pH-dependent electrochemical behavior of proteins with different isoelectric points on the nanostructured TiO2 surface

Journal of Electroanalytical Chemistry 642 (2010) 109–114

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pH-dependent electrochemical behavior of proteins with different isoelectric points on the nanostructured TiO2 surface Yongping Luo, Yang Tian *, Anwei Zhu, Haiqing Liu, Jinqing Zhou Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 30 November 2009 Received in revised form 7 February 2010 Accepted 10 February 2010 Available online 14 February 2010 Keywords: Electrochemical behavior Proteins Isoelectric points Nanostructured TiO2

a b s t r a c t Nanostructured materials are widely employed for facilitating electron transfer of proteins, and then constructing the third-generation biosensors. For further understanding the mechanism that nanomaterials facilitate electron transfer of proteins, nanostructured titanium dioxide (TiO2) film was prepared as an example for investigating the interaction between proteins and electrode surfaces. The TiO2 film was characterized by electrochemical methods, and the point of zero charge (pzc) was determined to be 6.27. On the other hand, cytochrome c (cyt. c) and superoxide dismutase (SOD) were raised as model proteins because they have different isoelectric points of 10.5 and 4.9, respectively. Direct electron transfer of cyt. c and SOD was achieved at the nanostructured TiO2 surfaces at pH 7.0 and the proteins were verified to be stably immobilized onto the TiO2 films. Furthermore, electrochemical behavior of cyt. c and SOD modified on nanostructured TiO2 film was investigated in a series of different pH PBS solutions based on cyclic voltammetry. It was found that electron transfer of proteins strongly depended on pH of solutions, i.e., the surface charge of TiO2 film. This observation could be ascribed to the electrostatic interaction between proteins and surfaces. This is the first report to provide the insight in the relationship between electron transfer of proteins and interaction of surfaces charge. Ó 2010 Elsevier B.V. All rights reserved.

1. Introduction Recently, it is considerable interesting to achieve direct electron transfer between electrodes and redox proteins. Understanding of these reactions can provide the insight into physiological electron transfer process as well as an impetus to the further development of biosensors and bioelectrocatalytical systems [1,2]. However, most of the proteins have a big 2D lipid structure, and their electroactive prosthetic group embeds deeply in the protein structure and unfavorable orientation of protein molecules on the electrode surface [3]. Therefore, it is still a challenging work to observe direct electron transfer of proteins. Up to now, a number of methods have been developed, such as mediators, promoters and nanomaterials, for realizing direct electron transfer of proteins [4,5]. With the development of nanoscience and nanotechnology, the nanostructured metal oxides, including SiO2 [6], TiO2 [7], ZnO [8], WO3 [9] and Nb2O5 [10], have been widely employed for achieving direct electron transfer of proteins. Nanostructured TiO2 is an electrode material of great importance in the fabrication of dye-sensitized solar cells and electrochromic devices [11]. It is also an efficient material for direct electron transfer of pro* Corresponding author. Tel.: +86 21 65987075; fax: +86 21 65981097. E-mail address: [email protected] (Y. Tian). 1572-6657/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2010.02.021

teins, due to its high specific surface area, good biocompatibility, stability and environmental safety. Luo et al. [12] first reported direct electron transfer of heme proteins assembled on nanocrystalline TiO2 film based on graphite substrate. Durrant and his coworkers have systematically investigated direct spectroelectrochemistry of proteins such as hemoglobin (Hb) and cytochrome c (cyt. c) on the nanostructured TiO2 film [13]. Recently, direct electron transfer of superoxide dismutase (SOD) has been realized at the nanospherical TiO2 film [14]. Meanwhile, direct electrochemistry of cyt. c has been achieved at highly conductive nanoneedles TiO2 films. On the basis of the direct electron transfer of protein, the biosensor for H2O2 has been developed with high analytical performance [15]. In the present work, in order to further understand the mechanism that the TiO2 surface facilitates the electron transfer of proteins, effect of pH on the electrochemical behavior of proteins with different isoelectric points were first systematically investigated, in which cyt. c with pI of 10.5 and SOD with pI of 4.9 were selected as model proteins. Firstly, the prepared nanostructred TiO2 films were characterized by electrochemical methods, and the surface pKa of the TiO2 film was estimated to be 6.27. At the nanostructured TiO2 surface, electron transfer of both cyt. c and SOD was greatly facilitated. In addition, the electrochemical behavior of proteins was found to be strongly dependent on pH of the solution, i.e., surface charge of TiO2 film. This observation

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could be ascribed to the electrostatic interaction between proteins and the nanostructured TiO2 surface.

were purged with purified nitrogen for at least 30 min to remove oxygen. And nitrogen was controlled during experiments.

