Enzymatic deposition of Au nanoparticles on the designed electrode surface and its application in glucose detection

Colloids and Surfaces B: Biointerfaces 82 (2011) 532–535

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Enzymatic deposition of Au nanoparticles on the designed electrode surface and its application in glucose detection Hongfang Zhang a , Ruixiao Liu b , Qinglin Sheng b , Jianbin Zheng b,∗ a b

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of Education, Department of Chemistry, Northwest University, Xi’an 710069, PR China Institute of Analytical Science/Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an 710069, PR China

a r t i c l e

i n f o

Article history: Received 6 April 2010 Received in revised form 7 September 2010 Accepted 7 October 2010 Available online 16 October 2010 Keywords: Au nanoparticles Glucose Oxidase Biocatalytic deposition Biosensors

a b s t r a c t This paper reported the enzymatic deposition of Au nanoparticles (AuNPs) on the designed 3-mercaptopropionic acid/glucose oxidase/chitosan (MPA/GOD/Chit) modified glassy carbon electrode and its application in glucose detection. Chit served as GOD immobilization matrix and interacted with MPA through electrostatic attraction. AuNPs, without nano-seeds presented on the electrode surface, was produced through the glucose oxidase catalyzed oxidation of glucose. The mechanism of production of AuNPs was confirmed to be that enzymatic reaction products H2 O2 in the solution reduce gold complex to AuNPs. The characterizations of the electrode modified after each assembly step was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. Scanning electron microscopy showed the average particle size of the AuNPs is 40 nm with a narrow particle size distribution. The content of AuNPs on the electrode surfaces was measured by differential pulse stripping voltammetry. The electrochemical signals on voltammogram showed a linear increase with the glucose concentration in the range of 0.010–0.12 mM with a detection limit of 4 ␮M. This provided a method to the determination of glucose. © 2010 Elsevier B.V. All rights reserved.

1. Introduction In recent years, many efforts have been made to develop reliable glucose biosensors using electrochemical methods, because simplicity, high selectivity and relative low cost determination of glucose concentration are very important in clinical applications [1]. A primary factor that affects the performance of a biosensor was the immobilization of glucose oxidase (GOD) on electrode surface [2]. Considerable attention has been devoted to improve the performance of enzyme-based glucose biosensor with novel immobilizing materials and techniques. Among various immobilizing materials, metal nanoparticles especially Au nanoparticles (AuNPs) have received considerable attention in electrochemical field because of their advantages of catalysis, mass transport, high effective surface area and excellent biological compatibility [3–7]. The ability of promoting electron transfer between the active centers of proteins and electrode makes AuNPs especially suitable for fabricating redox protein modified electrodes. Chen’s group [8,9] fabricated two simple glucose biosensor based on biocomposite containing AuNPs. Novel glucose biosensor based on the synergistic effects of AuNPs and the other materials such as Pb nanowires [2], multiwall carbon nanotubes [10,11] and polythiophene [12] was constructed, respectively.

∗ Corresponding author. E-mail address: [email protected] (J. Zheng). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.10.012

Using AuNPs as electron-transfer promoters, the direct electrochemical response of several proteins could be realized [13,14], which laid the foundation for understanding the redox properties of the proteins and developing the third generation biosensors. Therefore, AuNPs are considered to be an excellent candidate for replacing potentially harmful mediators in the construction of biosensors [15]. The most widely and traditionally used method for synthesis of AuNPs was electrochemical or chemical method [2,8–14]. Recently, the synthesis of AuNPs and the other nano-materials biomediated by enzymes has attracted great interest [15–18]. The enzymatic approach was biocompatible and environmentally benign process, and offer a higher degree of control over the kinetics of the reaction [17,18]. Besides, the enzymatic activity was reserved in the generation of AuNPs [19,20], which implied the direct application of the enzyme-AuNPs composite on the biosensor fabrication. Willner’s group [21–23] demonstrated the unique properties of the concept of integration of nanoparticles into biocatalytic reaction through several optical sensors for the detection of H2 O2 , NADH and glucose. Combining the character of AuNPs growth on the designed electrode surfaces with the biological catalytic reactions, a new way for designing simple and sensitive electrochemical biosensing platform was opened [23–25]. In this paper, we report a potential electrochemical glucose biosensing platform based on the biocatalytic growth of AuNPs on the designed electrode surface using chitosan (Chit) as templates and 3-mercapto-propionic acid (MPA) as attractor by virtue of the

H. Zhang et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 532–535

strong affinity of thiols to Au. Chit served as GOD immobilization matrix and interacted with MPA through electrostatic attraction. Characteristics of the electrode modified after each assembly step was studied in detail and the potential biosensor application of the modified electrode was investigated.

