Colloids and Surfaces B: Biointerfaces 82 (2011) 526531

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Electrochemical properties of myoglobin deposited on multi-walled carbon nanotube/ciprooxacin lm

S. Ashok Kumar a , Sea-Fue Wang a, , Yu-Tsern Chang b , His-Chuan Lu a , Chun-Ting Yeh a
a b

Department of Materials and Mineral Resources Engineering, No. 1, Sec. 3, Chung-Hsiao East Rd., National Taipei University of Technology, Taipei, Taiwan Department of Chemical and Materials Engineering, Nanya Institute of Technology, Jhongli 32091, Taiwan

a r t i c l e

i n f o

a b s t r a c t
We report the direct electrochemical and electrocatalytic properties of myoglobin (MB) on a multi-walled carbon nanotube/ciprooxacin (MWCNT/CF) lm-modied electrode. A highly homogeneous MWCNT thin-lm was prepared on an electrode surface using ciprooxacin (CF) as a dispersing agent. MB was then electrochemically deposited onto the MWCNT/CF-modied electrode. The MB/MWCNT/CF lm was characterized by scanning electron microscopy and UVvisible spectroscopy (UVvis). UVvis spectra conrmed that MB retained its original state on the MWCNT/CF lm. Direct electrochemical properties of MB on the MWCNT/CF lm were investigated by cyclic voltammetry. The formal potential and electron transfer rate constant were evaluated in pH 7.2 buffer solution as 0.327 V and 300 s1 , respectively. In addition, the MB/MWCNT/CF-modied electrode showed excellent electrocatalytic properties for the reduction of hydrogen peroxide (H2 O2 ). The MB/MWCNT/CF-modied electrode was used for the detection of H2 O2 at concentrations from 1 106 M to 7 104 M in pH 7.2 buffer solution. Overall, the MB/MWCNT/CF-modied electrode was very stable and has potential for development as a H2 O2 sensor. 2010 Elsevier B.V. All rights reserved.

Article history: Received 7 September 2009 Received in revised form 2 October 2010 Accepted 6 October 2010 Available online 15 October 2010 Keywords: Myoglobin CNTs Surfaces Thin lms Nanomaterials Direct electrochemistry

1. Introduction The immobilization of redox proteins on various nanomaterials and their direct electrochemical properties has attracted considerable attention in recent years due to their fundamental importance in the chemical and biological sciences [13]. Unfavorable orientation of protein molecules on an electrode surface may impede electron transfer between electrode and protein electroactive centers. In addition, adsorption of protein molecules onto a bare electrode surface can lead to denaturation, which also decreases the direct electron-transfer rate [3,4]. Carbon nanotubes (CNTs) have attracted enormous interest over the past few decades, mainly due to their exceptional electrical, chemical, and mechanical properties which make them attractive candidates for diverse applications such as nanoelectronics and biosensors [5,6]. Numerous studies have focused on the electrochemistry of myoglobin (MB) with biocompatible materials such as surfactants [710], ionic liquids [11,12], CNTs [1315], solgel materials [16,17], inorganic nanoparticles (NPs) [1821], polyelectrolytes [2225], claychitosangold NPs [26], zir-

Corresponding author. Tel.: +886 2 27712171x2735; fax: +886 2 27317185. E-mail addresses: sakumar80@gmail.com (S.A. Kumar), seafuewang@yahoo.com (S.-F. Wang). 0927-7765/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.10.011

conium phosphate nanosheets [27], and nonionic poly(ethylene glycol)/ZrO2 NPs [21]. Synthesized CNTs often form bundles due to strong van der Waals interactions between nanotubes. Therefore, the preparation of a homogenous dispersion of CNTs in an aqueous solution is difcult but can be achieved with organic dispersing agents. Recently, we reported a new method for the preparation of a multi-walled carbon nanotube (MWCNT) dispersion in an aqueous solution of ciprooxacin (CF) (a synthetic antibiotic) [28]. In this report, we describe a potential application of MWCNTs dispersed in CF solution. Dry lms of MWCNT/CF were prepared and used for the immobilization of MB on an electrode surface. Characterization of the modied electrode surface was performed by scanning electron microscopy (SEM), UVvisible spectroscopy (UVvis), and cyclic voltammetry. Direct electron transfer (DET) of MB was observed on the MWCNT/CF lms, and the electrochemical properties of MB were investigated. MB retained its original conformation in the MWCNT/CF lm, and the modied electrode has good stability in neutral buffer solution. Recently, Zhao et al. and Zhang et al. reported the DET of MB on a carboxylic acid-functionalized MWCNT-modied glassy carbon electrode (GCE) [29,30]. Further, ionic liquid/single-walled carbon nanotube- [31], gold-NPs/MWCNT- [32], and TiO2 -coated MWCNT- [33] modied electrodes were also used to examine the direct electrochemistry of MB. Interestingly, in the present study,

