Effect of N-Doping of Single-Walled Carbon Nanotubes on

Article pubs.acs.org/ac

Effect of N‑Doping of Single-Walled Carbon Nanotubes on Bioelectrocatalysis of Laccase Masato Tominaga,*,†,‡ Makoto Togami,† Masayuki Tsushida,§ and Daisuke Kawai∥ †

Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami Chuo-ku, Kumamoto 860-8555, Japan Kumamoto Institute for Photo-Electro Organics (Phoenics), 3-11-38 Higashi-machi, Kumamoto 862-0901, Japan § Faculty of Engineering, Kumamoto University, 2-40-1 Kurokami Chuo-ku, Kumamoto 860-8555, Japan ∥ Solution Technology Group, Technical Research Institute, Toho Gas Co., Ltd, 507-2 Shinpou-machi, Aichi 476-8501, Japan ‡

ABSTRACT: Nondoped and N-doped SWCNTs (N-SWCNTs) were used to clarify the effect of N-doping on a direct electron transfer (DET) reaction of laccase (Lac, from Trametes sp.). The level of N-doping in the carbon phase of the N-SWCNTs, which were synthesized by a CVD method, was determined to be 0.1, 2.4, and 4.1% from X-ray photoelectron spectroscopy measurements. The NSWCNTs were also carefully characterized using electron microscopy, Brunauer−Emmett−Teller (BET) specific surface area measurements, Raman spectroscopy, and electrochemistry. The bioelectrocatalytic current for the DET reaction of Lac immobilized onto the N-SWCNTs tended to decrease with increasing N dopant ratio, whereas the amount of Lac adsorbed per BET surface area of the N-SWCNTs did not depend on the N dopant ratio. There were two main explanations for this behavior. First, an electrostatic interaction between the positively charged interface of the N-SWCNTs due to nitrogen species surface functional groups and the negative charges of carboxylate residues surrounding the T1 site. Second, the surface potential of the N-SWCNTs during Lac modification, because the slope value of the surface potential versus N dopant ratio of the N-SWCNTs was about 53 mV/N%. From additional investigations into the surface potential effect and thermodynamic investigations, we carefully concluded that the above behaviors may be due to denaturation and/or decreasing of the DET reaction rate caused by the strong electrostatic interaction between Lac and the N-SWCNTs surface. end, including 4-aminothiophenol with α-lipoic acid modified gold electrodes,20 2-aminophenol and neocuproine modified graphite,18 and 1-aminophyrene and naphthalene functionalized multiwalled carbon nanotubes,15,21,26,27 which have all been reported as surfaces that enhanced the bioelectrocatalytic current of the DET of Lac. Furthermore, a pyrolitic graphite surface modified with anthracene, a polycyclic aromatic hydrocarbon, showed high current density and long-term stability for the electrocatalytic activity of Lac,24 indicating that Lac can be immobilized through affinity interactions between anthracene and the hydrophobic substrate binding pocket of the T1 site. Nanocarbons such as carbon nanotubes, graphene, acetylene black, and furnace black are attractive electrode materials for fabricating biological fuel cells, not only from the point of view of the cost of electrode preparation but also because of their excellent electric conductivity and high specific surface area. In particular, carbon nanotubes (CNTs) have the desirable combination of high aspect ratio, nanometer-sized dimensions,

L

accase (Lac) is a blue multicopper enzyme that catalyzes the four-electron reduction of molecular O2 to H2O, and it is coupled with the oxidation of a broad range of substrates such as polyphenols, methoxy-substituted phenols, diamines, and some inorganic compounds.1−6 Lac contains a mononuclear center (type 1, T1) near the substrate binding pocket, and a trinuclear (T2 and two T3) copper cluster buried further inside the protein structure. The T1 copper functions as the primary electron acceptor and shuttles electrons to the T2/T3 copper cluster, where O2 is fully reduced to water without the release of a H2O2 intermediate.1−6 Lac is of interest for technological applications such as wearable electrochemical biosensors and biocathode catalysts for enzymatic fuel cells.7−27 An important factor here is the preparation of an effective electrode interface for DET reaction of Lac. The reaction steps for the DET reaction of Lac are molecular oxygen binding, electron transfer reaction within the T2/T3 itself, intramolecular electron transfer between the T1 and T2/T3 sites, and the DET reaction with the electrode. Reports on the effective electrode interface at a particular orientation have been limited. Successful immobilization strategies for covalent bonding and noncovalent adsorption have used the binding affinity of Lac, and various compounds have been used to this © 2014 American Chemical Society