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A w = 1.0 Chit solution was prepared by dissolving certain amount of Chit into w = 0.5 acetic acid solution and diluted with ultrapure water. Prior to use, a GCE was polished by a polishing cloth with water slurry of alumina particles (0.05 ␮m diameter), rinsed with water, ultrasonicated in ethanol and ultrapure water, respectively. 5 ␮L of 2.5 mg mL−1 Chit (containing 2.0 mg mL−1 GOD) were spread on the GCE surface. After drying at 4 ◦ C in a refrigerator, the electrode was soaked in 50 mM MPA aqueous solution for 30 min at room temperature. The resulting electrode was thoroughly rinsed with water to remove all physically adsorbed MPA. The final electrode was noted as MPA/GOD/Chit electrode. Without use, the electrode was stored at 4 ◦ C in a refrigerator. The MPA/Chit electrode was also prepared without GOD in the Chit modified process. 2.3. Growth of AuNPs The AuNPs growth solution consists of 0.2 mM HAuCl4 in 0.05 M PBS (pH7.0), and different concentrations of H2 O2 for MPA/Chit electrode or 0.10 mM glucose (except explicitly demonstrated) for MPA/GOD/Chit electrode. Surfactant was not used in these experiments. The growth of AuNPs was performed at room temperature for both H2 O2 and glucose. After soaking in AuNPs growth solution for 10 min, AuNPs/MPA/GOD/Chit electrode was removed from the above solution and transferred into a 0.05 M PBS (pH7.0) after rinsed with PBS. 3. Results and discussion 3.1. The mechanisms of production of AuNPs AuNPs can be obtained by the reduction of HAuCl4 in the presence of GOD-glucose or cholesterol oxidase-cholesterol [23,25]. Zhou et al. [25] fabricated a cholesterol sensor on designed elec-

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GOD (>95%, 10,000 U, from Amresco), AuCl3 ·HCl·4H2 O (w(Au) = 48%) was obtained from Shanghai chemical reagent Co. Ltd. Chit (Mr = 1 × 106 , >90% deacetylation) was purchased from Shanghai Reagent Company (China). Glucose stock solution was allowed to mutarotate at room temperature overnight before use. The potassium hexacyanoferrate and potassium hexacyanoferrite were of analytical grades and used without further purification. 0.05 M phosphate buffer solution (PBS, pH7.0) made from Na2 HPO4 and KH2 PO4 was applied as supporting electrolyte. All other reagents were all of analytical grade and used as received. All solutions were prepared with deionized water. All electrochemical experiments were carried out in a three-electrode cell controlled by a CHI 660 electrochemical workstation (Chenhua Instruments, Shanghai, China). The modified glassy carbon electrode (GCE, 3 mm of diameter) acted as the working electrode. A saturated calomel electrode (SCE) and a platinum wire served as reference and counter electrode, respectively. Scanning electron microscopy (SEM) images were taken by using JSM-6460 SEM (JEOL, Japan).

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Fig. 1. DPSV response of MPA/Chit modified electrode in the absence (a) and presence of H2 O2 (b and c) or glucose (d) and MPA/GOD/Chit (d and e) modified electrode in the presence of glucose (e) after soaking in 0.05 M PBS (pH 7.0) containing 0.2 mM HAuCl4 . Preoxidation potential: 1.25 V, preoxidation time: 30 s.

trode surface in the presence of gold nano-seeds. They proposed that the enzymatic reaction products H2 O2 in the growth solution serves as reductant to convert Au(III) to Au(0) according to the following equation. HAuCl4 + 3/2H2 O2

