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Bioelectrochemistry
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Hybrid biobattery based on arylated carbon nanotubes and laccase
Krzysztof Stolarczyk a, Małgorzata Sepelowska a, Dominika Lyp a, Kamila Żelechowska b, Jan F. Biernat c,Jerzy Rogalski d, Kevin D. Farmer e, Ken N. Roberts e, Renata Bilewicz a,⁎a Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Polandb Faculty of Applied Physics and Mathematics, Gdansk University of Technology, Narutowicza 11/12; 80-233 Gdansk, Polandc Department of Chemistry, Gdansk University of Technology Narutowicza 11/12; 80-233 Gdańsk, Polandd Department of Biochemistry, Maria Curie Sklodowska University, Akademicka 19, 20-031 Lublin, Polande Department of Chemistry and Biochemistry, The University of Tulsa, 800 S. Tucker Dr., Tulsa, OK 74104, USA
Single-walled carbon nanotubes (SWCNT) were covalently modified with anthracene and anthraquinone andused for the construction of cathodes for biocatalytic reduction of dioxygen. The nanotubes with aromaticgroups casted onto the electrode increased the working surface of the electrode and enabled efficient directelectron transfer (DET) between the enzyme and the electrode. The aryl groups enter the hydrophobic pocketof the T1 center of laccase responsible for exchanging electrons with the substrate. Glassy carbon electrodecovered with arylated SWCNT and coated with a layer of neutralized Nafion containing laccase was foundto be a very efficient cathode in the hybrid battery. Zn wire covered with a Nafion film served as theanode. The cell parameters were determined: power density was 2 mW/cm2 and the open circuit potentialwas 1.5 V.
Biological fuel cells (BFCs) used to transform the chemical energyinto electrical energy employ enzymes as catalysts and natural com-pounds e.g. glucose or ethanol, as the fuels [1–9]. BFCs can utilize glu-cose and dioxygen dissolved in the body fluids as fuel and oxidant,respectively, and could serve as power source for implanted devicessuch as: microvalves, drug dispensers, pacemakers, sensors, etc.[10]. Important features of biofuel cells are the specificity and selec-tivity of processes occurring at the enzymatically modified electrodesand the ability to operate at room temperature and at pH close toneutral. The specificity of the immobilized enzyme allows the con-struction of enzymatic biofuel cells without a membrane separatingthe anode and cathode compartments, in particular as the open-type devices. Membrane-less biofuel cells can be easily miniaturizedand, in addition, open construction allows the utilization of oxidantand fuel from the surrounding environment.
The main advantage of BFCs over conventional fuel cells is a lowcost of their components. Problems to be solved are connected withthe limited stability of the modified electrodes, transport limitationsof the substrates and products of redox reactions, not efficient direct
ersity of Warsaw, ul. Pasteurafax: +48 22 8224889.cz).
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electron transfer due to large distance between the electrode surfaceand the active center of the enzyme, and limited number of enzymemolecules, that can be electrically connected with the electrode.
Carbon nanotubes (CNTs) possessing unique structural, mechani-cal and electronic properties can be used for the construction of ex-tended conducting networks, and at the same time, for increasingthe effective surface area of the electrode [11–14]. In addition, CNTscan be easily modified non-covalently or by covalent bonding at theedges or defect sites and side-walls of the carbon nanotubes [15–21]. Chemical modification of the nanotube side-wall or its terminusis generally needed to control dispersion of CNTs, which is crucialfor the construction of devices [16].
In the bioelectrocatalytic reduction of dioxygen catalyzed by lac-case or bilirubin oxidase, mediators are typically used to facilitatetransfer of electrons between the electrode and the active site of theredox center, T1, hidden inside the protein in the hydrophobic pocketarranged by aminoacid residues [4,7,13]. Leaching of the mediatorsinto the solution causes fast decrease of catalytic currents; and onthe other hand side reactions with the components of the biocathodeare the adverse effects of diffusing mediators. Adsorptive immobiliza-tion of mediators on carbon nanoparticles, nanotubes or graphenes, iscarried out to eliminate leakage of the mediator to the solution[18,20,21]. However, slow desorption of the mediator cannot beavoided, thus much better solution is the chemical immobilizationof the mediator on the nanotubes surface [17,19,22–31]. In our earlierreports, ABTS was chemically bonded to some extent preferentially to
155K. Stolarczyk et al. / Bioelectrochemistry 87 (2012) 154–163
the sides or ends of single-walled carbon nanotubes (SWNTs) to re-move leaching to the solution, that significantly improved the catalyticefficiency of the modified cathode [32–35].