Received: February 21, 2014 Accepted: April 19, 2014 Published: April 19, 2014 5053

dx.doi.org/10.1021/ac500700h | Anal. Chem. 2014, 86, 5053−5060

Analytical Chemistry

Article

consideration of its molar absorbance coefficient, assumed to be 93500 mol dm−3 cm−1 at 280 nm.36 Acetonitrile (AN, 99.8%) and ethanol (99.5%) were purchased from Nacalai Tesque (Japan). Water was purified with a Millipore Milli-Q water system. All other chemical reagents were of analytical grade and were used without further purification. Synthesis of N-SWCNTs and SWCNTs on a Gold Electrode. The SWCNTs and N-SWCNTs were synthesized onto a gold wire surface using a CVD method described in previous reports.37−39 Briefly, a gold wire (99.999%, 0.8 mm diameter) was modified with a catalytic ink-containing cobalt(II) acetate tetrahydrate and molybdenum(II) acetate dimer. The SWCNTs and N-SWCNTs were synthesized in a quartz tube (inner diameter: 35 mm; length: 840 mm) equipped with temperature and gas flow control systems. First, to prepare Co−Mo alloy nanoparticles as a catalyst, the modified gold wire was treated under a reducing atmosphere at ca. 850 °C for 10 min. Next, for SWCNT synthesis, ethanol (EtOH, 99.8%) was infused into the chamber at 850 °C for 10 min using H2 as a carrier gas. For the N-SWCNT synthesis, the N dopant concentration was controlled by the solution volume ratio of AN and EtOH. A mixed gas of EtOH and AN was infused into the chamber at 850 °C for 10 min using H2 as a carrier gas. Mixed solutions with EtOH/AN = 99:1, 50:50, and 0:100 were used. The N-SWCNTs synthesized using EtOH/ AN = 99:1, 50:50, and 0:100 were denoted as N1-, N50-, and N100-SWCNT, respectively. The gold wire surface was determined to be completely covered with N-SWCNTs and SWCNTs because the characteristic electrochemical behavior of a gold surface was not observed at any current scale used in this study. Immobilization of Lac onto the N-SWCNTs and SWCNTs. Immediately after synthesis, the fresh N-SWCNT and SWCNT electrodes were immersed into a 0.1 mol dm−3 acetate buffer solution containing 5 μmol dm−3 Lac for 30 min. The electrode surface was then rinsed gently with the buffer solution and used in measurements.

and high electrical conductivity, giving them great potential as electrode materials for electrochemistry.28−32 In fact, Lac has been successfully immobilized onto functionalized CNTs with chemical compounds having both affinity-binding sites with Lac and specific π−π interaction sites between an aromatic tether and the CNT surface.15,21,26,27 Furthermore, a molecular dynamic study between Lac and single-walled carbon nanotubes (SWCNTs) has been previously reported.23 Conversely, the local electron density in CNTs can be controlled by introducing heteroatoms such as nitrogen to substitute for carbon atoms in the graphene sheets that form CNTs.33 The change in local electron density on the electrode interface would be affect the DET reaction of enzymes immobilized on such modified CNTs.34 However, so far there has been no deep investigation of the effect of N-doping on the DET reaction of Lac. In the present study, we aimed to clarify the qualitative effects of the N-doping level of SWCNTs on the DET reaction of Lac in comparison with nondoped SWCNTs.

■

EXPERIMENTAL SECTION Instrumentation. The synthesized SWCNTs and NSWCNTs were characterized as follows. Transmission electron microscopy (TEM) observations were performed on a JEOL 2000FX electron microscope with an accelerating voltage of 200 kV. Field-emission scanning electron microscopy (FESEM) characterizations were performed on a Hitachi SU-8000. The Brunauer−Emmett−Teller (BET) specific surface area of the samples was determined using a Quantachrome Instruments NOVA2200e using N2. UV−vis spectroscopy measurements were performed using a Shimadzu UV3100 with a quartz cell of 0.2 cm path length. Cyclic voltammetry and controlled-potential coulometry measurements were performed with an electrochemical analyzer (ALS, Model 660A) using a conventional threeelectrode cell with Ag/AgCl (saturated KCl) as the reference electrode and a Pt plate as the counter electrode. Spontaneous potentials were evaluated using a potentiostat (Toho Technical Research PS-14). All potentials were reported with respect to the Ag/AgCl (saturated KCl) electrode. A 0.1 mol dm−3 acetate buffer solution (pH 5.0) was used as an electrolyte solution and was purged with high-purity argon before any measurements. Raman spectroscopy measurements were carried out using a Horiba (Jobin Yvon) LabRAM HR-800 instrument with 514 nm (2.41 eV) laser excitation. All images were captured with a digital charge-coupled device camera. Wavenumber calibration was performed using the 520 cm−1 emission of silica slides. The laser was focused at 2 mm with a laser power of 0.2 mW using a 50× long lens. X-ray photoelectron spectroscopy (XPS) measurements were performed using a Thermo VG Scientific, Sigma Probe HA6000II equipped with a focused monochromatic Al Kα Xray (1486.68 eV) source for excitation, a spherical section analyzer, and a six-element multichannel detection system. The X-ray beam was incident normal to the sample, and the detector was 37° offset from normal. The raw data were corrected for charging using the binding energy of graphite at 284.7 eV. The percentage of individual elements detected was determined by relative composition analysis of the peak band areas. Chemicals. Laccase (Lac, EC 1.10.3.2 from Trametes sp, ca. 70 kDa) was obtained from Daiwa Kasei (Japan). The Lac was purified by anion exchange chromatography with a DEAE Toyopeal column,35 and its concentration was determined in