2. Experimental

3. Results and discussion

2.1. Chemicals and materials

3.1. Characterization of the TiO2 film

Horse heart cyt. c and bovine erythrocyte Cu, Zn–SOD were purchased from Sigma and used without further purification. Phosphate buffered solution (PBS) was prepared with 10 mM KH2PO4–K2HPO4. The pH of the buffer solutions was adjusted to different value by a pH meter. Other reagents were analytical grade. All aqueous solutions were prepared with double distilled water. Indium tin oxide (ITO)-coated glass plates with a square resistance of 10–20 X cm2 were obtained from Shenzhen Nanbo Display Technology Co., Ltd. (China).

Fig. 1A shows a top-view SEM image of high-density TiO2 film. Spherical TiO2 nanoparticles with a mean diameter of 40 nm were clearly observed on the whole sheet, and the thickness of TiO2 film was estimated to be 3 lm. The crystalline orientation of TiO2 films was investigated by XRD, as shown in Fig. 1B. For the comparison, XRD pattern of ITO (a) was also given in Fig. 1B. The three intense diffraction peaks located at 25.36°, 38.03° and 48.21°, which correspond to the indices of (1 0 1), (0 0 4), (2 0 0) of anatase TiO2, are consistent with the reported values Joint Committee on Povder diffraction Standards JCPDS card (21-1272) [16]. In order to ascertain phase composition of TiO2 after immobilized on ITO-coated glass, the GAXRD pattern of TiO2 film (c) with a glancing angle of 0.1° was also given in Fig. 1B. This observation indicates that the prepared TiO2 film was confirmed to be seized of anatase crystal structure. The surface pzc of the nanostructured TiO2 film was determined by electrochemical method previously reported by Dong’s group [17]. Fig. 2 shows the relationship between anodic peak currents of an electron transfer indicator like K3Fe(CN)6 and pH value of bulk solution. From the peak of the differential curve, the surface pzc was estimated to be 6.27, which is in a good agreement with that reported previously [18]. This result suggests that the nanostructured TiO2 film shows negative charge at the neutral solution (pH 7.0). Electrochemical properties of the TiO2 film were also characterized by cyclic voltammetry and electrochemical impendence spectroscopy (EIS), as demonstrated in Fig. 3A and B, respectively. A decrease in the redox peak and a augment of the peak-to-peak separation (DEp) was observed, which may be ascribed to the elecor FeðCNÞ4 anion and the trostatic repulsion between FeðCNÞ3 6 6 negatively charged TiO2 surface. This observation was further confirmed by EIS. As shown in Fig. 3B, the charge-transfer resistance 3=4 redox couple is near 43.8 X at ITO substrate (Rct) of FeðCNÞ6 (a), but it increases to 304.8 X at the TiO2 surface (b). The remarkable increase in the charge-transfer resistance at the TiO2 film, not the resistance of film (Rfilm) itself suggests again that the negatively charged TiO2 surface inhibits the electron transfer of FeðCNÞ3 6 and/ or FeðCNÞ4 6 anion with same negative charge due to the electrostatic repulsion. These surface properties of the nanostructured TiO2 film can affect the electron transfer of proteins with different isoelectric points, which will be described later.