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MWCNT lms were prepared using a stable MWCNT/CF dispersion and rmly attached onto the GCE surface for use as a matrix to immobilize MB. The MWCNT/CF lm can be used without any other nanomaterials to study DET and other electrocatalytic properties of proteins. 2. Experimental 2.1. Reagents and chemicals MB (from horse heart) (MW 17,800), MWCNTs (purity = 99.8%), CF, and H2 O2 (35% w/w) were all purchased from Sigma and used as received without further purication. Na2 HPO4 , HCl, and NaOH were purchased from Wako Pure Chemicals. All chemicals were used as received without further purication. Buffer solutions (0.1 M) at various pH values were prepared by adjusting the pH with 0.1 M NaOH or 0.1 M HCl. Aqueous solutions were prepared with double distilled water. 2.2. Apparatus and instruments Electrochemical experiments were performed with a CH Instruments, Inc. Chi611c (Austin, TX). All electrochemical experiments were carried out with a conventional three-electrode system. A GCE or MWCNT/CF/GCE was used as a working electrode, and an indium tin oxide-coated glass electrode was used for the preparation of dry lms. Platinum wire and Ag/AgCl (3 M KCl) were used as the counter electrode and the reference electrode, respectively. The electrolyte solution was purged with high-purity argon gas for 10 min prior to each electrochemical experiment. The surface characterization was performed by SEM (Hitachi S-4700). Absorption spectra were recorded using a UV-vis spectrophotometer (PerkinElmer Lambda 900). 2.3. MB immobilization on MWCNT/CF lm A stable dispersion of MWCNTs (1 mg/mL) was prepared in 5 mM CF solution as reported elsewhere [28]. A quantity of 10 L of MWCNT dispersion was casted onto a GCE surface and dried at 60 C in an air oven. Then, MWCNT/CF lm-modied electrode was thoroughly washed with distilled water. Immobilization of MB on the MWCNT/CF-modied electrode was accomplished by potential cycling (between 0.2 and 0.6 V at a scan rate of 0.05 V/s) in pH 7.2 buffer solution containing 5 mM MB for 20 cycles. After the modication process, MB/MWCNT/CF/GCE was rinsed with distilled water. Electrochemical measurements were carried out in 0.1 M buffer solution (pH 7.2) using MB/MWCNT/CF/GCE as a working electrode. For SEM and UVvis studies, the MB/MWCNT/CF lm was similarly prepared on indium tin oxide-coated glass electrodes and used for characterization studies. The proposed modied electrode system is simple and can be prepared in less than 1 h. 3. Results and discussion 3.1. Direct electrochemistry of MB Fig. 1A shows the cyclic voltammograms (CVs) of the MWCNT/CF-modied GCE in 0.1 M buffer solution (pH 7.2) (curve a). A reversible redox peak was observed at (formal potential, E ) 0.04 V, which corresponds to the redox of carboxylic acid groups on the surface of the MWCNTs [34]. First, a clear solution of CF was prepared in 0.1 M HCl solution, and then a MWCNT/CF dispersion was prepared by the addition of CNTs and stirring for 3 h [28]. During this process, carboxyl groups appeared on surface of the MWCNTs. Fig. 1A, curve b, shows the CVs of the MB/MWCNT/CF-modied

Fig. 1. (A) CVs of MWCNT/CF/GCE (curve a) and MB/MWCNT/CF/GCE (curve b) recorded in 0.1 M buffer (pH 7.2). Scan rate = 0.05 V/s. (B) CVs of MB/MWCNT/CF/GCE at different scan rates in pH 7.2 buffer solution. Scan rates: (a) 0.02, (b) 0.04, (c) 0.06, (d) 0.08, (e) 0.10, (f) 0.12, (g) 0.14, (h) 0.16, (i) 0.18, (j) 0.20, (k) 0.25, (l) 0.30, (m) 0.35, (n) 0.40, (o) 0.45, and (p) 0.50 V/s. Inset of (B) shows a plot of Ipa and Ipc against scan rate.

