Direct electrochemistry of Cytochrome c on natural nano-attapulgite clay modified electrode and its electrocatalytic reduction for H2O2

Electrochimica Acta 52 (2007) 3601–3606

Direct electrochemistry of Cytochrome c on natural nano-attapulgite clay modified electrode and its electrocatalytic reduction for H2O2 Ji-ming Xu a,∗ , Wei Li b , Qi-fan Yin a,b , Yu-lan Zhu a,∗ a

Jiangsu Key Laboratory for Chemistry of Low-dimensional Materials, Huaiyin Teachers College, Jiangsu 223300, PR China b Department of Chemistry, College of Science, Yanbian University, Jilin 133002, PR China Received 20 July 2006; received in revised form 16 September 2006; accepted 15 October 2006 Available online 15 November 2006

Abstract Natural nano-structural attapulgite clay was purified by mechanical stirring with the aid of ultrasonic wave and its structure and morphology was investigated by XRD and transmission electron microscopy (TEM). Cytochrome c was immobilized on attapulgite modified glassy carbon electrode. The interaction between Cytochrome c and attapulgite clay was examined by using UV–vis spectroscopy and electrochemical methods. The direct electron transfer of the immobilized Cytochrome c exhibited a pair of redox peaks with formal potential (E0 ) of about 17 mV (versus SCE) in 0.1 mol/L, pH 7.0, PBS. The electrode reaction showed a surface-controlled process with the apparent heterogeneous electron transfer rate constant (ks ) of 7.05 s−1 and charge-transfer coefficient (α) of 0.49. Cytochrome c immobilized on the attapulgite modified electrode exhibits app a remarkable electrocatalytic activity for the reduction of hydrogen peroxide (H2 O2 ). The calculated apparent Michaelis–Menten constant (Km ) was 470 ␮mol/L, indicating a high catalytic activity of Cytochrome c immobilized on attapulgite modified electrode to the reduction of H2 O2 . Based on these, a third generation of reagentless biosensor can be constructed for the determination of H2 O2 . © 2006 Elsevier Ltd. All rights reserved. Keywords: Attapulgite clay; Cytochrome c; Direct electron transfer; Hydrogen peroxide

1. Introduction The feasibility of heterogeneous direct electron transfer (DET) reactions between redox proteins and electrode surface has attracted considerable interest and understanding of these reactions fundamentally can establish a desirable model for studying the redox behavior of the proteins in biological systems, which may elucidate the relationship between their structure and biological functions. On the other hand, studies of direct electron exchange between proteins and underlying electrodes can also provide a platform for fabricating biosensors, enzymatic bioreactors and biomedical devices [1–4]. Cytochrome c (Cyt c) is a very basic redox metalloprotein that is active in biological electron transfer chains. Under physiological conditions, it transfers electrons between Cyt c reductase and Cyt c oxidase, which are both embedded in the mitochondria membrane. The crucial role of Cyt c in the respiratory chain

∗

Corresponding author. Tel.: +86 517 3525085. E-mail address: [email protected] (J.-m. Xu).

0013-4686/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2006.10.020

of mitochondria has inspired biologists and biochemists to elucidate the mechanism of interprotein electron-transfer process. However, it is very difficult for Cyt c to exhibit a voltammetric response at a bare electrode because of its extremely slow electron transfer kinetics at the electrode/solution interface and its short-lived and transient response on a metal electrode surface [5,6]. Thus, considering that it is helpful to have a fundamental understanding of the structure–function relationship of Cyt c by the study of its direct electrochemistry, more and more researchers have focused their attentions on the enhancement of the electron transfer of Cyt c by using mediators or promoters, and some interesting results were obtained. Till now, the common modifiers including organic compounds, such as organosulphur compounds and pyridylthiol derivatives [7–11], carboxylic acid terminated alkanethiols [12–14], surfactants [15–16], inorganic porous materials, such as clay [17–19], porous alumina [20], zeolite [21], sol–gel matrix [22], nano-materials [23–25] and biomaterials [26–28] have been used to study the direct electron transfer between redox proteins and electrode surfaces. These modifiers may provide a favorable microenvironment for proteins and thus enhance the electron transfer rates.

3602

J.-m. Xu et al. / Electrochimica Acta 52 (2007) 3601–3606

Fig. 1. Crystalline structure of attapulgite from (0 0 1) plane.

Fig. 2. XRD pattern of ATP.

