multi-walled carbon nanotube nanocomposite

Materials Research Bulletin 45 (2010) 1855–1860

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Direct electrochemistry and electrocatalysis of myoglobin immobilized on zirconia/multi-walled carbon nanotube nanocomposite Ruping Liang, Minqiang Deng, Sanguan Cui, Hong Chen, Jianding Qiu * Department of Chemistry and Institute for Advanced Study, Nanchang University, Nanchang 330031, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 May 2010 Received in revised form 25 July 2010 Accepted 12 September 2010 Available online 17 September 2010

Zirconia/multi-walled carbon nanotube (ZrO2/MWCNT) nanocomposite was prepared by hydrothermal treatment of MWCNTs in ZrOCl28H2O aqueous solution. The morphology and structure of the synthesized ZrO2/MWCNT nanocomposite were characterized by transmission electron microscopy and X-ray diffraction analysis. It was found that ZrO2 nanoparticles homogeneously distributed on the sidewall of MWCNTs. Myoglobin (Mb), as a model protein to investigate the nanocomposite, was immobilized on ZrO2/MWCNT nanocomposite. Ultraviolet–visible spectroscopy and electrochemical measurements showed that the nanocomposite could retain the bioactivity of the immobilized Mb to a large extent. The Mb immobilized in the composite showed excellent direct electrochemistry and electrocatalytic activity to the reduction of hydrogen peroxide (H2O2). The linear response range of the biosensor to H2O2 concentration was from 1.0 to 116.0 mM with the limit of detection of 0.53 mM (S/ N = 3). The ZrO2/MWCNT nanocomposite provided a good biocompatible matrix for protein immobilization and biosensors preparation. ß 2010 Elsevier Ltd. All rights reserved.

Keywords: A. Nanostructures B. Chemical synthesis C. Electrochemical measurements D. Catalytic properties

1. Introduction Research concerning the acceleration of direct electron transfer (DET) between native redox proteins and an electrode surface is significant because it not only provides a model for the mechanistic study of biological electron transport but also enables the establishment of mediator-free (third-generation) biosensors and bioreactors [1,2]. However, since the electroactive centers of native redox proteins are buried deeply in the protein structure, as well as adsorptive denaturation and unfavorable orientations of proteins onto electrode surfaces [3], DET between proteins and bare electrode is much difficult. Hence, various materials, such as surfactants [4], polymers [5,6], nanomaterials [7,8], and inorganic mesoporous materials [9,10], were exploited to improve the immobilization efficiency of redox proteins on electrode surface, and the direct electrochemistry of some redox proteins was thus achieved for their improved nontoxicity, stability, biocompatibility, and conductivity. Nanostructured materials of metal oxide semiconductors are attracted considerable interest in the bioanalytical area as they can combine the properties of high surface area, non-toxicity, biocompatibility, ease of fabrication, and even good electrochemical catalytic activity. Zirconia (ZrO2) is one of the important metal oxide semiconductors due to its potential applications in catalysts,

* Corresponding author. Tel.: +86 791 3969518; fax: +86 791 3969963. E-mail address: [email protected] (J. Qiu). 0025-5408/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.materresbull.2010.09.016

