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Glass carbon electrode modified with horseradish peroxidase immobilized on partially reduced graphene oxide for detecting phenolic compounds
Journal of Electroanalytical Chemistry 681 (2012) 49–55
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Glass carbon electrode modified with horseradish peroxidase immobilized on partially reduced graphene oxide for detecting phenolic compounds Yan Zhang a, Jiali Zhang a, Haixia Wu a, Shouwu Guo a,⇑, Jingyan Zhang b,⇑ a National Key Laboratory of Micro/Nano Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of the Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, PR China b State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
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Article history: Received 5 December 2011 Received in revised form 31 May 2012 Accepted 6 June 2012 Available online 17 June 2012 Keywords: Partially reduced graphene oxide Horseradish peroxidase Glass carbon electrode Phenolic compounds
a b s t r a c t A glass carbon (GC) electrode has been modified with horseradish peroxidase (HRP) molecules, which are immobilized on the partially reduced graphene oxide (PCRG). The surface properties of the as-modified electrode are characterized with scanning electron microscopy (SEM), the electrochemical characteristics of the as-modified electrode are studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). We demonstrated that the PCRG can promote the electron transfer between HRP and GC electrode, and the immobilized HRP maintained its catalytic activity of the decomposition of phenol and p-chlorophenol. The GC electrode modified with PCRG immobilized HRP exhibits better electrochemical property over CRG, the modified electrode may find practical application as enzyme-based amperometric sensors used for detections of phenolic molecules or other permanent organic pollutants in water. The method provides a strategy for preparation of a sensitive amperometric sensor for the detection of phenolic compounds and other permanent organic pollutants. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Graphene has been considered as an ideal electrode material due to its ultra-large specific surface area, and excellent electrical conductivity [1,2]. However, the low solubility in water and most organic solvents, and the lack of surface functionality of graphene limit its practical application as biosensors. Differently, the partially reduced graphene oxide (PCRG), which can be prepared by chemical reduction of graphene oxide (GO) in aqueous solution using hydrazine [3,4], L-ascorbic acid [5], or hydroxylamine [6] in bulk scale production, has not only decent solubility in water and manifest electrical conductivity, but partially maintains surface functional groups of GO for external species tethering [7]. Thus, the PCRG should be appropriate to the electrodes used for construction of amperometric sensors [8–10]. In fact, it has been demonstrated recently that graphene and CRG both can accelerate the electron transfer between the substrate and enzyme, such as glucose oxidase [11], and promote the electrocatalytic performance to small molecules, such as H2O2 [12], NADH [13,14] and dopamine [15]. We previously demonstrated that PCRG exhibits excellent enzyme loading ability, the maximum loading for HRP was 1.3 mg per milligram of PCRG, which is much higher than that using GO and many other classical materials [16]. ⇑ Corresponding authors. Tel.: +86 21 34206915 (S. Guo). E-mail addresses: [email protected] (S. Guo), [email protected] (J. Zhang). 1572-6657/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2012.06.004
As an important heme-containing protein, horseradish peroxidase (HRP) has been applied widely in biotransformation, organic synthesis and treatment of waste waters [17]. However, due to the denaturalization of HRP adsorbed on the electrode surface, to study the HRP immobilized directly on a bare electrode is difficulty [18]. In addition, the active site of HRP is deep inside the protein, and the long distance between the active site and the surface of electrode may slow down the rate of electron transfer [19,20]. Hence, new materials or methodologies are required to modify electrode surface for HRP immobilization to obtain a more sensitive electrode. The modification of the electrode surface with PCRG could increase the conductance of the electrode, thus is possible to increase the electron transfer between enzyme and the electrode. Many works have been done to improve the sensitivity of the electrode with HRP immobilized, for instances, Serra et al. reported that the glucose oxidase and HRP could be co-immobilized by simple inclusion into the bulk of graphite-Teflon pellets as electrodes for detection of phenolic compounds [21]. Elyacoubi et al. used gold modified nanoporous silica as a substrate for the HRP immobilization, and used as the electrode to the detection of H2O2 and hydroquinone [22]. HRP could also be immobilized on nanoporous copper by adsorption as a biosensor that was very sensitive to o-phenylenediamine detection [23], and on chitosan for the detection of H2O2 [24]. Nevertheless, few studies about the GC electrode modified with HRP that was immobilized on graphene for the detection of phenolic compounds have been reported to date.
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Based on our previous work that HRP immobilized on PCRG with a high enzyme loading and exhibits high catalytic activity [16]. Herein, we describe a facile approach to modify the GC electrode with PCRG that immobilized HRP for the detection of phenolic compounds. We found that the GC electrode modified with PCRG immobilized HRP has better electrochemical properties over CRG because PCRG has a better water-solubility and relatively high conductance. The morphologies of the as-modified GC electrodes were characterized using scanning electron microscopy (SEM). The cyclic voltammetry (CV) and differential pulse voltammetry (DPV) data indicated that the HRP/PCRG/GC could promote the electron transfer of HRP, and can be used in H2O2, phenol and p-chlorophenol detection. 2. Experimental section 2.1. Materials HRP (E.C. 1.11.1.2) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and stored at 20 °C before using. The stock solution of HRP of 8 mg/mL was prepared in phosphate buffer and stored at 4 °C. All other chemicals were analytical grade and were used as purchased. Phosphate buffer containing 0.1 M K2HPO4, and 0.1 M KH2PO4 was adjusted to the desired pH. Diluted aqueous solutions of hydrogen peroxide, phenol and p-chlorophenol were freshly prepared as needed. 2.2. Preparation of partially reduced graphene oxide (PCRG) GO was prepared using natural graphite powder through a modified Hummer’s method that was described in details in our previous works [5]. The reduction of GO using L-AA as a reductant was performed in water at room temperature (23 °C). In a typical experiment, 50 mg of L-AA was added to 50 mL (0.1 mg/mL) of aqueous dispersion of GO under vigorous stirring for different time. The PCRG products were separated from the reaction mixtures through filtration, washed three times with ultrapure water, and finally redispersed in ultrapure water for further usage. The PCRGs reduced for 12 and 24 h, were used and named in this work as CRG12H and CRG24H, respectively. 2.3. Enzyme immobilization Typically, 100 lL PCRG (1.0 mg/mL) was mixed with 100 lL, HRP (8.0 mg/mL) in phosphate buffer, pH 7.4. The mixture was then incubated for 30 min on ice with shaking. The resulting solution was stored at 4 °C prior to use. 2.4. Modification of GC electrode A circle GC electrode, 3 mm in diameter, was polished sequentially by 1.0 lm, 0.3 lm, 0.05 lm of Al2O3 in microcloth, and ultrasonicated in ultrapure water and ethanol for 5 min, respectively. Finally, the electrode was dried with high pure nitrogen. The GC modification with PCRG immobilized with HRP was achieved through a solution casting procedure. Typically, 5 lL of aqueous dispersion of PCRG (0.5 mg/mL) with HRP (HRP/PCRG) was dropped at the center of GC electrode, and dried at 4 °C for 24 h. The modified electrode was soaked in phosphate buffer, pH 7.4, for 15 min to remove free enzymes before the electrochemical test. The modified electrode was stored in the phosphate buffer (pH 7.4) solution at 4 °C when not in use. 2.5. Electrochemical measurement The electrochemical measurements were performed on a CHI 660C electrochemical workstation (Chen Hua Co., China) using a
