Bioelectrochemistry 91 (2013) 52–61

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Bioelectrochemistry

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Plasma functionalized carbon electrode for laccase-catalyzed oxygenreduction by direct electron transfer

Malika Ardhaoui a,b,c,⁎, Meihui Zheng b, Jerome Pulpytel a, Denis Dowling c,Claude Jolivalt b, Farzaneh Arefi Khonsari a

a Laboratoire de Génie des Procédés Plasma et Traitements de Surface, Université Pierre et Marie Curie- Chimie ParisTech, 11 rue Pierre et Marie Curie, 75231 Paris, Franceb Laboratoire Charles Friedel, CNRS UMR 7223, Chimie ParisTech, 11 rue Pierre et Marie Curie, 75231 Paris cedex 05, Francec Surface Engineering Research Group, School of Electrical, Electronic and Mechanical Engineering, University College Dublin, Belfield, Dublin 4, Ireland

⁎ Corresponding author at: Chimie ParisTech, 11, rue Picedex 05, France. Tel.: +33 144276755.

E-mail address: malika.ardhaoui@ucd.ie (M. Ardhao

1567-5394/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.bioelechem.2012.12.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 March 2012Received in revised form 13 December 2012Accepted 18 December 2012Available online 12 January 2013

Keywords:Atmospheric pressure plasma jetCarbon functionalizationLaccase from Trametes versicolorDirect electron transferBiofuel cell cathode

For the first time, a fast and versatile technique, an atmospheric pressure plasma jet (APPJ), has been used tofunctionalise graphite carbon electrodes for biofuel cell applications. The bioelectrode was functionalized byan atmospheric pressure plasma jet (APPJ) system using air, oxygen (O2) and nitrogen (N2) plasmas appliedfor only a few seconds. XPS analysis showed that carboxylic groups were created on the carbon substratesusing both air and O2 plasmas, while mainly carbonyl and amine/amide functionalities were generated usingN2 plasmas. A purified laccase from Trametes versicolor was both adsorbed and covalently bound (NHS/EDCmethod) to the plasma modified carbon. Higher laccase activity was obtained for the covalently grafted laccasecompared to the physically adsorbed one: 13.2 (±2) 10−3U of laccase on air treated graphite and two-fold less(5.3 (±1.1) 10−3U) were obtained on N2 plasma treated surfaces (1 mM ABTS as a substrate, 30 °C, pH=3.0),one unit (U) being the quantity of ABTS (μmole) oxidized by laccase per minute. Dioxygen reduction wasperformed by direct electron transfer (DET). The highest current density, 108 μA/cm2 (at 0.2 V (vs. SCE), pH4.2, room temperature), was recorded for covalently immobilized laccase on N2 plasma treated surfaces (geo-metric surface=0.38 cm2). This could be explained by the fact that the highly conductive graphite structurewas retained in the case of this surface treatment and could also suggest a preferential orientation of the T1 Cucenter of the laccase toward the surface of the N2 plasma treated electrode.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

There has been an enormous increase in the global demand for ener-gy in recent years as a result of the industrial development and popula-tion growth. To face this demands and the eventual shortage in thetraditional fuel sources, alternative supply of sustainable electrical ener-gy, e.g., biofuel cells, has become a strategic research field. Biofuel cellsare energy conversion devices that use biocatalysts to convert chemicalenergy to electrical energy. Biofuel cells comprise two electrodesdesigned to harvest electric current from bioelectrocatalysed chemicalredox transformations. One of these electrodes is responsible for the ox-idation of the fuel (anode), while the other electrode is responsible forthe reduction of an oxidant (cathode). These redox reactions arebiocatalysed by different enzymes such as copper-containing oxidasesincluding laccases [1,2]. Laccases are convenient, industrially attractiveenzymes able to catalyze the four-electron reduction of dioxygen di-rectly to water without additional cofactors or the formation of reactive

erre et Marie Curie 75231 Paris

ui).

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intermediates. The active site of laccases comprises four copper ionsclassified into three types: type 1 (T1), type 2 (T2) and type 3 (T3). T1copper is recognized as the enzyme site for substrate oxidation, andthe T2/T3 cluster is recognized as the oxygen-reduction site [3,4]. Theredox potential of the T1 site ranges between +430 and +790 mV(vs. NHE) [3–6], with the highest redox potential for fungal laccases.Laccase from the white rot fungus Trametes versicolor was reported asan attractive biocatalyst for electrochemical applications such as enzy-matic biofuel cell cathodes [7–11] or biosensors [12,13] because of itshigh redox potential as well as its good stability [7,11,14–17].

The primary concern for selection of an electrode material is usu-ally based upon the electrical conductivity and the hardness of thematerial. Therefore, the electrode is conventionally made of solid sup-ports, such as: gold (foil and rod), platinum or platinum alloys (foiland rod), or carbon (paper, rod, paste, metallized carbon, glassy car-bon, carbon fiber, nanotubes film). However, most of the recent re-ports are based on laccase immobilized on carbonaceous electrodematerials. Few reports are available on laccase immobilized on otherelectrode materials, such as platinum or gold [18–21].