2.2. Preparation and modification ITO-coated glass plates were thoroughly cleaned by sonication for 20 min in the following solutions successively: neat ethanol, 1 M NaOH at 60 °C, rinsed by distilled, deionized water. The TiO2 sol (Ishihara Sangyo Kaisha, Ltd., Japan) was coated onto ITOcoated glass by spin-coating method, then annealed at 450 °C for 1 h. Protein-modified electrodes were prepared by immersing the above TiO2 film into the 0.2 mM protein PBS (pH 7.0). The immersion time for cyt. c and SOD are about 30 min and 3 h, respectively. The prepared protein-modified electrodes stored in 10 mM PBS (pH 7.0) at 4 °C while no use. 2.3. Apparatus and measurements An XRD pattern was performed by a D/max2550VB3 + /PC X-ray diffractometer using Cu Ka (40 kV, 100 mA). And structural character was studied by glancing angle X-ray diffraction (GAXRD) using Brucker D8 advanced diffractometer. Cu Ka was used as radiation (wavelength = 0.15418 nm) and a glancing angle of 0.1° for measurement. The UV–Vis absorption spectrum was collected by an Agilen 8453 UV–Vis near-infrared spectrophotometer (Agilent Instruments). The specific surface area surface area of TiO2 powder was calculated by the BET method with Micromeritics Tristar3000 instrument and was then employed to perform an appropriate normalization. All electrochemical measurements were performed in a conventional three-electrode cell with a CHI 660 electrochemical work station (Shanghai Chenhua Apparatus, China). The reference electrode was a KCl-saturated Ag|AgCl electrode, while the auxiliary electrode was a platinum wire. Prior to experiment, solutions

(B)

101

Intensity / a.u.

(A)

004

200

c b a

20

30

40

50

60

70

80

2θ / degree Fig. 1. (A) SEM of the nanostructured TiO2 film. (B) XRD patterns of ITO (a) and the TiO2 film (b), and GAXRD pattern of TiO2 film (c) with a glancing angle of 0.1°.

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40

(A)

0

8

38

-15

30

-20

4 0 -4

J / µA cm -2

a

32

a

34

di p / dpH

-10

J / µA cm -2

-5

36

I p / µA

12

-8

28 -25 26

-12

3 2 1 0 -1 -2 -3

Jpa

Jpc

0 100 200300400500 -1

V / mV s

3

4

5

6

7

8

9

-0.2

-0.1

pH

(B)

0.2

0.3

lg K s ¼ algð1  aÞ þ ð1  aÞlga  lgðRT=nF v Þ  að1  aÞnFDEp =2:3RT where a is the electron transfer coefficient, Ks is the standard rate 0 constant, v is the scan rate and E0 is the formal potential. Electron transfer rate constant (Ks) of cyt. c and SOD at the TiO2 surface were

0 12 8 4 0 -4 -8 -12

-5 -10 -15

Jpa

Jpc

0 100 200 300400 500 -1

V / mV s

-20 -0.3

-0.2

-0.1

0.0

0.1

0.2

E / V vs. Ag| AgCl Fig. 4. CVs obtained at (A) TiO2/cyt. c and (B) TiO2/SOD electrodes in 10 mM PBS (pH 7.0) at scan rate of 10–500 mV s1. The inset shows the relationship between peak currents and scan rates.

estimated to be 7.97 s1 and 3.83 s1, respectively. The Ks value indicated a quasi-reversible redox process of cyt. c and SOD at the nanostructured TiO2 film. Electrochemical impedance spectroscopic data also support this conclusion [20,21]. Fig. 6 shows Nyquist plots obtained at TiO2/cyt. c (A) and TiO2/SOD (B) electrodes in 0.1 M KCl solution containing 1 mM ½FeðCNÞ6 3=4 . The charge-transfer resistance of cyt. c and SOD immobilized at the TiO2 surfaces is estimated to be 1.067  104 X and 6.179  103 X, respectively. The charge-transfer resistances of protein-immobilized TiO2 electrodes have remarkable increases, compared with that obtained at bare TiO2 surface. It confirmed again that both cyt. c and SOD adsorbed onto electrode surfaces and inhibited the electrochemical communication between the electron transfer indicator (FeðCNÞ63=4 ) and the

(B)