The aryl functionalized carbon particles have been shown earlier toenter the hydrophobic pocket of T1 center of copper oxidoreductase fa-cilitating direct electron transfer from the electrode [36]. Blanford et al.coupled anthracene residue to the surface of pyrolytic graphite electrodeto facilitate the electrical contact with the enzyme active site and pro-vide hydrophobic interaction that holds the enzyme at the electrodesurface [36–38]. The modification required electrochemical generationof free radicals from anthracene diazonium salt. As shown by Sosna etal. [38] and Doppelt et al. [39], such modification led to the formationof a layer of branched anthracene structures, that are thicker than onemolecule. This would explain the high dioxygen catalytic currentsobtained and the appearance of additional voltammetric peaks [38].
Banks et al. used electrodes modified with nanotubes possessingcovalently bound residue of anthraquinone as an effective mediatorfor the electrocatalytic reduction of dioxygen [37]. The nanotubeswere attached to freshly prepared basal plane pyrolytic graphite viaabrasive immobilization. The catalytic reduction of dioxygen was ob-served at −0.29 V vs. SCE hence at the potentials of anthraquinoneredox processes. This proved that the mechanism was mediated elec-tron transfer (MET). The catalytic current density was 230 μA in phos-phate buffer solution, pH 2.5.
Sosna and coworkers prepared monolayers of anthraquinone or an-thracene covalently bonded to GC electrodes and used these structuresas platforms for the immobilization of Trametes hirsuta laccase [38].The GC electrodes were in the first step electrochemically modifiedwith diamines or diazonium linkers, followed by attachment of eitheranthraquinone or anthracene groups using solid phase chemical meth-odology. These well defined surfaces were found to adsorb laccase andto provide direct electrical contact with the enzyme active site, as evi-denced by XPS, EIS and voltammetry, respectively. The catalytic dioxy-gen reduction started at 0.6 V vs. SCE electrode, however, the catalyticcurrent density was less than 1 μA/cm2. Even the addition of a commonmediator, ABTS, to the solution did not increase the catalytic currentdensity indicating that the population of adsorbed and addressable lac-case molecules was low. This is not, however, surprising consideringthe size of the protein and only monolayer coverages. When the arylgroups attached to the electrode form a single monolayer, the popula-tion of laccasemolecules hydrophobically interactingwith themonolay-er modified electrode is small and the catalytic currents observed arealso not large. Nanostructuring the electrode with arylated single-walled carbonnanotubes (SWCNT)would transform the 2D type of elec-trode surface modification into the 3D assembly connected electricallywith the electrode. It allows significantly more laccase molecules to beconnected with the electrode surface, leading as the result to visible in-crease of the catalytic dioxygen reduction current.
Ramasamy and coworkers prepared an electrode modified withmulticopper oxidases: laccase or bilirubin oxidase, linked to multi-wall carbon nanotubes (MWCNTs) via the molecular tethering re-agent, 1-pyrenebutanoic acid, succinimidyl ester [40]. The bio-conjugates formed using PBSE effectively link of the enzyme withMWCNTs and facilitate DET. The current density of the catalytic re-duction of dioxygen was ca. 300 μA/cm2.
Rincon and coworkers reported a fully enzymatic biofuel cell operat-ing under a continuous flow— through regime [41]. A fungal laccasewasused as air-breathing cathode, and malate dehydrogenase (MDH) or al-cohol dehydrogenase (ADH) coupled with poly-methylene green(poly-MG) were employed to modify the anode. An open circuit voltage(OCV) was 0.584 V for MDH-laccase, and 0.618 V for the ADH-laccasebiofuel cells. Maximum power density was 20 μW/cm3.
In the present paper, we describe SWCNT modified with anthra-quinone or anthracene groups, either on the side or at the ends ofthe carbon nanotubes. We compare these two types of modificationin order to show that the redox properties of the group are not
obligatory for efficient electron transfer between the enzyme andthe electrode. Indeed, also in the case of anthraquinone, the directelectron transfer route is operative leading to large currents at poten-tials far from the formal potential of the redox processes of the an-thraquinone group. The electrodes were tested as cathodes inhybrid batteries. Laccase modified cathode nanostructured with ary-lated SWCNT were active towards dissolved dioxygen and theanode was Nafion-coated Zn wire covered by a protective film imper-meable to dioxygen. During discharging of Zn anode in buffer solutionZn3(PO4)2·4H2O (a hopeite-phase) is formed on its surface [29,42–47]. The film blocks the transport of dioxygen to the Zn surface,thus enabling the necessary transport of Zn2+ while preventing Zncorrosion. The open cell voltage for such hybrid battery system ex-ceeds 1 V, hence higher than that of glucose–O2 biobattery, due tonegative value of Zn/Zn2+ redox potential (E0=−0.76 V vs. NHE[48]). Fuel cell prototypes utilizing printed laccase–ABTS layer asthe cathode and printed Zn layer as the anode have been constructedby Smolander et al. [44]. Under 2.2 kOhm load, the cell voltage 0.8–0.6 V could be maintained for several days. Such alternative devicesmay produce much higher open circuit potentials in comparison tobiofuel cells constructed so far, and are proposed here as a reasonablechoice for testing new biocathodes.
