Direct Electrochemistry and Electrocatalytic Activity of Cytochrome c

Apr 30, 2008 - Chem. , 2008, 80 (11), pp 4141–4146 ... City University of Hong Kong. ... For a more comprehensive list of citations to this article,...
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Anal. Chem. 2008, 80, 4141–4146

Direct Electrochemistry and Electrocatalytic Activity of Cytochrome c Covalently Immobilized on a Boron-Doped Nanocrystalline Diamond Electrode Yanli Zhou,† Jinfang Zhi,*,† Yousheng Zou,‡ Wenjun Zhang,*,‡ and Shuit-Tong Lee†,‡ Laboratory of Organic Optoelectric Functional Materials and Molecular Engineering, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100080, China, and Center of Super-Diamond and Advanced Films (COSDAF) and Department of Physics and Materials Science, City University of Hong Kong, Hong Kong SAR, China Cytochrome c (Cyt c) was covalently immobilized on a boron-doped nanocrystalline diamond (BDND) electrode via surface functionalization with undecylenic acid methyl ester and subsequent removal of the protecting ester groups to produce a carboxyl-terminated surface. Cyt c-modified BDND electrode exhibited a pair of quasireversible and well-defined redox peaks with a formal potential (E0) of 0.061 V (vs Ag/AgCl) in 0.1 M phosphate buffer solution (pH 7.0) and a surface-controlled process with a high electron transfer constant (ks) of 5.2 ( 0.6 s-1. The electrochemical properties of as-deposited and Cyt c-modified boron-doped microcrystalline diamond (BDMD) electrodes were also studied for comparison. Investigation of the electrocatalytic activity of the Cyt c-modified BDND electrode toward hydrogen peroxide (H2O2) revealed a rapid amperometric response (5 s). The linear range of response to H2O2 concentration was from 1 to 450 µM, and the detection limit was 0.7 µM at a signal-to-noise ratio of 3. The stability of the Cyt c-modified BDND electrode, in comparison with that of the BDMD and glassy carbon counterpart electrodes, was also evaluated. Cytochrome c (Cyt c) is a basic redox heme protein which plays an important role in the biological respiratory chain by transferring electrons between membrane-bound enzyme complexes of Cyt c reductase and Cyt c oxidase.1 However, electrochemical detection of Cyt c using metal electrodes is very difficult because of the extremely slow electron-transfer kinetics by the denaturation of proteins at electrode surfaces. Electrodes modified with various electron transfer mediators have been used to investigate the electron transfer mechanism between Cyt c solution and electrodes,2–4 which leads to a better understanding of the natural redox properties of the protein and the interfacial charge transfer process. It is known that the heme-containing proteins such as Cyt c, myoglobin, and horseradish peroxidase, and so forth, have the ability to electrocatalyze the reduction of hydrogen * To whom correspondence should be addressed. Tel/Fax: +86-10-8254-3537. E-mail: [email protected] (J.Z.). E-mail: [email protected] (W.Z.). † Chinese Academy of Sciences. ‡ City University of Hong Kong. (1) Gong, J.; Yao, P.; Duan, H. W.; Jiang, M.; Gu, S. H.; Chunyu, L. J. Biomacromolecules 2003, 4, 1293–1300. 10.1021/ac702417x CCC: $40.75  2008 American Chemical Society Published on Web 04/30/2008