Attapulgite (ATP, or palygorskite as it often called) is a crystalline hydrated magnesium aluminum silicate with reactive –OH groups on its surface. ATP is a natural nano-structural material with formula Si8 O20 (Mg, Al, Fe)5 (OH)2 (OH2 )4 ·4H2 O and its ideal structure was studied by Bradley early in 1940 and shown in Fig. 1 [29]. The distinguishing feature of its structure is that the Si–O tetrahedron form long strips, each an amphibole unit wide on alternate sides of the oxygen sheet in a manner which confers a regular corrugated Si–O structure [30]. The structure of the mineral results in zeolite-like channels, which are ˚ × 6.0 A ˚ and 5.6 A ˚ × 11.0 A ˚ wide, respecapproximately 3.7 A tively [31]. ATP has advantages of specific features in dispersion, high temperature endurance, salt and alkali resistance and also high adsorption and penetrability due to its regular structure and large specific surface area. Because of its unique structure, ATP has been used as adsorbent [32], drilling muds [33], pharmaceutical preformulation study [34], animal feed supplement [35] and so on. In this paper, natural nano-structure attapulgite was used as a matrix for Cyt c immobilization, the direct electron exchange between Cyt c and electrode was investigated in detail. The results indicated that the construction of Cyt c immobilized on attapulgite modified glassy electrode is a very simple process. The biosensor has a high sensitivity and a good stability, indicating that ATP is a good matrix for protein immobilization and preparation of the third generation biosensor.

All experimental solutions were deaerated by bubbling nitrogen for at least 30 min and a nitrogen atmosphere was kept over the solution during measurements. Horse heart Cyt c was purchased from Sigma and used as received. Attapulgite clay was obtained from Hongqing Limited Company (Xuyi, Jiangsu, China), which should be purified prior to use. Phosphate buffer solutions were prepared by mixing stock standard solutions of Na2 HPO4 and KH2 PO4 and adjusting the pH with 0.1 M H3 PO4 and NaOH. The concentration of H2 O2 solution was determined by titration with standard KMnO4 solution. Other chemicals were of analytical reagent grade. All the solutions were prepared with doubly distilled water.

2. Experimental 2.1. Apparatus and reagents Electrochemical experiments were performed with CHI660B electrochemical analyzer (CHI, USA) with a conventional three-electrode cell. A Cyt c/ATP modified glassy carbon (GC) electrode was used as working electrode. A saturated calomel electrode (SCE) and a platinum wire were used as the reference and the auxiliary electrodes, respectively. UV–vis absorbance spectroscopy was performed using a UV-916 spectrophotometer (GBC, Australia). Transmission electron microscopy (TEM) observation of attapulgite was carried out with a JEM-200CX TEM instrument. XRD data were obtained using ARL diffractometer (Switzerland) with Cu K␣ radiation. All the measurements were carried out at room temperature.

2.2. Purification The crude attapulgite clay often contains some impurities, it should be purified prior to use. The purification procedure is given as follows: 10 g attapulgite and 200 mL H2 O were mixed in a flask and the mixture was stirred with the aid of ultrasonic wave for 3 h. The suspension colloid was then centrifuged. The solid was treated repeatedly for three times, finally and the pure attapulgite was obtained. The structure and morphology was examined with XRD and TEM. Fig. 2 shows the XRD patterns of ATP. The typical diffraction peaks at 2θ = 8.3◦ , 13.6◦ , 19.7◦ and 27.28◦ correspond to the primary diffraction of the (1 1 0), (2 0 0), (0 4 0) and (4 0 0) planes of the clay, respectively. The result clearly shows that the four-plane characteristic peaks for the ATP. Fig. 3 shows the dispersion of ATP by TEM. From Fig. 3, we can see that ATP emerged as rods (or fibers) and dispersed very well, the average diameter is about 20–50 nm. In addition, the result indicates that the aggregation of ATP is very weak, we can get better dispersion in water by mechanical stirring with the aid of ultrasonic wave. 2.3. Preparation of Cyt c immobilized ATP modified glassy carbon electrode A glassy carbon electrode (GCE, 3 mm in diameter) was polished to a mirror-like with fine emery papers and 1.0, 0.3 and 0.05 ␮m alumina slurry (Beuhler) followed by rinsing thoroughly with doubly distilled water. The electrode was then