2. Experimental
2.1. Materials and chemicals
Laccase Cerrena unicolorC-139was obtained from the culture collec-tion of the Regensberg University and deposited in the fungal collectionof the Department of Biochemistry (Maria Curie-Sklodowska Universi-ty, Poland) under the strain number 139. Laccase from the fermentorscale cultivation was obtained according to already reported procedureafter ion exchange chromatography onDEAE-Sepharose (fast flow) [49]and lyophilized on Labconco (Kansas City, USA, FreeZone 12 Lyophili-ser) in clear stoppering chambers. Enzyme activity wasmeasured spec-trophotometrically with syringaldazine as the substrate for laccase [50].The protein content was determined according to Bradford with bovinealbumin as the standard [51]. The concentration of isolated andfrozen (−18 °C) enzyme was Clacc=178 μg/cm3 and activity186,000 nkat/dm3. After lyophilizing, the laccase activity dissolvedin 1 ml of water was 3,000,000 nkat/dm3 and Clacc=1.5 mg/cm3.
Lecithin (3-sn-Phosphatidylocholine from fresh egg yolk) was fromFluka, Nafion from Aldrich (5% in alcohol/water mixture) and chitosan(from shrimp shells, ≥75%, deacetylated) was obtained from Sigma.The inorganic reagents were purchased from POCh (Gliwice, Poland),the organic reagents were purchased from Aldrich, and they wereused without further purification. Single-walled carbon nanotubes(>90%) were purchased from CheapTubes.com. and washed in nitricacid/hydrochloric acid mixture before using. Water was distilled andpassed through Milli-Q purification system.
2.2. Apparatus
Thermogravimetric analysis (TGA) was done using Universal V4.3ATA Instrument. The measurements were carried out in argon atmo-sphere at a heating rate of 10°/min. Raman spectra were collectedusing the Witec confocal Raman microscope system (Ulm, Germany)equipped with a fiber coupled Melles Griot (Carlsbad, CA) argon ionlaser operating at 514.5 nm focused through a 60× objective. Collectedlight was dispersed through a triple monochromator (600 g/mm,500 nm blaze angle) and detected with a thermoelectrically cooled(−60 °C) charge-coupled device. A small amount of carbon nanotubesin form of powder was placed on a microscope slide and covered witha coverslip. Laser power at the sample was approximately 5 mW. TheX-ray fluorescencemeasurementswere performed using the spectrom-eter consisting of the X-ray tube (IS601.5, Italstructures) producing a
A
O
O
ArylH2N-Arylamyl-nitrite
chlorobenzeneacetonitrile
Aryl:
from 2-aminoanthracene
from 2-aminoantraquinone
SWCNT-ANT-side
SWCNT-AQ-side
B
NN
and so on
C
OH
O
Cl
O NH
O
O
O
NH
OO
O
H2N
H2N
SOCl2
SWCNT-ANT-end
SWCNT-AQ-end
Fig. 1. A) Free radical syntheses of arylated SWCNT, B) Suggested structures of arylated SWCNT and C) Synthesis of SWCNT terminally modified with anthraquinone and anthracene.
156 K. Stolarczyk et al. / Bioelectrochemistry 87 (2012) 154–163
beam collimated to 4 mm in diameter by means of the Pb collimator,and the X-ray detection system (AXAS, Ketek) equipped with thermo-electrically cooled silicon drift detector of 10 mm2 surface (VITUS).Electrochemical experiments were done in three electrode arrange-ment with Ag/AgCl (KCl sat.) reference electrode, platinum foil as the
A
Procedure I Proced
B
Fig. 2. A) Three procedures used to modify the GCE electrode with matrix, laccase
counter electrode and glassy carbon electrode (GCE, BAS) as the work-ing electrodewith surface area of 0.071 cm2. Cyclic voltammetry exper-iments were carried out using ECO Chemie Autolab potentiostat. Allelectrochemical measurements were done at 22±2 °C. All current den-sities were calculated using geometrical area of the electrode.
ure II Procedure III
and arylated SWCNT and B) Schematic representation of the biobattery circuit.
Table 1TGA characteristics of the arylated SWCNTs.
a ML1 (%)=100%·(m0−m1)m0, where m0 is the initial mass of the sample and m1 is the residual mass after heating to 200 °C.b ML2 (%)=100%·(m1−m2)/m0, m2 is the residual mass after heating to 600 °C.c Residual mass after heating to 900 °C; mass of the SWCNTs.d Molar mass of detached substituent.e n(subst.)/(mmol) is the amount of substituent in mmol and m(SWCNT)/(mg) is the mass of SWCNTs in mg.f Molar ratio r/(mmol/mmol)=n(subst.)/n(SWCNT), where n(subst.)/(mmol) and n(SWCNT)/(mmol) is the amount of substituent and SWCNTs in mmols, respectively.