The two major problems of the enzymatic electrodes are the shortlifetime of the enzyme due to stability reduction in a foreign environ-ment and the low current densities resulting from the low electron

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transfer rate between the electrode and the enzyme active site. To over-come these problems, several enzyme immobilization approaches onelectrode surfaces were investigated. These includemainly physical ad-sorption and entrapment in conducting polymer [5,6,9,10]. To mediatethe electron transfer between the enzyme and the electrode surface,low molecular weight redox compounds called mediators have beenco-adsorbed or co-entrappedwith the enzymes. Thismediated electrontransfer (MET) approach is usually associated with problems such asthe mediator stability, selectivity, mass transport and leakage at theelectrode. To eliminate these issues, attention was recently given tothe development of direct electron transfer (DET) biofuel cells. Never-theless, current and power densities are usually lower for DET systemsthan mediated electron transfer (MET) systems [9,22]. This could bedue to the fact that the redox centers tend to be buried inside the pro-teins, far away from the electrode surface or to unfavorable orientationof the enzyme on the electrode surface. In addition, when adsorbed onthe electrode surface, the proteins may be denaturated [4,23]. Someredox enzymes are able to directly interact with an electrode due totheir short electron tunneling distances. Among them, one can citelaccases, oxidation active site of which is close to the protein surface(7 Å) [3,24]. Although DET using laccase has been reported since late1970, a mechanism of oxygen reduction catalysed by laccases adsorbedon a graphite surface was first proposed in 2005 [3,22,25]. Significantefforts have focused on developing a suitable connection between elec-trodes and laccases by adapting either the protein or the electrodestructure. To create a bridge between the enzyme and the electrodesurface, metallic electrode surfaces have been modified by depositionof self assembled monolayers (SAMs) [18], conductive polymers[26,27] or other conductivematrixes [9]. However, even if some reportsshowed high current density output and an extension of the lifetime ofthe bioelectrode by simple adsorption [28,29] and entrapment [30,31],stability data under working operating conditions are rarely reported.In some cases, this immobilization procedure suffers from a poorreproducibility [27,32,33]. A stronger linkage is then required for bio-electrode application. One approach is the covalent protein immobiliza-tion on functionalized electrodes, ideally at a site that does not affect theenzyme active center. To achieve this goal, it is compulsory to firstfunctionalise the electrode surface in a controlledway, inwhich specificcovalent bonds can be formed with the enzyme that lead to adequateorientation of its T1 site toward the electrode surface for efficient DETreactions. Covalent bonding has been used to immobilise laccases onself-assembledmonolayers (SAMs) [34] and electrochemically deposit-ed film [35,36]. The covalent laccase grafting on electrodes surfaces wasfound to be more efficient than the physical adsorption [19,35,37]. Theobtained current density outputs of these biocathodes vary from fewtens to fewhundreds of μA/cm2 [18,32,35]. However, despite the simplepreparation and the high density of the self-assembled monolayers,they are prone to instability [36,38]. Anodic oxidation could also beemployed however it is sometimes associated with surface structuredestruction [39] An alternative approach was to functionalise thesurface by aryldiazinium salts electroreduction on electrode carbon sur-faces [35,36,40]. Unfortunately, these surface modification approachesare also always associated with the difficulty of monitoring the layerthickness and the formation of current resistant multilayers [36,38].To overcome the limitations related to carbon electrode functionaliza-tion, the plasma activation method might be an alternative approachavoiding current resistant layer formation. Nitrogen-containing plas-mas generally give rise to a wide range of nitrogen functions such asamines, imine (CH_NH), cyano (\CN) and nitrile groups. Oxygen-containing plasmas can also functionalise the carbon electrode and in-corporate oxygen-containing groups such as hydroxyl, carbonyl andcarboxyl onto the surface. Depending on the plasma parameters, it canalso provide a wide range and controllable density of functional groups[41–49]. In addition, the plasma technique has the advantage of being arapid, non-polluting and low operating cost process. Plasma techniqueshave found widespread use for different industrial applications. In

particular, atmospheric pressure plasma jets (APPJ) gained large accep-tance, because they are easy to integrate into existing production linesand they can treat specific parts of a substrate selectively. APPJ are notlimited to flat and thin substrates but can also be used for three-dimensional structures [44]. Only few papers have been published,reporting the use of low pressure RF, plasma irradiation andmicrowaveplasmas to generate oxygen, amine and fluorine groups on carbon ma-terials [41,42,45,49]. The use of the atmospheric pressure plasma jetsystem (APPJ) for the treatment of polymers and metals has been alsoreported [46–48,50]. Only one paper reported the use of APPJ treatmentfor the functionalization of carbon nanotubes [42]. Recently, the laccasegrafting on low pressure plasma treated polymeric membranes forbiosenors application was investigated, demonstrating a good stabilityof the covalently immobilized enzymes on these surfaces [51,52].

The aim of this work is to achieve stable subsequent covalent immo-bilization of laccase from T. versicolor on carbon electrodeswhichwouldfavor high bioelectrocatalytic currents resulting from the reduction ofO2 based on DET. The effectiveness of atmospheric plasma treatmentsfor the surface functionalization of carbon electrodes was evaluated.The effect of various plasma parameters such as plasma cycle time(PCT), number of passes (Ps) and the type of the gas (N2, air and O2)on the carbon electrode surface chemistry and morphology was thenstudied. The bioelectrode efficiency was evaluated by measuring thelaccase activity using 2,2′-azobis(3-ethylbenzothiazole-6-sulfonic acid(ABTS) as a substrate and O2 biocatalysed reduction. Comparison ofthe laccase immobilization method (physical adsorption/covalentgrafting) on the biocathode efficiency was also investigated.