0.18

5

J / µ A cm -2

J / µA cm -2

Fig. 4 depicts typical CVs obtained on TiO2/cyt. c (A) and TiO2/ SOD (B) films in PBS (pH 7.0) at different scan rates. Well-defined redox peaks were clearly observed at both TiO2/cyt. c and TiO2/ SOD surfaces at the neutral solution, while no peaks were obtained 0 at bare TiO2 film (data not shown). The formal potential (E0 ) of cyt. c and SOD were estimated to be 36.6 ± 1.2 mV and 66.4 ± 2.3 mV versus Ag|AgCl at the nanostructured TiO2 film, respectively. It was found that both anodic and cathodic peak currents (Iap and Icp ) of cyt. c and SOD vary linearly with the potential scan rate (v) in the potential scan rate range of 10–500 mV s1 (shown in the inset of Fig. 4). This observation indicates that both the two kinds of proteins with different isoelectric points are successfully immobilized on the nanostructred TiO2 film surface. The Ep versus ln v plot gives a straight line at higher scan rates. Fig. 5A and B shows the relation of Ep versus ln v for TiO2/cyt. c and TiO2/SOD electrode, respectively. aa and ac are calculated from the slope RT/nFaa and RT/ nFac, respectively. Scan rate dependent potential shifting can be used for estimation of the standard rate constant of the surface reaction. While DEp > 200/n mV, based on the Laviron’s equation: [19]

15 10

3.2. Electrochemistry of proteins with different isoelectric points on the nanostructured TiO2 film

160 140

0.12

b

b

120

a

0.06

Z im / ohm

J / mA cm -2

0.1

E / V vs. Ag| AgCl

Fig. 2. Relationship between anodic peak currents of K3Fe(CN)6 and pH value of bulk solution. Black line: simulated curve of Iap , and red line: differential curve of Iap . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(A)

0.0

0.00 -0.06

100

a

80 60 40

-0.12

20 0

-0.18 0.0

0.1

0.2

0.3

0.4

E / V vs. Ag|AgCl

0.5

0.6

0

50

100 150 200 250 300 350

Zre / ohm

Fig. 3. (A) CVs obtained at ITO glass (a) and TiO2 film (b) in 0.1 M KCl solution containing 1 mM FeðCNÞ3=4 . Scan rate: 100 mV s1. (B) Nyquist plots of FeðCNÞ63=4 at ITO 6 glass (a) and TiO2 film (b).

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nanostructured surfaces. The total amount of charge (Q) passing the electrode for the redox reaction of electroactive proteins on the TiO2 films was obtained by integration of redox peaks at scan rate of 100 mV s1, and was estimated to be 2.437 and 1.648 lC for TiO2/cyt. c and TiO2/SOD, respectively. The surface concentration (C) of electroactive cyt. c on nanostructured TiO2 films is calculated to be 2.105  10–11 mol cm2, while that of SOD is estimated to be 1.423  10–11 mol cm2. It is based on the equation: C = Q/nFA, where n is the number of electrons transferred, A is the real electrode surface area (1.2 cm2), F is Faraday’s constant. Taking account of the size of cyt. c (2.6  3.2  3.0 nm) [22] and SOD molecules (3.0  3.3  3.6 nm) [23], it could be considered that cyt. c packed on the nanostructured TiO2 film in a monolayer with end-on, while SOD with side-on and end-on. 3.3. pH-dependent electrochemical behavior of proteins As demonstrated above, electron transfer of both cyt. c and SOD could be facilitated at the nanostructured TiO2 surface. In order to deeply understand the relationship between the electron transfer of proteins and the surface charge of TiO2 film, the electrochemical behavior of cyt. c and SOD at the nanostructred TiO2 film was investigated in 10 mM PBS with different pH values from 5.5 to 9.0. The relationship between either peak currents or peak separations of CVs obtained at TiO2/cyt. c (A) and TiO2/SOD (B) surfaces, and pH values of PBS was plotted in Fig. 7. Both anodic and cathodic peak currents of cyt. c increased with the increasing pH up to

(A)

around 7.0, and peak currents then gradually decreased when pH increased in the alkaline solution. Meanwhile, as expected, the peak separation (DEp) of cyt. c redox reaction at the nanostructured TiO2 film showed the opposite changes compared with peak currents, i.e., DEp decreased with the increasing pH up to around 7.0, then gradually increased when pH increased in the alkaline solution. On the other hand, peak currents of SOD increased with the decreasing pH values in the range of 5.5–9.0, and DEp of SOD redox reaction at the nanostructured TiO2 film increased with the increasing pH values. The electrochemical behavior of proteins was proposed to be dependent on the surface charge of the TiO2 films. As described above, the surface pzc was estimated to be 6.27 by the electrochemical method, meaning that TiO2 surface is negatively charged above pH 6.27 and positively charged below pH 6.27. Cyt. c and SOD have isoelectric points of 10.5 and 4.9, suggesting that cyt. c is positively charged below pH 10.5 and SOD is negatively charged above 4.9. As shown in Fig. 7A and C, in the case of cyt. c, at low pH (pH < 6), both cyt. c and the TiO2 film are positively charged and therefore electrostatic repulsion prevails. However, at pH 7–10, cyt. c is positively charged and a strong electrostatic interaction is expected. The maximum peak currents and minimum peak separation of cyt. c redox reaction were observed at pH 7.0, may due to the strong electrostatic interaction and favorable pH for electron transfer of proteins. On the other hand, in the case of SOD (Fig. 6B and D), at pH 7–10, both SOD and the TiO2 film are negatively charged and therefore electrostatic attraction is weakened.