three electrode system, with bare GC or modified GC as working electrode, platinum wire as counter electrode, and saturated calomel electrode as reference electrode. The electrolyte solution purged with pure N2 to remove oxygen for 15 min before the electrochemical measurement, and was maintained in nitrogen atmosphere during the experiment. Except for especially noted, the electrolyte is 0.05 M pH 7.4 phosphate buffer, and the scan rate is 50 mV/s. 2.6. Characterization Atomic force microscopic (AFM) images were taken on a MultiMode Nanoscope V scanning probe microscopy system (Veeco, USA). The commercially available AFM cantilever tips with a force constant of 48 N/m and resonance vibration frequency of 330 kHz were used. The scanning rate was set usually at 0.7– 1 Hz. Scanning electron microscopic (SEM) images of modified GC electrode were acquired on an Ultra 55 field emission scanning electron microscopy (Zeiss, Germany) with 3 kV of acceleration voltage. 3. Results and discussion As shown in Fig. 1, the AFM images and corresponding height profiles illustrate that the CRG12H and CRG24H have similar height of 0.9 nm, and reveal also their single atomic layered features. The reduction extents of GO were monitored by UV–Vis, FT– IR and Raman spectroscopy [5]. The morphology of the bared electrode (Fig. S1), HRP/CRG12H/GC and HRP/CRG24H/GC, were characterized using SEM. As depicted in Fig. 2a and b, homogeneous HRP/CRG12H and HRP/CRG24H thin films on the GC electrode surface were produced. Fig. 2c and d show that the morphologies of HRP/CRG12H/GC and HRP/CRG24H/GC were preserved well after recycling hundred times for the CV measurements. This result indicates that both HRP/CRG12H and HRP/CRG24H modified GC electrode should be stable enough for electrochemical study. The electrochemical properties of HRP/CRG12H/GC, HRP/ CRG24H/GC were first compared with that of HRP/GO/GC. As shown in Fig. 3a, in comparison to the system PCRG/GC without HRP (Fig. S2), the cathodic current peak appeared at 0.30 V in the CV curves of HRP/CRG12H/GC and HRP/CRG24H/GC, revealing that the direct electron transfer between electrode and HRP could be achieved [24]. In comparison with the CV curves of HRP/GO/GC and HRP/CRG12H/GC electrodes, the CV curve of HRP/CRG24H/GC electrode that acquired under the same conditions exhibited the largest cathodic peak current, suggesting that CRG24H has better conductivity. Hence, the as-prepared HRP/CRG24H/GC was used as the electrode for the following studies. Different from the GC electrode modified with graphene reported in the literatures [11,24], the background current and cathodic peak current of CV curves of HRP/CRG24H/GC electrode are relative small, due might to low conductivity of the partially reduction extent of CRG24H. Additionally, the immobilization of HRP molecules on both sides of the CRG24H sheets may decrease the conductivity of the HRP/ CRG24H/GC electrode as well. However, the high enzyme loading and stability of CRG24 make GC electrode modification with it is worthwhile. We also examined the effect of potential (voltage) scan rate on the current of the HRP/CRG24H/GC electrode. As shown in Fig. 3b, the peak current and the potential difference between cathodic and anodic peaks increased with increasing of scan rate from 10 to 300 mV/s. Inset in Fig. 3b shows a linear relationship between the cathodic peak current and square root of scan rate, the high correlation coefficient illustrates that the redox reaction of immobilized HRP on the HRP/CRG24H/GC electrode should be a diffusion controlled process [25]. Fig. 4a shows the effect of pH value on the
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Fig. 1. (a and c) tapping mode AFM images of GO sheets being reduced via L-AA for 12 and 24 h on mica surface. (b and d) the corresponding height profiles of the AFM images.
Fig. 2. SEM images of HRP/CRG12H/GC electrode and HRP/CRG24H/GC electrodes before (a and b), and after (c and d) performing 100 times continuous cycle of CV measurements, respectively.
performance of the HRP/CRG24H/GC electrode. The pH dependent peak current of the electrode is consisting with the pH dependence of the catalytic activity of free HRP. The lower or higher pH value
resulted in the loss of HRP catalytic activity, and consequently the decrease of peak current. Thus, we perform all other electrochemical experiments in the electrolyte solutions with pH 7.4.
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Fig. 3. (a) CVs of HRP/GO/GC, HRP/CRG12H/GC, HRP/CRG24H/GC electrodes in phosphate buffer. (b) CVs of HRP/CRG24H/GC electrode in phosphate buffer at various scan rates from 10, 25, 50, 100, 150, 200 and 250 to 300 mV/s, respectively. Inset in (b) shows plot of peak currents as a function of square root of scan rate.
The performance of the HRP/CRG24H/GC electrode was also examined in the presence of H2O2, which can be decomposed by HRP. When H2O2 was successively added to phosphate buffer, the cathodic peak current of HRP/CRG24H/GC at 0.3 V increased gradually as shown in Fig. 4b, with 0.5 mM H2O2, the peak current can be increased more than 10 times, suggesting that a fast direct electron transfer took place between the immobilized HRP and electrode surface with the assistance of CRG24H [26,27]. Because the differential pulse voltammetry (DPV) exhibits relatively lower background current and higher sensitivity than that of CV [28], we acquired DPV data of the HRP/CRG24H/GC electrode in phosphate buffer containing H2O2 with concentrations ranged from 1 lM to 10 mM (Fig. 5a). The typical current peaks of the HRP/CRG24H/GC electrode in the DPV is composed of two separated peaks at 0 and 0.18 V. The DPV curves of the HRP/ CRG24H/GC electrode show that the cathodic peak current gradually increased with increasing of H2O2 concentration. Comparably, the peak at 0.18 V is relatively more sensitive to the H2O2 concentration, especially at the lower concentration region. Therefore, the maximum cathodic peak currents at 0.18 V versus H2O2 concentrations were plotted in Fig. 5b. Overall I–C curve showed three very different response phases. The insets in Fig. 5b are the blow ups of each phases, illustrating that HRP/CRG24H/GC electrode has good linear responses to H2O2 in the concentration ranges of 0.001–0.09 mM, 0.1–0.9 mM, 1.0–10.0 mM, and the slopes are 24.8, 1.96 and 0.11 lA/mM, respectively. The modified electrode showed relatively high sensitivity in 1–100 lM H2O2 region. Lower sensitivity at high H2O2 concentration might due to the inactive
enzyme formed with the excess of H2O2 that inhibits the HRP catalysis [21,29]. Nevertheless, these results imply that the HRP/ CRG24H/GC electrode can efficiently detect H2O2, especially when its concentration is lower than 0.1 mM. The phenolic compounds detection, including phenol and p-chlorophenol, with HRP/CRG24H/GC was also investigated. The detection is based on the oxidation of phenolic compounds by HRP with H2O2, thus, the concentration of H2O2 was kept constant. As depicted in Fig. 6a, when the concentration of H2O2 was 0.02 mM, the cathodic peak current at 0.25 V increased with the addition of the phenol, indicating that HRP/CRG24H/GC electrode involved in the oxidization of phenol. More specifically, a redox peak near 0.25 V appeared, and the peak current of this peak is increased with increasing of phenol concentration too. We speculated that the reduction peak near 0.25 V corresponds to the reduction of o-quinone, the oxidation product of phenol by HRP [30,31]. Because the half-wave potential of o-quinone was observed at 0.2 V in phosphate buffer at pH 7.0 [32]. In order to confirm this assumption, we investigated the electrochemical redox reaction of 0.1 mM hydroquinone and 0.1 mM phenol using HRP/CRG24H/ GC electrode. For hydroquinone, as shown in Fig. S3a, there were two reduction peaks at 0.35 V and 0.02 V in negative scan direction, corresponding to the reduction peak of direct electron transfer between HRP and electrode, and benzoquinone, respectively. For phenol, there were also two reduction peaks 0.25 V and 0.25 V in negative scan direction, corresponding to the reduction peaks of HRP catalytic intermediate II [33,34] and o-quinone, as shown in Fig. S3b. We, therefore, believe that the phenol molecules
Fig. 4. (a) The plot of peak current as a function of pH. (b) CVs of HRP/CRG24H/GC electrode in phosphate buffer with successive addition of 0.05 mM H2O2 at 50 mV/s.
