A Comparative Study of Biocathodes Based on Multiwall CarbonNanotube Buckypapers Modified with Three DifferentMulticopper Oxidases

Dmitry V. Pankratov,a Yulia S. Zeifman,a Olga V. Morozova,a, b Galina P. Shumakovich,a, b Irina S. Vasil�eva,a, b

Sergey Shleev,b, c Vladimir O. Popov,a, b Alexander I. Yaropolov *a, b

a Kurchatov NBIC Centre, National Research Centre “Kurchatov Institute”, Akademika Kurchatova pl. 1, 123182 Moscow, Russiab A. N. Bach Institute of Biochemistry RAS, Leninsky prospekt 33,119071 Moscow, Russiac Biomedical Sciences, Faculty of Health and Society, Malmç University, Jan Waldenstrçms gata 25, 214 28 Malmç, Sweden*e-mail: yaropolov@inbi.ras.ru

Received: September 21, 2012Accepted: December 24, 2012Published online: March 19, 2013

Abstract14 Single- and multi-walled carbon nanotubes from different sources were characterized in detail, and the character-istics obtained were carefully analyzed. The carbon material with the highest capacitance, and also other superiorproperties (“Taunit-M” from “NanoTechCenter”, Russia), was chosen for further modification and fabrication ofbuckypaper based electrodes. These electrodes were biomodified with plant and fungal laccases, as well as fungal bi-lirubin oxidase. The designed biocathodes were investigated in simple buffers and also in a complex physiologicalfluid (human serum). Biocathodes based on immobilized fungal laccase were bioelectrocatalytically inactive in chlo-ride containing media at neutral pH. In spite of the quite high current densities realized using biodevices based onplant laccase and fungal bilirubin oxidase, the limited thermal stability of the enzymes renders the biocathodes inad-equate for practical applications in implanted situations.

Keywords: Biocathode, Bilirubin oxidase, Buckypaper, Laccase, Multicopper oxidase

DOI: 10.1002/elan.201200516

Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/elan.201200516

1 Introduction

Biological fuel cells or biofuel cells (BFCs) are devices,which transform chemical energy into electric energyusing biocatalysts. Contrary to fuel cells there is no actualneed to separate cathodic and anodic compartments inBFCs using a membrane because of the selectivity of bio-catalysts, which simplifies the construction and allowsminiaturization of these biodevices. In the last ten yearsmany papers were devoted to BFCs as possible powersources for implanted medical devices, cf. some famousreviews [1, 2]. Such a device can be potentially implantedinto a body and uses glucose and molecular oxygen (O2)readily available in human physiological fluids as fuel andoxidant, respectively. Power densities of already fabricat-ed and tested glucose/O2 BFCs is not so high and lies inthe mW-mW/cm2 ranges. However, these relatively lowvalues might still be enough to power useful biomedicaldevices, like self-contained biosensors, pacemakers, etc.

An open circuit voltage (OCV) of a BFC is determinedby the potential difference between the cathode andanode. Indeed, in order to activate O2 on the cathode dif-ferent catalysts including biological ones are used. Tworedox enzymes from the broad family of multicopper oxi-

dases (MCO) are the most popular biocatalysts to con-struct efficient and stable biocathodes, viz. laccase (Lc,benzenediol: oxygen oxidoreductases, EC 1.10.3.2) andbilirubin oxidase (BOx, bilirubin: oxygen oxidoreductase,EC 1.3.3.5) [3]. It was shown that these enzymes catalysethe reaction of O2 electroreduction by the mechanism ofdirect electron transfer between electrodes and activecentres of these proteins with concomitant reduction ofO2 directly to H2O [4]. The redox potential of the T1 site(ET1) of different Lcs and BOxs varies between 0.43 and0.78 V vs. NHE [4,5]. For instance, ET1 values of Lc fromthe basidiomycete Trametes hirsuta (ThLc) and BOx fromthe ascomycete Myrothecium verrucaria (MvBOx) are0.78 V [6] and 0.67 V [7,8], respectively. Indeed, thesefungal enzymes are high redox potential MCO, which isa very important property to construct high potential bio-cathodes. Contrary to fungal enzymes, plant Lcs are lowredox potential MCO, e.g. the ET1 value of Rhus vernici-fera Lc (RvLc) is 0.43 V vs. NHE [9]. Consequently,RvLc based biocathodes have quite low onset potentialsof O2 bioelectroreduction [10].