■

RESULTS AND DISCUSSION Characterization of N-SWCNTs via SEM and TEM. The SWCNT and N-SWCNT layers produced on the gold wire were visibly recognizable because the gold surface color changed to black. Figure 1A shows a representative FE-SEM image of the surface of the N100-SWCNT layer on the gold wire. N100-SWCNT bundles of 5−20 nm in diameter were observed. The thickness of the N100-SWCNT layer was estimated to be ca. 20 μm from cross-sectional FE-SEM images. The FE-SEM images of the N-SWCNTs on the gold wire were similar to those of the N100-SWCNTs, indicating that

Figure 1. SEM (A) and TEM (B) images of the surface layer of the N100-SWCNTs. 5054



Article

section). Furthermore, a down-shift of the G-band frequency was observed for the N-SWCNTs, which was similar to their previously reported behavior.44,45 Furthermore, the Raman spectrum of the N-SWCNTs showed the characteristic G-band peak, particularly the N50- and N100-SWCNTs. The peak positions of the G+ (higher frequency of the G-band) and G− (lower frequency of the G-band) modes of the N-SWCNTs showed no obvious shifts from those of the SWCNTs. However, the N-doping enhanced and broadened the G− peak, which occurs because the continuum electronic states around the Fermi level for metallic SWCNTs have a dynamic screening effect on longitudinal optical phonons.46−48 Furthermore, it is also known that the Breit−Wigner−Fano (BFW) peak is highly sensitive to the electronic density of states at the Fermi level for metallic SWCNTs, which results in a broadening of the BWF peaks by N-doping. These behaviors are similar to those observed in previous reports.48 Thus, Ndoped SWCNTs had been successfully synthesized, and from the appearance of the broad G− and BFW peaks, it was clear that the N dopant concentration increased with the volume ratio of AN in the mixed AN and EtOH solution. The Raman peak at around 2700 cm−1 corresponded to the second-order Raman spectrum of a crystalline sp2-hydribized structure carbon, which is termed the 2D (G′)-band.49,50 I2D /ID increased with nitrogen content; such behavior has also been observed in previous investigations on N-SWCNTs.51 The 2Dband intensity ratio, I2D/ID, can be used as a measure of the metallicity of SWCNTs, because it is stronger for metallic SWCNTs than that observed for semiconducting SWCNTs.52 The radial breathing mode (RBM) of the SWCNTs gives information on the diameter distribution of the SWCNTs. A diameter distribution range of 0.9−1.6 nm was observed for the synthesized SWCNTs, estimated using the equation: d/nm = 248/(v/cm−1), where d is the diameter of a SWCNT, and v is the Raman shift.53−55 The estimated diameter distribution (0.9−1.6 nm) of the N-SWCNTs became narrower (ca. 0.9 nm), and their diameter shrank with increasing N-doping. This change in diameter distribution agrees with the reported results,56,57 and is understood to be caused by inhibition of the formation of large diameter tubes. Characterization of N-SWCNTs via XPS. XPS analyses of the elemental composition of the N-SWCNTs were carried out to detect the content and bonding environment of the carbon and N species. Figure 3 shows high-resolution C(1s) and N(1s) peaks of the XPS spectra of the N-SWCNTs. The C(1s) peaks were centered at 284.7 eV for all N-SWCNTs, which is in good agreement with previously reported values for the binding energy of the sp2 hybridization of graphene sheets.58−60 This value was also obtained for the SWCNTs. The N/C atomic ratio in the N-SWCNTs was determined from the peak areas including the sensitivity factor. No nitrogen species were detected in the SWCNTs. The N/C ratios of the N1-, N50-, and N100-SWCNTs were evaluated to be 0.1, 2.4, and 4.1%, respectively. The N(1s) peaks in the N-SWCNTs were deconvoluted into contributions from pyridinic nitrogen (398.8 eV), pyrrolic nitrogen (399.0 eV), graphitic nitrogen (401.1 eV), amine-like nitrogen (399.9 eV), gaseous N2 sealed in the N-SWCNTs (404.4 eV), and two nitrogen oxides (NOX1: 402.5 eV; NOX2: 405.6 eV) by fitting the spectra with Gaussian curves.61−63 The results are summarized in Table 1. DET activities of Laccase on N-SWCNTs and SWCNTs. We hypothesized that SWCNTs are the best type of CNT for simplifying measurement of the N-doping effect, because we

their morphology was not dependent on the level of N-doping. Figure 1B shows a TEM image of the boundary interface at the surface of the N100-SWCNT layer as a representative example. Individual tube structures with estimated wall thicknesses of 0.3−0.35 nm were observed, indicating that these were singlewalled CNTs.28−32 Although some multiwalled, bamboo-like, structured CNTs (bamboo CNTs) with diameters of ca. 20 nm were observed by SEM and TEM measurements,40,41 observations of the whole surface confirmed that their numbers were very few in comparison with those of the N-SWCNTs. Therefore, we presumed that these bamboo CNTs had a negligible effect in the present study. Characterization of N-SWCNTs via Raman. The Raman spectra of the SWCNTs and N-SWCNTs are shown in Figure 2. Raman spectroscopy is useful for the characterization of sp2-