2. Materials and methods

2.1. Reagents

The reagents 2,2′-azobis(3-ethylbenzothiazole-6-sulfonic acid)(ABTS), toluidine blue-O (TBO) and N-hydroxysuccinimide (NHS)were purchased from Sigma–Aldrich. 2- 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was from Fluka. Buffer solutions were pre-pared with disodium hydrogen phosphate (Acros Organics), sodiumdihydrogen phosphate, sodium acetate and acetic acid (Prolabo) in de-ionized water (Milli-Q grade, Millipore). Sodium perchlorate (Sigma–Aldrich) was used as electrolyte.

2.2. Enzyme

Laccase was produced from T. versicolor (ATCC 32745) as previ-ously described [7,24]. Purified laccase (around 900 U mL−1 and3 mg total protein mL−1) was stored at −20 °C in 50 mM phosphatebuffer at pH 6.8, where the stability of the enzyme is maximal in pres-ence of glycerol (30% v/v final).

2.3. Plasma surface functionalization of the carbon electrode

2.3.1. Electrodes materials7 mm diameter spectrographic carbon–graphite rods (Mersen,

France) were used. Prior to surface modification, the carbon elec-trodes were ground with SiC paper (Buehler, Germany) with gritsizes 80, cleaned with Milli-Q water and dried by filtered compressedair. The roughness (Ra) of the resulting carbon coatings is approxi-mately 2500 (±300) nm. For all the experiments, after plasma treat-ment, the electrode rods were covered with thin Teflon in order toensure a constant circular cross section.

2.3.2. Plasma functionalization and surface characterization

2.3.2.1. Electrode plasma functionalization. A non-equilibrium atmo-spheric pressure plasma jet (APPJ) from Plasmatreat (PlasmatreaterAS400) was employed to activate and functionalise the rough carbon

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electrode surfaces using N2, O2 and air (Fig. 1). The plasma is generat-ed in a nozzle by an arc-like discharge with an excitation frequency inthe kHz regime. A high-frequency (21 kHz) pulsed voltage was ap-plied between two tubular electrodes separated by a dielectric mate-rial. The PT400 generator delivers a pulse-pause modulated current.The current modulation is controlled by adjusting the plasma cycletime (PCT). This parameter is a measure of the plasma intensity. Thejet was operated with either air (2400 L.h−1), N2 (purity 99.999%,Linde AG) and O2 (purity 99.999%, Linde AG) at an input pressure of5 bars.

All the experiments were carried out without a concealed envi-ronment around the sample and the plasma. Therefore, ambient airwas likely to interact with the sample and the plasma jet.

2.3.2.2. X-ray photoelectron spectroscopy (XPS). The samples were ana-lyzed in VG Microlab 310-F electron spectrometer at base pressures, inthe preparation and analysis chambers, of 2×10−8 and 1×10−8 Pa, re-spectively. The photoelectrons were excited with an x-ray source usingMgKα (hν=1253.6 eV) and the pass energy of the analyzer was 20 eVyielding a resolution of 1.1 eV. The C1s, N1s and O1s peaks wererecorded along with 50–1000 eV survey scans. The intensities of thepeakswere determined as the integrated peak areas assuming the back-ground to be linear.

2.3.2.3. Determination of the carboxylic group densities using TBO. Theamount of COOH was determined using the toluidine blue (TBO)method [53]. Plasma treated carbon rods were soaked in 1 ml of TBO(510−4 M) in NaOH (pH 10) during 6 h. After TBO binding, the super-natant was removed and the stained carbon surface was washed withNaOH solution (pH 10) and pure water for two times. 100 μl of 50%acetic acidwas added to remove the TBO dye from the carboxyl groupson the surfaces of carbon rods during 10 min. The optical density ofthe solution was measured at 633 nm. The concentration of releasedTBO was determined using a calibration plot. The concentration ofthe carboxyl groups on the surface was calculated based on the as-sumption that 1 mole of TBO has complexed exactly with 1 mole ofcarboxylic groups.

Fig. 1. Picture and schematic of the APPJ system used for the carbo

2.3.2.4. Surface wettability. The surface wettability was evaluated bymeasuring the sessile drop contact angle values by a video captureapparatus (Digidrop GBX-3S system, France) immediately after plasmatreatement. For each measurement, a 6 μL deionized water droplet wasdispensed onto the carbon electrode surface and images ofwater dropletwere captured continuously tomeasure the time resolved water contactangle values. The reportedwater contact angle (θ (degree)) values corre-spond to the average of three measurements, performed on differentparts of the samples.

2.3.2.5. Surface roughness. The surface roughness Rawas determined byVeeco Dektak 6 M Stylus surface profiler using a 12.5 μm tip radii. Thescanning scope was 1 mm. The average roughness (Ra (nm)) valuesfor each device were calculated using the Veeco 32 m profiler software.Three measurements were carried on each carbon sample before andafter plasma treatment.