To increase current densities of biocathodes, high sur-face area carbon matrixes, such as carbon black, activatedcharcoal, and carbon nanotubes (CNTs), immobilized on

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both rigid and flexible supports, are usually used [11,12].There are several reports in literature concerning usageof CNTs as a matrix for MCO immobilization and appli-cation of these biocomposites to construct biocathodesand functional BFCs [13–17] including air-breathing bio-devices [18,19].

One very interesting material based on CNTs isa buckypaper (BP) – a novel innovative nanomaterialwith superior properties for the construction of potential-ly implantable biodevices, such as very high surface area,porosity, chemical stability, flexibility, high mechanicalstrength, and, importantly, low toxicity both in vitro andin vivo [20]. BP is a plexiform film of densly packedCNTs maintaining close contact due to van der Waalsforces between nanotubes and it is produced by the filtra-tion of CNTs dispersions [21,22]. Recently, Hussein at al.has reported on the potential application of MCO modi-fied BPs for the construction of very efficient mediator-less biocathodes and BFCs [23–25].

Two major goals of our studies are (i) to compare theefficiency of biocathodes based on a BP modified withthree different MCO, i.e. ThLc, MvBOx, and RvLc, aswell as (ii) to clarify the perspective of their potentialusage to construct implantable BFCs.

2 Experimental

2.1 Reagents

Na2HPO4, KH2PO4, K4[Fe(CN)6], NaOH, HNO3, H3PO4,H3BO3, 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonicacid (ABTS), catechol, and cellulose acetate were ob-tained from Sigma-Aldrich (Seelze, Germany). Citric acidwas from AppliChem (Darmstadt, Germany). Silver-filledepoxy paste EPO-TEK was obtained from Epoxy Tech-nology (Billerica, MA, USA). All buffer solutions wereprepared using water (18 MW) purified with a Simplicitysystem (Millipore, Milford, CT, USA)

2.2 Enzymes

Bilirubin oxidase from Myrothecium verrucaria (MvBOx)was purchased from Amano Enzyme (Nagoya, Japan)and purified by means of DEAE-chromatography. Thespecific activity of the enzyme was 118 U/mg of proteinusing an ABTS assay at pH 7.4.

Fungal laccase from Trametes hirsuta (Wulfen) Pil�tCF-28 (ThLc) was purified to homogeneity as describedpreviously [26]. The specific activity of the enzyme prepa-ration was 198 U/mg of protein using catechol as sub-strate at pH 4.5.

Partly purified Rhus vernicifera laccase (RhLc) fromthe latex of the lacquer tree was kindly provided by Prof.B. Reinhammar (University of Gothenburg, Sweden).The final purification for the Lc was performed by meansof HPLC on TSK DEAE-2SW column (LKB, Sweden)using a Stayer HPLC system (Acvilon, Russia). Theenzyme was homogeneous as judged from SDS-PAGE.

The specific activity of the enzyme preparation was 14 U/mg of protein using K4[Fe(CN)6], as substrate at pH 4.5.

The preparations of all enzymes were stored at �18 8C.

2.3 Carbon Nanotubes

Commercially available CNTs, both single-walled andmulti-walled (SWCNTs and MWCNTs, respectively) wereused as received (Supporting Information, Table S1).MWCNTs “Taunit M” (NanoTechCentre Ltd, Tambov,Russia), generated via catalytic chemical vapour deposi-tion (purity of 95+%), had outer and inner diameters of20–70 and 5–10 nm, respectively, with lengths up to 2 mm.For functionalization the MWCNT samples were heatedat 90 8C with concentrated (65%) nitric acid for 5 h(100 mg MWCNT/20 ml HNO3), cooled, filtered, andwashed with deionized water until neutral, washed withacetone, and finally dried at 110 8C for 5 h.

As-received MWCNT, functionalized MWCNT (f-MWCNT), and BP samples were characterized by ther-mogravimetric analysis using a Thermoscan-2 (Analitpri-bor, St. Peterburg, Russia), as well as transmission (JEM-100 CX/SFG, “Jeol”, Japan) and scanning (Carl ZeissSupra 40 VP, Carl Zeiss Ltd., Cambridge, UK) electronmicroscopes.