Figure 2. Raman spectra of N-SWCNTs and SWCNTs synthesized on a gold surface. Excitation laser wavelength: 514.5 nm (2.41 eV). (A) Spectra of (a) N100-, (b) N50-, (c) N1−SWCNTs, and (d) SWCNTs, in the region of 100−3200 cm−1. (B) Spectra of (e) N100-, (f) N50-, (g) N1−SWCNTs, and (h) SWCNTs in the RBM region.

hybridized structures in carbon materials and yields information about defects and the crystalline structure.42,43 The prominent feature of the Raman spectra of the obtained CNTs is the Gband appearing at ca. 1590 cm−1. This G-band is a doubly degenerated phonon Raman active mode of a sp2-structured carbon network. The D-band at ca. 1350 cm−1 is localized where the lattice structure is not perfect, mostly at the edges and the defects of the sp2-hybridized carbon structure.42,43 The G/D intensity ratio (IG/ID) can be used to evaluate the quality of the crystals in the sp2-hybridized carbon structure. The IG/ID of the SWCNTs was evaluated to be ca. 35 when a 514.5 nm laser was used for excitation. A slight peak of the D-band was observed. The result indicated that the synthesized SWCNTs were highly crystalline in structure. In other words, the SWCNTs had few defects. In contrast, the IG/ID value decreased with increasing volume ratio of AN in the mixed solution. The IG/ID values for the N1-, N50-, and N100-SWCNTs were evaluated to be ca. 23, 7, and 5, respectively. This tendency is reasonable, because the crystalline translation symmetry is broken when the N atoms are doped in the sp2hybridized carbon structure (see XPS results in the next 5055



(1)

where Γ (mol cm−2) is the adsorbed amount of Lac per BET surface area of the electrode, determined from the change in concentration of Lac in a cuvette after electrode immersion 5056

2.3 1.1 0.0 0.0 0.5 0.2 0.0 0.0 0.2 0.2 0.1 0.0 0.8 0.7 0.0 0.0 0.3 0.2 0.0 0.0 17 16 14 14 79 81 86 86 N100-SWCNT N50-SWCNT N1-SWCNT SWCNT

ALac(U/(mol cm−2)) = [Q (c)/(96485 × 4)] × 106 /Γ

pyridinic N (398.8 eV) pyrrolic N (399.0 eV)

are interested primarily in surface interactions with Lac and its DET reaction behaviors. Bioelectrocatalytic currents caused by the DET reaction were observed in all N1-, N50-, and N100SWCNTs and SWCNT electrodes, as shown in Figure 4A. A catalytic oxygen reduction current was observed at a potential more negative than 0.64 V and was not dependent on the electrode used. It must be noted that the catalytic reduction current observed at potential more negative than 0.23−0.34 V was not due to the DET reaction of Lac but due to the catalytic activities of the N-SWCNTs and SWCNTs. In fact, as shown in Figure 4B, a catalytic oxygen reduction current was observed at around 0.3 V in the N-SWCNT and SWCNT electrodes, even if these electrodes were not modified with Lac. It is well-known that CNTs, especially N-doped CNTs, have great catalytic activity for oxygen reduction.64−66 The onset potentials for oxygen reduction at the N50- and N100-SWCNTs electrodes were 0.32−0.34 V in 0.1 mol dm−3 acetate buffer solution (pH 5.0), approximately 100 mV higher than those at the SWCNTs electrode. The observed potential shifts of up to ca. 22 mV per atom % N incorporated into the N-CNTs were reasonable in comparison with previously reported values.65 The catalytic current from the DET of Lac was dependent on electrode type and was found to decrease with increasing N dopant concentration. The current measured for the N1− SWCNT electrode was similar to the current observed at the SWCNT electrode. To clarify the effect of N-doping on the DET reaction of Lac, a controlled-potential electrolysis was carried out. A controlled-potential of 0.35 V was selected to eliminate the oxygen reduction reaction caused by the catalytic activity of the N-SWCNTs and SWCNTs. The charge flow was obtained from the oxygen reduction current from the DET. The activity (unit, U = μmol min−1) of Lac was estimated from the results of average charge flow (Q) per minute. Thus, the activity unit per surface area and the molar amount, ALac (U/ (mol cm−2)), for the oxygen reduction of the DET activated Lac is given by the following eq 1:

CC (284.7, 290.8 eV) C−C (285.3 eV) C−O (286.4 eV) CO (287.7 eV) OC−O (289.1 eV)

Table 1. Relative Percentages of Surface Functional Groups Containing Nitrogen Species in N-SWCNTs and SWCNTs

Figure 3. XPS results in the N(1s) region for (a) N100-, (b) N50-, (c) N1−SWCNTs, and (d) SWCNTs.

graphitic N (401.1 eV)

amine-like N (399.9 eV)

Noxl (402.5 eV) N2 sealed in Nox2 (405.6 eV) N-SWCNT (404.4 eV)

Article



5057

a

The amount of Lac immobilized per SBET, was determined from differences in the adsorption of the Lac solution at 280 nm before versus after electrode modification. Γ = (amount of Lac immobilized)/ SBET bLac activity was determined from the flowed Coulomb number for the DET reaction of Lac at 0.35 V in 0.1 mol dm−3 acetate buffer solution (pH 5.0). cThe surface electrode potential (open-circuit potential) was determined in 0.1 mol dm−3 acetate buffer solution (pH 5.0) purged with argon gas.

