Direct Electron Transfer of Trametes hirsuta Laccase in a Dual-Layer

Mar 10, 2011 - Direct Electron Transfer of Trametes hirsuta Laccase in a Dual-Layer Architecture of Poly(3,4-ethylenedioxythiophene) Films ... The enz...
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Direct Electron Transfer of Trametes hirsuta Laccase in a Dual-Layer Architecture of Poly(3,4-ethylenedioxythiophene) Films Xiaoju Wang,† Rose-Marie Latonen,‡ Pia Sj€oberg-Eerola,† Jan-Erik Eriksson,† Johan Bobacka,‡ Harry Boer,§ and Mikael Bergelin*,† †

Laboratory of Inorganic Chemistry, Process Chemistry Centre, Åbo Akademi University, Biskopsgatan 8, FIN-20500, Åbo/Turku, Finland ‡ Laboratory of Analytical Chemistry, Process Chemistry Centre, Åbo Akademi University, Bisopsgatan 8, FIN-20500, Åbo/Turku, Finland § VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland

bS Supporting Information ABSTRACT: Direct electron transfer (DET) type biocatalysis was accomplished for Trametes hirsuta laccase (ThL) on a glassy carbon (GC) electrode by immobilizing laccase into a well-designed duallayer architecture of poly(3,4-ethylenedioxythiophene) (PEDOT). PEDOT films were subsequently deposited on a GC electrode via electropolymerization, with NO3- as the counterion for the first accommodation layer and poly(styrene-sulfonate) anions (PSS-) for the second capping layer. The enzyme (ThL) was cast on top of the accommodation layer (PEDOT-NO3), and then the capping layer (PEDOT-PSS) was electrodeposited to entrap ThL between the layers. This enzyme electrode is reported to be able to promote DET between ThL and the GC electrode and catalyze the reduction of O2 into water. The influence of fabrication parameters on the enzyme electrode performance was investigated through chronoamperometric measurements. The investigated parameters included different combinations of PEDOT films, ThL loading, and the thicknesses of both PEDOT layers. As a representative, one optimized dual-layer-architecture enzyme electrode of PEDOT-NO3 (28 mC)/ThL (1.26 U)/ PEDOT-PSS (3.5 mC) performed fairly good reproducibility and operational stability. Its pH profile exhibited a bell-shape with an optimal pH in the range of 3.0-3.5. The influences of ionic strength and addition of a nonionic surfactant into the buffer solution on the enzyme electrode performance were also studied to obtain information about the DET mechanism of ThL in the dual-layer architecture. On the basis of the information obtained from different characterizations, π-π interaction between the PSS- ions and the hydrophobic substratebinding pocket in the vicinity of the T1 Cu site was proposed to result in a favorable location of the conducting polymer chain close to the T1 Cu site and thus facilitate DET of ThL within this particular architecture. The applicability of this approach to various electrode materials is also underlined, which makes it a favorable approach to construct an O2-consuming cathode for biofuel cells.

1. INTRODUCTION The “blue” multicopper oxidases refer to a class of coppercontaining oxidoreductases, which can catalyze the four-electron reduction of O2 into water, coupled to one-electron oxidation of a variety of small organic (generally aromatic) or inorganic substrates.1,2 The most commonly recognized multicopper oxidases include laccase,3,4 bilirubin oxidase,5 ceruloplasmin,6 and ascorbate oxidase.7,8 The catalytic sites of the multicopper oxidase consist of four copper ions, which can be classified in accordance with their spectroscopic characteristics as Type 1 (T1), Type 2 (T2), and Type 3 (T3) sites. A mononuclear T1 Cu is responsible for the blue color of the enzyme with a characteristic absorbance band at a wavelength around 610 nm in the UV-vis spectrum. T1 Cu is positioned near a wide, hydrophobic, substrate-binding pocket, rich in π electron density, to which a range of substrates can bind and undergo rapid, one-electron oxidation to radical products that dissociate before further reaction. One T2 Cu and two T3 Cu form the trinuclear cluster site, where O2 r 2011 American Chemical Society

is bound between the two T3 copper nuclear and reduced into water. T1 Cu extracts one electron from electron donors (substrates) with a subsequent intramolecular electron transfer via a His-Cys-His bridge to the T2-T3 Cu cluster, where O2 is reduced into water.8,9 Laccase (EC 1.10.3.2) widely distributes in fungi, in higher plants, and also in some bacteria.3,10,11 The enzymatic and physicochemical properties of laccase depend on the original source. Laccase has a relatively broad substrate spectrum and is a thermostable and environmentally friendly catalyst.12 Its industrial importance is reflected by its diverse applications, from textile bleaching to pulping and paper making,13 and from food applications14 to bioremediation processes.15 Recently, in development of bioelectronic devices, laccase has been used as the “recognition” component in the fabrication of amperometric biosensors for detecting a large number of phenolic Received: August 24, 2010 Revised: February 18, 2011 Published: March 10, 2011 5919