(B)

0.20 0.15

Epa

0.05

Epa

0.00

Ep / V

Ep / V

0.10 0.05

-0.05 -0.10

0.00

Epc

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Epc

-0.05

-0.20 -0.10 1.50 1.55 1.60 1.65 1.70 1.75 1.80

0.4

0.5

ln V

0.6

0.7

0.8

0.9

ln V

Fig. 5. Plot of Ep and ln v for (A) TiO2/cyt. c and (B) TiO2/SOD electrode.

(B)

(A) 6000

3500 3000

5000

b

b

Z im / ohm

3000

a

2000

Z im / ohm

2500 4000

2000

a

1500 1000

1000

500

a

0

a

0 0

2000 4000 6000 8000 10000 12000

Z re / ohm

0 1000 2000 3000 4000 5000 6000 7000 8000

Z re / ohm

Fig. 6. Nyquist plots obtained at bare TiO2 film (a), and at (A) TiO2/cyt. c (b) and (B) TiO2/SOD (b) electrodes in 0.1 M KCl solution (pH 7.0) containing 1 mM K3Fe(CN)6/ K4Fe(CN)6.

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1.2

+

J / µA cm -2

0.8 0.4

(B)

Surface charge of cyt. c

+

3 2

+ J / µA cm -2

(A)

+ +

0.0 -0.4

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Surface charge of SOD

1

-

-

-

0 -1

-0.8 -2 -1.2

Surface charge of the film

Surface charge of the film

+ +

-

-

-

-

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

pH

(C)

69.4 69.2 69.0

+ + Surface charge of cyt. c ΔE/ mV

ΔE/ mV

68.8 68.6

+

68.2

+

68.0 67.8 67.6

Surface charge of the film

+ +

-

-

pH

(D)

68.4

-

+ + -3 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

-

-

67.4 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

142 140 138 136 Surface charge of SOD 134 132 130 128 126 124 122 120 118 Surface charge of the film 116 + + 114 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5

pH

pH

Fig. 7. Relationship between peak currents (A and B) and peak separations (C and D) of (A and C) cyt. c, (B and D) SOD-modified TiO2 films and pH values of PBS solution. Potential scan rate: 100 mV s1.

However, at low pH (pH < 6), SOD is still negatively charged and TiO2 surface is positively charged, so a strong electrostatic interaction is expected to result in the larger peak currents and smaller peak separations. Thus, we can conclude that electrochemical behavior of proteins is strongly dependent on surface charge of TiO2 film, which may be ascribed to the electrostatic interaction between proteins and surfaces. In addition, the conformation of proteins varies with pH [24], thus pH possibly affects the charge transport pathway and kinetics. The proteins may adsorb onto the nanostructured TiO2 surface mainly through electrostatic and other interactions, and orient toward favorable for the direct electron transfer of themselves.

4. Conclusions The as-prepared TiO2 film was characterized by electrochemical methods, and the surface pzc of the TiO2 film was determined to be 6.27. The direct electron transfer of both cyt. c and SOD, which have different isoelectric points, was successfully realized at the nanostructured TiO2 film. Meanwhile, the proteins were found to be immobilized onto TiO2 surfaces. For further understanding the mechanism that the TiO2 surface facilitate the electron transfer of proteins, the electrochemical behavior of cyt. c and SOD were investigated in the range of pH from 5.5 to 9.0. Experimental results indicated that the electrochemical behavior of proteins is strongly dependent on pH of solution, i.e., the surface charge of TiO2 film, which may be attributed to the electrostatic interaction between proteins and electrode surfaces. This investigation not only first provided the new insight in the electrochemistry of proteins at the nanostructured TiO2 surface, but also opened up a way to design the electron transfer of proteins at the nanomaterials sur-