2.4. Laccase immobilization on the electrodes

The laccase was covalently immobilized on N2, air and O2 plasmatreated rough carbon electrodes. 50 μl of laccase solution (0.72 μg μl−1)prepared in 10 mM phosphate buffer, pH 7.0 was mixed with 22.5 μl ofa 20 mM NHS solution (in 10 mM phosphate buffer, pH 7.0) and27.5 μl of a 40 mM EDC solution (in 10 mM phosphate buffer, pH 7.0).The carboxyl group activation was allowed for 20 min. Covalent immo-bilization of the enzyme was allowed for 2 h at ambient temperature.Finally, the modified electrode was soaked 3 times in the phosphatebuffer solution (10 mM, pH 7.0) for 30 min with stirring to removeany unbound laccase. The same procedure in the absence of EDC/NHSwas performed to immobilise laccase by physical adsorption.

2.5. Determination of the immobilized laccase activity

The activity of the immobilized laccase was spectrophotometrical-ly determined at 420 nm with 2,2′-azobis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) as substrate. The electrode was soaked into2 mL of citrate phosphate buffer (25 mM, pH=3.0) containing

n electrode treatment with different gases (N2, air and/or O2).

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1 mM ABTS, at 30 °C. The formation of ABTS radicals was recorded bymeasuring the optical density (OD) at 420 nm during 1 min using aVarian Cary Spectrophotometer. The concentration of ABTS radical inthe solution, c (M), was calculated using the Beer Lambert relationship:OD=ε l c where OD is the measured absorbance (no unit), ε420nm(theextinction coefficient measured at 420 nm)=36,000 M.cm−1 and theabsorption path length l=1 cm. The immobilized laccase activity Ac(U Laccase), was then determined as follows: Ac=5.5610−2Δ (OD)/Δt with Δ (OD)/Δt: being the slope of absorbance as a function oftime (min). This corresponds to the immobilized laccase activity de-fined as the number of μmol of ABTS radical transformed in 1 min.One unit of activity (U) is the amount of enzyme that catalysed the for-mation of 1 μmol of ABTS radical/min.

2.6. Electrochemical measurements

Cyclic voltammetry was carried out with a Princeton AppliedResearch Model 263A Potentiostat/Galvanostat in an electrochemicalcell using three electrodes: the carbon working electrode, a saturatedcalomel electrode (SCE) was used as a reference and a platinum wireas the counter electrode. The three electrodes system was placed in30 mL of 50 mM acetate buffer, pH 4.2, containing 100 mM NaClO4.The solution was deaerated for 15 min with pure N2 gas which waskept flowing over the solution during the electrochemical measure-ments in the absence of oxygen. The potential was cycled between0.9 and −0.3 V (vs SCE) at 10 mV/s. The solution was then aerated for10 min. and cyclic voltammetry response in the presence of dioxygenwas recorded in the same conditions. All the tests were conducted atroom temperature. The current output values were determined at a po-tential of 0.2 V(vs. SCE) after subtraction of the current recorded at thesame potential under N2 gas.

For all the experiments, the laccase activity andelectrochemicalmea-surements were carried out on physically adsorbed laccase electrodes(2 samples) and covalently immobilized laccase electrodes (3 samples)and the experiment was reproduced at least three times.

3. Results and discussion

3.1. Influence of plasma treatment on the carbon electrode chemicalcomposition

The atmospheric plasma activation of the carbon electrodes usingN2, air and O2 gases is expected to functionalise surfaces with NH2

and/or COOH [41,42], without any formation of electrically resistivelayer. The effects of the plasma nature (N2, air and O2), the numberof passes (Ps) and the plasma cycle time (PCT) treatment conditionson the functionalized electrode surface chemistry were evaluated byXPS analysis, by the quantification of the density of the COOH andby measuring the surface wettability on treated and non treated car-bon. For all the experiments, the plasma jet nozzle height (h) fromthe carbon surface was fixed at 10 mm. A plasma voltage (PV) of90% of the maximum or nominal voltage (315 V) was applied. Thejet speed (S) was fixed at 15 m/min for N2 plasma treatment experi-ments (one pass corresponded to a treatment time of 7 s in this case)and at 5 m/min for air and also O2 plasma treatments.

3.1.1. N2 plasma treated carbon electrodeFrom the C1s and N1s core level spectra of the analyzed samples

(Fig. 2A and B) and Table 1, several observations are worth to be men-tioned: (i) the main peak at 284.6 eV corresponds to the graphite sig-nal. Table 1 indicates that the graphite content is around 70% for bothuntreated and N2 treated carbon demonstrating that the N2 type plas-ma retains the graphite original properties of the carbon electrode.One can notice that compared to the untreated carbon (Fig. 2A), theC1s peak of the nitrogen plasma treated carbon surface using a PCTof 50% is broadened on the high energy side (Fig. 2B). The broadening