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The Journal of Physical Chemistry C compounds.16-18 Moreover, laccase finds application as the catalyst in the O2-consuming cathode for biofuel cells after being integrated into the enzyme electrode.19-21 In the bioelectronics design, electron transfer (ET) between laccase and various electrode materials is typically realized in two manners. When a redox compound is introduced as a mediator to shuttle the electron between the electrode surface and the active centers, mediated electron transfer (MET) is to realize the ET. The synthetic mediator, 2,20 -azinobis(3-ethylbenzothiazoline-6sulfonate) (ABTS), and different types of Os-compounds in a form of redox hydrogel developed by Heller’s group are the most successful mediators so far for laccases in the enzyme electrode.22-25 Direct electron transfer (DET) can also be accomplished when the laccase molecules are oriented in a favorable manner to enable the electron tunneling from the electrode surface to the T1 Cu site since the T1 Cu is only positioned 6 Å away from the protein surface.26 To accomplish DET between the enzyme and electrode surface is of great significance for designing bioelectronic devices, such as the mediatorless biosensors and the ultimate biofuel cells. In particular, DET-type biocatalysis of laccase is desirable for the biocathode construction since it diminishes the potential loss caused by the mediated component and facilitates the prototype cell design, as the separator membrane is not required to prevent the crossover of the anodic and cathodic mediators, which is crucial for achieving a stable long-term operation. Significant research efforts have been made on realizing DET of laccase with the aid of self-assembled monolayers (SAMs), mainly on the Au electrode surface.27-29 Those studies are dedicated to understand the electron transfer mechanism between laccases from different origins and the electrode surface using electrochemical tools. This approach limits the type of electrode material that can be utilized as well as the mechanical strength and durability of the manufactured active layer, which may degrade the applicability of the electrode design. DET-type biocatalysis of laccase has also been reported for laccase absorbed on various carbon-based electrode materials, such as pyrolytic graphite,30 spectrographic graphite,31 carbon black, and carbon aerogel.32,33 One interesting approach, the “enhanced absorption” of laccase, was brought up by Blanford et al.,34 in which the polycyclic aromatic amines were chemically grafted on the electrode surface to enhance the absorption of laccase in a DET favorable manner by interacting more closely with the hydrophobic substrate-binding pocket in the vicinity of T1 Cu, due to the π-π affinity.35 This concept has been further explored in recent publications.36,37 A hot research trend on realizing DET biocatalysis of laccase is to integrate nanosized constructive components into the enzyme electrode design, to promote DET between laccase active centers and the current collector, such as integration of Au nanoparticles,38,39 carbon nanotubes,40-42 or conducting polymers into the enzyme electrode designing. Conducting polymers, such as polyaniline, polythiophene, and especially polypyrrole and its amphiphilic derivatives, have been widely used as matrices for enzyme immobilization.43-45 Enzymes can be immobilized into those polymer films by physical entrapment or covalent attachment.46 In most of the cases using conducting polymers as immobilization matrices, DET between the enzyme and the electrode surface is not accomplished, and a redox mediator is necessary. Exceptionally, in the polyaniline film with horseradish peroxidase,47 lactate dehydrogenase,48 cellobiose dehydrogenase immobilization49 and in a polypyrrole film with quinohemoprotein alcohol dehydrogenase immobilization,50

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conducting polymers have exhibited electrochemical-wiring ability to communicate ET between the enzyme and the current collector. Here, we present a novel approach of promoting DET between laccase and the GC electrode by integrating the enzyme into a duallayer architecture consisting of electrochemically synthesized conducting polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), films. Fungal laccase, Trametes hirsuta laccase (ThL) used in our study is produced by the white rot basidiomycete T. hirsuta. ThL has a > 90% sequence identity to the commercially available Trametes versicolor laccase, and it possesses a comparatively high redoxpotential of the T1 Cu site, 0.78 V vs. NHE, thermodynamically which is a measure for a high catalytic efficiency of laccase for oxidizing the substrate.51 PEDOT film was particularly highlighted from the conducting polymer family due to its outstanding properties of high electrical conductivity and excellent inherent environmental stability.52,53 In the present study, a novel approach to promote DET between ThL and the GC electrode via a dual-layer architecture of PEDOT film is proposed. The influence of fabrication parameters, which affect the enzyme electrode performance on the biocatalysis of O2 reduction, was investigated. On the basis of the information obtained from different characterizations, a feasible mechanism for the DET pathway within this particular dual-layer architecture is proposed. The applicability of this approach to various electrode materials is also underlined.

2. EXPERIMENTAL METHODS Materials. 3,4-Ethylenedioxythiophene (EDOT, >97%), poly(sodium 4-styrene-sulfonate) (NaPSS, molar mass = 70 000), and potassium nitrate (KNO3) were obtained from Aldrich. 2,20 Azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was also purchased from Sigma-Aldrich. All the other chemicals used to prepare buffer solutions were of analytical grade. Milli-Q Water was used to prepare all the solutions. Enzymes. T. hirsuta laccase (ThL) is produced and purified in two chromatographic steps at the VTT Technical research center of Finland as described in the literature54 (seen in Supporting Information: Section 1). Briefly, the concentrated and buffer exchanged culture filtrate from T. hirsute strain VTT D-95443 was applied to a DEAE Sepharose Fast Flow anion exchange column equilibrated with 15 mM sodium acetate pH 5.0. Proteins were eluted with a linear 50-150 mM NaCl gradient. Laccase positive fractions (on ABTS) were pooled, and Na2SO4 was added to the sample to a final concentration of 0.5 M. The sample was applied to a Phenyl Sepharose Fast Flow hydrophobic interaction column equilibrated with 20 mM citrate buffer pH 5.0 containing 0.5 M Na2SO4. Proteins were eluted with a linear decreasing Na2SO4 gradient (500-0 mM). Laccasecontaining fractions were pooled and concentrated (Millipore; PM10 membrane). The enzymatic activity of this purified protein preparation was 421 U 3 mL-1 (on ABTS at pH 4.5, 25 °C), and protein concentration was 3.9 mg 3 mL-1, measured with a Bio-Rad DC protein assay kit. The stock enzyme solution of ThL was divided into portions of 20 μL and stored in a freezer at -18 °C. Laccase-Based Enzyme Electrodes. A glassy carbon (GC) electrode (geometrical surface area = 0.28 cm2) was subsequently polished with 0.3 and 0.05 μm aluminum oxide powder, rinsed with water and ethanol, and cleaned in an ultrasonic bath before use. Electropolymerization of the PEDOT layer was performed in a one-compartment, three-electrode cell with the GC electrode as 5920