0.15 (±0.01) 0.07 (±0.03) −0.06 (±0.01) −0.04 (±0.01) (±0.04) (±0.06) (±0.10) (±0.05) 0.75 0.80 0.96 0.99 (±0.1) (±0.1) (±0.3) (±0.3) 2.1 1.8 1.8 1.8 (±0.2) (±0.0) (±0.3) (±0.2) 1.6 1.5 1.7 1.8 0.62 0.63 0.64 0.64 0.34 0.32 0.28 0.23 (±90) (±160) (±250) (±50) 790 870 950 660 4.1 2.4 0.1 0.0 N100-SWCNT N50-SWCNT N1-SWCNT SWCNT

surface potentialc (V vs Ag/AgCl) DET active Lacb (105 U (mol cm−2)) Γa (10−14 mol cm−2) Δ abs (10−3 abs) O2 reduction potential of Lac (V vs Ag/AgCl) O2 reduction potential (V vs Ag/AgCl)

using a molar absorption coefficient.36 Table 2 summarizes the relevant results. These results provide two pieces of evidence important for understanding the N-doping effect. First, the value of Γ did not depend on the N dopant content ratio in the SWCNTs, and Γ was estimated to be 1.8−2.1 × 10−14 mol cm−2, which indicates that much less than a monolayer of Lac was adsorbed. Assuming that Lac is spherical in shape with a diameter of 6 nm (6.5 × 5.5 × 4.5 nm),33 the theoretical number of Lac molecules in a monolayer, based on a simple model for monolayer packing on a planar substrate, is calculated to be ca. 5.3 × 10−12 mol cm−2. This result is of importance to simplify the discussion about the efficiency of the DET as follows. Here, we should note that Γ represents the total surface concentration including both the DET active and DET inactive Lac immobilized on the surface. Second, the value of ALac slightly decreased with increasing N dopant content ratio. The above results revealed that the efficiency of DET (meaning the ratio of DET activating Lac to adsorbed Lac) and/or the DET rate of Lac decreases with increasing N dopant concentration. We suggest two possible reasons for this Ndoping effect on the DET reaction. One possible explanation is the effect of nitrogen species surface functional groups on the DET reaction. Pyridine-like defects and amine-like defects were observed in the N50- and N100-SWCNTs. From the pKa values given in Table 3, these species would have a slightly positive charge under our experimental conditions. We should focus on pyridine-like defects around a divacancy as shown in Figure 5, in particular both the trimerized and the tetramerized pyridinelike defects, which are more energetically favorable than substitutional (graphitic) nitrogen defects. The large ovalshaped protrusion of the p-orbital-shaped electron density was localized toward the carbon defect vacancy, and we expect that such pyridine-like defects would have positive charge owing to proton binding to the large electron density site in the defects. It is known that the T1 site of Lac from Trametes versicolor is

SBET (104 cm2 g−1)

Figure 4. Voltammograms toward negative potential sweep for (A) NSWCNTs immobilized with Lac and (B) N-SWCNTs without Lac modification in a 0.1 mol dm−3 acetate buffer solution (pH 5.0) in the presence of O2. Potential sweep rate: 20 mV s−1. Electrode area: 0.25 cm2. In the figure, (a), (b), (c), and (d) represent N100-, N50-, N1SWCNTs, and SWCNTs, respectively. The dotted lines indicate anaerobic conditions.

N dopant (%)

Table 2. Evaluated N-Doping Ratio, BET Surface Area, O2 Reduction Potential, Amount of Lac Immobilized, Lac Activity Based on DET, and Surface Electrode Potential