157K. Stolarczyk et al. / Bioelectrochemistry 87 (2012) 154–163
2.3. Synthesis of arylated SWCNT
Pristine SWCNT were extracted with 4 M hydrochloric acid at60 °C for 4 h to remove traces of metallic impurities [52–54] TheCNTs were centrifuged and washed with water until the washingswere neutral. Then the residue was dried under reduced pressure at60 °C.
Synthesis was carried out by free radical reaction assumed to proceedon side walls [15, cf. 25] using solvent free modification [53].
Thefirst stepwas generation of diazoniumcompounds fromaromaticamines. The diazonium intermediates were thermally decomposed inthe presence of carbon nanotubes under permanent sonication at 60–65 °C (Fig. 1A):
The formulas in Fig. 1A may not reflect the real structures since theradicals generated are able to react not only with carbon nanotubesbut also with aryl groups that in the first step were bonded to theSWCNT side walls [38,39]. The attached aromatic residues react morereadily with free radicals or with diazonium salt than SWCNT. Hence,compounds presented in Fig. 1B can also be formed as the reaction by-products (c.f. Raman spectra below). The primary reaction productswere exhaustively washed with a range of hot solvents, each timeafter vigorous sonication. This procedure was repeated dozens of timesuntil the clear solution does not show fluorescence (aminoanthracene,
Raman
500 1000 1500
646.
9
1346
. 4
1583
.5
642.
2
1343
.5
1432
.51
1577
.7
INT
EN
SIT
Y
SWCNTs-ANT-side
SWCNTs-ANT-endD G
Fig. 3. Raman spectra of SWCNT modified with anthra
anthracene, aminoanthraquinone and anthraquinone show strong fluo-rescence). This confirms lack of species adsorbed on CNTs.
SWCNT were also oxidized by known procedures to obtain termi-nal SWCNT-COOH and with carboxyl groups also on defect sites. Thismaterial was in turn converted into acid chloride. The acid chloridewas reacted with 2-aminoanthraquinone or 2-aminoanthracene. Theadvantage of this approach is that branched residues of aromatic sys-tems could not be formed (Fig. 1C). In this case washing out excessivereagents was much easier.
2.4. Electrode modification procedures
Three procedures for the modification of electrodes were used(Fig. 2A):
I. GCE/SWCNT-aryl/laccase+matrix. GCE surface was covered withSWCNT–aryl by dropping a 10 μl suspension of nanotubes in ethanol(4 mg/ml). After drying, 10 μl of enzyme/matrix casting solution waspipetted onto the electrode and allowed to dry. Matrix was Nafionunmodified or modified, chitosan or lecithin,
II. GCE/SWCNT-aryl+laccase+matrix. GCE surface was coveredwith 10 μl of enzyme/matrix/SWCNT-aryl casting solution by pipettingonto the electrode and allowing to dry,
Shift (cm-1)
2000 2500 3000 3500
2689
.0
2936
.4
3177
.7
2682
.1
2938
.8
3168
.3
G’
cene residues on side walls and on the terminus.
A
B
E [V] vs. Ag/AgCl-0.8 -0.4 0.0 0.4 0.8
j [µ
A/c
m2 ]
-1600
-800
0
800
1600
INT
EN
SIT
Y
500 1000 1500 2000 2500 3000 3500 4000
202.
1 26
4.5
453.
8
836.
3 92
7.8
1146
.6
1334
.8
1585
.2
2683
.0
3175
.2
1338
.6
1568
.4
2662
.1
3148
. 2 SWCNTs-AQ-side
SWCNTs-AQ-end
Raman Shift (cm-1)
D
G
G’
Fig. 4. A) Raman spectra of SWCNT modified with anthraquinone residues on side walls and on the terminus and B) Cyclic voltammograms recorded in deoxygenated 0.1 M McIl-vaine buffer solution (pH 5.2) containing 0.2 M NaNO3 using electrodes modified with SWCNT-AQ-side (—), SWCNT-AQ-end (− −) and laccase/Nafion layer, scan rate 5 mV/s.
158 K. Stolarczyk et al. / Bioelectrochemistry 87 (2012) 154–163
III. GCE/SWCNT-aryl/SWCNT-aryl+laccase+matrix. GCE surfacewas coveredwithmodified SWCNT–aryl by dropping a 10 μl suspensionof nanotubes in ethanol (4 mg/ml). After drying, 10 μl of casting solu-tion containing enzyme and SWCNT-aryl in the matrix was pipettedonto the electrode and allowed to dry.