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The Journal of Physical Chemistry C the working electrode, a Pt coil as the counter electrode, and with a Ag|AgCl|KCl (saturated) electrode as the reference electrode. An aqueous solution containing 0.01 M EDOT and 0.1 M supporting electrolyte salt (KNO3 or NaPSS) was employed as the electrolyte for the electropolymerization. Before each electropolymerization, the electrolyte was thoroughly purged with N2 for 15 min. N2 supply was kept above the electrolyte during the electropolymerization. The first PEDOT film, PEDOT-NO3, was generated on the GC electrode by applying a constant potential of 0.95 V until a desired charge (typically 28 mC, this is varied) was passed. PEDOT-NO3 film was then rinsed with plenty of water to remove any excessive EDOT. Subsequently, ThL was evenly drop-cast onto the PEDOT-NO3 layer and dried under room ambient in half an hour. As the last step, this GC/ PEDOT-NO3/ThL electrode was promptly mounted into the cell for the electropolymerization of the second layer of PEDOT-PSS film. A constant potential of 0.95 V was applied until a desired charge (typically 3.5 mC, this is varied) was passed through the working electrode. When the GC/PEDOT-NO3/ThL/PEDOT-PSS electrode was taken out from the cell, it was ready for further characterizations and evaluations. Fabrication parameters, such as a combination of the duallayer films, ThL loading, and the thickness of the individual layer, were varied to investigate their influence on the enzyme electrode performance. Electrochemical Evaluations. Both cyclic voltammetric and chronoamperometric studies were performed using an IviumStat potentiostat (The Netherlands) in a conventional three-electrode cell equipped with a Ag|AgCl|KCl (saturated) reference electrode and a Pt coil as the counter electrode. The laccasebased enzyme electrode was used as the working electrode. The electrolyte solution used was 50 mM succinate buffer (pH = 4.5). The electrolyte was purged with O2 for half an hour before performing each measurement, and O2 supply was kept above the electrolyte level during the entire measurement. All the tests were conducted at room temperature. In determination of the pH profile for the enzyme electrode, a wide pH range buffer, McIIvaine buffer with a specific pH (from 2.2 to 6.0), prepared from 0.1 M citric acid and 0.2 M Na2HPO4, was used as the electrolyte. The polarization curve (j-E curve) for the enzyme electrodes was obtained by plotting the steady state current recorded under each constant potential step (typically 5 min to reach the steadystate current) vs the applied potential. The current densities are given according to the projected area of the GC electrode. Scanning Electron Microscopy. The morphology of PEDOT films was imaged using a scanning electron microscope, SEM model LEO 1530 (LEO Electron Microscopy Ltd., Germany). The sample was mounted with a conductive tape to a metal plate for direct observation without any coatings. The operating voltage was 2.7 kV.

3. RESULTS 3.1. Evidence of DET between ThL and the GC Electrode via a Conducting Polymer Chain Inside the Dual-Layer Architecture of PEDOT Films. In our previous work,55 we have

explored the possibility of utilizing conducting polymer, PEDOT, as the immobilization matrix for laccase for biofuel cell application. We demonstrated that laccase can be in situ entrapped into the PEDOT film from the electropolymerization solution and catalyze the reduction of O2 in the presence of ABTS as a mediator. However, the fact that only a limited amount of laccase can be entrapped compared with the total amount of laccase present in the electrolyte during the

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Figure 1. Cyclic voltammograms of dual-layer-architecture enzyme electrode PEDOT-NO3 (56 mC)/ThL (1.26 U)/PEDOT-PSS (14 mC) under anaerobic ambient (dashed line) and aerobic ambient (solid line). Electrolyte: 50 mM pH = 4.5 succinate buffer. Scan rate: 5 mV/s.