faces and then construct the third-generation biosensors with excellent analytical performance. Acknowledgments This work was financially supported by National Natural Science Foundation of China (20975075), the Program for New Century Excellent Talents in University (NCET-06-0380) and by the Scientific Research Foundation for the Returned Overseas Chinese Scholars from State Education Ministry, China, and Nanometer Science Foundation of Shanghai (0952nm04900). Tongji University is also greatly acknowledged. References [1] L.S. Duan, Q. Xu, F. Xie, S.F. Wang, Int. J. Electrochem. Sci. 3 (2008) 118–124. [2] J.M. Xu, W. Li, Q.F. Yin, H. Zhong, Y.L. Zhu, L.T. Jin, J. Colloid Interf. Sci. 315 (2007) 170–176. [3] N. Li, J.Z. Xu, H. Yao, J.J. Zhu, H.Y. Chen, J. Phys. Chem. B 110 (2006) 11561– 11565. [4] (a) K. Nakano, T. Yoshitake, Y. Yamashita, E.F. Bowden, Langmuir 23 (2007) 6270–6275; (b) Y. Tian, M. Shioda, S. Kasahara, T. Okajima, L. Mao, T. Hisabori, T. Ohsaka, Biochim. Biophys. Acta 1569 (2002) 151–158. [5] (a) S. Sergey, W. Jonas, M. Karl-Eric, R. Tautgirdas, Biosens. Bioelectron. 22 (2006) 213–219; (b) Y. Tian, L. Mao, T. Okajima, T. Ohsaka, Anal. Chem. 74 (2002) 2428–2432; (c) T. Ohsaka, Y. Tian, M. Shioda, S. Kasahara, T. Okajima, Chem. Commun. (2002) 990–991; (d) Y. Tian, L. Mao, T. Okajima, T. Ohsaka, Anal. Chem. 76 (2004) 4162–4168; (e) Y. Tian, T. Ariga, N. Takashima, T. Okajima, L. Mao, T. Ohsaka, Electrochem. Commun. 6 (2004) 609–614. [6] (a) G. Shi, Z. Sun, M. Liu, L. Zhang, Y. Liu, Y. Qu, L. Jin, Anal. Chem. 79 (2007) 3581–3588; (b) L. Zhu, X. Zhou, Y. Zhang, W. Xing, T. Lu, Electrochim. Acta 53 (2008) 7726– 7729; (c) H. Liu, N. Hu, Electroanalysis 19 (2007) 884–892.