Laccase solution was prepared by dissolving 1 mg of enzyme in0.64 ml 0.1 M McIlvaine buffer containing 0.2 M NaNO3 (pH 5.2). 1%Nafion was prepared by dissolution of 5% Nafion in ethanol (99.8%).Nafion was neutralized using the procedures described by Minteeret al. [55] using TBAB (tetrabutylammonium bromide) or Lo Gortonet al. using NH3 aqueous solution [56]. In the Minteer procedure qua-ternary ammonium salt with 5% by weight (0.41 mmoles salt/1 mlNafion) Nafion suspension was placed in the weighing boat. The
Table 2Ratios of the main intensities of modified SWCNTs.
concentration of quaternary ammonium salt is in three-fold excesscompared with concentration of sulfonic acid sites in the Nafion sus-pension. The polymer was dried overnight, 18 MΩ water was addedto the weighing boat and was allowed to soak overnight. Water waspipetted off and rinsed thoroughly (three times) with water. Thepolymer was again dried overnight. Then the film was re-suspendedin ethanol/water mixture (70%/30% alcohol/water).
Lecithin was prepared according to the procedure described earlier[57], by dissolving 2 mg lecithin in 1 ml methanol. 1% chitosan solutionwas prepared by dissolving chitosan in 2% acetic acid solution [58]. Mix-ture of laccase and the matrix was prepared bymixing 50 μl of the cho-sen matrix (Nafion, lecithin or chitosan solutions) and 50 μl laccasesolution. Mixture of laccase and SWCNT in the matrix was prepared
IG′/ID IG′/IG
9.34 0.541.01 0.720.49 0.583.61 0.452.52 0.42
A
B
0.0 0.3 0.6 0.9-270
-180
-90
0
E [V] vs. Ag/AgCl
E [V] vs. Ag/AgCl
0.0 0.3 0.6 0.9
j [µ
A/c
m2 ]
j [µ
A/c
m2 ]
-210
-140
-70
0
Fig. 5. Cyclic voltammograms recorded for catalyzed dioxygen reduction using elec-trodes modified with A) SWCNT-AQ-side and B) SWCNT-ANT-side covered with Nafionlayer containing laccase in 0.1 M McIlvaine buffer solution (pH 5.2)/0.2 M NaNO3: (—)deoxygenated and (− −) saturated with dioxygen, scan rate 1 mV/s.
159K. Stolarczyk et al. / Bioelectrochemistry 87 (2012) 154–163
by mixing 50 μl of the chosen matrix, 50 μl laccase solution and 25 μlsuspension of SWCNT in ethanol (4 mg/ml).
The hybrid battery parameters were examined in dioxygen saturat-ed 0.1 MMcIlvaine buffer solution (pH 5.2)/0.2 MNaNO3. Fig. 2B showsthe configuration of the cell. Open circuit voltage (OCV) was measuredin all experiments. The cell voltage (Vcell) weremeasured under varyingloadings in the range from 1 kΩ to 10 MΩ. Cell voltage was measuredafter 5 s after each load application. The anodes for the Zn–O2 hybridbattery were zinc wires (0.25 mm diameter, Goodfellow) coated witha Nafion film by dipping in 0.5% Nafion solution in ethanol and driedfor 5 min in ambient conditions. A hopeite layer was formed duringthe Zn electrode oxidation. The length of these electrodes was adjustedso as to obtain the same surface area as the cathode.
3. Results and discussion
3.1. Characterization of arylated SWCNT
The synthesis of single-walled carbon nanotubes functionalizedwith anthraquinone residues, i.e., chemical groups possessing redoxactivity was described in our recent paper [25]. TGA is always neces-sary in the studies of carbon nanotubes since it allows the determina-tion of the purity of the nanotubes employed (Table 1).
CNTs themselves do not have catalytic properties, however theimpurities and unintended functionalization may give rise to unex-pected behavior and misleading observations as shown e.g. byBanks et al. [54]. TGA is very useful as a general method to determinethe degree of nanotube modification expressed in the number ofmoles of substituent per mole of carbon [25]. Comparing the resultsobtained for the analyzed samples, we noticed, that the remainingmass for end-modified nanotubes is lower compared with those forside-modified nanotubes. This can be explained as follows. End-modified nanotubes are obtained by treatment with oxidizing acidsmixture, which introduces defects, shortens the nanotubes and in-serts oxygen atoms. Such partially destroyed/oxidized CNTs aremore sensitive to high temperature and they are destroyed in lowertemperatures during TGA analysis.
All samples were analyzed by X-ray fluorescence (XRF) method.The results revealed the presence of trace amounts of Fe, Co, Mo,Cu, Zn and Br. However the concentration of observed elements isbelow detection limit of TGA analysis, so their presence in the sam-ples is negligible. These elements probably could be introduced byusing metallic spatula for transferring the samples or they maycome from solvents used to wash the samples.