adsorbents, chemical sensors, and fuel cells [11–13]. With the thermal stability, chemical inertness and lack of toxicity and affinity for the groups containing oxygen, ZrO2 has become an ideal candidate of materials for immobilization biomolecules. It can provide a good compatible microenvironment to prevent proteins from aggregating and spontaneous denaturing to a significant extent, and an access to the immobilized proteins for cofactors, substrates, or redox reagents [14]. Some proteins immobilized in ZrO2 nanoparticles (NPs) have also been studied [15,16], those immobilized proteins could retain their conformations similar to the native states at room temperature and exhibited excellent direct electrochemistry at electrodes. The remarkable electrochemical properties and unique structure of carbon nanotube (CNT) offer the ability of promoting electron communication between redox enzymes and CNT modified electrodes. The small size and nanotube structure of CNT allow them to come close to the active centers of redox enzymes and offer possible direct electrochemistry of some proteins on the CNT modified electrodes [17–19]. Recently, the research interest has extended to modify CNT with nanomaterials to prepare nanohybrid materials. Up to now, some metal oxides NPs [20–22] and precious metal NPs [23–27] have been successfully decorated on CNT to synthesize CNT–nanohybrid materials. The nanohybrid materials possess the properties of each component and would be useful in studying the DET reaction of proteins. Titanium dioxide/MWCNT nanohybrid material has been used for immobilizing myoglobin (Mb) and realizing DET between Mb and electrodes [28]. The Au NPs/CNT-modified electrodes have

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also been used to study the DET of cytochrome c [29] and horseradish peroxidase (HPR) [30]. Taking advantage of the unique electronic properties of CNT and ZrO2, we expect that the combination of MWCNTs with ZrO2 NPs may induce interesting charge transfer and thus enhance the electrocatalytic activity of enzymatic bioelectrodes. Most recently, a simple one-step electrodeposition method for the construction of a ZrO2/ MWNTs/Mb electrochemical biosensor was proposed and the DET for the immobilized Mb was realized and high electrocatalytic efficiency toward H2O2 was observed [31]. In the present work, the ZrO2/MWCNT nanocomposite was synthesized via a simple hydrothermal treatment method and further applied in studying the immobilization and DET of Mb for the first time. The morphology and the electrochemistry of the composite film were imaged by transmission electron microscope and electrochemical techniques, respectively. UV–vis and electrochemical measurements displayed that the nanocomposite provides a biocompatible matrix for the immobilization of Mb and also facilitates DET between its active center and the surface of the electrode. Thus, the present biosensor exhibited good analytical performances such as low detection limit, low Michealis–Menten constant, favorable stability and reproducibility towards the quantification of H2O2.

18 h at 180 8C. The obtained products were filtered out using a 0.22 mm filter membrane and washed with distilled water and absolute alcohol repeatedly, then dried at 100 8C for 10 h. 2.3. Electrode modification Prior to use, glassy carbon electrode (3 mm, GCE) was polished on a polishing cloth with 1.0, 0.3, 0.05 mm alumina powder, respectively, and rinsed with deionized water followed by sonicating in acetone, ethanol and deionized water successively. ZrO2/MWCNT suspension was obtained by dispersing 4 mg ZrO2/ MWCNT powder in 1.0 mL 0.1 M phosphate buffer solution (PBS, pH 7.0). After mixing 5 mL 5.0 mg mL1 Mb solution (dissolved in 0.1 M pH 7.0 PBS) with 10 mL ZrO2/MWCNT suspension, 5 mL of the mixture was dropped onto the electrode surface and allowed to dry at room temperature overnight. The obtained sensor was denoted as Mb/ZrO2/MWCNT/GCE. For comparison, Mb/ZrO2 and Mb/ MWCNT modified electrodes were also prepared using the same procedure. Briefly, after mixing 5 mL 5.0 mg mL1 Mb solution with 10 mL ZrO2 or MWCNTs 4.0 mg mL1 suspension, 5 mL the mixture was cast onto the electrode to form the Mb/ZrO2 or Mb/ MWCNT modified electrode. 2.4. Apparatus and measurements

2. Materials and methods 2.1. Reagents and chemicals Myoglobin (Mb) from equine skeletal muscle (molecular weight, MW, 17,800) was purchased from Aldrich and used as received. Multi-walled carbon nanotubes (MWCNTs, with length of 5–15 mm, external diameter 10–20 nm and surface area 40– 300 m2 g1) were purchased from Shenzhen Nanotech Port Co. (Shenzhen, China) and purified using literature techniques [32]. ZrO2 NPs (about 20 nm) were prepared using literature procedure [33]. ZrOCl28H2O was purchased from Sinopharm Chemical Reagent CO. Ltd. China. All other chemicals were of analytical grade and doubly distilled water was used throughout.