Article



Article

Table 3. Surface Functional Groups of Induced Nitrogen Defects in N-SWCNTs and their pKa

concentration, thus increasing the rate of Lac denaturation on the N-SWCNT interface and/or decreasing the electron transfer rate. The other possible reason for the N-doping effect is the change in surface potential of the N-SWCNTs during Lac modification, which shifted positively with increasing N dopant concentration. The open-circuit potentials of the electrodes in 0.1 mol dm−3 acetate buffer solution (pH 5.0) are shown in Table 2. The slope of surface potential vs N dopant ratio in the N-SWCNTs was evaluated to be around 53 mV/N% up to 4% doping from several measurements. The difference in surface potential during Lac modification between the N1- SWCNTs, SWCNTs, and N100-SWCNTs was ca. 210 mV. This large difference in surface potential would be expected to affect the orientation of the Lac on the surface and/or change its binding strength. To clarify the above surface potential effects, Lac modification of N100-SWCNTs was carried out under potentialcontrolled conditions at −40 mV, which was the same as the surface potential of the SWCNTs. In addition, Lac modification of SWCNTs was performed under potential-controlled conditions at −100, 0, 100, and 200 mV. Both the resulting catalytic current from the DET reaction of Lac and the adsorbed amount of Lac were almost identical to that observed during Lac modification at the open-circuit potential. In other words, the amount, orientation, and activity of Lac were all independent of the electrode potential in the range of −100 to 200 mV. These results allow us to conclude that the effects of surface-functional-group-induced nitrogen defects on the DET reaction are larger than this surface potential range.

■

Figure 5. Schematic illustration of the atomic configuration of defects induced by N-doping: (a) nondefect, (b) substitutional (graphitic) nitrogen defect, (c) monomeric pyridine-like defect, (d) dimerizedpyridine-like defect, (e) trimerized-like defect, and (f) tetramerized pyridine-like defect.

CONCLUSIONS To clarify the effect of N-doping of SWCNTs on the DET reaction of Lac, N-SWCNTs with 0.1, 2.4, and 4.1% N dopant were synthesized directly onto a gold electrode. Although the amount of Lac adsorbed per BET surface area was not dependent on N dopant concentration, the bioelectrocatalytic current from the DET reaction of Lac immobilized onto the NSWCNTs decreased with increasing N dopant concentration. The preliminary results of a thermodynamic investigation suggested that this tendency of the bioelectrocatalytic current to decrease with increasing N dopant content in the NSWCNTs could be due to denaturation and/or decreasing of the DET reaction rate of Lac. This was derived from the strongly electrostatic interaction between the positively charged interface of the N-SWCNTs, caused by surface functional groups of aromatic nitrogen species and the T1 site surrounded by carboxylate residues with negative charges. This hypothesis was supported by the observation that the catalytic activity of Lac was independent of surface potential during Lac modification. This study has qualitatively demonstrated the

surrounded by negatively charged carboxylate residues at a pH value above 5, such as that used in the present experiments.6 Therefore, strong interactions could be expected to arise between the positively charged interface of the N-SWCNTs and the T1 site surrounding the carboxylate residues with negative charges. Such strong interactions may induce denaturation of the Lac at the electrode interface. The preliminary results of our thermodynamic investigations supported this expectation. The enthalpy change (ΔH) for the binding of Lac molecules with the SWCNTs was negative, indicating an exothermic reaction. Furthermore, ΔH became more negative with increasing N doping; the −ΔH value for NSWCNTs with 4% N dopant was approximately 1.3−1.5 fold higher than that in the case of 2% N dopant. These preliminary thermodynamic results suggest that the enzymes bound more strongly to the N-SWCNT surface with increasing N dopant 5058