electropolymerization makes in situ entrapment insufficient from the point of view of utilizing the enzyme to the largest extent. Presently, aiming toward more sufficient and tunable incorporation of laccase into the polymer film, a modified immobilization method is proposed, in which a dual-layer architecture of PEDOT films is finely designed as the matrix for laccase immobilization. A PEDOT-NO3 film was applied as the first layer to accommodate ThL molecules, which were applied by a solution-casting method. Subsequently, a more dense and compact PEDOT-PSS film was electrochemically synthesized on top of the PEDOT-NO3/ThL film to prevent leakage of the enzyme from the accommodation matrix. The dual-layer architecture of PEDOT films with ThL entrapped (GC/PEDOT-NO3/ThL/PEDOT-PSS) was then examined with cyclic voltammetry in pH 4.5 succinate buffer in the absence of any redox mediator. An apparent electrocatalytic reduction wave was observed in the cyclic voltammogram under aerobic ambient compared to that under anaerobic ambient. The voltammograms are shown in Figure 1. On the contrary, in the control sample without ThL, no cathodic shift was observed (seen in Supporting Information: Section 2). This provokes the speculation on the occurrence of DET of laccase in the dual-layer architecture of PEDOT films. To be convinced that the reduction current originated from the bioelectrocatalytic reduction of O2, the chronoamperometric response of this enzyme electrode was recorded at a potential of 0.35 V vs Ag|AgCl(s) with varying O2 concentration in the electrolyte. When O2 dissolved in the electrolyte was excluded by extensively purging with Ar, almost no reduction current was recorded. When Ar was replaced by O2 flow, the reduction current rapidly increased until it leveled off due to saturation of O2 gradient in the electrolyte. This serves as a direct proof to state that the reduction current originates from the reduction of O2. When Ar flow was switched on again, the reduction current disappeared, and after a second O2 purging it reappeared to almost the original value. This indicated a feasibly good stability of the electrode performance (seen in Supporting Information: Section 3). Cyclic voltammetry of the laccase enzyme electrode under anaerobic conditions did not reveal any nonturnover signal of 5921

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The Journal of Physical Chemistry C ThL in the potential window between 0.0 and 0.7 V vs Ag|AgCl(s), although the redox peaks for T1 Cu have been observed for laccases under anaerobic ambient on bare27 or SAM-modified Au electrodes.28 A few papers have reported a clear electrochemical response of the copper active sites of laccase on highly ordered pyrolytic graphite (HOPG) electrodes under anaerobic conditions.30,56 However, in the dual-layer architecture of PEDOT, the comparatively high capacitive current of PEDOT films57 severely hinders the observation of the noncatalytic electron transfer of ThL. Moreover, the primary electron acceptor, T1 Cu, has such a wide range of distances from the electron-transfer conduits that any nonturnover signal would be blurred by the effect of a wide range of electron transfer rates. As commonly recognized,30 the characteristics of the electroreduction of O2 at laccase-modified electrodes are dependent on the origin and also on the amount of enzyme on the electrode surface, which we have observed in our experiments. As displayed in Figure1, for the dual-layer-architecture enzyme electrode, the potential at which the reduction of O2 initiates is around 0.55 V vs Ag|AgCl(s) (0.75 V vs NHE), which is in the vicinity of the redox potential of the T1 Cu of ThL (0.78 V vs NHE). When the mechanism of DET between laccase and the GC electrode within this dual-layer architecture of PEDOT films is concerned, this is deemed as a piece of convincing supportive information for the involvement of the T1 Cu site in the ET process. Upon the biocatalysis of O2 reduction in this enzyme electrode, it is inferred that O2 was reduced into water with participation of four electrons in the ET process, which is the most common situation when the T1 Cu site is the primary electron acceptor in the ET process. To know how efficiently the dual-layer architecture of PEDOT films promotes DET between laccase and GC electrode, the current produced via the DET pathway was compared with that produced via the mediated electron transfer (MET) pathway. Figure 2 shows the chronoamperometric response of the enzyme electrode recorded in pH 4.5 succinate buffer solution at 0.35 V vs Ag|AgCl(s). A reduction current, originating from the biocatalysis of O2 reduction by ThL via the DET pathway, was recorded. ABTS was then added into the electrolyte, which is present at an adequate concentration in the electrolyte to shuttle the electron transfer sufficiently and while the current response was uninterruptedly recorded. Approximately a growth of 5% of the reduction current was detected in the MET mode. In other words, inside the dual-layer architecture, the majority of the entrapped ThL molecules can undergo DET. 3.2. Elaborate Study on the Fabrication of the ThL Enzyme Electrode. 3.2.1. Combinations of PEDOT Layers. As demonstrated,55 the dopant counterions incorporated into the PEDOT film remarkably affect the structural feature and morphology of the resulted polymer film. PEDOT-NO3 film shows a rather compact structure with fibrillar morphology, which is still quite open and porous on nanoscale, with a pore size of several tens of nanometers. In contrast with PEDOT-NO3 film, PEDOT-PSS film is denser and more compact with a grain-like appearance, which makes it ideal as a capping layer to maintain enzyme inside the porous PEDOT-NO3 film (seen in Supporting Information: Section 4). Two other combinations of dual layers of PEDOT films, GC/ PEDOT-NO3/ThL/PEDOT-NO3 and GC/PEDOT-PSS/ThL/ PEDOT-PSS, were also studied. As shown in Figure 3, both of the combinations displayed far poorer performance on the catalysis of O2 reduction into water via DET, compared with the combination of GC/PEDOT-NO3/ThL/PEDOT-PSS. No apparent reduction current was detected in the potential range of 600-500 mV, where the redox potential of the T1 Cu of ThL is located. No DET could be

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Figure 2. Chronoamperometric response (at 0.35 V vs Ag|AgCl(s)) of dual-layer-architecture enzyme electrode PEDOT-NO3(56 mC)/ThL (1.26 U)/PEDOT-PSS (14 mC) in the absence and presence of ABTS in the electrolyte: 50 mM pH = 4.5 succinate buffer.

