pH [3,8–10].One such enzyme is a fungal laccase sourced from Melanocarpus alb-omyces, which has been characterized by Kiiskinen and coworkers[11–15]. Studies have revealed that optimum guaiacol oxidationactivity for this enzyme is between pH 6.0–7.0 at 60–70 �C [11].The redox potential of the substrate oxidizing, T1, copper site for thislaccase is 0.26 V vs. Ag/AgCl at pH 7 [16],�0.25 V lower than that oflaccases sourced from Trametes versicolor or Coriolus hirsutus [6], pre-viously utilized in biocatalytic fuel cell assemblies [17–19]. None-theless, a fuel cell containing a mediated M. albomyces laccase(MaL) cathode coupled to a mediated glucose oxidase anode (operat-ing at��0.32 V vs. Ag/AgCl) could theoretically operate at a cell emfin excess of 0.5 V, with an appreciable power output, compared toother laccase-based biocatalytic cathodes, because of relativelyhigher laccase activity at neutral pH. Here we report on the perfor-mance of a MaL laccase cathode, under conditions of electrolyte pH

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density yet reported for laccase mediated oxygen reduction underphysiologically relevant conditions.

2. Experimental section

2.1. Materials

Synthesis of the redox polymers was achieved by adapting liter-ature procedures, using (NH4)2OsCl6 (Aldrich) as starting materialto prepare the cis-Os(2,20-bipyridine)Cl2 and cis-Os(4,40-dichloro-2,20-bipyridine)2Cl2 complexes, which were then complexed, via li-gand substitution reaction in ethanol/water solvent, to a previouslypresynthesized polyvinylimidazole polymer [20]. Poly(ethyleneglycol) diglycidyl ether (average Mn � 526) was purchased from Al-drich. Purified MaL was donated by VTT Technology, Finland (K.Kruus).

2.2. Apparatus

Electrochemical measurements were carried out with anEcoChemie Autolab PGSTAT10 potentiostat, using 3 mm glassy car-bon (GC), Ag/AgCl (3 M NaCl) and platinum wire as working, refer-ence and counter electrodes, respectively (all IJ Cambria), in athermostated, non-compartmentalized 3 electrode cell.

2.3. Methods

Electrodes were prepared, based on previously reported ratiosof redox polymer to enzyme optimized for T. versicolor laccase

-0.2 0.0 0.2 0.4 0.6

-4

-3

-2

-1

0

Cur

rent

Den

sity

(mA

/ cm

2 )

Potential / V (vs. Ag/AgCl)

Fig. 1. Cyclic voltammograms at electrodes modified with Polymer I (black) andPolymer II (red) and MaL in 0.05 M phosphate buffer containing dissolved O2, 0.1 MNaCl, pH 7.4, 37 �C. Scan rate 5 mV s�1. Solution agitated using magnetic stirrerrotating at 100 rpm.

4.5 5.0 5.5 6.0 6.5 7.0 7.540

50

60

70

80

90

100

Res

pons

e (%

)

pH

A

B

P. Kavanagh et al. / Electrochemistry Communications 10 (2008) 970–972 971

[21], by depositing a drop (12 lL) containing the enzyme (6 lL of860 nkat/ml MaL), the redox polymer (3 lL of a 8–10 mg/ml solu-tion/suspension in water) and poly(ethylene glycol) diglycidylether (3 lL of a 15 mg/ml solution in water) as a crosslinker ontoa polished 3 mm diameter glassy carbon electrode, followed byat least 24 h drying of the film. No attempt was made to increasethe electrochemically active area of the glassy carbon electrode.

3. Results and discussion

For O2 reduction to be thermodynamically driven downhill, seeScheme 1, the redox potential of the mediator should be negativeto that of the T1 substrate oxidizing site of the laccase enzyme.To test this, two osmium-based redox polymers were synthe-sized; [Os(2,20-bipyridine)2(polyvinylimidazole)10Cl]+/2+ (PolymerI) [20] (E00 0.22 V vs. Ag/AgCl), and [Os(4,40-dichloro-2,20-bipyri-dine)2(polyvinylimidazole)10Cl]+/2+ (Polymer II) [17] (E00 0.35 V vs.Ag/AgCl), and films of these polymers crosslinked with the MaL,were deposited onto previously polished glassy carbon electrodesurfaces. No attempt was made to roughen, or otherwise pre-treat,the glassy carbon electrode surface. Sigmoidal-shaped slow-scancyclic voltammograms (CVs), Fig. 1, characteristic of an EC’ scheme,indicating bioelectrocatalytic reduction of oxygen, were obtainedin all cases for the films prepared.

From CVs obtained in solutions under physiological conditionsagitated at 100 rpm using a magnetic stirrer, laccase cathodes ofPolymer I and Polymer II yield oxygen reduction current densitiesof 3.8 ± 0.2 mA cm�2 and 0.7 ± 0.5 mA cm�2 (n = 5 for both) atpotentials of 0.20 V and 0.30 V vs. Ag/AgCl, respectively. The rela-tively lower current density for Polymer II is due to the redox po-tential of Polymer II being more positive than that of the T1 site ofthe enzyme at pH 7, resulting in a thermodynamically unfavour-able uphill electron transfer reaction. The 3.8 mA cm�2 currentdensity at the Polymer I cathode in a physiological buffer solutionis the highest to date observed for laccase mediated systems underphysiological conditions. This result is particularly impressive, gi-ven that the films were deposited on smooth glassy carbon diskelectrodes, compared to deliberately roughened, or carbon fibre[26] surfaces which have either higher surface areas or improveddiffusion regimes, respectively.