Raman spectra of anthracene modified SWCNT are shown in Fig. 3.Several unique Raman modes are clearly observed for both modifica-tion schemes. The ID/IG ratios, commonly used as an indicator of nano-tube functionalization, are 1.18 and 0.72 for sidewall and endfunctionalized products, respectively. This suggests that both functio-nalization modes were successful. However, the large ID/IG could alsobe the result of the contribution to the disorder (D) band from strong\CC\ aromatic ring vibrations of anthracene at approximately1400 cm−1. As well, the Raman mode at approximately 1430 cm−1
can be attributed to \N_N\ and \N_N\ and phenyl ring modes.This peak was particularly pronounced in the sidewall modificationof SWCNT with anthracene. In addition, unique modes at approxi-mately 645 cm−1 were observed for both types of functionalizationwith anthracene. The mode has been previously reported in associa-tion with anthracene and SWCNT [59]. Further evidence for modifica-tion is observed. The harmonic overtone G′ peak at approximately2700 cm−1 can be bimodal in the case of mixed nanotubes (metallicand semiconducting). However, as shown here, the single mode peak in-dicates that the anthracene derivatized products are from a single type ofnanotube. Higher order Raman modes just above and below 3000 cm−1
referring to −Csp2H and −Csp3H bonding, further indicate partial disrup-tion of the nanotube structure from covalent binding of anthracene.
Raman spectra of anthraquinone modified SWCNT are shown inFig. 4A. As compared to anthracene modified SWCNT, the Ramanspectra for anthraquinone modified products revealed less disruptionof the D band, which indicates less efficient covalent binding to eitherthe nanotube ends or sidewalls. The ID/IG for sidewall and end modi-fied SWCNT were 0.12 and 0.17, respectively. However, severalmodes between ~400 and 1200 cm−1 in end-modified nanotubeswere observed that are not typical for SWCNT and were attributedto \CC\ vibrational modes observed in anthraquinone [60]. Aswell, radial breathing modes were observed in end-modifiedSWCNT which suggest less disturbance of the nanotube sidewalllong-range order, c.f. Raman spectrum of SWCNT functionalized onside walls by anthraquinone residue [25]. Although the ID/IG ratiowas relatively small, the ~20 cm−1 blue shift of the disorder D bandin sidewall modified tubes relative to end-modified nanotubes indi-cates potential coupling of the anthraquinone derivative to theSWCNT substrate. However, no other anthraquinone vibrationalmodeswere observedwhich are in agreementwith previously reportedfindings of SWCNT sidewall binding with anthraquinone [25].
Ratios of the indicative Raman intensities for anthracene andantraquinone modified SWCNT are summarized in Table 2.
3.2. Electrochemical studies
The reduction of dioxygen at a nonmodified glassy carbon electrode(GCE) proceedswith large overpotential (ca. -0.6 V vs. Ag/AgCl) both inthe absence and presence of laccase. In order to increase the efficiencyof the system, the GCEs were structured with nanotubes covalentlyderivatized with anthraquinone (AQ) moiety. We have recentlyshown thatmodification of electrodes with pristine SWCNT and laccase
Table 3Characteristics of electrodes modified with SWCNT-AQ and SWCNT-ANT and laccase/Nafion layer in deoxygenated and saturated with dioxygen in McIlvaine buffer solutions (pH5.2)/0.2 M NaNO3.
Table 4Characteristics of electrodes modified with SWCNT-AQ-end and laccase Nafion, lecithin or chitosan layer in deoxygenated and saturated with dioxygen McIlvaine buffer solution(pH 5.2)/0.2 M NaNO3.
Fig. 6. Catalytic current decrease with time measured for electrodes modified with( ) SWCNT-AQ-end/laccase in Nafion film (Procedure I), ( ) SWCNT-AQ-end/mixture of SWCNT-AQ-end with laccase in a film of Nafion neutralized by Minteermethod (Procedure III) and (■) SWCNT-AQ-end/laccase in a film of Nafion neutralizedby Minteer method (Procedure I).
160 K. Stolarczyk et al. / Bioelectrochemistry 87 (2012) 154–163
in a Nafion film leads to a decrease of the overpotential of dioxygen re-duction. The onset of reduction current is at ca. +0.612 V vs. Ag/AgCl(+0.812 V vs. NHE) and current density measured at 0.2 V is 186.7 μA/cm2 [34].
In the presence of SWCNT-AQ and laccase on the electrode, thedioxygen reduction wave appears at the same potential as on thepristine carbon nanotubes but the current density was higher [23].The electroactivity of AQ allows to determine number of AQ residuesin electrical contact with the electrode based on the charge of the vol-tammetric peaks (Fig. 4B).