Transmission electron microscopy (TEM) images were recorded on a Hitach-600 transmission electron microscope at an accelerating voltage of 75 kV. X-ray diffraction (XRD) patterns were recorded on a Rigaku powder diffractometer equipped with Cu Ka1 radiation (l = 1.5406 A˚). Ultraviolet–visible (Uv–vis) spectroscopy experiments were performed with a UV-2450 spectrophotometer (Shimadzu). Electrochemical measurements were performed on an Autolab PGSTAT30 electrochemical workstation (Eco Chemie). A three-electrode system was used including an Ag/AgCl (saturated KCl) reference electrode, a platinum wire auxiliary electrode, and the modified electrode as the working electrode. All experimental solutions were deoxygenated by bubbling highly pure nitrogen for 15 min and maintained under nitrogen atmosphere during the entire measurements.

2.2. Preparation of ZrO2/MWCNT composite 3. Results and discussion ZrO2/MWCNT composite was prepared using the Shan’s method [22]. In short, 70 mg of the purified MWCNTs were added into vigorously stirred 30 mL ZrOCl28H2O solution (0.03 M). After being ultrasonicated for 2 h, the black solution was put into a stainless steel Teflon-lined autoclave of 50 mL capacity and maintained for [(Fig._1)TD$IG]

3.1. Characteristics of the ZrO2/MWCNTs composite Fig. 1(A) shows the TEM image of MWCNTs before modification with ZrO2 NPs. It can be seen that the MWCNTs are 10–15 nm in

Fig. 1. TEM images of the pristine MWCNTs (A) and ZrO2/MWCNT nanocomposite (B).

[(Fig._3)TD$IG]

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3.2. UV–vis spectroscopic analysis ZrO2/MWCNT nanocomposite was used as a matrix to immobilize proteins and fabricate protein biofilms. Positions of the Soret absorption band of heme may provide the information about possible denaturation of heme proteins, especially about the conformational change in the heme group region [34]. To investigate native structures of Mb immobilized in ZrO2/MWCNT nanocomposite film, UV–vis absorption was carried out. As seen in Fig. 3, no prominent adsorption peak is observed in ZrO2/MWCNT film at 310–550 nm (curve a). However, after Mb immobilized in the ZrO2/MWCNT film, the absorption peak of 409 nm is obtained (curve b), which is identical to the position of the native Mb film (curve c), indicating that the native structure and conformation of the immobilized Mb in Mb/ZrO2/MWCNT nanocomposite film were well-retained. Thus, the ZrO2/MWCNT nanocomposite did

[(Fig._2)TD$IG] m

C--MWNTs m--monoclinic ZrO2 t--tetragonal ZrO2

tm

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2θ (degree) Fig. 2. X-ray diffraction patterns of pristine MWCNTs (a) and ZrO2/MWCNT nanocomposite (b).

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diameter with a rather smooth surface. After hydrothermal treatment of MWCNTs in ZrOCl28H2O aqueous solution, it can be clearly seen that the surface of MWCNTs become roughened and lots of ZrO2 NPs are spontaneously attached on the sidewalls of MWCNTs (Fig. 1(B)). Also, it is interesting that almost all the ZrO2 NPs preferentially adhere to the surfaces of MWCNTs rather than to other regions without MWCNTs. The process of the growth of ZrO2 NPs on the sidewalls of MWCNTs might be described as follows [22]: the positively charged precursors of ZrOCl28H2O are electrostatically adsorbed onto the surface of negatively charged MWCNTs, and these precursors act with the carboxylic acid groups on the surface of MWCNTs to form C–O–Zr bonds through esterification, and later ZrO2 NPs are spontaneously formed on the surface of MWCNTs. The formation of ZrO2/MWCNT nanocomposite was indicated by the characteristic reflections in the XRD patterns as shown in Fig. 2. The most intense peaks of MWCNTs correspond to the (0 0 2) and (1 1 0) reflection in Fig. 2(a), respectively. For sample ZrO2/ MWCNT nanocomposite, all the diffractive peaks in Fig. 2(b) belong to the characteristic peaks of m-ZrO2 (1 1 1), t-ZrO2 (1 1 1) and MWCNTs (0 0 2), but the peaks of m-ZrO2 are dominant. The characteristic peak of MWCNTs (0 0 2) is weaken seriously, which mainly results from the coverage of MWCNTs with ZrO2 NPs. Combining the results of TEM and XRD, it can be demonstrated that the ZrO2/MWNT composite were successfully prepared through hydrothermal treatment of MWCNTs in ZrOCl28H2O aqueous solution.