Figure 3. j-E curves of dual-layer-architecture enzyme electrodes with different combinations of dual layers: PEDOT-NO3 (56 mC)/ThL (1.26 U)/PEDOT-PSS (14 mC) (-9-); PEDOT-PSS (56 mC)/ThL (1.26 U)/PEDOT-PSS (14 mC) (-•-); PEDOT-NO3 (56 mC)/ThL (1.26 U)/PEDOT-NO3(14 mC) (-1-). Electrolyte: 50 mM pH = 4.5 succinate buffer.

accomplished in either of the combinations. This raises the speculation that the morphology of the PEDOT film is playing an important role in determining whether ThL entrapped can undergo DET or not and determining the efficiency on promoting DET between ThL and GC electrode via the dual-layer architecture of PEDOT films. 3.2.2. ThL Loading into the Dual-Layer Architecture. The combination of GC/PEDOT-NO3 (56 mC)/ThL/PEDOT-PSS (14 mC) was used in the fabrication of the dual-layer-architecture enzyme electrode. The ThL quantity cast into the dual-layer architecture was varied in the fabrication of enzyme electrodes, and their capability of catalyzing the reduction of O2 via the DET pathway was evaluatedby chronoamperometric measurements. As shown in Figure 4, in the potential range above the activation potential of ThL (>0.55 V), all four enzyme electrodes containing a different quantity of ThL gave 5922

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Figure 4. j-E curves of dual-layer-architecture enzyme electrode PEDOT-NO3 (56 mC)/ThL/PEDOT-PSS (14 mC) with different ThL loading: (a) 0.42 U; (b) 0.84 U; (c) 1.26 U; (d) 2.11 U. Electrolyte: 50 mM pH = 4.5 succinate buffer. Inset: Plot of current density j vs ThL quantity applied in a duallayer-architecture enzyme electrode and the linear fit to the plot set.

almost identical current response arising from the redox reaction of PEDOT. It may originate from the oxidation of the residue monomer or oligomer entrapped in the film. The catalytic activity of ThL initiated at the potential close to the redox potential of the T1 Cu, around 0.55 V vs Ag|AgCl(s). When more ThL was cast into the dual-layer architecture, a larger catalytic activity of ThL was observed, as indicated by the increased reduction current detected. In the inset of Figure 4, the current density, originating from O2 reduction catalyzed by ThL under a certain potential, was plotted versus the ThL quantity. The current density has a linear dependence on the ThL loading. It is also noted that the matrix of PEDOT-NO3 (56 mC)/ PEDOT-PSS (14 mC) can still sufficiently accommodate up to 2.11 U ThL and promote DET between laccase and the GC electrode. 3.2.3. Thicknesses of PEDOT Dual Layers. For a dosage of 1.26 U ThL into the dual-layer architecture, the thicknesses of the accommodation layer of PEDOT-NO3 and the capping layer of PEDOTPSS were tuned to investigate their influences on DET promoting capability. Here, the thickness of the PEDOT films is in terms of the deposition charge passed in the electropolymerization to generate the polymer, which is proportional to the polymer quantity generated by electropolymerization. First, the thickness of the PEDOT-PSS layer was kept constant by applying 14 mC deposition charge, and the thickness of the PEDOT-NO3 layer was varied by controlling the deposition charge during the electropolymerization. The j-E curves of these enzyme electrodes are displayed in Figure 5. As seen in the inset in Figure 5, an intermediate deposition charge for electropolymerization of PEDOT-NO3 film (the approximate thickness of this layer is in the order of 1 μm) was found optimal for accommodating and promoting 1.26 U of ThL. Second, with the optimal deposition charge of PEDOT-NO3 film (28 mC) consistently applied as the accommodation layer, PEDOT-PSS films resulted from variant deposition charge were tested as the capping layer: a rather thin layer of PEDOT-PSS, using a deposition charge of 3.5 mC during electropolymerization, offered the best performance on promoting

DET between laccase and the GC electrode, as shown in Figure 6. The origins of the optimal accommodation layer of PEDOT-NO3 still remain obscure; however, it is speculated that it is coresulted by the polymer film morphology control with increasing film thickness and variations of O2 concentration gradient and electron conductive distance through the PEDOT films. 3.3. Evaluation of Enzyme Electrode: GC/PEDOT-NO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS (3.5 mC). The enzyme electrode fabricated in this novel approach demonstrated a decent reproducibility. Nevertheless, based on our experience, two aspects are worthy to underline to obtain a good reproducibility of the enzyme electrode. One regards the rapidness of completing the electropolymerization of the capping layer PEDOT-PSS. If the electropolymerization of the capping layer is conducted in a sluggish manner, ThL can diffuse from the electrode surface into the electrolyte, which definitely causes a poor reproducibility of the enzyme electrode. The other aspect concerns the aging of the monomer solution used for electropolymerization. A fresh EDOT monomer solution prepared from argon-protected EDOT is crucial to ensure a good reproducibility. Employment of an aged EDOT monomer solution for electropolymerization causes a poor performance of the enzyme electrode (seen in Supporting Information: Section 5). This occurred probably due to the morphology variation of the resulted PEDOT films from the aged monomer solution, especially for the PEDOT-NO3 film. The operational stability of PEDOT-NO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS (3.5 mC) was evaluated under a continuous chronoamperometric measurement for 3 h. In general, the enzyme electrode displayed relatively good operation stability, especially in the initial half an hour of the test. The current arising from the O2 reduction decayed around 10% in 3 h of continuous operation. It is feasibly caused by either the O2 depletion in the electrolyte (the measurement was performed in a quiescent buffer solution with the blanket of a gentle O2 flux on top) or 5923

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Figure 5. j-E curves of dual-layer-architecture enzyme electrode PEDOT-NO3/ThL (1.26 U)/PEDOT-PSS (14 mC) with variation of the thickness of the accommodation layer: (a) 7 mC; (b) 14 mC; (c) 28 mC; (d) 56 mC; (e) 84 mC. Electrolyte: 50 mM pH = 4.5 succinate buffer. Inset: Plot of current density j under 0.35 V vs Ag|AgCl(s) versus the estimated thickness of the PEDOT-NO3 layer.