For the single-walled carbonnanotubesmodifiedwith anthraquinonethe reduction/oxidation peaks at ca.−0.4 V in 0.1 MMcIlvaine buffer so-lutions (pH 5.2)/0.2 M NaNO3 correspond to the quinone/hydroquinonecouple:(1)
O
O
OH
OH
+ 2H 2e+ -+ (1)
The surface concentration of the AQ residues present on the electrodecan be evaluated based on the charge of the voltammetric reduction peak:
Γ ¼ QnFA
� �ð2Þ
where Γ/(mol/cm2) — surface concentration of the electroactive compo-nent, Q/(C) — charge under cathodic peak and n — number of electronsexchanged.
The surface concentration of AQ for the electrode covered withSWCNT-AQ-end was 222.5±6.7 nmol/cm2 and for SWCNT-AQ-sideit was equal to 297.0±3.7 nmol/cm2.
Banks et al. [37] reported on derivatized multi-walled carbon nano-tubes obtained via the reduction of anthraquinone-1-diazonium chlo-ride with hypophosphorous acid. The authors modified basal planepyrolytic graphite electrodes with chemically modified MWCNTs,and found the surface coverage by the anthraquinone groups equalto 20.4 nmol/cm2. The surface concentration of anthraquinoneusing our electrodes is hence 10 times larger. Fig. 5 and Table 3
allow to compare the catalytic currents obtained using electrodesmodifiedwith the SWCNT arylated on the side-walls with anthraqui-none and anthracene (ANT) residues.
The jcat/Γ (Table 3) ratio is slightly higher for SWCNT-AQ-end thanSWCNT-AQ-side so the efficiency per mole of substituent is higher incase of its attachment at the ends of the nanotubes, however the totalcatalytic current density is higher for the nanotubes modified on theside-walls similarly to the case of non-electroactive substituent, anthra-cene (SWCNT-ANT) (Table 3).
The three procedures of the catalytic film preparation described inthe experimental part (procedures I, II and III) were compared for theSWCNT-AQ-end and different matrices (modified and nonmodifiedNafion, chitosan or lecithin) (Table 4).
Using nonmodified Nafion, the highest current density of dioxy-gen reduction was obtained for the electrodes structured withSWCNT-AQ-end and covered with Nafion film containing laccase.The catalytic current density, equal to −181.0 μA/cm2 was measuredat 0.2 V. Next, the same three procedures were tested for Nafion neu-tralized by the methods suggested byMinteer et al. [55] and Gorton etal. [56]. The current efficiency was improved by using the method of
Fig. 7. Dependence of ( ) power density and ( ) cell voltage on current density forthe best three systems: anode — Zn/Zn3(PO4)2, and cathode modified by: (A)SWCNT-AQ-end Procedure I, B) SWCNT-AQ-side — Procedure III, C) SWCNT-ANT-sideProcedure III. Electrolyte: McIlvaine buffer, pH 5.2/0.2 M NaNO3.
Table 5Characteristics of hybrid cell with anode–zinc wire and different cathodes in dioxygen satu
161K. Stolarczyk et al. / Bioelectrochemistry 87 (2012) 154–163
Minteer and the catalytic current reached the value of 246 μA/cm2,which is equal to 178 μA/cm2 after background current subtraction.
When lecithin or chitosan were used as the matrices the currentdensities were lower than in case of Nafion films. Best results arealso shown in Table 4.
As shown above, from the matrices studied: Nafion, chitosan and lec-ithin, Nafion was found most useful when the height of the catalytic cur-rent is considered. Comparison of unmodified Nafion, Nafion modifiedwith TBAB according to the Minteer procedure and Nafion modifiedwith NH3 according to Gorton procedure, led us to choose the Minteerprocedure as the more suitable because of higher catalytic current effi-ciency. This is probably due to the sizes of channels in Nafion preparedby the procedure of Minteer since when TBABwas employed as the neu-tralizing agent its large size and steric requirements lead to larger widthsof the channels, which in turn improved the transport of molecules with-in the film. Among the three different procedures used to prepare themodified electrode, the finally selected one was: electrode first modifiedwith arylated SWCNT and then covered with a Nafion matrix layer con-taining both laccase and the same arylated SWCNT.
Comparison of time stability of all of the electrodes (Fig. 6) indicatesthat catalytic current density first decreases more rapidly and then sta-bilizes for several days. In case of the selected electrode modificationafter 12 days 40% of the catalytic current remained.
The electrodes prepared using procedures I and III were tested in thehybrid cell. The cover of the cell used in our experiments was made ofglass, and solution volume was 20 ml. Variable loads, in the rangefrom 1 kΩ to 10 MΩ, were applied between the anode and the cathodeto determine the cell voltage (Vcell). Plots of power density vs. currentdensity and voltage vs. current density are presented in Fig. 7.