114

Y. Luo et al. / Journal of Electroanalytical Chemistry 642 (2010) 109–114

[7] (a) F. Wu, J. Xu, Y. Tian, Z. Hu, L. Wang, Y. Xian, L. Jin, Biosens. Bioelectron. 24 (2008) 198–203; (b) H. Cao, Y. Zhu, L. Tang, X. Yang, C. Li, Electroanalysis 20 (2008) 2223–2228; (c) S. Bao, C.M. Li, J. Zang, X. Cui, Y. Qiao, J. Guo, Adv. Funct. Mater. 18 (2008) 591–599; (d) A. Liu, M. Wei, I. Honma, H. Zhou, Anal. Chem. 77 (2005) 8068–8074. [8] (a) Z. Deng, Q. Rui, X. Yin, H. Liu, Y. Tian, Anal. Chem. 80 (2008) 5839–5846; (b) W. Sun, Z. Zhai, D. Wang, S. Liu, K. Jiao, Bioelectrochemistry 74 (2009) 295– 300; (c) Z. Dai, G. Shao, J. Hong, J. Bao, J. Shen, Biosens. Bioelectron. 24 (2009) 1286– 1291; (d) X. Lu, H. Zhang, Y. Ni, Q. Zhang, J. Chen, Biosens. Bioelectron. 24 (2008) 93– 98; (e) A. Umar, M.M. Rahman, S.H. Kim, Y.B. Hahn, J. Nanosci. Nanotechnol. 8 (2008) 3216–3221. [9] (a) Z. Deng, Y. Gong, Y. Luo, Y. Tian, Biosens. Bioelectron. 24 (2009) 2465–2469; (b) J. Feng, J. Xu, H. Chen, Electrochem. Commun. 8 (2006) 77–82. [10] (a) X. Xu, B. Tian, S. Zhang, J. Kong, D. Zhao, B. Liu, Anal. Chim. Acta 519 (2004) 31–38; (b) S.S. Rosatto, P.T. Sotomayor, L.T. Kubota, Y. Gushikem, Electrochim. Acta 47 (2002) 4451–4458. [11] (a) H.J. Snaith, A. Petrozza, S. Ito, H. Miura, M. Gratzel, Adv. Funct. Mater. 19 (2009) 1810–1818; (b) P. Chen, J.H. Yum, F. De Angelis, E. Mosconi, S. Fantacci, S. Moon, R.H. Baker, J. Ko, M.K. Nazeeruddin, M. Gratzel, Nano Lett. 9 (2009) 2487–2492; (c) T. Bessho, E. Yoneda, J. Yum, M. Guglielmi, I. Tavernelli, H. Imai, U. Rothlisberger, M.K. Nazeeruddin, M. Gratzel, J. Am. Chem. Soc. 131 (2009) 5930–5934; (d) F. Fabregat-Santiago, J. Bisquert, L. Cevey, P. Chen, M. Wang, S. Zakeeruddin, M. Gratzel, J. Am. Chem. Soc. 131 (2009) 558–562; (e) Y. Tian, T. Tatsuma, Chem. Commun. 16 (2004) 1810–1811; (f) Y. Tian, T. Tatsuma, J. Am. Chem. Soc. 127 (2005) 7632–7637;

[12] [13]

[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]

(g) Y. Tian, H. Notsu, T. Tatsuma, Photochem. Photobiol. Sci. 8 (2005) 598–601; (h) Y. Tian, X. Wang, D. Zhang, X. Shi, S. Wang, J. Photochem. Photobiol: A. Chem. 199 (2008) 224–229; (i) Y. Tian, X. Shi, C. Lu, X. Wang, S. Wang, Electrochem. Commun. 11 (2009) 1603–1605. Q. Li, G. Luo, J. Feng, Electroanalysis 13 (2001) 359–363. (a) E. Topoglidis, T. Lutz, J.R. Durrant, E. Palomares, Bioelectrochemistry 74 (2008) 142–148; (b) E. Topoglidis, T. Lutz, R.L. Willis, C.J. Barnett, A.E. Cass, J.R. Durrant, Faraday Discuss. 116 (2000) 35–46; (c) E. Topoglidis, C.J. Campbell, A.E.G. Cass, J.R. Durrant, Electroanalysis 18 (2006) 882–887; (d) E. Topoglidis, C.J. Campbell, A.E.G. Cass, J.R. Durrant, Proc. Electrochem. Soc. 18 (2001) 196–202. Y. Luo, Y. Tian, A. Zhu, Q. Rui, H. Liu, Electrochem. Commun. 11 (2009) 174– 176. Y. Luo, H. Liu, Q. Rui, Y. Tian, Anal. Chem. 81 (2009) 3035–3041. D. Wang, B. Yu, J. Hao, W. Liu, Mater. Lett. 62 (2008) 2036–2038. J.W. Zhao, L.Q. Luo, X.R. Yang, E.K. Wang, S.J. Dong, Electroanalysis 11 (1999) 1108–1111. E. Vassileva, I. Proinova, K. Hadjiivanov, Analyst 121 (1996) 607–612. E. Laviron, J. Electroanal. Chem. 101 (1979) 19–28. C. Fernández-Sánchez, C.J. McNeil, K. Rawson, TrAC, Trends Anal. Chem. 24 (2005) 37–48. E. Katz, I. Willner, Electroanalysis 15 (2003) 913–947. X. Zhang, J. Wang, W. Wu, S. Qian, Y. Man, Electrochem. Commun. 9 (2007) 2098–2104. Z. Deng, Y. Tian, X. Yin, Q. Rui, H. Liu, Y. Luo, Electrochem. Commun. 10 (2008) 818–820. I. Chiara, D.S. Giampiero, N. Francesca, S. Helena, S. giulietta, C. Massimo, Biochemistry 39 (2000) 8234–8242.