Y. Zhang et al. / Journal of Electroanalytical Chemistry 681 (2012) 49–55
Fig. 5. (a) DPVs of HRP/CRG24H/GC electrode in phosphate buffer with successive addition of H2O2. (b) The plot of peak currents at H2O2. Inset in (b) shows relation of peak current versus the concentration of H2O2.
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0.2 V as a function of concentration of
Fig. 6. (a) CVs of HRP/CRG24H/GC electrode in phosphate buffer containing phenol with different concentration at 50 mV/s. (b) DPVs of HRP/CRG24H/GC electrode in phosphate buffer acquired during the successive addition of phenol.
were oxidized by the HRP intermediate forming o-quinone, and the reduction peaks appeared near 0.25 V and 0.25 V are characteristic peaks for phenol using HRP/CRG24H/GC electrode. Consequently, HRP/CRG24H/GC electrode can be used as a sensor to identify phenol and hydroquinone based on their different reduction potentials. To elucidate the detection sensitivity of HRP/CRG24H/GC electrode to phenol and p-chlorophenol, the DPV data were acquired from the electrolyte solutions containing phenol or p-chlorophenol molecules with different concentrations of 1 lM to 10 mM. As shown in Fig. 6b, the current of cathodic peak at 0.2 V increased with increasing of concentration of phenol. A reduction peak of oquinone at 0.27 V appeared in the DPV, also increased with increasing of concentration of phenol. Hence, the reduction peaks at 0.27 V and 0.2 V should be both diagnostic for phenol detection. The curve of cathodic peak current of o-quinone at 0.27 V versus phenol concentration, as shown in Fig. 7a, could be fitted into three linear phases corresponding to the phenol concentration ranges of 0.05–0.1 mM, 0.2–1.0 mM, 2.0–10.0 mM, and the slopes were 61, 9.1 and 0.8 lA/mM, respectively. The limit of detection was 4.4 lM at a signal to noise ratio of 3. Similarly, Fig. 7b shows that the current of the reduction peak at 0.2 V versus phenol concentrations could also be divided into three linear parts in the ranges of 0.001–0.09 mM, 0.1–0.9 mM, 1.0–10.0 mM, and the slopes were 32, 1.7 and 0.05 lA/mM, respectively. The limit of
detection was 12.8 lM. Apparently, the reduction peaks at 0.27 V is more diagnostic for phenol detection in terms of the detection limit and sensitivity. These results demonstrated that the HRP/ CRG24H/GC electrode could be used as a sensor to detect phenol for a wide concentration range. Similarly, the applicability of using HRP/CRG24H/GC electrode to detect p-chlorophenol was also investigated. There is no other redox peak appeared in CV spectra (Fig. S4). The DPV data showed that the reduction peak at 0.27 V is much weaker than that of the phenol (Fig. S5), and a reduction peak near 0 V increased remarkably when the concentration of p-chlorophenol was increased, suggesting that the electrochemical oxidation product of p-chlorophenol is different from that of the phenol [35]. The HRP/CRG24H/GC electrode had a linear response to p-chlorophenol at 0.2 V in the concentration ranges of 1 lM to 0.8 mM with correlation coefficients were 0.997. The limit of detection was 15.2 lM at a signal to noise ratio of 3 (Fig. S4). These results demonstrated that the HRP/CRG24H/ GC electrode could be used as a sensor to detect phenolic compounds with a high sensitivity and lower detection limit. To elucidate the practical applicability of the HRP/CRG24H/GC electrode as a sensor in detecting phenol and p-chlorophenol, the currents of the reduction peak of the mixture sample at 0.3 V and 0.25 V versus the time were recorded, respectively. As shown in Fig. S6, when phenol (10 lM) or p-chlorophenol (10 lM) solution was added in the electrolyte solution containing 5 lM of H2O2, the
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Fig. 7. Plots of peak currents versus concentration of phenol in phosphate buffer, (a) the peak at 0.27 V, (b) the peak at peak current and the concentration of phenol.
current of the reduction peak varied instantly, and reached the steady state within 25 s for phenol (95%), and 40 s for p-chlorophenol (95%). The reusability and stability of HRP/CRG24H/GC electrode used for phenolic compound detection were also established. After 10 times detection using the HRP/CRG24H/GC electrode in the electrolyte containing 5 lM of H2O2 and 10 lM of phenol, the relative standard deviation (RSD) was 5.38%. Under the same condition, the RSD for p-chlorophenol detection was 6.19%. These results illustrate that the HRP/CRG24H/GC electrode has good reusability as a sensor. Moreover, we studied the long-term stability of HRP/ CRG24H/GC electrode stored at 4 °C. For electrolyte solution containing 5 lM of H2O2 and 10 lM of phenol, the detected current of the reduction peak of phenol with HRP/CRG24H/GC electrode stored at 4 °C for 3 and 7 days decreased by 5.2% and 7.0% in comparison to the data obtained using an instantly prepared electrode. For electrolyte solution containing 10 lM of p-chlorophenol, the peak current decreased by 3.4% after 3 days, and by 7.3% after 7 days. The stability of the prepared electrode is superior to the sol–gel derived HRP sensor, its activity decreased by 50% after three day storage [36]. So the better reusability and stability make this electrode as a promise biosensor. 4. Conclusions In summary, we have demonstrated that GC electrode can be modified by HRP immobilized on PCRG. The as-modified electrodes have good stability, reusability, and high catalytic activity for the decompositions of H2O2, phenol and p-chlorophenol in water. Given the simple and inexpensive advantages, this method may find practical application in the preparation of enzyme-based amperometric sensors used for detections of phenolic molecules or other permanent organic pollutants in water. Acknowledgments This work was financially supported by the NSFC (No. 91123011, 90923041, 31070742), Shanghai Committee of Science and Technology (Grant 11DZ2260600), and the National ‘‘973’’ Program (2010CB933900) of China.
0.2 V. Inset in (b) shows the linear relationships of the
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jelechem.2012. 06.004.