can be ascribed to the formation of both C-N and/or C-O bonds corre-sponding to the possible presence of amines/amides, hydroxyls, alde-hydes and ketones. It has already been reported that the nitrogenplasma treatment of carbon increases the amount the chemicalgroups such as — C\OH, C\O\C\, O_C\O, pyridine and quaterna-ry nitrogen [47]. The aldehyde groups are particularly attractive be-cause of their high reactivity toward the laccase NH2 groups evenwithout coupling agents leading to the formation of a Schiff base[38,54,55]. (ii) As summarized in Table 1, the N2 plasma treatmenthas led to a slight increase of the surface nitrogen content from0.76% (untreated carbon) to 1.01%. There is no major difference be-tween the N1s spectra of the N2 plasma treated carbon and theuntreated one and they are both centered at 399.6 eV. This low nitro-gen amount introduced during nitrogen plasma treatment may becaused by the low reactivity of nitrogen in the plasma. iii) The O1sspectrum of the treated and untreated samples was centered at533.4 eV. In the case of the nitrogen plasma, the oxygen content is in-creased by two-fold from 5.24% to 11.66% (Table 1). This oxygen uptakeof the plasma treated surfaces is a well-known process [42,56–58],which in this particular case could be due to the diffusion of oxygeninto the nitrogen plasma jet and/or post-oxidation of the plasma acti-vated surface.

The contact angles of N2 plasma treated samples were below 20°(Table 2) whereas the untreated carbon water contact angle was130±3°, which is another indication of a successful functionalizationof the surface. This hydrophilicity depends on the applied plasma PCT.The higher is the latter, the more hydrophilic is the carbon surface.The water contact angle drops from 130±3° for the untreated roughcarbon electrode to only 18±3° once treated using PCT=30% (treat-ment time=21 s). It falls down to 5±3° when a PCT of 100% wasapplied. For this latter sample, the decrease of the water contact angleis due to both surface functionalization and smoothening by plasmaetching. As illustrated in Table 2, the surface roughnesses of the carbonelectrodes were evaluated before and after N2 plasma treatment at dif-ferent PCT. When treated with N2 plasma at a PCT of 100%, the surfaceroughness decreased from 2500 (±300) nm to 500 (±100) nm. How-ever, no change in the roughness was observed for samples treated at apower of 50% and 30%. This etching phenomenon has been observed inthe case of both low pressure and atmospheric plasmas [41,42,45,48].Lommatzsch et al. [46] showed that the nitrogen plasma applied usingthe same Plasmatreat system increased the roughness of the polyethyl-ene. Other authors have observed an increase of the roughness of car-bon fibers treated by a low pressure nitrogen plasma [48]. Therefore,the increase of the PCT gives rise to an increase of the energetic charac-ter of the discharge and the different reactive species formed, leading tomore chances to react with the carbon surface and cause the elimina-tion of the amorphous carbon domains which are more easily etched.

3.1.2. Air plasma treated carbon electrodeAiming at creating COOH groups on the carbon electrode surface

for subsequent coupling with the amine groups of the protein, airand O2 plasmas were tested. The effect of the gas nature on the chem-ical composition, COOH group density, wettability and surface rough-ness of the plasma treated carbon surfaces has been investigated.

The C1s XPS spectrum of the air treated carbon (Fig. 2C) shows thatcompared to the untreated sample, the C1 peak of the plasma treatedcarbon is broadened to high energy values. The main peak centered at284.6 eV corresponded to the graphite signal. However, contrarily tothe carbon electrode treated by N2 plasma, there was an additionalpeak at 285.2±0.2 eVwhich is attributed to the sp3 carbon atom. In ad-dition one very important point that can be noted, as shown in theTable 1, compared to the untreated and N2 treated carbon, the graphitecontent was lower. It decreased from around 70% to only 58% when airplasma was used. The broadening on the high energy side showedclearly three peaks that can be ascribed to the formation of carbon-oxygen bonds. Indeed peaks at 286.01 ±0.2 eV, 287.03 ±0.2 eV, and

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Table 1Chemical composition (XPS analysis) of the untreated, nitrogen and air plasma treated carbon electrodes.

Peak BE eV (±0.2) Untreated carbon Nitrogen plasma treated carbon Air plasma treated carbon

% % Peak BE eV (±0.2) %

C1s 284.6 71.83 70.01 284.36 58.36C1s A 285.2 14.37 0 285.1 10.33C1s B 285.8 6.12 8.18 286.01 5.95C1s C 286.9 1.69 9.14 287.03 6.87C1s D – – – 288.7 3.1O1s 532.4 5.24 11.66 532.4 14.47N1s 399.6 0.76 1.01 400.4 0.65N1s A – – – 406.2 0.27

BE: binding energy.

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288.7±0.2 eV correspond to hydroxyl, carbonyl and carboxyl groups,respectively [41,47]. The air plasma treatment using a PCT of 50% hasled to an increase of the surface oxygen content from 5.24% to 14.47%(Table 1). The N1s XPS spectrum after air plasma treatment showsalso a peak at 400.4 eV (imine and amide species) and a second peakat higher energy corresponding probably to the oxidized nitrogen spe-cies (around 406.2 eV) attributed to NO2 functional groups (Fig. 2C)[46]. The formation of those functional groups can be readily explainedby the simultaneous existence of reactive oxygen and nitrogen species,as well as NO and NO2 species, in the air plasma.