Figure 6. j-E curves of dual-layer-architecture enzyme electrode PEDOT-NO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS with the variant deposition charge of the capping layer during electropolymerization: (a) 2.5 mC; (b) 3.5 mC; (c) 7 mC; (d) 14 mC. Electrolyte: 50 mM pH = 4.5 succinate buffer.

desorption of ThL from the dual-layer architecture into the electrolyte solution. To better understand the working conditions of the enzyme electrode, as a representative, the pH profile of GC/PEDOT-NO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS (3.5 mC) was determined in Figure 7. The pH profile appears as a bell shape with a maximum current density in the range of pH 3.0-3.5, which is in good agreement with the pH profiles for the high-potential fungal laccases undergoing DET as reported previously.30,54 All laccase substrates can in principle be divided into two groups: (i) electron-no-proton donors, including simple inorganic compounds, e.g., ferrocyanide,

and organic compounds which are oxidized by laccase through cation radical mechanism, e.g., ABTS, and (ii) electron-proton donors, including the phenols and aromatic amines. Depending on the type of electron donors, the pH profile of the laccase catalysis is determined by different factors, such as the inhibition of OH- at alkaline pHs to the copper active centers (T2 Cu site) and the dependence of the redox potential of electron donors on pH, which results in the loss of the thermodynamic driving force at acidic pHs in the cases of electron-proton donors. Specifically for ThL, when electron-no-proton donors are used as mediators, the pH optimum is found to be in a rather acidic region, around pH 3.0-3.5; 5924

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Figure 7. pH profile of dual-layer-architecture enzyme electrode PEDOT-NO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS (3.5 mC): plot of current density j generated at 0.35 V vs Ag|AgCl(s) versus the pH of the McIIvaine buffer solution used as the electrolyte for the measurements.

meanwhile, when electron-proton donors are used as mediators, the pH optimum is usually shifted one unit more, close to pH 4-4.5. For the dual-layer-architecture ThL enzyme electrode, the pH optimum is coincident with that when utilizing electron-no-proton donors like ABTS as a mediator. The redox potential of the T1 Cu of laccase is not significantly affected by pH.58 Nevertheless, with increasing pH close to the neutral range, the complex formation between the T2 Cu site and OH- gradually inhibits the intramolecular electron transfer between the T1 and T2 Cu site of laccase, resulting in the appearance of lower catalytic current at higher pH value. Concomitantly, the decrease of laccase activity in the low pH region (from pH e 3 and lower) is likely due to the loss of the stability of laccases at low pH values. The dual-layer architecture of PEDOT films works, to some extent, like an electron-nonproton donor, and the protons involved in O2 reduction are taken from the surrounding electrolyte. Here, the effect of pH on the redox potential of the O2/H2O couple should also be taken into consideration. As reported,4 for the four-electron, four-proton reduction of O2 into water, the onset of the catalysis should shift by approximately -60 mV per pH unit. One should keep in mind that the pH profile has reflected this pH effect on the overall biocatalysis of O2 reduction, both on the laccase catalytic activity and on the driving force needed for O2 reduction. The effect of the ionic strength of the buffer solution was also investigated to obtain information about the mechanism of DET of ThL inside the dual-layer architecture of PEDOT films. Figure 8 shows the chronoamperometric curves of GC/PEDOTNO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS (3.5 mC) recorded in pH = 4.5 succinate buffer with variant ionic strength. The noninhibiting ion of laccase, NO3-, was chosen to adjust the ion strength of the electrolyte. When the concentration of KNO3 was increased from 0 to 100 mM, the reduction current density dramatically decayed. This phenomenon may suggest that the electrostatic interaction between the conducting polymer network and the ThL molecules is playing a relevant role on promoting DET in the dual-layer architecture. On the other hand, further addition of KNO3 up to 250 mM seemed to retard the enzyme electrode performance far less severely, and the

reduction current did not vanish totally. Even the local charge compensation is sufficient enough to shield the electrostatic interactions. Herein, the electrostatic interaction between the polymer chains (positively charged) and ThL is not exclusively responsible for the mechanism behind DET of ThL inside the dual-layer architecture of PEDOT films. A nonionic surfactant, Tween-20, was added into the succinate buffer to evaluate the performance of the dual-layer-architecture enzyme electrode. The generated current density decayed severely when Tween-20 was present in the electrolyte as shown in Figure 9. By increasing the concentration of Tween-20, the electrode performance retarded more dramatically. When 2.5% Tween-20 was present, only 25% of the current density remained at the potential of 300 mV. Herein, it is believed that Tween-20 absorbs onto the dual-layer architecture and perturbs the enzyme electrode by varying its microstructure. One feasible assumption is that the hydrophobic interaction between PSS- entrapped in the conducting polymer chain as counterions in the capping layer and the substrate-binding pocket surrounded the T1 Cu site. It can be inferred that this hydrophobic interaction contributes more to the functioning DET inside the dual-layer architecture of PEDOT films.