References [1] M. Pumera, Electrochemistry of graphene: new horizons for sensing and energy storage, Chem. Rec. 9 (2009) 211–223. [2] M. Liang, L. Zhi, Graphene-based electrode materials for rechargeable lithium batteries, J. Mater. Chem. 19 (2009) 5871–5878. [3] S. Park, R.S. Ruoff, Chemical methods for the production of graphenes, Nat. Nanotechnol. 4 (2009) 217–224. [4] S. Stankovich, D.A. Dikin, R.D. Piner, K.A. Kohlhaas, A. Kleinhammes, Y. Jia, Y. Wu, S.T. Nguyen, R.S. Ruoff, Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon 45 (2007) 1558–1565. [5] J. Zhang, H. Yang, G. Shen, P. Cheng, J. Zhang, S. Guo, Reduction of graphene oxide via L-ascorbic acid, Chem. Commun. 46 (2010) 1112–1114. [6] X. Zhou, J. Zhang, H. Wu, H. Yang, J. Zhang, S. Guo, Reducing graphene oxide via hydroxylamine: a simple and efficient route to graphene, J. Phys. Chem. C 115 (2011) 11957–11961. [7] H.C. Schniepp, J.-L. Li, M.J. McAllister, H. Sai, M. Herrera-Alonso, D.H. Adamson, R.K. Prud’homme, R. Car, D.A. Saville, I.A. Aksay, Functionalized single graphene sheets derived from splitting graphite oxide, J. Phys. Chem. B 110 (2006) 8535–8539. [8] R.L. McCreery, Advanced carbon electrode materials for molecular electrochemistry, Chem. Rev. 108 (2008) 2646–2687. [9] I. Dumitrescu, P.R. Unwin, J.V. Macpherson, Electrochemistry at carbon nanotubes: perspective and issues, Chem. Commun. (2009) 6886–6901. [10] Y. Shao, J. Wang, H. Wu, J. Liu, I.A. Aksay, Y. Lin, Graphene based electrochemical sensors and biosensors: a review, Electroanalysis 22 (2010) 1027–1036. [11] C. Shan, H. Yang, J. Song, D. Han, A. Ivaska, L. Niu, Direct electrochemistry of glucose oxidase and biosensing for glucose based on graphene, Anal. Chem. 81 (2009) 2378–2382. [12] M. Zhou, Y. Zhai, S. Dong, Electrochemical sensing and biosensing platform based on chemically reduced graphene oxide, Anal. Chem. 81 (2009) 5603– 5613. [13] L. Tang, Y. Wang, Y. Li, H. Feng, J. Lu, J. Li, Preparation, structure, and electrochemical properties of reduced graphene sheet films, Adv. Funct. Mater. 19 (2009) 2782–2789. [14] H. Liu, J. Gao, M. Xue, N. Zhu, M. Zhang, T. Cao, Processing of graphene for electrochemical application: noncovalently functionalize graphene sheets with water-soluble electroactive methylene green, Langmuir 25 (2009) 12006–12010. [15] Y. Wang, Y. Li, L. Tang, J. Lu, J. Li, Application of graphene-modified electrode for selective detection of dopamine, Electrochem. Commun. 11 (2009) 889– 892.
Y. Zhang et al. / Journal of Electroanalytical Chemistry 681 (2012) 49–55 [16] Y. Zhang, J. Zhang, X. Huang, X. Zhou, H. Wu, S. Guo, Assembly of graphene oxide-enzyme conjugates through hydrophobic interaction, Small 8 (2012) 154–159. [17] N.C. Veitch, Horseradish peroxidase: a modern view of a classic enzyme, Phytochemistry 65 (2004) 249–259. [18] J. Hong, A.A. Moosavi-Movahedi, H. Ghourchian, A.M. Rad, S. Rezaei-Zarchi, Direct electron transfer of horseradish peroxidase on Nafion-cysteine modified gold electrode, Electrochim. Acta 52 (2007) 6261–6267. [19] J. Hong, H. Ghourchian, A.A. Moosavi-Movahedi, Direct electron transfer of redox proteins on a Nafion-cysteine modified gold electrode, Electrochem. Commun. 8 (2006) 1572–1576. [20] Y. Tian, L. Mao, T. Okajima, T. Ohsaka, Superoxide dismutase-based thirdgeneration biosensor for superoxide anion, Anal. Chem. 74 (2002) 2428– 2434. [21] B. Serra, A.J. Reviejo, J.M. Pingarron, Composite multienzyme amperometric biosensors for an improved detection of phenolic compounds, Electroanalysis 15 (2003) 1737–1744. [22] A. Elyacoubi, S.I.M. Zayed, B. Blankert, J.-M. Kauffmann, Development of an amperometric enzymatic biosensor based on gold modified magnetic nanoporous microparticles, Electroanalysis 18 (2006) 345–350. [23] H. Qiu, L. Lua, X. Huang, Z. Zhang, Y. Qu, Immobilization of horseradish peroxidase on nanoporous copper and its potential applications, Bioresource. Technol. 101 (2010) 9415–9420. [24] K. Zhou, Y. Zhu, X. Yang, J. Luo, C. Li, S. Luan, A novel hydrogen peroxide biosensor based on Au–graphene–HRP–chitosan biocomposites, Electrochim. Acta 55 (2010) 3055–3060. [25] H. Yin, Q. Ma, Y. Zhou, S. Ai, L. Zhu, Electrochemical behavior and voltammetric determination of 4-aminophenol based on graphene–chitosan composite film modified glassy carbon electrode, Electrochim. Acta 55 (2010) 7102–7108.
55
[26] M. Pumera, A. Ambrosi, A. Bonanni, E.L.K. Chng, H.L. Poh, Graphene for electrochemical sensing and biosensing, Trends Anal. Chem. 29 (2010) 954–965. [27] C. Fu, W. Yang, X. Chen, D.G. Evans, Direct electrochemistry of glucose oxidase on a graphite nanosheet–Nafion composite film modified electrode, Electrochem. Commun. 11 (2009) 997–1000. [28] H. Du, J. Ye, J. Zhang, X. Huang, C. Yu, A voltammetric sensor based on graphene-modified electrode for simultaneous determination of catechol and hydroquinone, J. Electroanal. Chem. 650 (2010) 209–213. [29] V.A. Bogdanovskay, V.A. Fridman, M.R. Tarasevich, F. Scheller, Bioelectrocatalysis by immobilized peroxidase: the reaction mechanism and the possibility of electroanalytical detection of both inhibitors and activators of enzyme, Anal. Lett. 27 (1994) 2823–2847. [30] H. Sharifian, D.W. Kirk, Electrochemical oxidation of phenol, J. Electrochem. Soc. 133 (1986) 921–924. [31] M. Gattrell, D.W. Kirk, A study of the oxidation of phenol at platinum and preoxidized platinum surfaces, J. Electrochem. Soc. 140 (1993) 1534–1540. [32] J.A. Dean, Lange’s Handbook of Chemistry, Science Press, BeiJing, 2003. [33] X. Yi, J. Huang-Xian, C. Hong-Yuan, Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode, Anal. Biochem. 278 (2000) 22. [34] H. Yin, Q. Ma, Y. Zhou, S. Ai, L. Zhu, Electrochemical behavior and voltammetric determination of 4-aminophenol based on graphene-chitosan composite film modified glassy carbon electrode, Electrochim. Acta 55 (2010) 7102–7108. [35] R.A. Torres, W. Torres, P. Peringer, C. Pulgarin, Electrochemical degradation of p-substituted phenols of industrial interest on Pt electrodes. Attempt of a structure–reactivity relationship assessment, Chemosphere 50 (2003) 97–104. [36] S.A. Kane, E.I. Iwuoha, M.R. Smyth, Development of a sol–gel based amperometric biosensor for the determination of phenolics, Analyst 123 (1998) 2001–2006.