GCE in pH 7.2 buffer solution. The electrochemically immobilized MB protein on the electrode surface generates a new redox peak [MB (Fe(III)Fe(II)] [35]. The anodic and the cathodic peaks of MB are located at 0.3085 V and 0.3450 V, with an apparent E of 0.327 V. The peak-to-peak separation ( Ep = Epa Epc ) was found to be 36.5 mV, which is anticipated for a reversible surfaceconned redox couple. The shapes of the cathodic and anodic waves were nearly symmetric; the reduction and oxidation peaks of MB have the same heights (Fig. 1A, curve b). We conclude that the MWCNT/CF lm must have an effect on the kinetics of the electrode reaction with the proteins, providing a favorable microenvironment for the electron exchange between the adsorbed MB protein and the underlying electrode. In addition, the redox peak of the carboxylic group on the MWCNT/CF lm almost disappeared after adsorption of the MB protein (Fig. 1A, curve b). 3.2. Electrochemical properties of the biosensor CVs were recorded in 0.1 M KCl containing 5 mM [Fe(CN)6 ]3/4 using a MWCNT/CF-modied GCE, a MWCNT/GCE, and an unmodied GCE, respectively. As shown in Fig. 2A, curve a, highly enhanced anodic and cathodic peak currents were observed at the MWCNT/CF-modied electrode. As expected, the MWCNT/GCE (curve b) and unmodied GCE showed lower redox peak currents for the redox probe (Fig. 2A, curve c). The increased electroactive surface area of the electrode allows high peak currents to occur at the MWCNT/CF-modied electrode. In addition, as conrmed from these observations, the COOH groups of the MWCNTs are not negatively charged at neutral pH, because a negative charge would prevent observation of redox peak currents for the negatively charged redox probe. CF, which is attached to the surface

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thus, the neutral nonionic form spontaneously forms the zwitterion (Fig. 2B). The pKa1 of CF is around 6, and the pKa2 , which is due to the presence of an ionizable proton on the secondary piperazinyl nitrogen, is around 8.5 [36]. CF molecules in neutral form on the MWCNT lm might help to avoid aggregation of MWCNTs [28]. The COOH groups of MWCNT/CF are not charged at pH 7.2, so repulsion between the COOH groups of MWCNT/CF and MB was not expected. This phenomenon was also supported by earlier reports where carboxylic acid-functionalized MWCNTs were used as the matrix for immobilization of MB to study its direct electrochemistry and electrocatalytic properties [29,30]. Overall, these results indicate that MB protein adsorbs on the MWCNT/CF lm and that the carboxylic groups might react with the MB macromolecule. Above the isoelectric point (pH 6.8), MB is negatively charged while the MWCNT/CF lm is essentially neutral or has no net charge [28]. Thus, the driving force for the binding of MB to the MWCNT/CF lm would be mainly hydrophobic interaction between the MB macromolecule and the hydrocarbon backbone of the MWCNT/CF lm. Thus, the role of MWCNT/CF lm for facilitating electron transfer may be ascribed to good biocompatibility. 3.3. Effect of scan rate and Lavirons treatment Cyclic voltammetry showed that the cathodic and anodic peak currents of MB increased linearly with the scan rate in the range of 20500 mV/s as expected for thin-layer electrochemistry [37] (Fig. 1B). According to Eq. (1), an average surface concentration ( ) of electroactive MB adsorbed on the MWCNT/CF lm is given as:
Fig. 2. (A) CVs recorded in 5 mM [Fe(CN)6 )3/4 ] + 0.1 M KCl using a MWCNT/CF/GCE (curve a), MWCNT/GCE (curve b), and bare-GCE (curve c). Scan rate = 0.05 V/s. (B) Chemical formula of CF and its zwitterion.

Ip =

n2 F 2 A 4RT

(1)

of the MWCNTs, can exist as a zwitterion HQ and a neutral nonionized species HQ, depending on the pH [36]. At low pH values, both the 7-piperazinyl group and 3-carboxyl group are protonated, whereas at high pH values, neither is protonated. The carboxyl group is normally a stronger acid than the ammonium group;

where Ip , , A, n, T, , F, and R represent the peak current (A), scan rate (0.02 V/s), surface area of the electrode (cm2 ), charge transfer number (n = 1), temperature (298 K), surface concentration (mol/cm2 ), the Faraday constant, and gas constant, respectively [37]. The value of MB was about 1.263 1010 mol/cm2 , indicating an approximate monolayer adsorption. According to Lavirons equation [38], for the present electrode system with

Fig. 3. SEM images of (a) pure-MWCNTs, (b) a MWCNT/CF lm, and (c) a MB/MWCNT/CF lm. (d) UVvis spectrum of MB/MWCNT/CF lm attached to the electrode.