peroxide (H2O2). Recently, amperometric biosensors have been developed on the basis of Cyt c immobilized on a self-assembled monolayer,5 conductive polymer membranes,6 and nanomaterial modified/incorporated electrodes.7,8 These modified electrodes have shown the capability to immobilize Cyt c, and their direct electrochemistry and electrocatalysis toward H2O2 have also been studied. Nevertheless, some inherent defects such as low stability, complexity of the preparation process, and low conductivity limited their applications in amperometric biosensors. The search for reliable electrode material and a simple method to immobilize Cyt c is still of considerable interest. Boron-doped microcrystalline diamond (BDMD) thin films have been reported to be ideal substrates for amperometric biosensors.9–13 BDMD-based thin-film electrodes have a unique combination of electrochemical properties, for example, (i) wide electrochemical potential window, (ii) low and stable background current, (iii) high resistance to deactivation by fouling, and (iv) biocompatibility.12,13 It has been demonstrated that surface smoothness of BDMD electrodes affect their amperometric response significantly. Mechanical polishing of a BDMD electrode to a nanometer-scale finish has been shown to result in a welldefined voltammetric response of Cyt c in solution.14 Compared with BDMD (grain sizes in µm range) electrodes, boron-doped nanocrystalline diamond (BDND, grain sizes below 100 nm) thinfilm electrodes have a smoother surface while maintaining the intact diamond properties. Moreover, as discussed in ref 15, in (2) Lojou, E´.; Bianco, P. J. Electroanal. Chem. 2000, 485, 71–80. (3) Ion, A.; Banica, F. G. J. Solid State Electrochem. 2001, 5, 431–436. (4) Wang, J. X.; Li, M. X.; Shi, Z. J.; Li, N. Q.; Gu, Z. N. Anal. Chem. 2002, 74, 1993–1997. (5) Ge, B.; Lisdat, F. Anal. Chim. Acta 2002, 454, 53–64. (6) Jiang, X.; Zhang, L.; Dong, S. J. Electrochem. Commun. 2006, 8, 1137– 1141. (7) Xu, J.-M.; Li, W.; Yin, Q.-F.; Zhu, Y.-L. Electrochim. Acta 2007, 52, 3601– 3606. (8) Ju, H. X.; Liu, S. Q.; Ge, B. X.; Lisdat, F.; Scheller, F. W. Electroanalysis 2002, 14, 141–147. (9) Popa, E.; Notsu, H.; Miwa, T.; Tryk, D. A.; Fujishima, A. Electrochem. Solid State Lett. 1999, 2, 49–51. (10) Lee, J.; Park, S.-M. Anal. Chim. Acta 2005, 545, 27–32. (11) Zhou, Y. L.; Zhi, J. F. Electrochem. Commun. 2006, 8, 1811–1816. (12) Fujishima, A.; Rao, T. N.; Popa, E.; Sarada, B. V.; Yagi, I.; Tryk, D. A. J. Electroanal. Chem. 1999, 473, 179–185. (13) Xu, J. S.; Chen, Q. Y.; Swain, G. M. Anal. Chem. 1998, 70, 3146–3154. (14) Marken, F.; Paddon, C. A.; Asogan, D. Electrochem. Commun. 2002, 4, 62–66.

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addition to boron doping, the sp2-bonded carbon phase on grain boundaries of BDND films can provide charge carriers and high carrier mobility pathways, which may lead to better reversible properties of electrodes for redox systems. Thus, the BDND thin film is expected to be a more suitable candidate to provide a high activity for biosensors.16 In fact, it has been reported that direct electron transfer could occur between Cyt c existing in solution and the as-deposited BDND electrode with quasi-reversible, diffusion-controlled electron transfer kinetics.17 In addition, BDMD electrodes modified by IrOx electrodeposition and Pt implantation have been applied for electrochemical oxidation of H2O2, and a low detection limit (10-8 M) was obtained.18,19 However, in real biological matrices, a major problem for electrochemical detection of H2O2 is the coexistence of many interfering compounds including ascorbic acid, uric acid, dopamine, and so forth. These compounds can be oxidized at a potential close to that of H2O2, which results in the overlap of voltammetric response. Because Cyt c is able to electrocatalyze the reduction of H2O2, amperometric biosensors based on Cyt c immobilization on electrode surfaces, in particular via covalent modification, which is prone to obtain the direct electron transfer between protein and electrode surfaces, can be used to detect H2O2 selectively. Therefore, the biosensors based on Cyt c immobilized covalently onto BDND electrodes are expected to have good electrochemical redox behavior and excellent eletrocatalytic activity for the reduction of H2O2 due to the above distinguished chemical and electrochemical properties. Herein, we used a photochemical reaction scheme to modify hydrogen-terminated BDND surfaces chemically using a vinyl group of undecylenic acid methyl esters, followed by hydrolyzation of the esters under basic conditions to produce a monolayer of carboxylic groups on the surfaces. Cyt c was covalently immobilized for the first time on the resulting carboxyl-terminated BDND electrode through carbodiimide coupling reaction. We further investigated direct electrochemistry of Cyt c and electrocatalytic activity for reduction of H2O2 at the Cyt c-modified BDND electrode. EXPERIMENTAL SECTION Reagents. Cyt c (from horse heart, molecular weight 12384 g mol-1) and H2O2 were supplied by sigma. N-Hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Acros organics Co. All other chemicals were of analytical grade, and the water used was obtained from a Millipore M-Q purification system (M-Q water, >18 MΩ cm). The phosphate buffer solution (PBS) was prepared with KH2PO4 and K2HPO4. (15) Bennett, J. A.; Wang, J.; Show, Y.; Swain, G. M. J. Electrochem. Soc. 2004, 151, E306-E313. (16) Rubio-Retama, J.; Hernando, J.; Lo´pez-Ruiz, B.; Ha¨rtl, A.; Steinmu ¨ ller, D.; Stutzmann, M.; Lo´pez-Cabarcos, E.; Garrido, J. A. Langmuir 2006, 22, 5837–5842. (17) Haymond, S.; Babcock, G. T.; Swain, G. M. J. Am. Chem. Soc. 2002, 124, 10634–10635. (18) Terashima, C.; Rao, T. N.; Sarada, B. V.; Spataru, N.; Fujishima, A. J. Electroanal. Chem. 2003, 544, 65–74. (19) Ivandini, T. A.; Sato, R.; Makide, Y.; Fujishima, A.; Einaga, Y. Diamond Relat. Mater. 2005, 14, 2133–2138. (20) Zhang, R. J.; Lee, S. T.; Lam, Y. W. Diamond Relat. Mater. 1996, 5, 1288– 1294. (21) Le´vy-Cle´ment, C.; Ndao, N. A.; Katty, A.; Bernard, M.; Deneuville, A.; Comninellis, C.; Fujishima, A. Diamond Relat. Mater. 2003, 12, 606–612.