FTIR spectra were recorded using KBr pellets with anIR Prestige Fourier transform spectrophotometer (Shi-madzu, Japan) at room temperature in the 400–4000 cm�1

range.Four-point conductivity measurements were carried out

with a Loresta GP instrument (Mitsubishi, Japan).

2.4 Fabrication of Buckypaper Based Electrode

f-MWCNT (10 mg) were dispersed in 10 mL of ethanolfollowed by ultrasonication for 3 h. The stable suspen-sions were filtered through a fluoroplastic membranefilter with a pore size of 0.2 mm (BioChemMac, Moscow,Russia) using a 13 mm Millex Syringe Filter unit (Milli-pore, Carrigtwohill, Ireland). The samples were dried atroom temperature and peeled off afterwards. Samples ofBP were dried under a coverslip.

A copper wire was glued to the surface of BP using thesilver-filled epoxy paste. To insulate the location of thecontact site, the contact was covered with cellulose ace-tate.

Enzyme modification of the electrode was performedby applying a drop (20 mL) of an enzyme solution to thesurface of a BP, and drying the drop for 20 minutes in air.Then, electrodes were rinsed with water and immersed ina buffer solution to avoid complete drying and possibledenaturation of the enzyme.

2.5 Buffers (Electrolytes)

Five different electrolytes were used in these studies, viz.0.1 M phosphate buffer, pH 7.4 (PB), 0.1 M phosphatebuffer containing 0.15 NaCl, pH 7.4 (PBS), 0.1 M citrate-

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phosphate buffer, pH 4.5 (CPB), 40 mM universal buffer(mixture of 40 mM of phosphoric acid, boric acid, andacetic acid adjusted to the desired pH using NaOH), anda human serum. The serum was prepared as known in theart and stored at �18 8C until use.

2.6 Electrochemical Measurements

Linear sweep voltammograms (LSVs) and cyclic voltam-mograms (CVs) were recorded with a scan rate of 10 mV/s on a PC controlled electrochemical analyzer (CV-50W,Bioanalytical Systems, BAS, West Lafayette, IN, USA) ina one-compartment three-electrode cell with a totalvolume of 10 mL. Glassy carbon electrodes from BAS(GCEs) modified with CNTs, bare and enzyme modifiedspectrographic graphite and BP based electrodes wereused as working electrodes. A platinum wire (1 mm diam-eter) and Ag jAgCl j3 M NaCl (BAS) were used as coun-ter and reference electrodes, respectively. All potentialsin the present work are given vs. NHE.

2.7 Spectrophotometric Measurements of EnzymeStability in Homogeneous Solution

The activities of the enzymes were measured using a UV-1240 spectrophotometer from Shimadzu (Suzhou, China).MvBOx, ThLc, and RvLc activities were determined inPB, pH 7.4 using ABTS assay (l=405 nm), in CPBpH 4.5 and in PB pH 7.4 using cathehol (l=405 nm) asa substrate, respectively.

3 Results and Discussion

3.1 Physicochemical Characteristics of CNTs

In order to produce a BP with a highly developed surface,CNTs from different sources were tested at the beginningof our studies (Supporting Information (SI), Table S1),viz. specific capacitance of carbon materials was estimat-ed by cyclic voltammetry (SI, Figure S1). Based on thehighest specific capacitance recorded, MWCNTs “TaunitM” from NanoTechCentre Ltd. were used in further stud-ies. Prior to production, the nanomaterial “Taunit M” wascharacterized in detail. It consists of cylindrical nanotubes(6–10 carbon atoms) with a length of 2 mm, forming bun-dles. For compatibility with matrixes of a polar nature,e.g. redox proteins and enzymes, chemical modification ofMWCNTs was done as descried above. In Figure 1 TEMimages of the nanomaterial before (a) and after (b) treat-ment with nitric acid are shown. It can be seen that treat-ment with nitric acid results in removal of amorphouscarbon and disalignment of bundles.