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b 0.4

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360

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Wavelength / nm Fig. 3. UV–vis spectra of the ZrO2/MWCNT film (a), Mb/ZrO2/MWCNT film (b) and Mb film (c) deposited on a quartz glass side.

not change the fundamental microenvironment of Mb and retained the natural secondary structure of Mb. 3.3. Direct electrochemistry of Mb/ZrO2/MWCNT modified electrode Fig. 4(A) shows typical cyclic voltammograms of different modified electrodes at 100 mV s1 in 0.1 M PBS solution. The Mb/ ZrO2/MWCNT modified electrode gave a couple of stable and welldefined redox peaks at 312 and 375 mV (curve a), while no redox peaks were observable at ZrO2/MWCNT modified electrode (curve d). Obviously, the response of the Mb/ZrO2/MWCNT modified electrode was attributed to the redox of the electroactive centers in the immobilized Mb. The Mb/ZrO2 (curve b) and Mb/ MWCNT (curve c) modified electrodes also displayed a couple of redox peaks of Mb. Experimental results suggested that both MWCNTs and ZrO2 NPs would realize the DET between Mb and the electrode, consistent with the literature reports [35,36]. However, the anodic and cathodic peak currents at the Mb/ZrO2 and Mb/ MWCNT modified electrodes were smaller than those at the Mb/ ZrO2/MWCNT modified electrode. It suggested that integration of MWCNTs and ZrO2 NPs offered a synergetic effect for the DET of Mb. On the one hand, MWCNTs could not only enhance the conductivity of the matrix but also provide conductive pathway to accelerate electron transfer between the immobilized Mb and the electrode. On the other hand, ZrO2 NPs provided a microenvironment for preserving the natural structure of the immobilized protein molecules and more spatial freedom for Mb in its orientation also favored the DET between the protein molecules and the conductor surface. The formal potential E1/2 of the heme FeIII/II couple in Mb/ZrO2/MWCNT modified electrode, estimated as the midpoint of reduction and oxidation potentials, was 344 mV in 0.1 M pH 7.0 PBS. This value was similar to those of 342 mV at Mb–silk fibroin [37] and 342 mV at Mb–colloidal gold [38], suggesting that most molecules preserved their native structure after being immobilized in the Mb/ZrO2/MWCNT film. The effect of scan rate on electrochemistry of the immobilized Mb is shown in Fig. 4(B). With an increasing scan rate from 50 to 1000 mV s1, the redox peak currents increased linearly (inset in Fig. 4(B)), indicating a surface-controlled process. From the integration of the reduction peak of Mb/ZrO2/MWCNT modified electrode at different scan rates [39], an average surface coverage (G*) of Mb was calculated to be 1.36  1010 mol cm2, which was much larger than 5.18  1011 mol cm2 at Mb–agarose hydrogel electrode [6] and 8.85  1011 mol cm2 at {[SiO2–(Mb/PSS)m]/ PEI}n [40]. It was possible that the ZrO2/MWCNT nanocomposite provided a larger surface area available for protein binding and

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Fig. 4. (A) Cyclic voltammograms of Mb/ZrO2/MWCNT (a), Mb/ZrO2 (b), MWCNT/Mb (c), ZrO2/MWCNT (d) modified electrodes in 0.1 M PBS (pH 7.0) at 100 mV s1. (B) Cyclic voltammograms of Mb/ZrO2/MWCNT modified electrode in 0.1 M PBS (pH 7.0) at 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mV s1 (from lowest to highest current). Inset: plot of peak current vs. scan rate.