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Preparation of BDND Thin Films. BDND thin films were prepared by hot-filament chemical vapor deposition (HFCVD) on silicon (100) wafers. To enhance nucleation, Si substrates were abraded with 1 µM diamond powder and then ultrasonically cleaned successively in acetone and in M-Q water for 1 min. Ta wires of 0.6 mm in diameter and 14 cm in length were used as filaments. The experimental conditions were filament-substrate distance, 6 mm; Tfil, 2100 ± 200 °C; Tsub, 850-950 °C; vapor pressure, 0.7 KPa; H2 flow rate, 200 standard cubic centimeters per minute (sccm). Acetone was used as the carbon source and its concentration (CH3COCH3/H2, V/V) was 3%, while trimethyl borate dissolved in the acetone was used as boron source at a B/C molar ratio of 0.5%. The surface morphologies of the BDND films were observed by scanning electron microscopy (SEM, Hitachi S-4800). The phase composition of the BDND films was evaluated by Raman spectroscopy (Renishaw 1000) using a He-Ne laser (632.8 nm) as an excitation source. For comparison, BDMD thin films were also prepared under conditions similar to those for depositing BDND thin films except that the vapor pressure was elevated to 1.7 KPa. Preparation of the Cyt c-Modified BDND Electrode. The as-deposited BDND films were heated to approximately 700 °C in 2.45 GHz inductively coupled hydrogen plasma (45 Torr H2) for 45 min and subsequently cooled in hydrogen plasma to terminate the surface with hydrogen. In a Teflon reactor with constant N2 gas purging, the hydrogen-terminated BDND surface was covered with a thin layer of undecylenic acid methyl ester and exposed to a 254 nm UV lamp (20 W) for 12 h. The ester group-modified BDND surface was hydrolyzed by immersion in a 250 mM solution of potassium tert-butoxide (t-BuOK) in dimethyl sulfoxide (DMSO) for 3 min at room temperature, followed by rinsing with 100 mM HCl solution to obtain a carboxyl-terminated BDND surface. Reflection-absorption infrared (RAIR) spectroscopy obtained using a Perkin-Elmer PC 16 FT-IR spectrophotometer was performed to study the surface functionalization process. To immobilize Cyt c, the carboxyl-terminated BDND samples were treated with 1:1 of 30 mM EDC and 15 mM NHS (in 0.01 M PBS, pH 7.0) for 1 h to activate carboxylic groups through the formation of a NHS-ester intermediate. After rinsing with buffer solution, 20 µL of 100 µM solution of Cyt c in 0.01 M PBS (pH 7.0) was dropped onto the surface of active carboxyl-terminated BDND electrodes (D ) 0.56 cm). After reaction for 90 min, the Cyt c-modified BDND electrodes were washed with buffer solution and preserved in a refrigerator at 4 °C until use. For comparison, counterpart biosensors in which Cyt c was immobilized on BDMD and glassy carbon (GC) electrodes were also prepared following the same procedure. Electrochemical Measurements. A CHI 802 electrochemical workstation (Shanghai Chenhua, China) was used for electrochemical analysis. Electrochemical impedance spectroscopy measurements were carried out with a 263A Potentiostat/Galvanostat and a FRD 100 frequency response detector (Princeton, USA). All experiments were performed in a three-electrode cell system with the Cyt c-modified BDND electrode, platinum wire, and Ag/ AgCl (saturated KCl) electrode as working electrode, auxiliary electrode, and reference electrode, respectively. Amperometric experiments were carried out in a stirred cell by applying a potential of -0.05 V (vs Ag/AgCl) to the working electrode, and