For the qualitative analysis of functional groups on thesurface of MWCNT FTIR spectra were recorded (SI, Fig-ure S2). The characteristic oscillatory bands at 1580 and1160 cm�1 suggest the presence of S=S and S�S bonds, re-spectively, for both f-MWCNT and the initial nanomater-al. The peak at 1620 cm�1 in the FTIR spectrum of f-MWCNT points to the presence of S=O bonds due to theoxidation of carbonaceous material. A broad peak at3406 cm�1 could be attributed to ON-groups on the sur-face of f-MWCNT, or to H2O molecules inside the poresof the modified samples (SI, Figure S2). Thus, it wasshown that treatment of MWCNTs with nitric acid athigh temperatures resulted in at least partial oxidation ofthe surfaces of nanotubes and significant hydrophilizationof the nanomaterial.

Fig. 1. TEM images of MWCNTs and f-MWCNTs “Taunit-M”.

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The thermostability of the samples was studied usingdifferential thermal analysis (DTA). The data suggestedthe presence of several fractions, with three differentcharacteristic temperatures of degradation, viz. under100 8C, 100–300 8C, and 300–700 8C (SI, Figure S3). In thetemperature interval below 100 8C evaporation of H2Ooccurred from the surface of the nanomaterial, as well asfrom the inner surfaces of opened nanotubes, whereas inthe 100–300 8C temperature region, oxidation of amor-phous carbon was taking place.

The studies of elemental compositions of MWCNTsshowed that the treatment of the nanomaterial with nitricacid resulted in significant decrease of metal content inthe samples and a concomitant increase in oxygen content(SI, Table S2).

A typical SEM image of f-BP fabricated form f-MWCNTs is shown in Figure 2. It can be seen that theelectrode material has a close-packed porous structure.The specific electric conductivity of BPs prepared fromMWCNTs and f-MWCNTs measured by a four-point con-ductivity method were 15.4 and 16.2 S/cm, respectively.

To conclude, because of the highest specific capaci-tance, highest conductivity and the presence of a hydro-philic surface, f-MWCNTs “Taunit M” from NanoTech-Centre Ltd. were used to fabricate BP based electrodes.

3.2 Bioelectrocatalytic Reduction of Oxygen on MCOModified BP Based Electrodes

BP electrodes based on f-MWCNTs were placed in N2-,air-, and O2-saturated acidic, neutral, and alkaline solu-tions and the catalytic current related to electroreduction

of O2 was clearly visible under aerobic conditions forboth biomodified and bare electrodes (Figure 3). At400 mV and below, non-enzymatic electrocatalytic reduc-tion of O2 on CNTs could be observed, i.e. the onset po-tential of the electrocatalytic process was at least 150 mVlower compared to the onset potentials of bioelectrocata-lytic reduction of O2 (cf. curve 4 with other curves inFigure 3). Moreover, the onset potentials of the bioelec-trocatalytic reaction correlate with the redox potentials ofthe T1 site of the bioelements used, i.e. RhLc, MvBOx,and ThLc equal to 430 mV, 670 mV, and 780 mV, respec-tively [6, 9,27]. The shape of the CVs (almost steady-statepotential-current curves with peaks at ca. 480 mV,710 mV, and 810 mV for RhLc, MvBOx, and ThLc modi-fied BP based electrodes, respectively) and the observeddependence of biocatalytic currents on the stirring, repre-sents strong evidence for mass-transfer limitations in ourstudies (Figure 3). Significantly higher values of the peakpotentials (ca. 40–50 mV higher) compared to the redoxpotential of the first electron acceptor of the enzymes, CuT1 sites (vide supra), suggest quite fast rates of DET reac-tions for MCO modified BP based electrodes. Also, itshould be emphasized that all three fabricated, BP basedbioelectrodes, were very efficient in bioelectroreductionof O2, working at a diffusion-limited regime under theseconditions. Moreover, enzymatic BP electrodes showedone order of magnitude higher bioelectrocatalytic currentdensities, compared to the MCO modified spectrographicgraphite (SPG) based biocathodes (120–220 mA/cm2 forBP vs. 12–35 mA/cm2 for SPG; cf. Figure 3 and Figure S4).

One of the main parameters of potentially implantablebioelectrodes is their functionality under physiological

Fig. 2. SEM image of BP fabricated from f-MWCNTs.