The open circuit potential measured for all systems containingarylated SWCNT was ca. 1.52 V. For the electrode modified accordingto Procedure III: SWCNT-AQ-side/laccase+Nafion+SWCNT-AQ-side, the maximal power density is 3.50±0.07 mW/cm2 at 0.50±0.01 V. If Nafion was neutralized by the Minteer method themaximal power density is 2.74±0.18 mW/cm2 at 0.44±0.01 V. Thecharacteristics of the batteries consisting of zinc wire anode anddifferent cathodes are presented in Table 5. The stabilities in time ofthe studied cell were measured (Fig. 8).
In the cell, an abrupt decrease of initial power was observed inthe first 64 min of continuous work under 10 kΩ load; at longertime scale, the power density stabilizes. Continuous supply ofdioxygen was needed to stabilize the biobattery. The best powerdensities together with best stabilities were obtained for the bat-teries consisting of the cathode prepared by Procedure III wherethe surface is nanostrucured with SWCNT-AQ-side and coveredwith neutralized Nafion layer containing laccase and SWCNT-AQ-side.
The choice of time following each load application is importantsince for the Zn/Zn3(PO4)2 anode at longer times corrosion cannotbe fully avoided and leads to a decrease of current density and nonli-nearity of the dependence of voltage on current density (Fig. 9A).
rated McIlvaine buffer solution (pH 5.2)/0.2 M NaNO3.
Fig. 8. Dependence of power density on working time for best three systems: anode —
Zn/Zn3(PO4)2, cathode — ( ) SWCNT-AQ-end/Procedure I, ( ) SWCNT-AQ-side/Procedure III, (▲) SWCNT-ANT-side — Procedure III. Electrolyte: McIlvaine buffer, pH5.2/0.2 M NaNO3.
162 K. Stolarczyk et al. / Bioelectrochemistry 87 (2012) 154–163
The cell efficiency strongly depends on the external dioxygen partialpressure. The maximum power decreases when air saturated solutionsare used instead of dioxygen saturated as shown in Fig. 9B for theSWCNT-AQ-side/laccase modified cathode. Under very high currents(small loads) also the nanotube based cathode is not stable andmeasure-ments taken 1 s, 5 s, 60 s after application of each load are not identical.
A
j [mA/cm2]
j [mA/cm2]
0.0 2.5 5.0 7.5
P [
mW
/cm
2 ]P
[m
W/c
m2 ]
0.0
1.5
3.0
4.5
B
0.0 0.8 1.6 2.40.0
0.6
1.2
1.8
Fig. 9. Dependence of power density on current density for the biobattery: Zn/Zn3(PO4)2,anode and SWCNT-AQ-side/laccase cathode (prepared by Procedure I) A) voltage mea-surement (●) 1 s, ( ) 5 s, ( ) 60 s after application of each load R, B) 60 s after applica-tion of each load R in (●) dioxygen, ( ) air and ( ) argon saturated McIlvaine buffersolution, pH 5.2/0.2 M NaNO3.
4. Conclusions
Efficient and reproducible reduction of dioxygen catalyzed by laccasehas been observed using GCE electrodes modified with SWCNT-AQ andSWCNT-ANT. Dioxygen reduction catalyzed by laccase proceeds close tothe formal potential of laccase T1 site. Direct electron transfer to the lac-case active site is facilitated by the aryl group attached to the SWCNTand the population of laccase molecules addressed is much larger thanin the case of monolayer modified electrodes reported earlier [38, 42].The highest initial catalytic dioxygen reduction current is observed forthe electrodes modified first with a layer of SWCNT arylated on theside-walls and then covered with Nafion layer containing the sameSWCNT and laccase. Nafion neutralized using TBAB according the proce-dure of Topcagic and Minteer [55] was found most suitable in terms ofelectrode stability and efficiency of catalytic current. Continuous supplyof dioxygen is needed and clearly, the cell efficiency strongly depends onthe external dioxygen partial pressure. The highest power densitiesobtained for the hybrid biobattery with zinc wire anode and GCE cath-ode covered with arylated SWCNT and laccase in Nafion film exceeded2 mW/cm2. Open circuit potential was ca. 1.5 V. Such hybrid cell is,therefore, advantageous compared to biofuel cells constructed so far,and is proposed here as a useful device for testing new biocathodes.Search for new bioanodematerials— enzymes and synthetic complexesundergoing electrode processes at similarly to Zn/Zn2+ negative poten-tials is continued in our laboratory.
Acknowledgments
This work was supported by the Polish Ministry of Sciences andHigher Education and The National Center for Research and Develop-ment (NCBiR), grant NR05-0017-10/2010 (PBR-11) and by Faculty ofChemistry, University of Warsaw, grant BW 501/68-191210.
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