4. DISCUSSION DET of laccase was not observed in the enzyme electrode when ThL was immobilized into the PEDOT-NO3 film through in situ entrapment, as shown in a previous study.35 Moreover, failure to observe DET in both GC/PEDOT-NO3/ThL/PEDOT-NO3 and GC/PEDOT-PSS/ThL/PEDOT-PSS combinations also underlines the critically functioning role of the GC/PEDOT-NO3/ ThL/PEDOT-PSS combination to accomplish DET. In the case of GC/PEDOT-PSS/ThL/PEDOT-PSS, the failure to accomplish DET may be due to the compact and dense microstructure of PEDOT-PSS featuring a grain-like morphology, which makes it unfavorable on capturing enzyme clusters after the drop-casting of ThL and retaining them from diffusing off during the synthesis of the capping layer. On the contrary, the pore structure of PEDOT-NO3 in an order of several tens of nanometers provides the adequate 5925

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Figure 8. j-E curves of a dual-layer-architecture enzyme electrode PEDOT-NO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS (3.5 mC) in buffer solutions of variant ionic strength: (a) 50 mM pH = 4.5 succinate buffer; (b) 50 mM pH = 4.5 succinate buffer containing 50 mM KNO3; (c) 50 mM pH = 4.5 succinate buffer containing 100 mM KNO3; and (d) 50 mM pH = 4.5 succinate buffer containing 250 mM KNO3.

Figure 9. j-E curves of dual-layer-architecture enzyme electrode PEDOT-NO3 (28 mC)/ThL (1.26 U)/PEDOT-PSS (3.5 mC) in buffer solutions in the absence and presence of Tween 20: (a) 50 mM pH = 4.5 succinate buffer; (b) 50 mM pH = 4.5 succinate buffer containing 0.5% Tween 20; (c) 50 mM pH = 4.5 succinate buffer containing 2.5% Tween 20.

accommodation to a single ThL molecule or to enzyme clusters of agglomerated ThL molecules since the ThL molecule theoretically has a crystalline dimension close to that of Trametes versicolor laccase, 65  55  45 Å (the soluble enzyme has a little bit larger hydrodynamic volume).59 As a side proof, the profound influence of an aged EDOT monomer solution on the enzyme electrode performance also claims the strict requisite of the proper morphology of PEDOT-NO3 film to obtain a well-functioning dual-layer architecture for the enzyme electrode. Above all, no DET occurs in the combination of GC/PEDOT-NO3/ThL/PEDOT-NO3, which

strongly suggests that the PEDOT-PSS capping layer is playing an exclusively decisive role on promoting DET of ThL inside the dual-layer architecture. Another conducting polymer, polypyrrole (Ppy), was reported to promote DET between quinohemoprotein alcohol dehydrogenase entrapped into the Ppy film and the electrode surface.50 The authors proposed that Ppy chains in close proximity to the heme sites of the enzyme offered an ET pathway from the heme active sites to the electrode surface. The electrostatic interaction between the conducting polymer chain (positively charged) and the enzyme mainly 5926

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Scheme 1. Schematic ET Pathway in the Dual-Layer-Architecture Enzyme Electrode PEDOT-NO3/ThL/PEDOT-PSS

contributes to resulting in the preferable location of conducting polymer chains. However, in our study, the electrostatic interaction between the polymer chains (positively charged) and ThL is inferred as nonexclusive “driving force” for DET between ThL and the GC electrode. The results obtained by the addition of nonionic surfactant support the speculation that the interaction between PSS- and the hydrophobic substrate-binding pocket close to the T1 Cu appears to be mainly responsible for resulting in such a preferable location of conducting polymer chains close to the T1 Cu site. This is also the main driving force to the occurrence of DET in this particular system, which is holding an explanation similar to that Blandford et al. has put forward for the realization of DET of laccases with the “enhanced absorption method”.34,35 When PEDOT-PSS film was electrosynthesized as the capping layer, the PEDOT-PSS chains preferably grow toward the vicinity of the substrate binding pockets feasibly due to π-π interaction between the aromatic benzene rings of PSS (as seen in the molecular structure of PEDOT-PSS in Scheme1) and the hydrophobic substrate-binding pockets, which brings the polymer chain closer to the T1 Cu site of ThL. In this manner, the PEDOTPSS chain provides the ET pathway from the electrode surface to the T1 Cu site. The electron is then transferred through an internal ET mechanism to the T2/T3 cluster (the distance is 12-13 Å), where O2 is reduced into water. The realization of DET for the redox proteins typically relyies on the intrinsic properties of the electrode material itself, which, in some cases, makes it less adaptable for a specific application purpose. Regarding the approach we present in this paper, it is worth acknowledging the merit of the universal applicability of this novel methodology on different electrode materials. Besides on the GC electrode, the applicability of this dual-layer architecture to promote DET of ThL has been explored on different electrode materials, aiming to develop a mediatorless cathode for a biofuel cell. The electrode materials under investigation include different types of carbon-based porous materials, e.g., carbon paper and carbon veil. The outcome here is that the incorporation of porous material with large surface area into an enzyme

electrode construction promisingly benefits us to enlarge the current output generated by ThL in a manner of DET.