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
Glass carbon electrode modified with horseradish peroxidase immobilized on partially reduced graphene oxide for detecting phenolic compounds Yan Zhang a, Jiali Zhang a, Haixia Wu a, Shouwu Guo a,⇑, Jingyan Zhang b,⇑ a National Key Laboratory of Micro/Nano Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of the Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, PR China b State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
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Article history: Received 5 December 2011 Received in revised form 31 May 2012 Accepted 6 June 2012 Available online 17 June 2012 Keywords: Partially reduced graphene oxide Horseradish peroxidase Glass carbon electrode Phenolic compounds
a b s t r a c t A glass carbon (GC) electrode has been modified with horseradish peroxidase (HRP) molecules, which are immobilized on the partially reduced graphene oxide (PCRG). The surface properties of the as-modified electrode are characterized with scanning electron microscopy (SEM), the electrochemical characteristics of the as-modified electrode are studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). We demonstrated that the PCRG can promote the electron transfer between HRP and GC electrode, and the immobilized HRP maintained its catalytic activity of the decomposition of phenol and p-chlorophenol. The GC electrode modified with PCRG immobilized HRP exhibits better electrochemical property over CRG, the modified electrode may find practical application as enzyme-based amperometric sensors used for detections of phenolic molecules or other permanent organic pollutants in water. The method provides a strategy for preparation of a sensitive amperometric sensor for the detection of phenolic compounds and other permanent organic pollutants. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Graphene has been considered as an ideal electrode material due to its ultra-large specific surface area, and excellent electrical conductivity [1,2]. However, the low solubility in water and most organic solvents, and the lack of surface functionality of graphene limit its practical application as biosensors. Differently, the partially reduced graphene oxide (PCRG), which can be prepared by chemical reduction of graphene oxide (GO) in aqueous solution using hydrazine [3,4], L-ascorbic acid [5], or hydroxylamine [6] in bulk scale production, has not only decent solubility in water and manifest electrical conductivity, but partially maintains surface functional groups of GO for external species tethering [7]. Thus, the PCRG should be appropriate to the electrodes used for construction of amperometric sensors [8–10]. In fact, it has been demonstrated recently that graphene and CRG both can accelerate the electron transfer between the substrate and enzyme, such as glucose oxidase [11], and promote the electrocatalytic performance to small molecules, such as H2O2 [12], NADH [13,14] and dopamine [15]. We previously demonstrated that PCRG exhibits excellent enzyme loading ability, the maximum loading for HRP was 1.3 mg per milligram of PCRG, which is much higher than that using GO and many other classical materials [16]. ⇑ Corresponding authors. Tel.: +86 21 34206915 (S. Guo). E-mail addresses: [email protected] (S. Guo), [email protected] (J. Zhang). 1572-6657/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jelechem.2012.06.004
As an important heme-containing protein, horseradish peroxidase (HRP) has been applied widely in biotransformation, organic synthesis and treatment of waste waters [17]. However, due to the denaturalization of HRP adsorbed on the electrode surface, to study the HRP immobilized directly on a bare electrode is difficulty [18]. In addition, the active site of HRP is deep inside the protein, and the long distance between the active site and the surface of electrode may slow down the rate of electron transfer [19,20]. Hence, new materials or methodologies are required to modify electrode surface for HRP immobilization to obtain a more sensitive electrode. The modification of the electrode surface with PCRG could increase the conductance of the electrode, thus is possible to increase the electron transfer between enzyme and the electrode. Many works have been done to improve the sensitivity of the electrode with HRP immobilized, for instances, Serra et al. reported that the glucose oxidase and HRP could be co-immobilized by simple inclusion into the bulk of graphite-Teflon pellets as electrodes for detection of phenolic compounds [21]. Elyacoubi et al. used gold modified nanoporous silica as a substrate for the HRP immobilization, and used as the electrode to the detection of H2O2 and hydroquinone [22]. HRP could also be immobilized on nanoporous copper by adsorption as a biosensor that was very sensitive to o-phenylenediamine detection [23], and on chitosan for the detection of H2O2 [24]. Nevertheless, few studies about the GC electrode modified with HRP that was immobilized on graphene for the detection of phenolic compounds have been reported to date.
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Based on our previous work that HRP immobilized on PCRG with a high enzyme loading and exhibits high catalytic activity [16]. Herein, we describe a facile approach to modify the GC electrode with PCRG that immobilized HRP for the detection of phenolic compounds. We found that the GC electrode modified with PCRG immobilized HRP has better electrochemical properties over CRG because PCRG has a better water-solubility and relatively high conductance. The morphologies of the as-modified GC electrodes were characterized using scanning electron microscopy (SEM). The cyclic voltammetry (CV) and differential pulse voltammetry (DPV) data indicated that the HRP/PCRG/GC could promote the electron transfer of HRP, and can be used in H2O2, phenol and p-chlorophenol detection. 2. Experimental section 2.1. Materials HRP (E.C. 1.11.1.2) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China), and stored at 20 °C before using. The stock solution of HRP of 8 mg/mL was prepared in phosphate buffer and stored at 4 °C. All other chemicals were analytical grade and were used as purchased. Phosphate buffer containing 0.1 M K2HPO4, and 0.1 M KH2PO4 was adjusted to the desired pH. Diluted aqueous solutions of hydrogen peroxide, phenol and p-chlorophenol were freshly prepared as needed. 2.2. Preparation of partially reduced graphene oxide (PCRG) GO was prepared using natural graphite powder through a modified Hummer’s method that was described in details in our previous works [5]. The reduction of GO using L-AA as a reductant was performed in water at room temperature (23 °C). In a typical experiment, 50 mg of L-AA was added to 50 mL (0.1 mg/mL) of aqueous dispersion of GO under vigorous stirring for different time. The PCRG products were separated from the reaction mixtures through filtration, washed three times with ultrapure water, and finally redispersed in ultrapure water for further usage. The PCRGs reduced for 12 and 24 h, were used and named in this work as CRG12H and CRG24H, respectively. 2.3. Enzyme immobilization Typically, 100 lL PCRG (1.0 mg/mL) was mixed with 100 lL, HRP (8.0 mg/mL) in phosphate buffer, pH 7.4. The mixture was then incubated for 30 min on ice with shaking. The resulting solution was stored at 4 °C prior to use. 2.4. Modification of GC electrode A circle GC electrode, 3 mm in diameter, was polished sequentially by 1.0 lm, 0.3 lm, 0.05 lm of Al2O3 in microcloth, and ultrasonicated in ultrapure water and ethanol for 5 min, respectively. Finally, the electrode was dried with high pure nitrogen. The GC modification with PCRG immobilized with HRP was achieved through a solution casting procedure. Typically, 5 lL of aqueous dispersion of PCRG (0.5 mg/mL) with HRP (HRP/PCRG) was dropped at the center of GC electrode, and dried at 4 °C for 24 h. The modified electrode was soaked in phosphate buffer, pH 7.4, for 15 min to remove free enzymes before the electrochemical test. The modified electrode was stored in the phosphate buffer (pH 7.4) solution at 4 °C when not in use. 2.5. Electrochemical measurement The electrochemical measurements were performed on a CHI 660C electrochemical workstation (Chen Hua Co., China) using a