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To confirm the effect of H2 O2 on the formation of AuNPs, the performance of the MPA/Chit electrode for employing the AuNPs growth solution containing H2 O2 was investigated. Stripping voltammetric method after electrochemical oxidation at 1.25 V was frequently applied to detect of AuNPs and further the quantitative analysis of the substrate involved in the biocatalytic growth of AuNPs [26,27]. Fig. 1 gives the differential pulse stripping voltammetric (DPSV) response of the MPA/Chit electrode before (curve a) and after (curve b and c) soaking in the growth solution for 10 min. No obvious redox peaks were observed for MPA/Chit electrode in the absence of H2 O2 (curve a in Fig. 1), suggesting that both the MPA and Chit are electro-inactive in this potential window. Obviously, the peak at 0.46 V (curve b and c in Fig. 1) is ascribed to the reduction peak of Au(III) obtained by electrochemical oxidation of AuNPs under polarization [26,27]. The peak current increased with the increasing concentration of H2 O2 . These results demonstrated the importance of H2 O2 in the production of AuNPs, and also the growth of AuNPs on this designed electrode surface without the need of nano-seeds [25]. To prove the enzymatic nature of AuNPs on the designed electrode surface, the DPV response of MPA/Chit and MPA/GOD/Chit

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Z'/ohm Fig. 2. CVs (A) and EIS (B) of bare GCE (a), GOD/Chit electrode (b), MPA/GOD/Chit electrode (c) and AuNPs/MPA/GOD/Chit electrode (d). CVs was recorded in a solution of KCl containing 5 mM Fe(CN)6 3− at a scan rate of 100 mV s−1 ; EIS was obtained in a solution of KCl containing 1 mM Fe(CN)6 3− + 1 mM Fe(CN)6 4− . The frequencies swept from 105 to 10−2 Hz at the formal potential of the redox couple.

modified electrode after soaking in the growth solution in the presence glucose instead of H2 O2 was recorded, respectively. As shown in Fig. 1d, no peaks appeared at ca. 0.46 V for MPA/Chit electrode in the presence of glucose, which indicated that glucose itself could not convert Au(III) to Au(0) at this experimental conditions. By comparison, a sharp peak was observed when the electrode was modified with GOD. Naturally enough, the proposed mechanism for the biocatalytic growth of AuNPs could be expected as follows. The flavin group (FAD) in the enzyme was reduced by reaction with glucose to the reduced form of the enzyme GOD(FADH2 ), then, GOD(FADH2 ) was oxidized by molecular oxygen and, H2 O2 were produced [28]. Finally, Au(III) was reduced by H2 O2 to generate AuNPs. 3.2. Characterizations of the modified electrode The cyclic voltammetry of ferricyanide was chosen as a marker to investigate the changes of electrode behavior after each assembly step. It was relied on that the electron transfer between the solution species and the electrode must occur by tunneling either through the barrier or through the defects in the barrier [29,30]. For bare GCE, one pair of well-defined redox wave of Fe(CN)6 3− with the peak separation of 77 mV was observed (curve a in Fig. 2A). When the electrode was assembled on Chit and GOD mixture, the peak current decreased slightly and the peak separation increased obviously (curve b in Fig. 2A). The enlarged peak separation indicated an insulating barrier to the interfacial electron transfer, which might mainly be ascribed to the effect of GOD [29]. Chit owns many primary amino groups, and has a pKa value of about 6.3 [31]. At pH sufficiently below the pKa , most of the amino groups