J.-m. Xu et al. / Electrochimica Acta 52 (2007) 3601–3606

3603

than 3.0, the current does not change obviously, which suggests that the best Cyt c incorporation was obtained. In this paper, the ratio of 3 was selected for the preparation of Cyt c/ATP/GC modified electrode. At first, 5 ␮L 2.0 mg/mL ATP colloid solution was spread onto the pretreated GC electrode surface and allowed to dry for 1 h, then, 5 ␮L 6.0 mg/mL Cyt c (in pH 7.0 PBS) was dropped on the surface of ATP modified GC electrode, the modified electrode was dried under ambient conditions and rinsed with doubly distilled water for twice to remove the non-firmly adsorbed Cyt c. The obtained Cyt c/ATP/GC modified electrode was stored dry at 4 ◦ C in a refrigerator when not in use. 3. Results and discussion 3.1. UV–vis absorption spectroscopic characterization Fig. 3. TEM micrographs of ATP.

Fig. 4. Current response of CVs at different ratios of Cyt c to ATP films modified GC electrode in pH 7.0 PBS. ATP 2 ␮g and scan rate 0.1 V s−1 .

successively sonicated in alcohol and doubly distilled water and allowed to dry at room temperature. To get the optimum current response, the ratios of Cyt c to ATP on GC electrode surface were investigated by cyclic voltammetry. The results were presented in Fig. 4. From Fig. 4, we can see that the current increases with the increase of the ratio. When the ratio of Cyt c to ATP is greater

Fig. 5. UV–vis absorption spectra of Cyt c in 0.1 M pH 7.0 PBS (a) and Cyt c/ATP composite in 0.1 M pH 7.0 PBS (b).

As a matrix for immobilization of protein, the material must maintain the properties and reactivity of the protein as it has in its physiological environment. UV–vis spectroscopy is a useful tool for monitoring the possible change of the Soret absorption band of Cyt c [36]. Fig. 5 shows the UV–vis spectra of Cyt c and Cyt c/ATP cross-linked composite in pH 7.0 PBS solution. The native Cyt c gave a heme band at 408.6 nm (curve a in Fig. 5). For Cyt c/ATP cross-linked composite, the absorption band was red shifted by only 0.7 nm at 409.3 nm (curve b in Fig. 5). The slight shift may be due to the interaction between ATP and Cyt c. Such interaction did not destroy the proteins’ structure and change the fundamental microenvironment. A shift of only 0.7 nm suggests that the environment of the protein is slightly changed and no significant denaturation occurred [37]. 3.2. Electrochemical behavior of Fe(CN)6 3− in ATP/GC electrode: determination of the porosity of ATP film As the CV current observed at an ATP-modified electrode mainly depends on the porosity of the ATP film, Fe(CN)6 3− was chosen as an electrochemically active probe ion for an indirect measure of the porosity of the ATP film [38]. Typical CV curves obtained in 1.0 mmol/L Fe(CN)6 3− in 1.0 mol/L PBS solution at bare or at ATP modified electrode are shown in

Fig. 6. Cyclic voltammograms of 1.0 mM Fe(CN)6 3− at bare GC electrode (a) and ATP/GC electrode (b). Scan rate 0.1 mV s−1 .

3604

J.-m. Xu et al. / Electrochimica Acta 52 (2007) 3601–3606

Fig. 7. Cyclic voltammograms of bare GCE (a); ATP/GCE (b); Cyt c/ATP/GCE (c) in 0.1 M pH 7.0 PBS. Scan rate 0.1 V s−1 .

