Electrochimica Acta 53 (2008) 7726–7729

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Electrochimica Acta

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A bifunctional structure for the direct electron transfer of Cytochrome c

, Tiol of th

ioxidn on

baseure. Tr and

Lin Zhu, Xiaochun Zhou, Yuwei Zhang, Wei Xing ∗,1

Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Graduate Scho5625 Remin Street, Changchun, 130022, PR China

a r t i c l e i n f o

Article history:Received 1 April 2008Received in revised form 12 May 2008Accepted 12 May 2008Available online 21 May 2008

Keywords:Cytochrome cSilicon dioxide nanoparticleDirect electron transferBiosensorBifunctional structure

a b s t r a c t

It was found that silicon dited a dramatic promotiomechanism was discussedof the bifunctional structwithout chemical mediato

1. Introduction

Inorganic nanoparticles have been investigated as substrates forimmobilizing redox proteins and enzymes thanks to their supe-

rior structural stability and small sizes [1–4]. Lvov et al. reportedheme protein films assembled layer-by-layer with MnO2 or silicondioxide (SiO2) that provide favorable microenvironment for directelectron transfer between the redox proteins and underlying elec-trodes [5]. Hu and co-workers demonstrated that the driving forcesbetween the nanoparticles and the redox proteins were hydro-gen bonds, electrostatic and hydrophobic interactions [6]. Zhu andco-workers showed that the smaller SiO2 exhibited increased elec-trochemical response with respect to their larger counterparts [7,8].In addition, Wang and Wang obtained a pair of quasi-reversiblepeaks of Cytochrome c (Cyt c) by assembling gold nanoparticlesonto silica gel, which act as a bridge for electron transfer betweenprotein and electrode [9]. Ugo et al. achieved the direct electro-chemistry of Cyt c on Au nanodisks in the case of no promoteradded even from unpurified commercial preparations [10,11]. Inthis communication, we discovered a spatial effect of SiO2 on pro-moting the direct electron transfer from glassy carbon (GC) to Cyt cand proposed a spatial geometrical model to explain the observedphenomena [7,8].

∗ Corresponding author. Tel.: +86 431 85262225; fax: +86 431 85685653.E-mail address: xingwei@ciac.jl.cn (W. Xing).

1 ISE Member.

0013-4686/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.electacta.2008.05.034

anhong Lue Chinese Academy of Sciences,

e (SiO2) nanoparticles modified onto glassy carbon (GC) electrode exhib-the direct electron transfer of Cytochrome c (Cyt c). The correspondingd on the electrochemical characteristics and a spatial geometrical modelhe model could offer insight to the study of biosensors and bioreactorsserve as a basis for their fabrication.

© 2008 Elsevier Ltd. All rights reserved.

2. Experimental

2.1. Materials

Horse heart Cyt c was purchased from Sigma Co. and usedas received. The solution for the electrochemical measurementis 0.1 M phosphate buffer solution (PBS, pH 6.8) containing0.24 mmol/l Cyt c. The buffer solutions (0.1 M) were prepared

from NaH2PO4 and Na2HPO4, and other reagents were of ana-lytical reagent grade. The electrochemical measurements wereperformed with an EG&G PAR 273 potentiostat/galvanostat (Prince-ton Applied Research, USA) and a conventional three-electrode cell.Electrochemical measurements were performed at 25 ◦C in a glassmicro-electrochemical cell equipped with a reference Ag/AgCl(SATKCl) electrode, a Pt foil counter electrode and a GC working elec-trode with 3 mm diameter, respectively. Before the measurements,oxygen was purged from the bottom of the solution by bubblingwith highly purified nitrogen for 30 min and a nitrogen environ-ment of the cell was kept during the experiments in the upperpart.

The environment scanning electron microscope (ESEM) imageswere observed with XL-30 ESEM (FEI Company, USA) using anaccelerating voltage of 15 kV with 45◦ angle. We use chrono-coulometry [12] to calculate charge quantity obtained from experi-ments.