are protonated. Thus, Chit exists as a positively charged polyelectrolyte at pH 5.0 [32]. Because the isoelectric point, Ip of GOD is 4.2, it is part negatively charged at pH 5.0 [33], and would neutralize partial charge of Chit. Anyway, the GOD–Chit mixture coated on the electrode surface is still a positively charged matrix, and electrostatically adsorbed Fe(CN)6 3− [34]. Thus the peak current did not shown apparent decrease on the curve b by comparison with curve a in Fig. 2A. After immersing GOD/Chit modified electrode in the MPA solution, a marked decrease in the peak current was observed (curve c in Fig. 2), meaning that a blocking layer for the diffusion of Fe(CN)6 3− toward the electrode surface was introduced [29]. It had been reported that the pKa of MPA is 4.3 and the acid strength of COOH-terminated alkanethiols decreases significantly after adsorption [35], which ensured the interaction of negatively charged MPA with the GOD–Chit matrix. MPA on the electrode surface repulse intensively Fe(CN)6 3− to the electrode surface, which leads to the great decrease of peak current on the cyclic voltammogram (curve c in Fig. 2A). Therefore, it was concluded that MPA were successfully integrated by means of electrostatic interaction. Usually, MPA is used to construct selfassembled monolayers on gold electrode surface [36,37]. In this work, MPA, modified on the electrode surface, would attract the AuNPs produced in the solution on the electrode surface by virtue of Au-S bonding [37]. In comparison with the CV at the MPA/GOD/Chit electrode, a significant reduce of peak potential and enhancement of peak currents was obtained at the AuNPs/MPA/GOD/Chit electrode (curve d in Fig. 2A), which demonstrated an effective facilitation of electron transfer rate constant of Fe(CN)6 3−/4− redox reaction at the electrode surface [38], indicating the successful deposition of AuNPs. Electrochemical impedance spectroscopy (EIS) is also a powerful technique for studying the interface properties of electrode surfaces. The EIS spectra, presented as Nyquist plots (Z versus Z ), of bare GCE, GOD/Chit electrode, MPA/GOD/Chit electrode and AuNPs/MPA/GOD/Chit electrode in equivalent Fe(CN)6 3−/4− solution were shown in Fig. 2B. It can be observed that the bare GCE exhibit an almost straight line (curve a in Fig. 2B), indicating a diffusion limiting step of the electrochemical process [38]. Spreading of GOD/Chit on the electrode surface generated an insulating layer on the electrode that functioned as a barrier to the interfacial electron transfer. This is reflected by the appearance of the semicircular part of the spectrum (curve b in Fig. 2B). The diameter of the respective semicircular element corresponds to the electron transfer resistance (Ret ) at the electrode surface [39]. As shown in curve c of Fig. 2B, Ret increased dramatically when MPA was assembled on the electrode, indicating formation of a ferrocyanide transport blocking layer on the electrode, which is consistent with the result obtained from CV experiment. After incubating the MPA/GOD/Chit electrode in an AuNPs growth solution for 10 min, the diameter of the semicircular impedance spectrum shows a marked decrease (curve d in Fig. 2B). The achieved low Ret of AuNPs/MPA/GOD/Chit electrode indicates that AuNPs were self-assembled successfully at the surface of the designed electrode. This conclusion was supported by the good conductivity of AuNPs [40,41]. From the SEM image of the AuNPs/MPA/GOD/Chit film (not shown), the average particle size of the AuNPs is 40 nm with a narrow particle size distribution. This image suggests also that AuNPs could be produced on the proposed growth solution without need of surfactant. It had been reported that GOD evidently terminated propagation of Pt particle growth, which is attributed to surface groups (for example, COOH of aspartic and glutamic acid, NH2 of lysine and arginine) binding to the Pt(IV)-complex or to the surface of growing Pt nanoparticles [42]. The reason could also explain the nano-particle size of Au(0) obtained in the MPA/GOD/Chit composition film.

H. Zhang et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 532–535

based on the DPSV response of AuNPs deposited in different concentration of glucose. By immobilization of fresh GOD, the AuNPs/MPA/GOD/Chit electrode was expected to be an amperometric glucose biosensor [44]. Due to the involvement of H2 O2 , this study may provide a universal method to design AuNPs based biosensors for other substrates by using various corresponding O2 dependent oxidases.

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Fig. 3. DPSV response of MPA/GOD/Chit electrodes after treated with 0.2 mM HAuCl4 in air-saturated 0.05 M PBS (pH7.0) containing 0.010, 0.050, 0.060, 0.10, 0.12 and 0.20 mM glucose. Preoxidation potential: 1.25 V, preoxidation time: 30 s. The inset shows the peak current on DPSV response to glucose concentration.