GC electrodes modified with films prepared with Polymer I andMaL were examined further under varying conditions of pH andNaCl concentration. It is evident that mediated biocatalytic activityincreases with increasing pH at these films, reaching an optimumat pH 6.5–7.0 (Fig. 2A), in agreement with previous studies of solu-tion phase MaL activity [11]. At pH 7.4, we observe a slight increasein oxygen reduction currents at these films on addition of[NaCl] 6 0.05 M (Fig. 2B), also observed for T. versicolor laccase in

Scheme 1. Schematic depiction of a mediated (Med’) biocatalytic reduction of di-oxygen using laccase (Lac) as a biocatalyst. Potentials are quoted vs. Ag/AgCl at pH7.4.

films of Polymer I [21]. It is possible that a Donnan potential effect,due to the high local concentration of ions present in the film (esti-mated at >0.1 M) is responsible for this behaviour [20,21]. Addition

0 100 200 300 400 50070

80

90

100

Res

pons

e (%

)

[NaCl] / mM

Fig. 2. Dependence of response for Polymer I/MaL on (A) pH, and (B) [NaCl] inquiescent 0.05 M phosphate buffer solution. Scan rate 5 mV s�1. Currents are nor-malized to the maximum current obtained for each parameter.

972 P. Kavanagh et al. / Electrochemistry Communications 10 (2008) 970–972

of [NaCl] > 0.5 M resulted in a decrease in oxygen reduction cur-rent, although 75% is still retained at 0.5 M NaCl. In addition, asphysiological conditions of 0.1 M NaCl and 0.15 M NaCl concentra-tions are approached, only a slight drop in oxygen reduction cur-rent (3% and 5%, respectively) is evident compared to solutionswith no added NaCl.

Few studies specifically describing the operation of a laccase-based oxygen cathode under physiological conditions are available.Barton et al. [22] reported on the electroreduction of O2 at a med-iated Pleurotus ostreatus laccase cathode. Although P. ostreatus wasshown to retain as much as 70% of its pH 5 syringaldazine oxida-tion activity at pH 7 [23], only 10 % of the current density observedat pH 5 was evident at pH 7 when immobilized in an osmium redoxpolymer, with maximum current density of 55 lA cm�2 reported at900 rpm in the presence of 0.1 M NaCl at 37 �C. Barrière et al. [24]reported a similar observation for a T. versicolor laccase-based oxy-gen cathode using an osmium redox polymer mediating film, withonly 7% of the current density observed at pH 5 present at pH 7,and a maximum current density of �17 lA cm�2 reported at thatpH, in the presence of 0.1 M NaCl at 37 �C, without stirring. Fur-thermore, the current density displayed by the Polymer I-basedcathode at pH 7.4 in Fig. 1, is comparable to that observed usinga C. hirsutus laccase [25,26] operating in pH 5 buffer, or a bilirubinoxidase [27,28] operating in neutral pH buffer. Increasing the rateof O2 mass transport to the electrode would likely yield greatercurrent densities [25,27,29]. For example, Barton et al. [25] reportthat the current density (6.0 ± 0.8 mA cm�2) of a mediated C. hirsu-tus laccase cathode in pH 5 buffer becomes limited by kinetics ofthe electrocatalyst at 4000 rpm, with the stability of the films,however, being compromised. In addition, the current density ofthis mediated C. hirsutus laccase cathode was recorded in the ab-sence of NaCl [25,26], whereas the current density of the mediatedM. albomyces laccase electrode (3.8 mA cm�2) was recorded at NaClconcentrations (0.1 M) approaching physiological conditions. Thisis an important consideration in the development of the cathodefor a prospective enzymatic biofuel cell operating in a physiologicalenvironment.

4. Conclusion

MaL appears to be an excellent candidate for the mediated bio-electrocatalytic reduction of O2 at the cathode of an implantablebiofuel cell assembly, yielding current densities of 3.8 mA cm�2

at 0.2 V vs. Ag/AgCl in physiological buffer. This represents thelargest current density achieved to date using a mediated laccasecathode in a physiological buffer. Lower, though still appreciable,current densities can be obtained using redox polymers of higherpotentials. We are currently in the process of examining the per-formance of prototype glucose/O2 biofuel cells operating in physi-

ological buffer using biocatalytic cathodes containing MaL. Aninvestigation into various strategies to improve film stability isunderway, as are means to increase the microscopic surface areaof the electrodes, thus increasing the current density.

Acknowledgements

This research was funded by the European Union through con-tract NMP4-CT-2006-017350, and supported by the EnvironmentalChange Institute, NUI Galway. The donation of Melanocarpus alb-omyces laccase from VTT Technology, Finland (K. Kruus) is grate-fully acknowledged.

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