The synthesis of the silicon dioxide nanoparticles was the sameas that reported in the literatures [13–15]. Briefly, the sizes of SiO2were adjusted by varying the reaction time and the amounts oftetraethyl orthosilicate (TEOS), ethanol and ammonia.

L. Zhu et al. / Electrochimica Acta 53 (2008) 7726–7729 7727

Fig. 1. ESEM images of 4 mg ml−1 90 nm SiO2 nanoparticles (a) before (b) after adsorbedadsorbed on the SiO2, green is adsorbed on the GC electrode and the red is adsorbed betweto color in this figure legend, the reader is referred to the web version of the article.)

2.2. Preparation of SiO2/GC electrode

2.0, 4.0 or 6.0 mg of SiO2 nanoparticles were dispersed in 1 mlwater to obtain 2.0, 4.0 or 6.0 mg ml−1 of SiO2 suspension. The GCelectrode with 3 mm diameter was polished to a mirror-like statewith 0.3 and 0.05 �m alumina slurry successively. Then, it was son-icated and washed with the doubly distilled water. Then 5 �l SiO2suspension with the concentration of 2.0, 4.0 or 6.0 mg ml−1 wascast on the surface of the GC electrode. After drying at least 24 h,the SiO2/GC electrode was obtained. The stability and the repro-ducibility of the modified electrode were all excellent.

3. Results and discussion

3.1. Adsorption model of Cyt c on the modified electrode

Fig. 1 shows the ESEM images of SiO2 before and after adsorptionby Cyt c. We speculate on that there are three kinds of adsorption

Fig. 2. Cyclic voltammograms of GC electrodes modified with (a) 0 mg ml−1, (b) 2 mg ml−

size of the SiO2 nanoparticles: 90 nm, scan rate: � = 100 mV s−1. Inset sketch maps are cor

by Cyt c. Three adsorbed forms of Cyt c onto the modified GC electrode: blue isen the on the interface of SiO2 and GC electrode. (For interpretation of the references

forms of Cyt c on the modified GC electrode as shown in Fig. 1b.The blue small balls are those balls adsorbed on the surface of SiO2through the electrostatic interaction between them [16], while thegreen small balls are adsorbed on the GC electrode surface includingabundant hydroxyl and carboxyl groups [6]. The red small balls areadsorbed on the interface between SiO2 and GC electrode.

3.2. Direct electron transfer of Cyt c on the modified electrode

Fig. 2 shows the cyclic voltammograms (CV) of Cyt c at themodified GC electrode. Peak 1 decreases with increasing the con-centration of SiO2. However, peak 2 increases initially and thendecreases, the drop magnitude is related to the concentration ofSiO2 (as seen in Fig. 3). There is only one pair of redox peaks at thebare GC electrode (Fig. 2a). When the concentration is too low forSiO2 to cover the surface of the GC electrode (Fig. 2b), there are twopairs of peaks in the CV curve. Peak 1 belongs to the SiO2-uncovered

1, (c) 4 mg ml−1 and (d) 6 mg ml−1 SiO2 in 3 mg ml−1 Cyt c in PBS (pH 6.8); averageresponding to the CVs.

ica Acta 53 (2008) 7726–7729

) 4 m

7728 L. Zhu et al. / Electrochim

Fig. 3. ESEM images of GC electrodes modified with (a) 2 mg ml−1, (b

part of the electrode while peak 2 to the SiO2-covered part whichis similar to the completely modified electrode. The optimal SiO2concentration of around 4 mg ml−1 (Fig. 2c) results in a pair of well-defined quasi-reversible redox peaks. When the concentration of