Figure 1. Raman spectrum of the as-deposited BDND film. Insets are the corresponding (A) top-view and (B) cross-sectional SEM images of the film.

the current-time curve was recorded after a steady-state current was achieved. The electrolyte solutions were purged with highpurity N2 gas for 15 min to reduce the level of oxygen dissolved before each electrochemical measurement. RESULTS AND DISCUSSION Characterization of As-Deposited BDND Film. Insets (A) and (B) of Figure 1 show the top-view and cross-sectional SEM images obtained from the as-deposited BDND films. A smooth and uniform BDND film with grain sizes of 20-100 nm and a thickness of about 13 µm was deposited after growth for 7 h. There are four features in the Raman spectrum of the BDND film as shown in Figure 1. The 1332 cm-1 diamond peak widens and shifts to a lower wavenumber (1313 cm-1), which is consistent with the Raman result of conductive diamond in refs 20 and 21. A broad peak at 500 cm-1 and a shoulder peak around 1200 cm-1 are associated with the Fano effect due to the doped boron.22 A shoulder peak around 1150 cm-1 is often used as a signature for nanocrystalline diamond and is attributed to a surface phonon mode of diamond.23 The signal with a maximum at 1540 cm-1 is assigned to disordered sp2-bonded carbon in the grain boundaries, and the signal is weak indicating the low proportion of sp2-bonded carbon.24 The above results demonstrate that a high-quality BDND film has formed on silicon by HFCVD. To compare the electrochemical properties of BDMD and BDND electrodes, the cyclic voltammograms (CVs) for 0.5 mM [Fe(CN)6]3-/4- in 0.1 M PBS and Nyquist plots of impedance measurements for 10 mM [Fe(CN)6]3-/4- in 0.1 M KCl were performed at BDND and BDMD electrodes, as shown in Figure 2. In Figure 2A, well-defined CV curves of [Fe(CN)6]3-/4- on the two electrodes were obtained, indicating nearly reversible or quasireversible electron-transfer kinetics for both electrode interfaces. However, compared with the BDMD electrode, there was an (22) Ushizawa, K.; Watanabe, K.; Ando, T.; Sakaguchi, I.; Nishitani-Gamo, M.; Sato, Y.; Kanda, H. Diamond Relat. Mater. 1998, 7, 1719–1722. (23) Show, Y.; Witek, M. A.; Sonthalia, P.; Swain, G. M. Chem. Mater. 2003, 15, 879–888. (24) Prawer, S.; Nugent, K. W.; Jamieson, D. N.; Orwa, J. O.; Bursill, L. A.; Peng, J. L. Chem. Phys. Lett. 2000, 332, 93–97.