three electrode system, with bare GC or modified GC as working electrode, platinum wire as counter electrode, and saturated calomel electrode as reference electrode. The electrolyte solution purged with pure N2 to remove oxygen for 15 min before the electrochemical measurement, and was maintained in nitrogen atmosphere during the experiment. Except for especially noted, the electrolyte is 0.05 M pH 7.4 phosphate buffer, and the scan rate is 50 mV/s. 2.6. Characterization Atomic force microscopic (AFM) images were taken on a MultiMode Nanoscope V scanning probe microscopy system (Veeco, USA). The commercially available AFM cantilever tips with a force constant of 48 N/m and resonance vibration frequency of 330 kHz were used. The scanning rate was set usually at 0.7– 1 Hz. Scanning electron microscopic (SEM) images of modified GC electrode were acquired on an Ultra 55 field emission scanning electron microscopy (Zeiss, Germany) with 3 kV of acceleration voltage. 3. Results and discussion As shown in Fig. 1, the AFM images and corresponding height profiles illustrate that the CRG12H and CRG24H have similar height of 0.9 nm, and reveal also their single atomic layered features. The reduction extents of GO were monitored by UV–Vis, FT– IR and Raman spectroscopy [5]. The morphology of the bared electrode (Fig. S1), HRP/CRG12H/GC and HRP/CRG24H/GC, were characterized using SEM. As depicted in Fig. 2a and b, homogeneous HRP/CRG12H and HRP/CRG24H thin films on the GC electrode surface were produced. Fig. 2c and d show that the morphologies of HRP/CRG12H/GC and HRP/CRG24H/GC were preserved well after recycling hundred times for the CV measurements. This result indicates that both HRP/CRG12H and HRP/CRG24H modified GC electrode should be stable enough for electrochemical study. The electrochemical properties of HRP/CRG12H/GC, HRP/ CRG24H/GC were first compared with that of HRP/GO/GC. As shown in Fig. 3a, in comparison to the system PCRG/GC without HRP (Fig. S2), the cathodic current peak appeared at 0.30 V in the CV curves of HRP/CRG12H/GC and HRP/CRG24H/GC, revealing that the direct electron transfer between electrode and HRP could be achieved [24]. In comparison with the CV curves of HRP/GO/GC and HRP/CRG12H/GC electrodes, the CV curve of HRP/CRG24H/GC electrode that acquired under the same conditions exhibited the largest cathodic peak current, suggesting that CRG24H has better conductivity. Hence, the as-prepared HRP/CRG24H/GC was used as the electrode for the following studies. Different from the GC electrode modified with graphene reported in the literatures [11,24], the background current and cathodic peak current of CV curves of HRP/CRG24H/GC electrode are relative small, due might to low conductivity of the partially reduction extent of CRG24H. Additionally, the immobilization of HRP molecules on both sides of the CRG24H sheets may decrease the conductivity of the HRP/ CRG24H/GC electrode as well. However, the high enzyme loading and stability of CRG24 make GC electrode modification with it is worthwhile. We also examined the effect of potential (voltage) scan rate on the current of the HRP/CRG24H/GC electrode. As shown in Fig. 3b, the peak current and the potential difference between cathodic and anodic peaks increased with increasing of scan rate from 10 to 300 mV/s. Inset in Fig. 3b shows a linear relationship between the cathodic peak current and square root of scan rate, the high correlation coefficient illustrates that the redox reaction of immobilized HRP on the HRP/CRG24H/GC electrode should be a diffusion controlled process [25]. Fig. 4a shows the effect of pH value on the
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Fig. 1. (a and c) tapping mode AFM images of GO sheets being reduced via L-AA for 12 and 24 h on mica surface. (b and d) the corresponding height profiles of the AFM images.
Fig. 2. SEM images of HRP/CRG12H/GC electrode and HRP/CRG24H/GC electrodes before (a and b), and after (c and d) performing 100 times continuous cycle of CV measurements, respectively.
performance of the HRP/CRG24H/GC electrode. The pH dependent peak current of the electrode is consisting with the pH dependence of the catalytic activity of free HRP. The lower or higher pH value
resulted in the loss of HRP catalytic activity, and consequently the decrease of peak current. Thus, we perform all other electrochemical experiments in the electrolyte solutions with pH 7.4.
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Fig. 3. (a) CVs of HRP/GO/GC, HRP/CRG12H/GC, HRP/CRG24H/GC electrodes in phosphate buffer. (b) CVs of HRP/CRG24H/GC electrode in phosphate buffer at various scan rates from 10, 25, 50, 100, 150, 200 and 250 to 300 mV/s, respectively. Inset in (b) shows plot of peak currents as a function of square root of scan rate.
The performance of the HRP/CRG24H/GC electrode was also examined in the presence of H2O2, which can be decomposed by HRP. When H2O2 was successively added to phosphate buffer, the cathodic peak current of HRP/CRG24H/GC at 0.3 V increased gradually as shown in Fig. 4b, with 0.5 mM H2O2, the peak current can be increased more than 10 times, suggesting that a fast direct electron transfer took place between the immobilized HRP and electrode surface with the assistance of CRG24H [26,27]. Because the differential pulse voltammetry (DPV) exhibits relatively lower background current and higher sensitivity than that of CV [28], we acquired DPV data of the HRP/CRG24H/GC electrode in phosphate buffer containing H2O2 with concentrations ranged from 1 lM to 10 mM (Fig. 5a). The typical current peaks of the HRP/CRG24H/GC electrode in the DPV is composed of two separated peaks at 0 and 0.18 V. The DPV curves of the HRP/ CRG24H/GC electrode show that the cathodic peak current gradually increased with increasing of H2O2 concentration. Comparably, the peak at 0.18 V is relatively more sensitive to the H2O2 concentration, especially at the lower concentration region. Therefore, the maximum cathodic peak currents at 0.18 V versus H2O2 concentrations were plotted in Fig. 5b. Overall I–C curve showed three very different response phases. The insets in Fig. 5b are the blow ups of each phases, illustrating that HRP/CRG24H/GC electrode has good linear responses to H2O2 in the concentration ranges of 0.001–0.09 mM, 0.1–0.9 mM, 1.0–10.0 mM, and the slopes are 24.8, 1.96 and 0.11 lA/mM, respectively. The modified electrode showed relatively high sensitivity in 1–100 lM H2O2 region. Lower sensitivity at high H2O2 concentration might due to the inactive
enzyme formed with the excess of H2O2 that inhibits the HRP catalysis [21,29]. Nevertheless, these results imply that the HRP/ CRG24H/GC electrode can efficiently detect H2O2, especially when its concentration is lower than 0.1 mM. The phenolic compounds detection, including phenol and p-chlorophenol, with HRP/CRG24H/GC was also investigated. The detection is based on the oxidation of phenolic compounds by HRP with H2O2, thus, the concentration of H2O2 was kept constant. As depicted in Fig. 6a, when the concentration of H2O2 was 0.02 mM, the cathodic peak current at 0.25 V increased with the addition of the phenol, indicating that HRP/CRG24H/GC electrode involved in the oxidization of phenol. More specifically, a redox peak near 0.25 V appeared, and the peak current of this peak is increased with increasing of phenol concentration too. We speculated that the reduction peak near 0.25 V corresponds to the reduction of o-quinone, the oxidation product of phenol by HRP [30,31]. Because the half-wave potential of o-quinone was observed at 0.2 V in phosphate buffer at pH 7.0 [32]. In order to confirm this assumption, we investigated the electrochemical redox reaction of 0.1 mM hydroquinone and 0.1 mM phenol using HRP/CRG24H/ GC electrode. For hydroquinone, as shown in Fig. S3a, there were two reduction peaks at 0.35 V and 0.02 V in negative scan direction, corresponding to the reduction peak of direct electron transfer between HRP and electrode, and benzoquinone, respectively. For phenol, there were also two reduction peaks 0.25 V and 0.25 V in negative scan direction, corresponding to the reduction peaks of HRP catalytic intermediate II [33,34] and o-quinone, as shown in Fig. S3b. We, therefore, believe that the phenol molecules
Fig. 4. (a) The plot of peak current as a function of pH. (b) CVs of HRP/CRG24H/GC electrode in phosphate buffer with successive addition of 0.05 mM H2O2 at 50 mV/s.