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n Ep > 200 mV, Eq. (2) can be used. log ks = log(1 ) + (1 ) log log RT nF (1 )nF Ep 2.3RT (2)

Here, the electrochemical transfer coefcient () is assumed to be 0.5, and n = 1 for MB. The symbols R, T, and F have typical values [37]. The heterogeneous electron-transfer rate constant (Ks ) was calculated to be 300 s1 , suggesting a fast electron-transfer kinetics process. 3.4. SEM and UVvis studies Fig. 3 shows the SEM image of pure-MWCNTs (a) and a dry lm of MWCNT/CF (b). The SEM image of the MWCNT/CF lm showed that CF molecules were adsorbed on the nanotubes. In addition, the MWCNT/CF lm surface was analyzed after MB protein adsorption. Clearly, MB molecules were adsorbed on the MWCNT/CF surface (Fig. 3c); however, it was unclear whether or not the protein had been denatured after adsorption on the MWCNT/CF lm. It is well known that the UVvis absorptive bands (Soret band) of the heme group can indicate possible denaturation of heme proteins [3,4]. Fig. 3(d) shows the UVvis spectrum of the MB/MWCNT/CF lm. The absorptive peak is located at 408 nm which is the same wavelength as that of MB in solution [21,26,27]. It was expected that a new absorption peak at 363 nm in UVvis spectrum was due to conformational changes in the entrapped protein or to unfolding of the native structure of the protein [39,40]. However, the extent of denaturation or the unfolding process of the protein was not observed in the MWCNT/CF lm, suggesting that MB was in the native state on the modied electrode. In addition, the effect of pH of the supporting electrolyte on the redox signal of MB was investigated. The voltammetric response of MB/MWCNT/CF/GCE was obtained in solutions of different pH values in a range of 211. As shown in Fig. 4A, the E of the MB redox peak was pH dependent with a slope of 48 mV per pH unit, which is very close to the anticipated Nernstian behavior (Eq. (3)). MB (FeIII ) + H+ + e MB (FeII ) (3)

Fig. 4. (A) CVs of MB/MWCNT/CF/GCE recorded in solution at different pH values: (a) pH 2.2, (b) pH 7.2, and (c) pH 11.0. (B) CVs of the MB/MWCNT/CF/GCE in pH 7.2 buffer: (a) 1st cycle and (b) 200th cycle. Scan rate = 0.1 V/s.

3.5. Stability test of the biosensor We performed a stability test using a MB/MWCNT/CF-modied electrode in 0.1 M buffer solution (pH 7.2). A MB/MWCNT/CF/GCE was cycled in 0.1 M buffer solution between 0.0 and 0.6 V at a scan rate of 0.1 V/s. The MB redox peak current remained almost constant after 200 cycles (Fig. 4B), which indicates that the MB molecules were strongly attached onto the MWCNT/CF lm. The fabrication reproducibility of the biosensor (MB/MWCNT/CF/GCE) was tested for ve electrodes made independently and showed reproducibility with a relative standard deviation (RSD) of 3.2% for the redox peak of MB. 3.6. Electrocatalytic reduction of H2 O2 The possible application of the MB/MWCNT/CF-modied electrode as a biosensor for H2 O2 sensing was investigated. Fig. 5 (curves a and b) depicts the CVs of the MB/MWCNT/CF/GCE in 0.1 M buffer solution (pH 7.2) in the absence (curve a) and presence (curve b) of 5 104 M H2 O2 at a scan rate of 50 mV/s. In the absence of H2 O2 , a pair of the redox peaks of MB was retained (curve a). However, in the presence of 5 104 M H2 O2 , the voltammetric behavior changes drastically, conrming the electrocatalytic effect of MB. A large cathodic current for the reduction of H2 O2 appears, and the anodic peak completely disappears (Fig. 5, curve

b). Further, the cathodic current for the reduction of H2 O2 increased with respect to the increased concentration of H2 O2 in pH 7.2 buffer solution (Fig. 5, curves bd). Catalytic reduction of H2 O2 was observed at 0.2 V, which is lower than the formal potential of

Fig. 5. CVs recorded in the absence (curve a) and presence of 5 104 M (curve b), 6 104 M (curve c), and 7 104 M H2 O2 (curve d) in pH 7.2 buffer solution using a MB/MWCNT/CF-modied electrode. CVs of a bare-GCE (curve e) and MWCNT/CF/GCE (curve f) under the same conditions with 7 104 M H2 O2 . Scan rate = 0.02 V/s.