Article

(26) Vaz-Dominguez, C.; Campuzano, S.; Rüdiger, O.; Pita, M.; Gorbacheva, M.; Shleev, S.; Fernandez, V. M.; Lacey, A. L. D. Biosens. Bioelectron. 2008, 24, 531−537. (27) Pang, H. L.; Liu, J.; Hu, D.; Zang, X. H.; Chen, J. H. Electrochim. Acta 2010, 55, 6611−6616. (28) Iijima, S. Nature 1991, 354, 56−58. (29) Geim, A. K.; Novoselov, K. S. Nat. Mater. 2007, 6, 183−191. (30) Wallace, P. R. Phys. Rev. 1947, 71, 622−634. (31) Saito, R.; Dresselhaus, G.; Dresselhaus, M. S. Physical Properties of Carbon Nanotubes; Imperial College Press: London, 1998. (32) McCreery, R. L. Chem. Rev. 2008, 108, 2646−2687. (33) Fujimoto, Y.; Saito, S. Phys. Rev. B 2011, 84, 245446. (34) Wang, Y.; Ai, H. J. Phys. Chem. B 2009, 113, 9620−9627. (35) Kamitaka, Y.; Tsujimura, S.; Setoyama, N.; Kajino, T.; Kano, K. Phys. Chem. Chem. Phys. 2007, 9, 1793−1801. (36) Santucci, R.; Ferri, T.; Morpurgo, L.; Savini, I.; Avigliano, L. Biochem. J. 1998, 332, 611−615. (37) Tominaga, M.; Sakamoto, S.; Yamaguchi, H. J. Phys. Chem. C 2012, 116, 9498−9506. (38) Tominaga, M.; Iwaoka, A.; Kawai, D.; Sakamoto, S. Electrochem. Commun. 2013, 31, 76−79. (39) Sakamoto, S.; Tominaga, M. Chem.− Asian J. 2013, 8, 2680− 2684. (40) Glerup, M.; Castignolles, M.; Holzinger, M.; Hug, G.; Loiseau, A.; Bernier, P. Chem. Commun. 2003, 2542−2543. (41) Ayala, P.; Grüneis, A.; Kramberger, C.; Rümmeli, M. H.; Solórzano, I. G.; Freire, F. L., Jr; Pichler, T. J. Chem. Phys. 2007, 127, 184709. (42) Tuinstra, F.; Koenig, J. L. J. Chem. Phys. 1970, 53, 1126−1130. (43) Tuinstra, F.; Koenig, J. L. J. Composite Mater. 1970, 4, 492−499. (44) Yang, Q.-H.; Hou, P.-X.; Unno, M.; Yamauchi, S.; Saito, R.; Kyotani, T. Nano Lett. 2005, 5, 2465−2469. (45) Villalpando-Paez, F.; Zamudio, A.; Elias, A. L.; Son, H.; Barros, E. B.; Chou, S. G.; Kim, Y. A.; Muramatsu, H.; Hayashi, T.; Kong, J.; Terrones, H.; Dresselhaus, G.; Endo, M.; Terrones, M.; Dresselhaus, M. S. Chem. Phys. Lett. 2006, 424, 345−352. (46) Brown, S. D. M.; Jorio, A.; Corio, P.; Dresselhaus, M. S.; Dresselhaus, G.; Saito, R.; Kneipp, K. Phys. Rev. B 2001, 63, 155414. (47) Fouquet, M.; Telg, H.; Maultzsch, J.; Wu, Y.; Chandra, B.; Hone, J.; Heinz, T. F.; Thomsen, C. Phys. Rev. Lett. 2009, 102, 075501. (48) Liu, Y.; Jin, Z.; Wang, J.; Cui, R.; Sun, H.; Peng, F.; Wei, L.; Wang, Z.; Liang, X.; Peng, L.; Li, Y. Adv. Funct. Mater. 2011, 21, 986− 992. (49) Ferrari, A. C.; Meyer, J. C.; Scardaci, V.; Casiraghi, C.; Lazzeri, M.; Mauri, F.; Piscanec, S.; Jiang, D.; Novoselov, K. S.; Roth, S.; et al. Phys. Rev. Lett. 2006, 97, 187401−187404. (50) Pimenta, M. A.; Dresselhaus, G.; Dresselhaus, M. S.; Cançado, L. G.; Jorio, A.; Saito, R. Phys. Chem. Chem. Phys. 2007, 9, 1276−1291. (51) Ibrahim, E. M. M.; Khavrus, V. O.; Leonhardt, A.; Hampel, S.; Oswald, S.; Rümmeli, M. H.; Büchner, B. Diamond Relat. Mater. 2010, 19, 1199−1206. (52) Kim, K. K.; Park, J. S.; Kim, S. J.; Geng, H. Z.; An, K. H.; Yang, C.-M.; Sato, K.; Saito, R.; Lee, Y. H. Phys. Rev. B 2007, 76, 205426. (53) Saito, R.; Dresselhaus, G.; Dresselhaus, M. S. Phys. Rev. B 2000, 61, 2981−2990. (54) Jorio, A.; Saito, R.; Hafner, J. H.; Lieber, C. M.; Hunter, M.; McClure, T.; Dresselhaus, G.; Dresselhaus, M. S. Phys. Rev. Lett. 2001, 86, 1118−1121. (55) Kataura, H.; Kumazawa, Y.; Maniwa, Y.; Umezu, I.; Suzuki, S.; Ohtsuka, Y.; Achiba, Y. Synth. Met. 1999, 103, 2555−2558. (56) Wiltshire, J. G.; Li, L.-J.; Herz, L. M.; Nicolas, R. J.; Glerup, M.; Sauvajol, J.-L.; Khlobystov, A. N. Phys. Rev. B 2005, 72, 205431. (57) Villalpando-Paez, F.; Zamudio, A.; Elias, A. L.; Son, H.; Barros, E. B.; Chou, S. G.; Kim, Y. A.; Muramatsu, H.; Hayashi, T.; Kong, J.; Terrones, H.; Dresselhaus, G.; Endo, M.; Terrones, M.; Dresselhaus, M. S. Chem. Phys. Lett. 2006, 424, 345−352. (58) Liu, J.; Webster, S.; Carroll, D. L. J. Phys. Chem. B 2005, 109, 15769−15774.

effects of N-doping level in SWCNTs on the bioelectrocatalytic behavior of Lac.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./Fax: +81-96342-3655. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Prof. Naotoshi Nakashima and his group at Kyushu University for microcalorimetric measurements. M.T. also acknowledges a Research Foundation Grant from Toho Gas Co., Ltd. This work was financially supported by a Grant-in-Aid for Scientific Research (no. 24550159) from the Ministry of Education, Culture, Science, Sports and Technology, Japan.