Fig. 6. From Fig. 6, we can see that the redox potentials shift slightly and that the peak heights are lowered due to the restricted active electrode surface area. From the ratio of the peak currents obtained at the modified to bare electrode, an estimation of the void space of ATP film was calculated, which included both porosity and tortuosity of the coating. The total pore space of the ATP/GCE is about 70%, which suggests that electron transfer may occur between the electroactive probe and GC electrode surface through the pore space of the ATP film. 3.3. Direct electrochemistry of Cyt c at the ATP/GC electrode Fig. 7 shows the cyclic voltammograms of different electrode in 0.1 mol/L pH 7.0 PBS. From Fig. 7, we can see that no voltammetric responses can be observed at both bare and ATP modified electrode, which indicates that ATP is electroinactive in the potential window. A well-defined redox peaks were observed at the Cyt c/ATP/GC electrode, with the reductive peak potential at −0.023 V, the corresponding oxidative peak potential at 0.057 V and Ep = 80 mV (Fig. 7c). The formal potential (E0 ) for the redox reaction of Cyt c is 0.017 V (versus SCE), which is consistent with that of Cyt c in solution [39]. Obviously, these peaks are attributed to the redox reaction of the electroactive center of Cyt c. The results may be caused by the electrostatic interaction between Cyt c and ATP film. In pH 7.0 solution, positively charged Cyt c (pI = 10.5) and negatively charged ATP [40] may electrostatically bind each other, the positive lysine residues in Cyt c coordinate with oxygen atoms of the ATP, which make the electroactive sites of Cyt c closer to the surface of the electrode and facilitate the electron exchange between Cyt c and GC electrode. To further investigate the characteristics of Cyt c at ATP/GC electrode surface, the effect of scan rates on the voltammetric behavior of Cyt c was studied in detail. Fig. 8 displays cyclic voltammograms for immobilized Cyt c at various scan rates. It can be seen that the redox peak currents increase linearly with scan rates, suggesting that the redox process is surfacecontrolled electrode process. On the other hand, with the increase of scan rates, the oxidation peak shifts to more positive poten-

Fig. 8. (A) Cyclic voltammograms of Cyt c/ATP/GCE in 0.1 M pH 7.0 PBS at different scan rates. Scan rates from a to n are: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, and 3000 mVs−1 , respectively. Inset is the plot of cathodic and anodic peak currents vs. scan rates. (B) Various of peak potentials vs. the logarithm of the scan rates.

tials, while the reduction peak shifts to more negative potentials. The anodic and cathodic peak potentials are linearly dependent on the logarithm of scan rates (v) when v > 1 V s−1 , which is in agreement with the Laviron theory [41]: a plot of Ep versus log v yields two straight lines with slopes of −2.3RT/␣nF and 2.3RT/(1 − α)nF for the cathodic peak and for the anodic peak, respectively. So the value of α can be estimated as 0.49 from the slopes of the straight lines based on the equation: va va α α log or = log = vc 1−α vc 1−α The average kinetic parameter ks = 7.05 s−1 can be obtained according to the following equation: log ks = α log(1 − α) + (1 − α) log α − log −

RT nFv

α(1 − α)nF Ep 2.3RT

The larger value of ks indicates that the Cyt c on ATP/GC electrode surface shows good reversibility of the electron transfer process. According to Laviron’s equation [42], Ip = n2 F 2 AΓv/4RT , the surface coverage (Γ ) of Cyt c on and/or inside the ATP film is 8.7 × 10−11 mol cm−2 , suggesting an approximate monolayer of Cyt c on the surface of ATP modified GC electrode.

J.-m. Xu et al. / Electrochimica Acta 52 (2007) 3601–3606

Fig. 9. Cyclic voltammograms of Cyt c/ATP/GCE in 0.1 M PBS (curve a), in the presence of 2.0 × 10−3 M H2 O2 (curve b) and 4.0 × 10−3 M H2 O2 (curve c). Scan rate 0.1 V s−1 .

3.4. Electrocatalytic behavior of Cyt c/ATP/GC electrode to the reduction of H2 O2 Electrocatalytic reduction of H2 O2 using Cyt c/ATP/GC electrode was studied by CVs. Fig. 9 shows the cyclic votammograms in the presence and absence of H2 O2 at Cyt c/ATP/GC electrode in 0.1 M PBS. As can be seen, when H2 O2 was added to the buffer solution, the voltammetric behavior of the Cyt c-immobilized ATP/GC electrode changes obviously, with an increase of the reduction peak current and the disappearance of the oxidation peak and the reduction peak current increases with the increasing concentration of H2 O2 in solution. The disappearance of the oxidation peak shows that the oxidation rate of Cyt c by H2 O2 is very fast, indicating the pseudo peroxidase activity of the Cyt c immobilized in the ATP film. To evaluate the dependence of the electrocatalytic current on the concentration of H2 O2 , the characteristics of the Cyt c/ATP/GC electrode were investigated by chronoamperometric measurement under the optimum conditions obtained above. The results presented in Fig. 10 indicate that the electrocatalytic current increases with increasing concentration of H2 O2 . The linear