5. CONCLUSIONS A well-designed dual-layer architecture of PEDOT films, GC/ PEDOT-NO3/ThL/PEDOT-PSS, is evidenced to promote DET between ThL and the GC electrode. The T1 Cu was deemed as the primary electron acceptor. The efficiency of DET in the dual-layer-architecture enzyme electrode is comparable to MET via ABTS, with regard to the magnitude of the catalytic current generated. For a specific dual-layer architecture, the catalytic current density has a linear dependence on ThL loading inside the dual-layer architecture. For a certain quantity of enzyme, the thicknesses of both PEDOT-NO3 and PEDOTPSS layers affect DET promoting capability on the biocatalysis of O2 reduction. The combination of the PEDOT-NO3 layer with an intermediate thickness and a thin PEDOT-PSS layer offered the optimal performance. The morphology of PEDOT films has a profound influence on the electrode performance, and the capping layer of PEDOT-PSS is believed to play an exclusively decisive role on resulting DET. The “freshness” of the monomer solution used for the electropolymerizations is crucial to obtain a good reproducibility of the enzyme electrode performance by avoiding the variation of morphology caused by self-polymerization in the “aged” monomer solution. π-π interaction between the benzene rings of PSS and the hydrophobic substrate-binding pocket most likely contributes more on resulting in a preferable location of PEDOT-PSS chain close to the T1 Cu site. As a result, the conductive distance to the current collector is shortened, and the conducting polymer chains function as the electron relay from the current collector to the primary electron acceptor, the T1 Cu site. On the basis of the above-stated considerations, a schematic representation of the dual-layer-architecture enzyme electrode is presented in Scheme 1, which illustrates the proposed ET pathway 5927

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The Journal of Physical Chemistry C inside the system. The PEDOT-PSS chains grow close to the mononuclear of the T1 Cu site due to the preferable affinity arising from π-π interaction between the aromatic benzene rings of PSS- and the hydrophobic substrate-binding pockets. In this manner, PEDOT-PSS chains provide the ET pathway from the electrode surface to the T1 Cu site of ThL along the conducting polymer chain network of the dual-layer architecture. The electron is then transferred through an internal ET mechanism to the T2/T3 Cu cluster, where molecular O2 is reduced into water.

’ ASSOCIATED CONTENT

bS Supporting Information. Coomassie stained SDS-PAGE gel of purified ThL; Cyclic voltammograms of the control sample PEDOT-NO3/PEDOT-PSS; chronoamperometric response of ThL immobilized in PEDOT-NO3/ThL/PEDOT-PSS electrode; SEM images of PEDOT-NO3 and of PEDOT-PSS films; chronoamperometric response of dual-layer-architecture enzyme electrode PEDOT-NO3/ThL/PEDOT-PSS. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author

*Tel.:þ358 2215 4928. Fax: þ358 2215 4962. E-mail: mikael. bergelin@abo.fi.

’ ACKNOWLEDGMENT Finnish national project ‘Tekes-PEPSic’ (1681/31/07) is gratefully acknowledged for the funding to this work, and Ms. Xiaoju Wang would like to thank Finnish Graduate School of Chemical Engineering (GSCE) for their financial support to her Ph.D study and Dr. Marco Frasconi (Sapienza Universita di Roma) for his valuable discussions concerning the mechanism of DET for ThL in this particular system. ’ REFERENCES (1) Nakamura, K.; Go, N. Cell. Mol. Life Sci. 2005, 62, 2050–2066. (2) Kosman, D. J. J. Biol. Inorg. Chem. 2010, 15, 15–28. (3) Riva, S. Trends Biotechnol. 2006, 24, 219–225. (4) Rodgers, C. J.; Blanford, C. F.; Giddens, S. R.; Skamnioti, P.; Armstrong, F. A.; Gurr, S. J. Trends Biotechnol. 2009, 28, 63–72. (5) Santos, L.; Climent, V.; Blanford, C. F.; Armstrong, F. A. Phys. Chem. Chem. Phys. 2010, 12, 13962–13974. (6) Bielli, P.; Calabrese, L. Cell. Mol. Life Sci. 2002, 59, 1413–1427. (7) Messerschmidt, A. Ascorbate oxidase. In Handbook of Metalloproteins; Messerschmidt, A., Huber, R., Poulos, T., Wieghardt, K., Eds.; Wiley, Ltd: New York, 2001; Vol. 2, pp 1345-1358 (8) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. Rev. 1996, 96, 2563–2606. (9) Shleev, S.; Tkac, J.; Christenson, A.; Ruzgas, T.; Yaropolov, A. I.; Whittaker, J. W.; Gorton, L. Biosens. Bioelectron. 2005, 20, 2517–2554. (10) Yaropolov, A. I.; Skorobogat’ko, O. V.; Vartanov, S. S.; Varfolomeyev, S. D. Appl. Biochem. Biotechnol. 1994, 49. (11) Sharma, P.; Goel, R.; Capalash, N. World J. Microbiol. Biotechnol. 2007, 23, 823–832. (12) Witayakran, S.; Ragauskas, A. J. Adv. Synth. Catal. 2009, 351, 1187–1209. (13) Widsten, P.; Kandelbauer, A. Enzyme. Microb. Technol. 2008, 42, 293–307. (14) Minussi, R. C.; Pastore, G. M.; Duran, N. Trends Food Sci. Technol. 2002, 13, 205–216. (15) Mayer, A. M.; Staples, R. C. Phytochemistry 2002, 60, 551–565.

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