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Table 1 Comparisons of analytical parameters of the proposed method with earlier reports. Modied electrode [DHP]/MB]6 MB-MWCNTs MWCNTs-Naon-MB Naon/MWCNTs/CILE/MB {ZrO2 /MB}10 MBPNM lm Naon/MB/CGNs/GCE MB-ZnO MB/MWCNTs/CF pH 7.0 7.0 7.0 7.0 7.0 7.0 6.9 7.0 7.2 Formal potential (E )/V 0.35 0.347 0.32 0.32 0.337 0.36 0.373 0.339 0.327 Ks s1 43 50 6 1.42 0.332 21 4 20 1 300 Linear range for H2 O2 (M) 265 160 680 8.0196 0.490 0.191.53 1.590 4.8200 1700 Reference [10] [13] [14] [15] [16] [17] [18] [19] Proposed method

DHP, anionic surfactant dihexadecyl phosphate; CILE, carbon ionic liquid electrode; PNM, poly(N-isopropylac-yamide-co-3-methacryloxy-propyl-trimethoxysilane); CGNs, colloidal gold nanoparticles.

the MB/MWCNT/CF-modied electrode, conrming that the MBattached MWCNT/CF electrode has a potent electrocatalytic effect for H2 O2 . To further demonstrate the application of these electrodes as biosensors for H2 O2 , the electrocatalytic reduction of H2 O2 was also performed on the MWCNT/CF-modied electrode (Fig. 5, curve f) and on a bare GCE (curve e) under the same conditions. As expected, negligible responses were observed on these surfaces, indicating that the observed electrocatalytic effect for H2 O2 on the MB/MWCNT/CF-modied electrode is due solely to the presence of MB. In the course of evolution, MB is a protein that has been naturally selected for oxygen storage. It contains a single, noncovalently bonded iron protoporphyrin IX as the prosthetic group in a hydrophobic pocket [41]. MB is known to have some intrinsic peroxidase activity due to its close structural similarity to peroxidase. Therefore, it appears reasonable to use the heme group of MB to serve as an active center to catalyze the reduction of H2 O2 (Eq. (4)) [18]. According to the previous reports, the possible electrode reaction between MB and H2 O2 could be described as follows [27,42,43]. 2MB Fe(II) + 2H+ + H2 O2 2MB Fe(III) + 2H2 O (4)

4. Conclusions In summary, the direct electrochemistry of MB was examined on a MWCNT/CF-modied GCE after immobilization of MB. The immobilized MB retained its redox activity because it was biocompatible with the MWCNT/CF lm. Further, the MB/MWCNT/CF lm showed excellent stability in neutral buffer solution. A high Ks value for MB was also observed on the MB/MWCNT/CF-modied electrode, which is probably due to the good biocompatibility of the host lm. Moreover, the MB/MWCNT/CF-modied electrode showed a linear current response for the reduction of H2 O2 at concentrations from 1 106 to 7 104 M. We expect that, in the future, MB/MWCNT/CF lms will be developed as biosensors for H2 O2 sensing. References
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The electrocatalytic reduction peak of H2 O2 by MB/MWCNT/CFmodied electrode was used to quantitatively determine the concentration of H2 O2 . The catalytic peak currents are proportional to the concentration of H2 O2 with a linear range from 1 106 to 7 104 M H2 O2 in neutral buffer solution (Fig. 5). The detection limit is estimated to be 1 106 M when the signal to noise ratio is 3. Reproducibility of the MB/MWCNT/CF-modied electrode was tested for the reduction of H2 O2 . The RSD was 2.3% for six measurements at 5 104 M H2 O2 . Further, three independent GCEs were modied by MB/MWCNT/CF lm and used to measure the catalytic current at 5 104 M H2 O2 . An acceptable reproducibility was achieved with an RSD of 3.5%. As shown in Fig. 4B, the MB/MWCNT/CF-modied electrode was very stable and also showed a good catalytic effect toward H2 O2 . The stability of the MB/MWCNT/CF/GCE biosensor was also tested. When the electrode was stored in pH 7.2 buffer solution at 4 C for four weeks, the peak currents and potentials were stable. As shown in Table 1, electroanalytical parameters of the proposed electrode system such as pH, formal potential, Ks , and linear range for H2 O2 are compared with parameters from earlier reports. The E of MB closely matched the values reported in the literature [10,1319], and a considerably high Ks value was obtained for MB on the MWCNT/CF lm-modied electrode, which might be due to the fast electron transfer between MB and the electrode through the MWCNT/CF lm. In addition, our proposed method showed a wide linear range for detection of H2 O2 in neutral buffer solution [10,1319].

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