The direct electrochemistry of the Mb/ZrO2/MWCNT modified electrode exhibited a strong dependence on solution pH, as shown in Fig. 5. With the increasing of solution pH from 5.3 to 9.2, the negative shifts of both reduction and oxidation peak potentials were observed. In general, all changes in the peak potentials and currents with solution pH were reversible in the pH range from 5.3 to 9.2, which was the same CVs could be obtained if the electrode was transferred from a solution with a different pH value to its original solution. The plot of formal potential vs. pH showed a slope of 47.9 mV pH1 (inset in Fig. 5), which was close to 48.7 mV pH1 for Mb–clay films [45] and the expected value of 57.8 mV pH1 for a single proton transfer coupled to a reversible single electron transfer at 291 K [46]. It suggests that a single proton participated in the electron transfer process [47,48], which is represented in general terms by Mb heme Fe(III) + H+ + e fi Mbheme Fe(II). 3.5. Electrocatalysis of Mb/ZrO2/MWCNT modified electrode to reduction of H2O2 Mb immobilized on an electrode surface normally exhibits electrocatalytic activity for oxygen, H2O2, trichloroacetic acid

MbFeðIIIÞ þ e ! MbHFeðIIÞ

(1)

H2 O2 þ 2MbHFeðIIÞ ! 2MbFeðIIIÞ þ 2H2 O

(2)

In the presence of H2O2, MbHFe(II) was efficiently converted to its oxidized form, MbFe(III). Consequently, more MbFe(III) molecules were reduced at the electrode surface by the DET. Thus, the reduction current shown in the CVs increased for this reaction. The reduction peak current increased linearly with increasing H2O2 concentration over a wide range. Based on the transduction mechanism of the peroxide sensor and the linear

[(Fig._5)TD$IG]

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pH 5.3 E/V

3.4. Effect of solution pH on the DET of immobilized Mb

(TCA), and NO2 [49,50]. By using H2O2 as a model, the biocatalytic ability of the protein is examined. Upon addition of H2O2 to 0.1 M pH 7.0 PBS, the electrochemical behavior of Mb immobilized in Mb/ZrO2/MWCNT modified electrode changed evidently. The reduction peak current of immobilized Mb increased dramatically and oxidation peak current decreased (Fig. 6A), the reduction peak current increased with H2O2 concentration in solution (curves b– h), displaying an obvious electrocatalytic behavior of the Mb to the reduction of H2O2. The electrocatalytic process can be expressed as follows:

Current /μA

increased the loading of Mb. The electroactive Mb was about 2.1% of the total Mb content deposited on the Mb/ZrO2/MWCNT composite modified electrode, which was higher than 1.1% at Mb–TNS electrode [41]. Obviously, not all protein molecules could achieve the DET with the electrode surface. Maybe just those in the inner layers of the film closest to the electrode surface has optimized position to establish electrical contacting of the active centers with the electrode surface and contribute to the observed redox responses. On the other hand, the anodic peak potentials slightly shifted to the positive direction and the cathodic peak potentials slightly shifted to the negative direction at higher scan rate, which resulted in an increase of the peak separation between anodic and cathodic peak. The peak separation at higher scan rate could be used to estimate the heterogeneous electron transfer rate constant (ks). According to the method of Laviron [42], the average value of (ks) was estimated to be 1.52 s1, which was larger than those of 0.93 s1 for Mb immobilized on DL-homocysteine selfassembled gold electrode [43], 1.34 s1 for Mb immobilized in silk fibroin film [37], 1.2 s1 for Mb entrapped in agarose hydrogel film in room-temperature ionic liquid [44], suggesting a reasonably fast electron transfer between the immobilized Mb and the electrode due to the presence of ZrO2/MWCNT nanocomposite.