■

REFERENCES

(1) Yoshida, H. J. Chem. Soc. 1883, 43, 472−486. (2) Sakurai, T. Biochem. J. 1992, 284, 681−685. (3) Yaropolov, A. I.; Skorobogat’ko, O. V.; Vartanov, S. S.; Varfolomeev, S. D. Appl. Biochem. Biotechnol. 1994, 49, 257−280. (4) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. Rev. 1996, 96, 2563−2605. (5) Sakurai, T.; Kataoka, K. Cell. Mol. Life Sci. 2007, 64, 2642−2656. (6) Piontek, K.; Antorini, M.; Choinowski, T. J. Biol. Chem. 2002, 277, 37663−37669. (7) Windmiller, J. R.; Wang, J. Electroanalysis 2013, 25 (1), 29−46. (8) Barton, S. C.; Gallaway, J.; Atanassov, P. Chem. Rev. 2004, 104, 4867−4886. (9) Shleev, S.; Tkac, J.; Christenson, A.; Ruzgas, T.; Yaropolov, A. I.; Whittaker, J. W.; Gorton, L. Biosens. Bioelectron. 2005, 20, 2517−2554. (10) Yan, Y.; Zheng, W.; Su, L.; Mao, L. Adv. Mater. 2006, 18, 2639− 2643. (11) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A. Chem. Rev. 2008, 108, 2439−2461. (12) Gellett, W.; Schumacher, J.; Kesmez, M.; Le, D.; Minteer, S. D. J. Electrochem. Soc. 2010, 157, B557−B562. (13) Ding, S.-N.; Holzinger, M.; Mousty, C.; Cosnier, S. J. Power Sources 2010, 195, 4714−4717. (14) Miyake, T.; Yoshino, S.; Yamada, T.; Hata, K.; Nishizawa, M. J. Am. Chem. Soc. 2011, 133, 5129−5134. (15) Karaskiewicz, M.; Nazaruk, E.; Zelechowska, K.; Biernat, J. F.; Rogalski, J.; Bilewicz, R. Electrochem. Commun. 2012, 20, 124−127. (16) Nazaruk, E.; Karaskiewicz, M.; Zelechowska, K.; Biernat, J. F.; Rogalski, J.; Bilewicz, R. Electrochem. Commun. 2012, 14, 67−70. (17) Wei, W.; Li, P.; Li, Y.; Cao, X.; Liu, S. Electrochem. Commun. 2012, 22, 181−184. (18) Lörcher, S.; Lopes, P.; Kartashov, A.; Ferapontova, E. E. ChemPhysChem 2013, 14, 2112−2124. (19) Portaccio, M.; Martino, S. D.; Maiuri, P.; Durante, D.; Luca, P. D.; Lepore, M.; Bencivenga, U.; Rossi, S.; Maio, A. D.; Mita, D. G. J. Mol. Catal. B 2006, 41, 97−102. (20) Qiu, H.; Xu, C.; Huang, X.; Ding, Y.; Qu, Y.; Gao, P. J. Phys. Chem. C 2009, 113, 2521−2525. (21) Ramasamy, R. P.; Luckarift, H. R.; Ivnitski, D. M.; Atanassov, P. B.; Johnson, G. R. Chem. Commun. 2010, 46, 6045−6047. (22) Lee, J. Y.; Shin, H. Y.; Kang, S. W.; Park, C.; Kim, S. W. Enzyme Microb. Technol. 2011, 48, 80−84. (23) Trohalaki, S.; Pachter, R.; Luckarift, H. R.; Johnson, G. R. Fuel Cells 2012, 12, 656−664. (24) Blanford, C. F.; Heath, R. S.; Armstrong, F. A. Chem. Commun. 2007, 1710−1712. (25) Shleev, S.; Christenson, A.; Serezhenkov, V.; Burbaev, D.; Yaropolov, A.; Gorton, L.; Ruzgas, T. Biochem. J. 2005, 385, 745−754. 5059



Article

(59) Maldonado, S.; Morin, S.; Stevenson, K. J. Carbon 2006, 44, 1429−1437. (60) Kim, S. Y.; Lee, J.; Na, C. W.; Park, J.; Seo, K.; Kim, B. Chem. Phys. Lett. 2005, 413, 300−305. (61) Dommele, S. V.; Romero-Izquirdo, A.; Brydson, R.; Jong, K. P. D.; Bitter, J. H. Carbon 2008, 46, 138−148. (62) Lee, D. H.; Lee, W. J.; Kim, S. O. Nano Lett. 2009, 9, 1427− 1432. (63) Jian, G.; Zhao, Y.; Wu, Q.; Yang, L.; Wang, X.; Hu, Z. J. Phys. Chem. C 2013, 117, 7811−7817. (64) Gong, K.; Du, F.; Xia, Z.; Durstock, M.; Dai, L. Science 2009, 323, 760−764. (65) Wiggins-Camacho, J. D.; Stevenson, K. J. J. Phys. Chem. C 2011, 115, 20002−20010. (66) Alexeyeva, N.; Shulga, E.; Kisand, V.; Kink, I.; Tammeveski, K. J. Electroanal. Chem. 2010, 648, 169−175.

5060


Effect of N-Doping of Single-Walled Carbon Nanotubes on

Recommend Documents