Figure 2. (A) CVs of 0.5 mM [Fe(CN)6]3-/4- in 0.1 M PBS (pH 7.0) at the scan rate of 100 mV s-1 and (B) Nyquist plots of 10 mM [Fe(CN)6]3-/4- in 0.1 M KCl from 10 KHz to 0.1 Hz at an ac amplitude of 10 mV under open-circuit potential conditions, obtained at (a) BDND and (b) BDMD electrodes.

increase in the cathodic and anodic current responses and a decrease in the separation of peak to peak (∆Ep from 92 to 73 mV) at BDND electrodes. In Figure 2B, the semicircular portion at higher frequencies represents the electron-transfer-limited process, and the electron transfer resistance (Ret) is equal to the semicircle diameter. The BDND electrode shows a lower interfacial electron transfer resistance (98 ± 5 Ω) than that of the BDMD (260 ± 19 Ω) electrode. These facts suggest that the reversibility of electrode reaction becomes better and that electron transfer is faster at the BDND electrode in comparison with that at the BDMD electrode. The superior properties of the BDND electrode are believed to be ascribed to the increased sp2-bonded carbon phase on grain boundaries of BDND films, which may provide charge carriers and high carrier mobility pathways.15 Therefore, BDND film is an ideal substrate to study the redox properties of proteins because of not only its flat surface but also its rapid electron transfer process compared to that of the BDMD electrode. Characterization of Carboxyl-Terminated BDND Surface. The procedures of photochemical functionalization and subsequent covalent attachment of Cyt c to a BDND surface are depicted in Scheme 1. Figure 3 shows the RAIR spectra obtained from before and after deprotection of the functionalized BDND surfaces. The spectrum of methyl ester-terminated surface shows a strong peak at 1740 cm-1 arising from the CdO stretching mode of the methyl ester. The two peaks at 2854 and 2930 cm-1 are assigned to the CH2 symmetric and asymmetric stretching modes, while the shoulder located at 2960 cm-1 corresponds to the CH3 vibration of the terminal methyl group. After deprotection, the peak of CdO stretching mode shifts to a lower wavenumber at 1700 cm-1, and the shoulder peak (2960 cm-1) disappears because of the change of methyl ester groups to carboxylic groups. Thus, the functionalization process has produced a high density of carboxylic groups on the BDND surface. Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

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Scheme 1. Schematic Illustration of the Photochemical Functionalization and Subsequent Covalent Attachment of Cyt c to a BDND Surface

Electrochemical Impedance Response of Cyt c Immobilization. Electrochemical impedance spectroscopy is an effective method to probe the change of interface properties of an electrode surface during the modification process. Typical Nyquist plots for the modified BDND electrodes in the presence of 10 mM [Fe(CN)6]3-/4- in 0.1 M KCl are shown in Figure 4. Ret could be estimated to be 97 ± 6, 298 ± 19, and 404 ± 32 Ω for the hydrogenterminated, carboxyl-terminated, and Cyt c-modified BDND electrodes, respectively. The value of Ret at the carboxyl-terminated BDND electrode evidently increases compared with that of the hydrogen-terminated BDND electrode, indicating that the modification of long chain fatty acid on the electrode blocks the electrochemical reaction by an electrostatic repulsion between the negatively charged carboxylic groups and the negative redox couple ions. After the immobilization of Cyt c, the value of Ret has a further increase, resulting from the barrier layer of the protein. These data show that Cyt c has been successfully immobilized on the BDND electrode by the bonding of negatively charged carboxylic groups and positively charged lysine residue of Cyt c (pI 10).

Figure 3. RAIR spectra obtained from (a) methyl ester-terminated and (b) carboxyl-terminated BDND surfaces. The reference of each spectrum is the hydrogen-terminated BDND surface.

Figure 4. Nyquist plots for (a) hydrogen-terminated, (b) carboxylterminated, and (c) Cyt c-modified BDND electrodes in the presence of 10 mM [Fe(CN)6]3-/4- in 0.1 M KCl from 10 KHz to 0.1 Hz at an ac amplitude of 10 mV under open-circuit potential conditions. 4144

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Figure 5. (A) CVs of the Cyt c-modified BDND electrode in 0.1 M PBS (pH 7.0) at different scan rates: (a) 50, (b) 80, (c) 100, (d) 150, (e) 200, (f) 250, (g) 300, (h) 350, (i) 400, (j) 450, and (k) 500 mV s-1. (B) The plots of (a) anodic and (b) cathodic peak currents vs scan rates.