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E / V vs. Ag/AgCl Fig. 5. Cyclic voltammograms of Mb/ZrO2/MWCNT modified electrode in a buffer solution at pH values of 5.3, 6.2, 7.0, 8.0, and 9.2 (from left to right) at 100 mV s1. Inset: Plot of formal potential vs. pH.

[(Fig._6)TD$IG]

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resulted from the interaction between Fe3+ and H2O2 was similar to that reported previously [54] and could be excluded by adding EDTA in the sample solution.

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3.7. Stability and reproducibility of the H2O2 biosensor

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t/s Fig. 6. Amperometric response of the sensor at an applied potential of 400 mV to successive additions of H2O2 in 0.1 M PBS (pH 7.0). Inset: H2O2 concentration dependence of the CV response of the biosensor in 0.1 M PBS (pH 7.0) at a scan rate of 100 mV s1. From (a–h) the H2O2 concentrations are 0, 10, 15, 25, 45, 65 and 115 mM, respectively (A) and plot of steady-state current vs. H2O2 concentration (B).

The Mb/ZrO2/MWCNT modified electrode could retain the direct electrochemistry of the immobilized Mb at constant current values in 0.1 M pH 7.0 PBS upon the continuous CV sweep over the potential range from 0.8 to +0.2 V at 100 mV s1. After cyclically swept at 100 mV s1 for 50 times the immobilized Mb lost only 5.0% of its initial activity. When the sensor was not in use, it was stored in 0.1 M pH 7.0 PBS at 4 8C. A storage period of a week almost did not change the currents of the DET and the responses to H2O2. The sensor could retain 95% of its initial response to H2O2 after two months, demonstrating good long-term stability. The fabrication of five electrodes, made independently, showed an acceptable reproducibility with the R.S.D of 1.5% for the current determination of 50 mM H2O2. Thus, the good long-term stability and acceptable reproducibility can be attributed to the good biocompatibility of the ZrO2/MWCNT nanocomposite, which can provide a favorable microenvironment for Mb to retain its bioactivity. 4. Conclusions

response of the catalytic current with H2O2 concentration, H2O2 could be detected quantitatively. The amperometric response of Mb/ZrO2/MWCNT modified electrode with successive addition of H2O2 to 0.1 M pH 7.0 PBS at an applied potential of 400 mV is shown in Fig. 6. It was observed that the current value achieved 95% of the steady state response within 10 s, indicating a fast response. The fast response can be mainly attributed to the fast diffusion process and high electronic conductivity of the ZrO2/MWCNT nanocomposite. The current response of the modified increased along with H2O2 concentration. The linear response range of the modified electrode to H2O2 concentration was from 1.0 to 116.0 mM with a linear regression equation of I (mA) = 0.061 + 0.017c (mM) (R = 0.9999, n = 30) (Fig. 6B), and the detection limit was estimated to be 0.53 mM at S/N of 3s, which was lower than those of 8.0 mM for Mb immobilized in hydroxyethylcellulose [51] and 4.0 mM for Mb in ZrO2/chitosan [16]. An enzymatic saturation response was observed when the concentration of H2O2 was higher than 116.0 mM, showing a characteristic of the Michaelis–Menten kinetic mechanism. The app apparent Michaelis–Menten constant (KM ) was obtained to be 0.085 mM for H2O2 from the electrochemical version of the app Linweaver–Burk equation [52]. The KM value was much smaller than those of 1.3 mM for Mb immobilized on silver nanoparticles [53], 1.53 mM for Mb/ZrO2/chitosan [16], 0.65 mM for Mb immobilized on colloidal gold nanoparticles [38] and 0.26 mM for ZrO2/MWNTs/Mb nanobiocomposite film [31]. Thus, the good biocompatibility of ZrO2/MWCNT nanocomposite increased affinity of the immobilized Mb for H2O2. 3.6. Interference study Possible interference that might occur in real samples was tested. No significant interference (less than 2.6%) could be observed for matters, such as SO42, CO32, ClO3, Cl, Br, I, glycin and ascorbic acid, at concentrations 10 times that of H2O2 at 50.0 mM, indicating these matters coexisting in the sample matrix did not affect the determination of H2O2. However, Fe3+ might be a main interference to Mb for the electrocatalytic reduction of H2O2. When the concentration of Fe3+ was increased to 5 times that of H2O2, the peak current changed approximately 6.5%. This possibly