Under otherwise identical process parameters, the O2 plasma treat-ment leads to lower contact angle than the nitrogen plasma (Table 2).This seems to be caused by the high total concentration of heteroatoms(O, N), especially oxygen, incorporated into the surface of the O2 plasmatreated sample. Another explanation could be that it is a consequence ofa higher surface etching that could occur under air or O2 plasma treat-ment compared to N2 plasma treatment [41,48]. Under a PCT of 50%,the N2 plasma didn't etch at all the carbon surface while the air plasmadecreased the Ra from 2500 (±300) nm to 430 (±100).nm.

Toluidine blue method has been used to evaluate the amount ofCOOH created after plasma treatment using air and O2. As shown inTable 2 a higher COOH group density (1.610−9 (±0.3) mol/cm2) wasobtained when an air plasma was employed. It decreases down to0.510−9 (±0.1) mol/cm2 when O2 plasma was applied. This behaviorcould be due to the O2 plasma surface etching discussed above.

3.2. Laccase carbon electrode efficiency

T. versicolor laccase was covalently bound or physically adsorbed onthe N2, air or O2 plasma functionalized carbon electrodes. The manu-factured bioelectrodes efficiency has been evaluated by measuringboth the laccase activity and the O2 biocatalysed reduction.

3.2.1. Bioelectrode ABTS laccase activityOn N2 plasma treated carbon, the initial objective was to covalent-

ly immobilise laccase on generated amine groups. In the case of N2

plasma-treated carbon, if we do have some NH2 functions, theirdensity is below the detection limit as measured with the probe

Table 2Chemical and morphology properties of the untreated, nitrogen, air and oxygen plasma tre

Sample description Plasma treatmentconditions (PCT; Ps)

θ (d

Untreated carbon – 130Nitrogen plasma treated carbon 100; 3 5 (±

50; 3 12 (50; 1 14 (30; 3 18.3

Air plasma treated carbon 50; 3 11(Oxygen plasma treated carbon 50; 3 0

Ra: surface roughness; θ: water contact angle; PCT: plasma cycle time; Ps: number of jet pa

which wasSulfo-LC-SPDP (sulfosuccinimidyl 6-[3′-(2-pyridyldithio)-propionamido] hexanoate) [54]. Covalent binding could also occurbetween the plasma generated aldehyde on the carbon electrodeand the amine groups of the laccase. On air and O2 plasma treated car-bon electrodes, the covalent binding took place between the plasmagenerated carboxylic groups and the laccase amines. The covalent im-mobilization efficiency, which depends on the density and reactivityof the groups on the surface, and the physical adsorption of thelaccase on these plasma treated surfaces, may be different.

The immobilized laccase activity on the electrodes was estimatedwith ABTS as substrate. This activity allowed estimating the enzymeloading assuming that the immobilization procedure did not lead toany partial denaturation of the enzyme and that the substrate diffu-sion to the enzyme catalytic site is similar to that of free enzymes.Fig. 3A summarizes the laccase activity tested on both physicallyadsorbed and covalently immobilized laccase on N2, air and O2 treat-ed carbon electrodes.

For the N2 plasma treated carbon, the obtained results showed thathigher covalently immobilized laccase activity (5.3 (±1.1) 10−3 Ulaccase) was obtained when a PCT of 50% was applied. This could beexplained by a better surface activation which gave rise to higher func-tionalities when a higher plasma cycle time was applied. This is also inagreement with the wettability tendency of these nitrogen plasmatreated carbon discussed earlier (refer to text). A slightly lower activityof 4 (±0.9) 10−3 U laccase was measured on carbon treated under aPCT of 100%. The treatment duration has also an influence on thefunctionalization efficiency and the subsequent laccase loading. Whenthree passes of the N2 plasma jet was performed, at a PCT of 50% (treat-ment time of 21 s), the laccase activitywas two times higher thanwhenjust one pass was applied.

The activity of the physically adsorbed laccase is much lower thanthe covalently immobilized laccase, consistent with the formation of acovalent bond in the presence of the coupling agents EDC/NHS. Howev-er, we can notice that the carbon surface treated at PCT=30% showed aten-fold higher laccase adsorption (0.556 (±0.006) 10−3U laccase)than when a PCT of 100% was applied (0.055 (±0.02) 10−3U laccase).This discrepancy could be attributed to the effect of the surface rough-ness. Indeed, at PCT 30%, the surface roughness remained similar to

ated carbon electrodes.

egree) Ra (nm) Plasma generated COOH groupdensity (10−9 mol/cm2)-TBO

(±3) 2500 (±300) –

3) 500 (±100) –

±3) 2400 (±350) –

±3) – –

(±3) 2600 (±300) –

±3) 430 (±100) 1.6 (±0.3)– 0.5 (±0.1)

sses, TBO: toluidine blue O method.

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

AB

TS

Act

ivit

y/10

-3 U

Lac

case

Adsorption

A

B

Covalent

0

20

40

60

80

100

120

140

j/ (µ

A/c

m2 )

Adsorption

Covalent

N2 (100, 3p) N2 (50, 3p) N2 (50, 1p) N2 (30, 3p) air (50, 3p) O2 (50, 3p)

N2 (100, 3p) N2 (50, 3p) N2 (50, 1p) N2 (30, 3p) air (50, 3p) O2 (50, 3p)

Fig. 3. Effect of the plasma type (N2, air, O2), plasma cycle time (PCT) and passes number (Ps) on adsorbed and covalently immobilized (A) laccase ABTS activity (10−3 U laccase)and (B) current density output (μA/cm2).