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Fig. 5. (a) DPVs of HRP/CRG24H/GC electrode in phosphate buffer with successive addition of H2O2. (b) The plot of peak currents at H2O2. Inset in (b) shows relation of peak current versus the concentration of H2O2.
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0.2 V as a function of concentration of
Fig. 6. (a) CVs of HRP/CRG24H/GC electrode in phosphate buffer containing phenol with different concentration at 50 mV/s. (b) DPVs of HRP/CRG24H/GC electrode in phosphate buffer acquired during the successive addition of phenol.
were oxidized by the HRP intermediate forming o-quinone, and the reduction peaks appeared near 0.25 V and 0.25 V are characteristic peaks for phenol using HRP/CRG24H/GC electrode. Consequently, HRP/CRG24H/GC electrode can be used as a sensor to identify phenol and hydroquinone based on their different reduction potentials. To elucidate the detection sensitivity of HRP/CRG24H/GC electrode to phenol and p-chlorophenol, the DPV data were acquired from the electrolyte solutions containing phenol or p-chlorophenol molecules with different concentrations of 1 lM to 10 mM. As shown in Fig. 6b, the current of cathodic peak at 0.2 V increased with increasing of concentration of phenol. A reduction peak of oquinone at 0.27 V appeared in the DPV, also increased with increasing of concentration of phenol. Hence, the reduction peaks at 0.27 V and 0.2 V should be both diagnostic for phenol detection. The curve of cathodic peak current of o-quinone at 0.27 V versus phenol concentration, as shown in Fig. 7a, could be fitted into three linear phases corresponding to the phenol concentration ranges of 0.05–0.1 mM, 0.2–1.0 mM, 2.0–10.0 mM, and the slopes were 61, 9.1 and 0.8 lA/mM, respectively. The limit of detection was 4.4 lM at a signal to noise ratio of 3. Similarly, Fig. 7b shows that the current of the reduction peak at 0.2 V versus phenol concentrations could also be divided into three linear parts in the ranges of 0.001–0.09 mM, 0.1–0.9 mM, 1.0–10.0 mM, and the slopes were 32, 1.7 and 0.05 lA/mM, respectively. The limit of
detection was 12.8 lM. Apparently, the reduction peaks at 0.27 V is more diagnostic for phenol detection in terms of the detection limit and sensitivity. These results demonstrated that the HRP/ CRG24H/GC electrode could be used as a sensor to detect phenol for a wide concentration range. Similarly, the applicability of using HRP/CRG24H/GC electrode to detect p-chlorophenol was also investigated. There is no other redox peak appeared in CV spectra (Fig. S4). The DPV data showed that the reduction peak at 0.27 V is much weaker than that of the phenol (Fig. S5), and a reduction peak near 0 V increased remarkably when the concentration of p-chlorophenol was increased, suggesting that the electrochemical oxidation product of p-chlorophenol is different from that of the phenol [35]. The HRP/CRG24H/GC electrode had a linear response to p-chlorophenol at 0.2 V in the concentration ranges of 1 lM to 0.8 mM with correlation coefficients were 0.997. The limit of detection was 15.2 lM at a signal to noise ratio of 3 (Fig. S4). These results demonstrated that the HRP/CRG24H/ GC electrode could be used as a sensor to detect phenolic compounds with a high sensitivity and lower detection limit. To elucidate the practical applicability of the HRP/CRG24H/GC electrode as a sensor in detecting phenol and p-chlorophenol, the currents of the reduction peak of the mixture sample at 0.3 V and 0.25 V versus the time were recorded, respectively. As shown in Fig. S6, when phenol (10 lM) or p-chlorophenol (10 lM) solution was added in the electrolyte solution containing 5 lM of H2O2, the
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Fig. 7. Plots of peak currents versus concentration of phenol in phosphate buffer, (a) the peak at 0.27 V, (b) the peak at peak current and the concentration of phenol.
current of the reduction peak varied instantly, and reached the steady state within 25 s for phenol (95%), and 40 s for p-chlorophenol (95%). The reusability and stability of HRP/CRG24H/GC electrode used for phenolic compound detection were also established. After 10 times detection using the HRP/CRG24H/GC electrode in the electrolyte containing 5 lM of H2O2 and 10 lM of phenol, the relative standard deviation (RSD) was 5.38%. Under the same condition, the RSD for p-chlorophenol detection was 6.19%. These results illustrate that the HRP/CRG24H/GC electrode has good reusability as a sensor. Moreover, we studied the long-term stability of HRP/ CRG24H/GC electrode stored at 4 °C. For electrolyte solution containing 5 lM of H2O2 and 10 lM of phenol, the detected current of the reduction peak of phenol with HRP/CRG24H/GC electrode stored at 4 °C for 3 and 7 days decreased by 5.2% and 7.0% in comparison to the data obtained using an instantly prepared electrode. For electrolyte solution containing 10 lM of p-chlorophenol, the peak current decreased by 3.4% after 3 days, and by 7.3% after 7 days. The stability of the prepared electrode is superior to the sol–gel derived HRP sensor, its activity decreased by 50% after three day storage [36]. So the better reusability and stability make this electrode as a promise biosensor. 4. Conclusions In summary, we have demonstrated that GC electrode can be modified by HRP immobilized on PCRG. The as-modified electrodes have good stability, reusability, and high catalytic activity for the decompositions of H2O2, phenol and p-chlorophenol in water. Given the simple and inexpensive advantages, this method may find practical application in the preparation of enzyme-based amperometric sensors used for detections of phenolic molecules or other permanent organic pollutants in water. Acknowledgments This work was financially supported by the NSFC (No. 91123011, 90923041, 31070742), Shanghai Committee of Science and Technology (Grant 11DZ2260600), and the National ‘‘973’’ Program (2010CB933900) of China.
0.2 V. Inset in (b) shows the linear relationships of the
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jelechem.2012. 06.004.