In conclusion, we synthesized ZrO2/MWCNT nanocomposite by hydrothermal treatment of MWCNTs in ZrOCl28H2O aqueous solution, by which stronger bonds of Zr–O–C were formed. ZrO2/ MWCNT nanocomposite was successfully used to immobilize the model protein Mb as an immobilization matrix. The obtained results revealed that the ZrO2/MWCNT nanocomposite film possessed excellent biocompatibility to retain the activity of the immobilized Mb and facilitated the direct electron exchange between Mb and electrode. The H2O2 biosensor based on the fast DET of the Mb immobilized in ZrO2/MWCNT nanocomposite film exhibited wide linear detection range, acceptable reproducibility, storage stability. The novel ZrO2/MWCNT nanocomposite can provide a good platform for redox-active proteins and enzymes, and may find wide potential applications in direct electrochemistry, biosensors, biocatalysis and biomedical devices. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (20605010; 20865003; 20805023), the Jiangxi Province Natural Science Foundation (0620039) and the Opening Foundation of State Key Laboratory of Chem/Biosensing and Chemometrics of Hunan University (2006022; 2007012). References [1] C.C. Page, C.C. Moser, X.X. Chen, P.L. Dutton, Nature 402 (1999) 47. [2] V.V. Shumyantseva, Y.D. Ivanov, N. Bistolas, F.W. Scheller, A.I. Archakov, U. Wollenberger, Anal. Chem. 76 (2004) 6046. [3] J.J. Feng, J.J. Xu, H.Y. Chen, J. Electroanal. Chem. 585 (2005) 44. [4] X.Y. He, L. Zhu, Electrochem. Commun. 8 (2006) 615. [5] Q. Lu, T. Zhou, S.S. Hu, Biosens. Bioelectron. 22 (2007) 899. [6] H.H. Liu, Z.Q. Tian, Z.X. Lu, Z.L. Zhang, M. Zhang, D.W. Pang, Biosens. Bioelectron. 20 (2004) 294. [7] L.Y. Zhao, H.Y. Liu, N.F. Hu, Anal. Bioanal. Chem. 384 (2006) 414. [8] P.L. He, N.F. Hu, J.F. Rusling, Langmuir 20 (2004) 722. [9] Z.H. Dai, S.Q. Liu, H.X. Ju, H.Y. Chen, Biosens. Bioelectron. 19 (2004) 861. [10] Y.Z. Xian, Y. Xia, L.H. Zhou, F.H. Wu, Y. Liang, L.T. Jin, Electrochem. Commun. 9 (2007) 142. [11] E.L. Crepaldi, G.J.D.A. Soler-Illia, D. Grosso, P.A. Albouy, Chem. Commun. 17 (2001) 1582. [12] M. Vallet-Regı´, S. Nicolopoulos, J. Roma´n, J.L. Martı´nez, J.M. Gonza´lez-Calbet, J. Mater. Chem. 7 (1997) 1017. [13] Z.Y. Zhang, C. Zhang, X.R. Zhang, Analyst 127 (2002) 792.

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