58 M. Ardhaoui et al. / Bioelectrochemistry 91 (2013) 52–61

that of the untreated surface (2600 (±300) nm), as compared to the100% PCT treated carbon surfaces (500 (±100) nm), thus the surfaceavailable for the protein adsorption is higher.

As for the N2 treated carbon, the highest laccase activities of airand O2 plasma treated electrodes were obtained for covalentlyimmobilized laccase electrodes compared to the immobilization byadsorption. The comparison with N2 plasma treated samples showedthat the activity was at least twice higher when laccase was graftedon COOH functionalized electrode surfaces treated by air and O2 plas-ma. This could be due to the presence of more functional groups onthe air plasma treated surfaces than on the nitrogen treated ones, aswas shown by XPS characterization.

Fig. 3A showed also that on physically adsorbed laccase electrodes,the activities measured on air and O2 plasma treated carbon weregenerally ten times higher than on N2 plasma treated ones. Thiscould be explained by both the superhydrophilicity of these surfaces

promoting a better laccase adhesion and also by the orientation ofthe laccases. Indeed, since the graphite like surface was retainedwhen the carbon electrodes were treated by N2 plasma, the laccasemolecule could be oriented with the T1 site facing toward the graph-ite surface. This orientation could then probably prevent the ABTS dif-fusion to the T1 active site [3,59], leading to an underestimation of themeasured laccase activity, compared to air plasma treated surfaceswhere laccases were randomly orientated on the surface.

Based on these laccase activity results, the electrode coverage rateby the enzyme can be estimated assuming that the immobilizedlaccases were fully active and knowing the molecular surface of onelaccase molecule (SL=3510−14 cm2), as deduced from dimensions oflaccase from crystallographic data [24]. In this case, we find that an en-zyme activity of 12.3 (±0.7) 10−3 U laccase for laccase covalentlyimmobilized on O2 plasma treated carbon corresponded to an electrodesurface coverage rate of 36%, as calculated according to the following

59M. Ardhaoui et al. / Bioelectrochemistry 91 (2013) 52–61

relationship: Г=100(NL SL)/SE with Г: surface coverage rate (%), NL:number of laccases molecules immobilized on the surface, SL: surface ofone laccase molecule (cm2) and SE: electrode surface area (0.384 cm2).Knowing that the laccase molecular mass is around 60 kDa, NL was cal-culated as follow: NL=(Ac NA)/(As ML) with NA: Avogadro constant(6,022 1023 mol−1), As: laccase specific activity (300 U/mg) and ML:laccase molar mass (6 107 mg/mol). Therefore, one can conclude thatthe electrodes are only partially covered, regardless of the immobiliza-tion conditions.

3.2.2. Bioelectrode O2 reductionFor biofuel cell applications not only the enzyme activity, but also

the direct electron transfer ability, and consequently, the O2 reduc-tion current intensity are crucial parameters (Figs. 3B and 4).

The currents of O2 biocatalytic reduction were measured by cyclicvoltammetry. The onset of the catalytic waves for oxygen reductionstarts at 600 mV vs SCE (Fig. 4), which is in good agreementwith previ-ous works on laccase-modified graphite electrodes [18,35]. On theuntreated rough carbon electrodes, insignificant current was recordedfor both adsorbed and covalently immobilized laccase electrodes.As expected, the results demonstrate a higher current for covalentlybiofunctionalized laccase electrodes compared to the adsorbed ones(Figs. 3B and 4).

The current output at 0.2 V/SCE is at least three times higher withcovalent immobilization than with adsorption. For the N2 treated elec-trode the highest current, 108 μA/cm2 (±10%) was obtained using aPCT of 50% and 3 passes and subsequent covalent immobilization oflaccase (Figs. 3B and 4A). On covalently immobilized laccase electrodes,

A

B

-80

-60

-40

-20

0

20

40

60

80

-0,4 -0,2 0 0,2 0,4 0,6 0,8 1

I/ (µ

A)

I/ (µ

A)

E/V (vs.SCE)

-0,4 -0,2 0 0,2 0,4 0,6 0,8 1

E/V (vs.SCE)

-80

-60

-40

-20

0

20

40

60

80

Fig. 4. Cyclic voltammograms under N2 (grey line) and under O2 of an adsorbed (blackline) and covalently immobilized laccase (black dashed line) on N2 plasma (A) and airplasma (B) treated carbon electrodes. Measured in 50 mM acetate buffer, pH 4.2,100 mM NaClO4, room temperature. Scan rate was 10 mV s−1.

the highest current outputs were obtained for the carbon electrodesexhibiting the highest laccase activities.

On physically adsorbed laccase electrodes a significant currentwas also observed, although the laccase activity of these electrodesis low. For all the N2 plasma treated samples, the current measuredwith adsorbed laccase was one third of that observed with covalentlyimmobilized enzyme. It's around 21 μA/cm2 (±10%) when the car-bon electrodes were treated by N2 plasma under a PCT of 50%.

Fig. 4B shows the cyclic voltammograms of adsorbed and covalentlyimmobilized laccase on air plasma treated carbon electrodes. Thecurrent output is higher for covalently biofunctionalized electrodesthan the adsorbed laccase electrodes. A current output of 51 μA/cm2

(±10%) was measured for the air plasma treated carbon having thehighest density of carboxylic functional groups. A lower current outputof 36 μA/cm2 (±10%) was registered on O2 plasma treated carbon.