Direct Electrochemistry of the Cyt c-Modified BDND Electrode. Figure 5A shows the CVs of Cyt c-modified BDND electrode in 0.1 M PBS (pH 7.0) at different scan rates. The Cyt c-modified BDND electrode shows a pair of well-defined redox peaks with a ∆Ep of 69 mV at the scan rate of 50 mV s-1, and ∆Ep increases with the increase of the scan rate. The formal potential (E0 ) (Epa + Epc)/2) is around 0.061 V (vs Ag/AgCl), which is consistent with the formal potential of Cyt c present in solution at the BDND electrode.17 As show in Figure 5B, a linearity of the cathodic or anodic peak currents with scan rates suggests that the reaction is a surface-controlled process, verifying that Cyt c was stably immobilized on the electrode surface. When n∆Ep < 200 mV, the electron transfer rate constant (ks) could be obtained according to Laviron’s formula, m ) (RT/F)(ks/nv).25 Assuming the electron rate coefficient R ) 0.5, ks of the Cyt c-modified BDND electrode was estimated to be 5.2 ± 0.6 s-1 for the scan rates ranging from 350 to 500 mV s-1. The value of ks obtained in the present BDND electrode is higher than those of Cyt c immobilized on a colloidal gold-modified carbon paste electrode,8 a multiwalled carbon nanotube-modified electrode,26 and a L-cysteine-modified gold electrode.27 The high value of ks suggests that good quasi(25) Laviron, E. J. Electroanal. Chem. 1979, 101, 19–28.

Figure 6. CVs of the Cyt c-modified (a) BDND and (b) BDMD electrodes in 0.1 M PBS (pH 7.0) at a scan rate of 100 mV s-1.

reversible electron-transfer kinetics is observed for Cyt c immobilized on the BDND electrode surface. According to Laviron’s equation,28 Ip ) n2F2Av/4RT, surface coverage (v) of the Cyt c-modified BDND electrode was calculated to be 1.2 × 10-11 mol cm-2, where A (the real electrode surface area) was estimated to be 0.27 cm2 including the contribution of surface roughness.29 The shape of the Cyt c molecule is roughly spherical with a diameter of ca. 3.4 nm,30 indicating that the theoretical monolayer coverage is about 1.7 × 10-11 mol cm-2 based on the assumption that the molecules reached hexagonal monolayer packing. It can be concluded that Cyt c modification on the surface of functionalized BDND electrodes is an approximate monolayer. Furthermore, BDND thin films are expected to be better substrates than BDMD to study the redox properties of protein. To demonstrate this, a comparison of CVs for Cyt c-modified BDND and BDMD electrodes was measured in 0.1 M PBS (pH 7.0) at the scan rate of 100 mV s-1 as shown in Figure 6. Compared with the Cyt c-modified BDMD electrode, as expected, an increase in the cathodic and anodic current responses and a decrease in the ∆Ep were observed at the Cyt c-modified BDND electrode, which supports the above claim that the electron transfer of the Cyt c-modified BDND electrode is faster than that of the Cyt c-modified BDMD electrode. Electrocatalytic Behavior of the Cyt c-Modified BDND Electrode. In order to investigate the electrocatalytic activity of Cyt c on the BDND electrode, its response to the reduction of H2O2 was explored. The CVs of Cyt c-modified BDND electrode in the presence (curve a) and absence (curve b) of 100 µM H2O2 are shown in Figure 7. In the absence of H2O2, only the redox peaks of Cyt c are observed. Upon the addition of 100 µM H2O2 to the buffer solution, the voltammetric behavior of the Cyt c-modified BDND electrode changes dramatically, with an increase of the obvious catalytic reduction peak located at -0.05 V (vs Ag/ AgCl) and a disappearance of the oxidation peak due to the fast electron transfer of the oxidation of Cyt c by H2O2. For comparison, no evident reduction peak of H2O2 is observed on the carboxylterminated BDND electrode (curve c in Figure 7). These results show that the immobilized Cyt c retains its electrocatalytic activity to reduce H2O2 diffusing from solution onto the BDND electrode surface. (26) Zhao, G.-C.; Yin, Z.-Z.; Zhang, L.; Wei, X.-W. Electrochem. Commun. 2005, 7, 256–260. (27) Liu, Y.-C.; Cui, S.-Q.; Zhao, J.; Yang, Z.-S. Bioelectrochem. 2007, 70, 416– 420. (28) Laviron, E. J. Electroanal. Chem. 1979, 100, 263–270. (29) Tian, R.-H.; Zhi, J.-F. Electrochem. Commun. 2007, 9, 1120–1126. (30) D’Souza, F.; Rogers, L. M.; O’Dell, E. S.; Kochman, A.; Kutner, W. Bioelectrochem. 2005, 66, 35–40.