3.3. Effect of experimental conditions on the AuNPs growth It is expected that the growth of AuNPs on the designed MPA/GOD/Chit electrode would be affected by the incubation time, the concentration of HAuCl4 and the pH of the growth solution. The incubation time was selected as 10 min according to the reported procedure [22]. The concentration of HAuCl4 and the pH of the growth solution were chosen to be 0.2 mM HAuCl4 in 0.05 M PBS (pH7.0) through consulting references [22,43]. The detailed influences were not investigated in this work. The effect of glucose concentration on growth of AuNPs was investigated by the DPSV signal of AuNPs/MPA/GOD/Chit electrode. The MPA/GOD/Chit electrode was soaking in air saturated 0.05 M PBS (pH7.0) consisted of 0.2 mM HAuCl4 . Different aliquots (10.0, 50.0, 60.0, 100, 120, 200 ␮L) of 20 mM glucose solution were added to 10 mL of the solution. The ultimate concentration of glucose is 0.010, 0.050, 0.060, 0.10, 0.12 and 0.20 mM. After 10 min of growth, the AuNPs/MPA/GOD/Chit electrode was removed from the above solution, and transferred into a 0.05 M PBS (pH7.0) after rinsed with PBS. Then, DPSV signal was obtained in the potential scanned from 0.8 to −0.1 V, resulting in an analytical signal due to the reduction of AuCl4 − at potential about 0.4 V [26,27]. Fig. 3 gives the DPSV responses of AuNPs/MPA/GOD/Chit electrodes for different concentration of glucose. The modified electrode exhibited a linear response to glucose in the concentration range of 0.010–0.12 mM, with a correlation coefficient of 0.997, the sensitivity is 15.6 ␮A/mM. The peak current increased little when the concentration of glucose was increased from 0.12 mM to 0.20 mM, mainly because of the exhaustion of HAuCl4 . This provided a determination method for glucose. The detection limit of the method is determined to be 4 ␮M with the signal-to-noise ratio of 3. The method might be used to detect blood sugar of human in daily life. The reproducibility was evaluated by examining the DPSV current response of five times of fabrication of AuNPs/MPA/GOD/Chit electrode in AuNPs growth solution containing 0.10 mM glucose solution. The relative standard deviation (RSD) is 3.9% for the five modified electrodes, showing a good fabrication and determination reproducibility. 4. Conclusions AuNPs was produced on the MPA/GOD/Chit electrode surface with the catalytic reaction of the substrate by the corresponding enzyme. A novel method of glucose detection was constructed