3605

range between the electrocatalytic current and the concentration of H2 O2 can extend to 0.50 mmol/L. When the concentration of H2 O2 was higher than 0.60 mmol/L, a plateau was observed, showing the characteristics of Michaelis–Menten kinetics. The app apparent Michaelis–Menten constant (Km ) which gives an indication of the enzyme–substrate kinetics, could be obtained from the electrochemical version of the Lineweaver–Burk equaapp tion [43]: 1/Iss = (1/Imax ) + (Km /Imax c), where Iss is the steady-state current after the addition of a substrate, which can be obtained from amperometric experiments, Imax the maximum current under saturated substrate conditions and c is the bulk app concentrate of the substrate. Km can be obtained by analysis of the slope and intercept of the plot of the reciprocals of app the steady state current versus H2 O2 concentration. Km value for this Cyt c/ATP modified GC electrode was estimated to be 470 ␮mol/L. This value is markedly smaller than 2.1 mmol/L [44], 898 ␮mol/L [45] and 857 ␮mol/L [25], which implies that the Cyt c/ATP modified electrode exhibits a higher affinity for H2 O2 . The Cyt c/ATP/GC electrode shows good reproducibility for the determination of H2 O2 in the linear range. For instance, the variation coefficient is 4.5% for 11 successive assays at H2 O2 concentration of 2.0 × 10−3 mol/L on the Cyt c/ATP/GC electrode. The fabrication reproducibility of three electrodes, prepared independently, shows an acceptable extent with a variation coefficient of 4.9% for the determination of 2.0 × 10−3 mol/L H2 O2 . The long-term stability of Cyt c/ATP/GC electrode was studied ca. 2 weeks. When the electrode was stored dry at 4 ◦ C and measured intermittently (every 2 days), there is unnoticeable change with 2.0 × 10−3 mol/L H2 O2 within this period. Thus, the modified electrode was very efficient for retaining the electrocatalytic activity of Cyt c and preventing it from leaking out of the electrode. The influences of foreign species were investigated by analyzing a standard solution of 1.0 × 10−4 mol/L hydrogen peroxide to which interfering species were added. 0.2 mmol/L ascorbic acid and the same concentration of dopamine did not interfere hydrogen peroxide determination. One millimole per liter K+ , Na+ , NH4 + , Mg2+ , Al3+ , Cl− , Br− , NO3 − , CO3 2− , SO4 2− , 0.2 mmol/L Zn2+ , Cu2+ and 0.1 mmol/L Fe3+ produced the relative response of <3%, respectively, indicating that these substances coexisting in the sample matrix did not affect the determination of hydrogen peroxide. 4. Conclusions

Fig. 10. Dependence of the electrocatalytic current on the concentration of H2 O2 . Inset is the plot of Lineweaver–Burk equation.

Natural nano-structural attapulgite was purified and used as a matrix to modify glassy carbon electrode. Cyt c could be effectively immobilized on attapulgite modified electrode to produce a fast direct electron transfer. On the surface of ATP/GC electrode, Cyt c retained its bioactivity and native structure. The immobilized Cyt c displayed a high affinity and sensitivity to H2 O2 , which allowed producing a novel H2 O2 sensor for a quick measurement of H2 O2 . This work offers a way to build the new mediator-free sensors by immobilizing proteins or enzymes on natural attapulgite clay for the determination of different substrates, such as glucose using glucose oxidase. This may also be