Figure 7. CVs of the Cyt c-modified BDND electrode in 0.1 M PBS (pH 7.0) in the (a) presence and (b) absence of 100 µ H2O2. Curve (c) shows the CV of the carboxyl-terminated BDND electrode in 0.1 M PBS (pH 7.0) containing 100 µM H2O2. Scan rate is 25 mV s-1.

Figure 8. Amperometric response of the Cyt c-modified BDND electrode to H2O2 in 0.1 M PBS (pH 7.0) at -0.05 V vs Ag/AgCl. Inset shows the calibration curve.

As shown in Figure 8, the amperometric response of the Cyt c-modified BDND electrode was recorded through successive addition of H2O2 into a continuously stirred buffer solution at an applied potential of -0.05 V(vs Ag/AgCl). Upon the addition of H2O2, the steady-state current of Cyt c-modified BDND electrode achieved 95% in less than 5 s. Such a fast response is attributed to a direct electron transfer between Cyt c and the BDND electrode. The inset of Figure 8 illustrates the dependence of the electrocatalytic current on the concentration of H2O2. The response to H2O2 is linear in the range from 1 to 450 µM (R ) 0.999), and a sensitivity of 75.6 mA M-1 cm-2 can be calculated from the slope of the linear range. The detection limit is 0.7 µM at a signal-to-noise ratio of 3, which is lower than those of the conductive polymer membrane-modified ITO electrode6 and the nanohybrid film-modified GC electrode.31 The low detection limit of the Cyt c-modified BDND electrode could be ascribed to the high loading of Cyt c by the present method and the rapid electron transfer between Cyt c and the BDND electrode. The reproducibility and stability of the Cyt c-modified BDND electrode was also investigated by the measurement of the response to 100 µM H2O2 in 0.1 M PBS (pH 7.0). The relative standard deviation (RSD) is 2.1% for 10 successive assays. The fabrication reproducibility was examined at five different Cyt c-modified BDND electrodes prepared under the same conditions, and the RSD is 6.9%. The long-term stability of the developed enzyme electrode was determined every 3 days, and 85% of its initial activity can be obtained after 6 weeks. Moreover, for comparison, the stability of Cyt c-modified BDMD and GC electrodes were also explored simultaneously. It was revealed that (31) Xiang, C. L.; Zou, Y. J.; Sun, L.-X.; Xu, F. Talanta 2007, 74, 206–211.

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Cyt c-modified BDMD and GC electrodes retained 80% and 25% of their initial activity after 6 weeks, suggesting that the stability of Cyt c-modified BDND electrode is nearly equivalent to that of the Cyt c-modified BDMD electrode but is significantly higher than that of the Cyt c-modified GC electrode. The superior stability of the Cyt c-modified BDND electrode may be the result of the biocompatible microenvironment for the enzyme provided by the BDND electrode and the high chemical and electrochemical stability of the BDND electrode. CONCLUSIONS In summary, this study has introduced a novel method for immobilizing Cyt c covalently on the carboxyl-terminated BDND surfaces. Direct electron transfer between Cyt c and the electrode was obtained on the Cyt c-modified BDND electrode, and the electron transfer is faster than that of the Cyt c-modified BDMD electrode because of the incorporated sp2 state carbon as charge transfer mediators on BDND surface. The prepared Cyt c-modified

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BDND electrode showed excellent electrocatalytic performance in terms of fast response, low detection limit, and high stability toward the reduction of H2O2. Therefore, the outstanding electrochemical properties, together with its inherent biocompatibility and flat surface, make the BDND thin film an interesting candidate for the study of the direct electrochemistry of redox proteins and their sensing applications. ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (Project No. 20773150) and the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. CityU 122805 and CityU 123806). Support from the Croucher Foundation is also gratefully acknowledged. Received for review November 26, 2007. Accepted March 29, 2008. AC702417X