This work was financially supported by the National Natural Science Foundation of China (Nos. 20905061, 20875076), the Scientific Initializing Foundation (No. PR09018), the Doctorate Dissertation of Excellence Funds (No. 08YYB06) and the Science Foundation (No. 09NW02) of Northwest University. References [1] A. Heller, B. Feldman, Chem. Rev. 108 (2008) 2482. [2] H. Wang, X. Wang, X. Zhang, X. Qin, Z. Zhao, Z. Miao, N. Huang, Q. Chen, Biosens. Bioelectron. 25 (2009) 142. [3] F. Wang, S. Hu, Microchim. Acta 165 (2009) 1. [4] J.-J. Yu, S. Lu, J.-W. Li, F.-Q. Zhao, B.-Z. Zeng, J. Solid State Electrochem. 11 (2007) 1211. [5] S. Ma, W. Lu, J. Mu, F. Liu, L. Jiang, Colloids Surf. A 324 (2008) 9. [6] Q. Xu, Y. Zhao, J.Z. Xu, J.-J. Zhu, Sens. Actuators B 114 (2006) 379. [7] P. Santhosh, K.M. Manesh, S. Uthayakumar, S. Komathi, A.I. Gopalan, K.-P. Lee, Bioelectrochemistry 75 (2009) 61. [8] Y. Du, X.-L. Luo, J.-J. Xu, H.-Y. Chen, Bioelectrochemistry 70 (2007) 342. [9] X.-L. Luo, J.-J. Xu, Y. Du, H.-Y. Chen, Anal. Biochem. 334 (2004) 284. [10] D. Ragupathy, A.I. Gopalan, K.-P. Lee, Electrochem. Commun. 11 (2009) 397. [11] Y. Zhang, G. Guo, F. Zhao, Z. Mo, F. Xiao, B. Zeng, Electroanalysis 22 (2010) 223. [12] P. Pandey, S.K. Arya, Z. Matharu, S.P. Singh, M. Datta, B.D. Malhotra, J. Appl. Polym. Sci. 110 (2008) 988. [13] J. Li, L. Zhou, X. Han, H. Liu, Sens. Actuators B 135 (2008) 322. [14] M.A. Rahman, H.-B. Noh, Y.-B. Shim, Anal. Chem. 80 (2008) 8020. [15] F.H. Zhang, S.S. Cho, S.H. Yang, S.S. Seo, G.S. Cha, H. Nam, Electroanalysis 18 (2006) 217. [16] S.Y. Lim, J.S. Lee, C.B. Park, Biotechnol. Bioeng. 105 (2010) 210. [17] Q. Sheng, K. Luo, J. Zheng, H. Zhang, Biosens. Bioelectron. 24 (2008) 429. [18] L. Caseli, D.S. dos Santos Jr., R.F. Aroca, O.N. Oliveira Jr., Mater. Sci. Eng. C 29 (2009) 1687. [19] I. Willner, R. Baron, B. Willner, Adv. Mater. 18 (2006) 1109. [20] K. Kalishwaralal, S. Gopalram, R. Vaidyanathan, V. Deepak, S.R.K. Pandian, S. Gurunathan, Colloids Surf. B 77 (2010) 174. [21] M. Zayats, R. Baron, I. Popov, I. Willner, Nano Lett. 5 (2005) 21. [22] Y. Xiao, V. Pavlov, S. Levine, T. Niazov, G. Markovitch, I. Willner, Angew. Chem. Int. Ed. 116 (2004) 4619. [23] Y.M. Yan, R. Tel-Vered, O. Yehezkeli, Z. Cheglakov, I. Willner, Adv. Mater. 20 (2008) 2365. [24] D. Du, J. Ding, J. Cai, A. Zhang, Sens. Actuators B 127 (2007) 317. [25] N.D. Zhou, J. Wang, T. Chen, Z.G. Yu, G.X. Li, Anal. Chem. 78 (2006) 5227. ˜ [26] M.T. Castaneda, A. Merkoc¸i, M. Pumera, S. Alegret, Biosens. Bioelectron. 22 (2007) 1961. [27] K. Kerman, M. Chikae, S. Yamamura, E. Tamiya, Anal. Chim. Acta 588 (2007) 26. [28] Q.-L. Sheng, Y. Shen, H.-F. Zhang, J.-B. Zheng, Chin. J. Chem. 26 (2008) 1244. [29] S. Zhang, N. Wang, Y. Niu, C. Sun, Sens. Actuators B 109 (2005) 367. [30] G. Yang, R. Yuan, Y.-Q. Chai, Colloids Surf. B 61 (2008) 93. [31] J. Yang, R. Zhang, Y. Xu, P. He, Y. Fang, Electrochem. Commun. 10 (2008) 1889. [32] M. Zhang, A. Smith, W. Gorski, Anal. Chem. 76 (2004) 5045. [33] B.-Y. Wu, S.-H. Hou, F. Yin, J. Li, Z.-X. Zhao, J.-D. Huang, Q. Chen, Biosens. Bioelectron. 22 (2007) 838. [34] F. Li, Z. Wang, W. Chen, S. Zhang, Biosens. Bioelectron. 24 (2009) 3030. [35] A. Kudelski, A. Michota, J. Bukowska, J. Raman Spectrosc. 36 (2005) 709. [36] J. Strutwolf, C.K. Sullivan, Electroanalysis 19 (2007) 1467. [37] R.F. Carvalhal, R.S. Freire, L.T. Kubota, Electroanalysis 17 (2005) 1251. [38] P. Wang, Z. Mai, Z. Dai, Y. Li, X. Zou, Biosens. Bioelectron. 24 (2009) 3242. [39] X. Zhang, G. Wang, W. Zhang, N. Hu, H. Wu, B. Fang, J. Phys. Chem. C 112 (2008) 8856. [40] I.I. Suni, Trends Anal. Chem. 27 (2008) 604. [41] X.-L. Luo, J.-J. Xu, Q. Zhang, G.-J. Yang, H.-Y. Chen, Biosens. Bioelectron. 21 (2005) 190. [42] P. Karam, Y. Xin, S. Jaber, L.I. Halaoui, J. Phys. Chem. C 112 (2008) 13846. [43] L. Shang, H. Chen, L. Deng, S. Dong, Biosens. Bioelectron. 23 (2008) 1180. [44] X. Pan, J. Kan, L. Yuan, Sens. Actuators B 102 (2004) 325.