3606

J.-m. Xu et al. / Electrochimica Acta 52 (2007) 3601–3606

a good alternative method for further study on the direct electrochemistry of redox proteins and their sensing application. Acknowledgements This work was supported financially by the National Natural Science Foundation of China (No. 20571029), the Natural Science Foundation of Jiangsu Education Committee (06KJB150011), and the Science and Technology Bureau of Huai’an city, Jiangsu (HAG06049). References [1] C.C. Moser, C.C. Page, R. Farid, P.L. Dutton, J. Bioenerg. Biomembr. 27 (1995) 263. [2] F. Scheller, F. Schubert, Biosensor, Elsevier, New York, 1992. [3] C.X. Cai, J. Chen, Anal. Biochem. 325 (2004) 285. [4] D.M. Sun, C.X. Cai, X.G. Li, W. Xing, T.H. Lu, J. Electroanal. Chem. 566 (2004) 415. [5] P. Yeh, T. Kuwana, Chem. Lett. (1977) 1145. [6] M.J. Eddowes, H.A.O. Hill, J. Chem. Soc. Chem. Commun. (1977) 771. [7] I. Taniguchi, M. Iseki, H. Yamaguchi, K. Yasukouchi, J. Chem. Soc. Chem. Commun. (1982) 1032. [8] H.A.O. Hill, D.J. Page, N.J. Walton, J. Electroanal. Chem. 217 (1987) 129. [9] F.A. Armstrong, A.M. Bond, H.A.O. Hill, I.S.M. Psalti, C.G. Zoski, J. Phys. Chem. 93 (1989) 6485. [10] T. Sagara, K. Niwa, A. Sone, C. Hinnen, K. Niki, Langmuir 6 (1990) 254. [11] M. L-Dagan, I. B-Dov, I. Willner, Colloids Surf. B 8 (1997) 251. [12] Y. Maeda, H. Yamamoto, H. Kitano, J. Phys. Chem. 99 (1995) 4837. [13] S. Song, R.A. Clark, E.F. Bowden, M.J. Tarlov, Langmuir 97 (1993) 6564. [14] J.D.H. Glenn, E.F. Bowden, Chem. Lett. 399 (1996). [15] H.Y. Liu, L.W. Wang, N.F. Hu, Electrochim. Acta 47 (2002) 2515. [16] E.V. Ivanova, E. Magner, Electrochem. Commun. 7 (2005) 323. [17] C. Lei, F. Lisdat, U. Wollenberger, F.W. Scheller, Electroanalysis 11 (1999) 274.

[18] Y. Sallez, P. Bianco, E. Lojou, J. Electroanal. Chem. 491 (2000) 37. [19] E. Lojou, M.T. Giudici-Orticoni, P. Bianco, J. Electroanal. Chem. 579 (2005) 199. [20] O. Ikeda, M. Ohtani, T. Yamaguchi, A. Komura, Elctrochim. Acta 43 (1998) 833. [21] Z.H. Dai, S.Q. Liu, H.X. Ju, Electrochim. Acta 49 (2004) 2139. [22] J. Yu, H. Ju, Anal. Chem. 74 (2002) 3579. [23] J.J. Feng, G. Zhao, J.J. Xu, H.Y. Chen, Anal. Biochem. 342 (2005) 280. [24] L. Wang, E.K. Wang, Electrochem. Commun. 6 (2004) 49. [25] G.C. Zhao, Z.Z. Yin, L. Zhang, X.W. Wei, Electrochem. Commun. 7 (2005) 256. [26] Y. Sato, F. Mizutani, Electrochim. Acta 45 (2000) 2869. [27] X.Q. Ding, J.H. Lin, J.B. Hu, Q.L. Li, Anal. Biochem. 339 (2005) 46. [28] H.H. Liu, J.L. Lu, M. Zhang, D.W. Pang, H.D. Abruna, J. Electroanal. Chem. 54 (2003) 93. [29] W.F. Bradley, Am Miner. 25 (1940) 405. [30] E. Cao, R. Bryant, D.J.A. Williams, J. Colloid Interface Sci. 179 (1996) 143. [31] R.L. Frost, G.A. Cash, T.J. Kloprogge, Vib. Spectrosc. 16 (1998) 173. [32] J.H. Potgieter, S.S. Potgieter-Vermaak, P.D. Kalibantong, Miner. Eng. 19 (2006) 463. [33] A. Neaman, A. Singer, Appli. Clay Sci. 25 (2004) 121. [34] C. Viseras, A. Lopez-Galindo, Appli. Clay Sci. 14 (1999) 69. [35] E. Galan, Clay Miner. 31 (1996) 443. [36] P. George, G. Hanania, J. Biochem. 55 (1953) 236. [37] P.L. He, N.F. Hu, J.F. Rusling, Langmuir 20 (2004) 722. [38] S. Lee, A. Fitch, J. Phys. Chem. 94 (1990) 4998. [39] F.M. Hawkridge, T. Kuwana, Anal. Chem. 45 (1973) 1021. [40] E. Cao, R. Bryant, D.J.A. Williams, J. Colloid Interface Sci. 179 (1996) 143. [41] E. Laviron, J. Electroanal. Chem. 101 (1979) 19. [42] E. Laviron, J. Electroanal. Chem. 100 (1979) 263. [43] R.A. Kamin, G.S. Wilson, Anal. Chem. 52 (1980) 1198. [44] B.Q. Wang, S.J. Dong, Talanta 51 (2000) 565. [45] Q.L. Wang, G.X. Lu, B.J. Yang, Biosens. Bioelectron. 19 (2004) 1269.