One can notice that even if these air and O2 plasma treated bio-electrodes exhibited high activity, they didn't show a high current out-put compared to the N2 treated samples. The current output of the airtreated electrodes is about half that of the N2 treated ones. This couldbe probably explained by (i) a high adsorption of laccases on suchsmooth and hydrophilic surfaces confirmed by a highmeasured laccaseactivity, which can limit the electron transfer (ii) This is clearly consis-tent with a suitable laccase orientation on N2 treated surfaces: the en-zyme is preferentially adsorbed with the T1 site facing the surface,thus enhancing the direct electron transferwhereas the activity appearsto be low due to the hindered diffusion of the substrate. On air plasmatreated electrodes, the enzyme is randomly orientated, leading to ahigher activity but a lower current which is probably due to a highlyaltered graphite surface shown by XPS measurements.

In the literature, several papers have reported on the covalent im-mobilization of laccase on COOH functionalized electrodes with a cur-rent of a few nA were measured on MET laccase electrodes [33,60].Compared to the current output of around 40 μA/cm2 recently reportedfor laccase covalently bound to NH2 functionalized gold planar elec-trodes in the absence of redox mediator, as well as earlier work [3,33],the APPJ treatment used in this work that is a simple and rapid (fewseconds) surface modification approach led to comparable currentoutputs of the functionalized bioelectrodes. For functional groupselectrochemically generated on low density graphite (LDG) electrodesby electroreduction of diazonium salts, Vaz-Domínguez et al. [35]reported a maximal current densities of 500 μA/cm2 and current densi-ties around 300 μA/cm2 at 0.2 V/Ag/AgCl as deduced from cyclicvoltammograms,which is about 3–4 times higher than the one reportedin this paper. This difference could be due to (i) different surface rough-ness of the electrodes (ii) the laccase type tested (laccase from Trameteshirsuta). Even if the two laccases genomic sequences (T. versicolor andT. hirsuta) are rather similar (87% identity of their primary sequence),there are 3 mores lysine residues on T. hirsuta laccase, which could en-hance the protein covalent immobilization efficiency (iii) the betterelectroactivity of the carbon electrodes: the electroactive surface ofthe Sigma rods used by Vaz dominguez et al. [35] was 1 (±0.3) cm2

for a geometric surface of 0.07 cm2 whereas for the spectrographic car-bon (geometrical area=0.384 cm2) we used, the electroactive surfacewas only 0.5 cm2. If we compare the current densities calculatedusing the electroactive surface rather than the geometric one, our plas-ma treated electrodes appear to be more efficient.

To date, to the best of our knowledge, the highest DET bioelectrodecurrent outputs reported in the literature were obtained for physicallyadsorbed laccase on nanoporous–nanostuctured tridimensional carbonelectrodes [9,10]. However, in all likelihood the achieved current outputcan be attributed to the very high specific area of the electrode used. Forthese bioelectrodes however, long term stability remains an importantissue. The present work shows the proof of concept that laccase canbe covalently immobilized on plasma functionalized carbon electrodesby a simple and very fast technique. Further improvements of thecurrent output densities will probably be obtained on nanoporous/

60 M. Ardhaoui et al. / Bioelectrochemistry 91 (2013) 52–61

nanostructured functionalized carbon electrodes with high specificsurface.

4. Conclusions

In this paper, we successfully demonstrated that the surface func-tionalization of graphite carbon electrodes, without creating an elec-trically resistive layer, was possible owing to an atmospheric plasmajet system which gave rise to tailored functionalized surfaces in afew seconds, allowing subsequent laccase covalent immobilization.The nature and density of the functional groups as well as the surfaceroughness could be controlled by monitoring the plasma treatmentconditions (treatment time, gas nature, PCT, etc.). Contrarily to theair plasma, the N2 plasma treatment created mainly carbonyl groups(probably aldehyde groups), with only a slight nitrogen uptake with-out forming an electrically resistive layer by maintaining the graphitenature of the carbon electrodes. The surface roughness remainedunchanged when a N2 plasma was applied using a PCT≤50% whereasthe air and O2 plasma treatment lead to carbon surface etching. Theseresults will be used for further research work to optimize the elec-trode surface characteristics that are crucial to succeed a covalentlaccase immobilization. The covalent immobilization showed a betterlaccase activity and current output compared to mere physical ad-sorption of the enzyme. The highest laccase activities were measuredon air and O2 plasma treated carbon. However, the highest currentoutput was obtained on N2 plasma treated surfaces. Current densityoutput of more than 108 μA/cm2 was obtained on planar carbon sur-face by N2 plasma treatments as short as a few seconds. Based onthese preliminary results, deeper study will be carried out in orderto correlate between the surface properties and the laccase distribu-tion, conformation and orientation of both adsorbed and covalentlyimmobilized and its bioelectrochemical behavior. Plasma functional-ized nanoporous electrodes will also be explored in order to loadmore laccase and then improve the current output.

Acknowledgments

The Authors would like to acknowledge the financial support ofthe Irish Research Council for Science, Engineering and Technology(IRCSET).

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