References [1] M. Pumera, Electrochemistry of graphene: new horizons for sensing and energy storage, Chem. Rec. 9 (2009) 211–223. [2] M. Liang, L. Zhi, Graphene-based electrode materials for rechargeable lithium batteries, J. Mater. Chem. 19 (2009) 5871–5878. [3] S. Park, R.S. Ruoff, Chemical methods for the production of graphenes, Nat. Nanotechnol. 4 (2009) 217–224. [4] S. Stankovich, D.A. Dikin, R.D. Piner, K.A. Kohlhaas, A. Kleinhammes, Y. Jia, Y. Wu, S.T. Nguyen, R.S. Ruoff, Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon 45 (2007) 1558–1565. [5] J. Zhang, H. Yang, G. Shen, P. Cheng, J. Zhang, S. Guo, Reduction of graphene oxide via L-ascorbic acid, Chem. Commun. 46 (2010) 1112–1114. [6] X. Zhou, J. Zhang, H. Wu, H. Yang, J. Zhang, S. Guo, Reducing graphene oxide via hydroxylamine: a simple and efficient route to graphene, J. Phys. Chem. C 115 (2011) 11957–11961. [7] H.C. Schniepp, J.-L. Li, M.J. McAllister, H. Sai, M. Herrera-Alonso, D.H. Adamson, R.K. Prud’homme, R. Car, D.A. Saville, I.A. Aksay, Functionalized single graphene sheets derived from splitting graphite oxide, J. Phys. Chem. B 110 (2006) 8535–8539. [8] R.L. McCreery, Advanced carbon electrode materials for molecular electrochemistry, Chem. Rev. 108 (2008) 2646–2687. [9] I. Dumitrescu, P.R. Unwin, J.V. Macpherson, Electrochemistry at carbon nanotubes: perspective and issues, Chem. Commun. (2009) 6886–6901. [10] Y. Shao, J. Wang, H. Wu, J. Liu, I.A. Aksay, Y. Lin, Graphene based electrochemical sensors and biosensors: a review, Electroanalysis 22 (2010) 1027–1036. [11] C. Shan, H. Yang, J. Song, D. Han, A. Ivaska, L. Niu, Direct electrochemistry of glucose oxidase and biosensing for glucose based on graphene, Anal. Chem. 81 (2009) 2378–2382. [12] M. Zhou, Y. Zhai, S. Dong, Electrochemical sensing and biosensing platform based on chemically reduced graphene oxide, Anal. Chem. 81 (2009) 5603– 5613. [13] L. Tang, Y. Wang, Y. Li, H. Feng, J. Lu, J. Li, Preparation, structure, and electrochemical properties of reduced graphene sheet films, Adv. Funct. Mater. 19 (2009) 2782–2789. [14] H. Liu, J. Gao, M. Xue, N. Zhu, M. Zhang, T. Cao, Processing of graphene for electrochemical application: noncovalently functionalize graphene sheets with water-soluble electroactive methylene green, Langmuir 25 (2009) 12006–12010. [15] Y. Wang, Y. Li, L. Tang, J. Lu, J. Li, Application of graphene-modified electrode for selective detection of dopamine, Electrochem. Commun. 11 (2009) 889– 892.
Y. Zhang et al. / Journal of Electroanalytical Chemistry 681 (2012) 49–55 [16] Y. Zhang, J. Zhang, X. Huang, X. Zhou, H. Wu, S. Guo, Assembly of graphene oxide-enzyme conjugates through hydrophobic interaction, Small 8 (2012) 154–159. [17] N.C. Veitch, Horseradish peroxidase: a modern view of a classic enzyme, Phytochemistry 65 (2004) 249–259. [18] J. Hong, A.A. Moosavi-Movahedi, H. Ghourchian, A.M. Rad, S. Rezaei-Zarchi, Direct electron transfer of horseradish peroxidase on Nafion-cysteine modified gold electrode, Electrochim. Acta 52 (2007) 6261–6267. [19] J. Hong, H. Ghourchian, A.A. Moosavi-Movahedi, Direct electron transfer of redox proteins on a Nafion-cysteine modified gold electrode, Electrochem. Commun. 8 (2006) 1572–1576. [20] Y. Tian, L. Mao, T. Okajima, T. Ohsaka, Superoxide dismutase-based thirdgeneration biosensor for superoxide anion, Anal. Chem. 74 (2002) 2428– 2434. [21] B. Serra, A.J. Reviejo, J.M. Pingarron, Composite multienzyme amperometric biosensors for an improved detection of phenolic compounds, Electroanalysis 15 (2003) 1737–1744. [22] A. Elyacoubi, S.I.M. Zayed, B. Blankert, J.-M. Kauffmann, Development of an amperometric enzymatic biosensor based on gold modified magnetic nanoporous microparticles, Electroanalysis 18 (2006) 345–350. [23] H. Qiu, L. Lua, X. Huang, Z. Zhang, Y. Qu, Immobilization of horseradish peroxidase on nanoporous copper and its potential applications, Bioresource. Technol. 101 (2010) 9415–9420. [24] K. Zhou, Y. Zhu, X. Yang, J. Luo, C. Li, S. Luan, A novel hydrogen peroxide biosensor based on Au–graphene–HRP–chitosan biocomposites, Electrochim. Acta 55 (2010) 3055–3060. [25] H. Yin, Q. Ma, Y. Zhou, S. Ai, L. Zhu, Electrochemical behavior and voltammetric determination of 4-aminophenol based on graphene–chitosan composite film modified glassy carbon electrode, Electrochim. Acta 55 (2010) 7102–7108.
55
[26] M. Pumera, A. Ambrosi, A. Bonanni, E.L.K. Chng, H.L. Poh, Graphene for electrochemical sensing and biosensing, Trends Anal. Chem. 29 (2010) 954–965. [27] C. Fu, W. Yang, X. Chen, D.G. Evans, Direct electrochemistry of glucose oxidase on a graphite nanosheet–Nafion composite film modified electrode, Electrochem. Commun. 11 (2009) 997–1000. [28] H. Du, J. Ye, J. Zhang, X. Huang, C. Yu, A voltammetric sensor based on graphene-modified electrode for simultaneous determination of catechol and hydroquinone, J. Electroanal. Chem. 650 (2010) 209–213. [29] V.A. Bogdanovskay, V.A. Fridman, M.R. Tarasevich, F. Scheller, Bioelectrocatalysis by immobilized peroxidase: the reaction mechanism and the possibility of electroanalytical detection of both inhibitors and activators of enzyme, Anal. Lett. 27 (1994) 2823–2847. [30] H. Sharifian, D.W. Kirk, Electrochemical oxidation of phenol, J. Electrochem. Soc. 133 (1986) 921–924. [31] M. Gattrell, D.W. Kirk, A study of the oxidation of phenol at platinum and preoxidized platinum surfaces, J. Electrochem. Soc. 140 (1993) 1534–1540. [32] J.A. Dean, Lange’s Handbook of Chemistry, Science Press, BeiJing, 2003. [33] X. Yi, J. Huang-Xian, C. Hong-Yuan, Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode, Anal. Biochem. 278 (2000) 22. [34] H. Yin, Q. Ma, Y. Zhou, S. Ai, L. Zhu, Electrochemical behavior and voltammetric determination of 4-aminophenol based on graphene-chitosan composite film modified glassy carbon electrode, Electrochim. Acta 55 (2010) 7102–7108. [35] R.A. Torres, W. Torres, P. Peringer, C. Pulgarin, Electrochemical degradation of p-substituted phenols of industrial interest on Pt electrodes. Attempt of a structure–reactivity relationship assessment, Chemosphere 50 (2003) 97–104. [36] S.A. Kane, E.I. Iwuoha, M.R. Smyth, Development of a sol–gel based amperometric biosensor for the determination of phenolics, Analyst 123 (1998) 2001–2006.