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conditions. Thus, the dependence of bioelectrocatalyticactivity of MCO modified BP based electrodes on solu-tion pH was investigated and the results are presented inFigure S5 (SI). The ThLc based BP electrode showedhigh bioelectrocatalytic activity only in acidic media,whereas RvLc and MvBOx modified BP electrodes werehighly active at physiological pH values, i.e. pH 6–8. Thus,based on the criterion concerning pH optima of biocatho-des, both MvBOx and RhLc based BP electrodes can beused as biocathodes for potentially implantable BFCs.

It should be emphasized that registered onset poten-tials of O2 bioelectroreduction for MvBOx modified BPbased biocathodes were ca. 200 mV higher compared tothe onset potentials of RvLc based biodevices (cf.curves 2 and 3 in Figure 3). Thus, BFCs based on BOxmodified cathodes will show ca. 200 mV higher operatingvoltages compared to the devices modified with the plantLc.

Another important characteristic of biocathodes istheir operational and long-term stabilities. It should bementioned that stability of an enzyme based biocathodedepends on several major factors, viz. intrinsic stability ofa redox enzyme, the method of immobilization on theelectrode surface, the properties of the electroconductivematrix, etc. In Figure 4 A long-term (storage) stabilities ofenzymatic biocathodes based on BP at 4 8C are shown. Itis clearly seen that the ThLc modified BP electrode pre-serves 70 % of its initial activity after storage for 10 h at4 8C. Indeed, this electrode is the most stable biocathodeamong all biodevices fabricated and investigated in thepresent work. Both RvLc and MvBOx modified BP elec-trodes preserve only ca. 40% of their initial activitiesunder the same conditions.

The long-term stability of bioelements used in the pres-ent work was also investigated (Figure 4B). These studieswere done in homogeneous systems at 37 8C, i.e. at a tem-perature appropriate when a BFC is implanted in the

Fig. 3. LSVs of BP electrodes based on f-MWCNTs. 1) ThLc(CPB, pH 4.5); 2) MvBOx (PB, pH 7.4); 3) RvLc (PB, pH 7.4);4) without enzymes (PB, pH 7.4); scan rate: 10 mV/s.

Fig. 4. Stability of bioelements and biomodified electrodes. (A)Long-term stability of BP based biocathodes at 4 8C. 1) ThLc; 2)MvBOx; 3) RvLc. Conditions: batch system, air saturated CPB,pH 4.5 (ThLc), PB, pH 7.4 (MvBOx, RvLc); scan rate: 10 mV/s.The stability of bioelectrodes were measured as a decay of theircurrent obtained from CVs at E=400 mV vs. NHE (MvBOx,RvLc) or at E=500 mV vs. NHE (ThLc). (B) Storage stability ofnative enzymes at 37 8C in PB, pH 7.4. 1) ThLc; 2) MvBOx; 3)RvLc. (C) Long-term stability of BP electrodes modified withMvBOx. The activity of the enzyme was measured based on thebioelectrocatalytic reduction of O2 at 37 8C. Conditions: air satu-rated PBS, pH 7.4; scan rate 10 mV/s. The currents were ob-tained from CVs at E=650 mV vs. NHE.

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human body. The activities of the enzymes in homogene-ous solutions were measured at optimum pH values ob-tained from the results presented in Figure S5 (SI). Halfinactivation times for ThLc, MvBOx, and RvLc, stored at37 8S in PB, pH 7.4, were measured to be only 32, 17, and18 h, respectively. One can assume, however, a significantincrease in thermal stability of immobilized enzymescompared to the bioelements in homogeneous systems.Indeed, the long-term stability of BP based electrodeswith immobilized MvBOx was also studied (Figure 4 C).

It could be clearly seen that MvLc modified BP elec-trode preserved only ca. 10 % of its initial activity afterstorage in PBS at 37 8C for 10 h. Thus, enzyme immobili-zation has decreased the stability of the bioelement, i.e.the half-inactivation time dropped from 18 h in the homo-geneous reaction to only 3 h in the heterogeneous system(cf. curve 2 in Figures 4 B and 4C). On the other hand,the remaining activity of MvBOx under these close tophysiological conditions (PBS, pH 7.4, 37 8C) for immobi-lized enzyme and for BOx in homogeneous solution wasmaintained for a long time, e.g. 5–10% after several daysof storage (Figure 4B).

To conclude, our studies in a model physiological elec-trolyte (PBS, pH 7.4 at 37 8C) showed rather poor stabili-ty of the bioelements, as well as insufficient stability ofenzyme modified BP based biocathodes under physiologi-cal conditions with regard to practical usage in implanta-ble BFCs. To clarify the situation further additional invitro studies were performed using a real physiologicalfluid, viz. human serum (vide infra).

3.3 Function of BP Based Biocathodes in a BiologicalFluid

To understand the capacity of enzyme modified BP basedelectrodes for usage in implantable BFCs, additional stud-ies of the biocathodes in human serum were performed(Figure 5). Contrary to simple buffers, serum contains lowand high molecular weight compounds, which can influ-ence MCO, changing their specific biocatalytic activities.ThLc modified BP based biocathodes submerged intoserum did not reveal any bioelectrocatalytic currents, inall likelihood, due the pH profile of the enzyme, which isalmost inactive at pH above 7 (SI, Figure S5), as well asreversible enzyme inhibition by chloride ions. In contrast,MvBOx and RvLc modified electrodes showed well-pro-nounced bioelectrocatalytic currents of O2 reduction,when submerged into air saturated human serum(Figure 5). It could be clearly seen, however, that after1 h of incubation in human serum both biocathodesshowed significant reduction of their activities, which re-sulted in negative changes of current densities, as well asdecrease of onset potentials (cf. curves 2 and 3 inFigure 5). When biocathodes were carried from serum toPB their bioelectrocatalytic activity was completely re-stored (Figure 5).

Based on the obtained results, as well as literature dataconcerning the performance of MCO based biocathodes

in different physiological fluids, e.g. human blood andplasma [28], serum [29], as well as lachrymal liquid(tears) and saliva [3,30], we can suggest that the signifi-cant drop of the performance of MvBOx and RvLc modi-fied BP based biocathodes in human serum occurred be-cause of electrochemical oxidation processes taking placeon nanostructured electrode surfaces. It was shown thatthe limited performance of biocathodes in human physio-logical fluids including serum can be expected because ofthe electrochemical oxidation of different interferingcompounds, primarily ascorbate, on the developed elec-trode surfaces [29, 30].

4 Conclusions

Enzymatic cathodes based on BP showed high currentdensities compared to other MCO based electrodes, e.g.modified SPG, because of a high loading capacity of

Fig. 5. LSVs of BP based electrodes modified with MvBOx (A)and RvLc (B), as well as without enzymes (control curves). 1, 1)control curves without MCO; 2, 2’) directly after submerging ofbiocathodes into serum; 3, 3’) after incubation in serum for 1 (3)and 2 (3’) h; 4, 4’) after submerging electrodes into PB. Condi-tions: batch system; air saturated human serum; scan rate:10 mV/s.

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CNTs with regard to redox enzymes, as well as enhancedmass transfer of O2 to the electrode surface due to itsnanostructured nature. In spite of quite high bioelectro-catalytic activity and good stability, ThLc based bioca-thode cannot be used to design implantable BFCs sincethe enzyme is almost inactive at physiological conditions,i.e. in solutions with neutral pH and high concentrationsof chloride ions. On contrast, MvBOx and RvLc can bepotentially exploited as biocatalysts to create implantablebiodevices because these MCO do function in both buf-fers with neutral pH and physiological fluids. However,the thermostability of both enzymes seems to be insuffi-cient for practical application of MCO based biocathodesin implanted situations. Moreover, the bioelectrocatalyticactivity of both MvBOx and RvLc based biocathodesdropped significantly, when the biodevices were operatingin a real physiological fluid, i.e. human serum.

Supporting Information

Additional studies of carbon nanotubes, biomodifiedgraphite and backypaper based electrodes. This materialis available free of charge via the Internet.

Acknowledgements

The authors thank Amano Enzyme Inc. for the kind giftof Amano 3 preparation of M. verrucaria bilirubin ox-idase. The work has been supported financially by theMinistry of Education and Science of the Russian Federa-tion (Russian State contract No. 16.512.11.2001; fromFebruary 1, 2011) and by the Russian Foundation forBasic Research (Research Project No. 12–04–33102).

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Biocathodes Based on MWCNT Buckypapers