SiO2 is too high to allow complete access of Cyt c through the SiO2layer to GC electrode (Fig. 2d), the peak current is dramaticallydecreased. It is reasonable that only the Cyt c adsorbed onto theinterface between SiO2 and GC electrode could exhibit enhancedelectrochemical activities. This is because adsorption sites possessthe bifunctional structure suitable for facilitating both the electrontransfer and the immobilization. The electrons can be transferreddirectly to the GC electrode from the contacted Cyt c. The effect ofimmobilization results from the strong adsorption of Cyt c on theSiO2. The Cyt c can keep its native activity after adsorbed on theinterface between SiO2 and GC electrode compared with the casewhere it is being adsorbed on the bare GC electrode. Thus, it can beconcluded that the different electrochemical responses in CV wereattributed to the different adsorption forms of Cyt c.

3.3. Role of the bifunctional structure

3.3.1. SiO2 of different sizes with the same concentrationsAs shown in Fig. 4, a spatial geometrical model based on the

structure was proposed to illustrate the microenvironment where

Fig. 4. Spatial geometrical model for the bifunctional structure for direct electrontransfer of Cyt c.

g ml−1 and (c) 6 mg ml−1 of suspension of SiO2 with 90 nm diameter.

Table 1Theoretical and practical ratio of charge quantity of SiO2 (4 mg ml−1)

Practicle diameter, a/ba (nm) 90/110 90/150 90/200Theoretical ratio of charge quantity 1.35 2.14 3.30Practical ratio of charge quantity 1.56 1.76 2.82

a a and b are the diameters of different size of SiO2.

the direct electron transfer of Cyt c was promoted by the interactionbetween SiO2 and Cyt c. The following are the special geometrical

parameters and the charge quantity related to different sizes of SiO2with the same concentrations on identical GC electrodes and in thesame electrochemical microenvironments.

The theoretical values can be calculated by the followed equa-tions.

When the surface coverage of Cyt c on interface between SiO2and GC electrode is 100%, the amount of Cyt c adsorbed on theinterface per SiO2 grain is

n = 2�

2˛(1)

where n is the amount of Cyt c adsorbed on the interface betweenSiO2 and GC electrode. ˛ is the angle labeled in Fig. 4.

˛ = arcsin12

√r

R(2)

where r and R are the radius of Cyt c and SiO2, respectively.The total amount of Cyt c on the whole surface of GC electrode

is

N = n

SA (3)

Fig. 5. Cyclic voltammograms of GC electrodes modified with 5 �l 4 mg ml−1 (a)90 nm, (b) 110 nm, (c) 150 nm and (d) 200 nm SiO2 in 3 mg ml−1 Cyt c in PBS (pH6.8); scan rate: � = 100 mV s−1. Insets plot of chronocoulometric charge quantity vs.time.

ica Ac

l−1, (b

L. Zhu et al. / Electrochim

Fig. 6. (a) The sketch map of the concentration lower than 4 mg m

where S and A are the section of SiO2 grain and the area of the GCelectrode, respectively. Because SiO2 is a hexagonal assembly (asseen in Fig. 3), the surface area of SiO2 is 2

√3R2.

The total amount of charge quantities of the Cyt c at the wholeGC electrode are

Q = kAn

S(4)

where k is the charge quantity per Cyt c molecule. Because there isonly one electron transfer in this reaction, k = 1.6 × 10−19 C.

Combine Eqs. (1)–(4), we can obtain that

Q = 1.6 × 10−19A�

2√

3R2 arcsin 1/2√

r/R(5)

For the different size (a and b) of SiO2, the ratio of charge quantityis

Qa

Qb= arcsin 1/2

√r/Rb

arcsin 1/2√

r/Ra

(Rb

Ra

)2(6)

It can be seen from Table 1 that the practical values from theCV curves in Fig. 5 fit well with the theoretical ones. The resultsdemonstrated that the red balls in the model are effective Cyt c,

namely, Fig. 4 quantitatively interprets the effect of bifunctionalstructure on the direct electron transfer between Cyt c and the GCelectrode.

3.3.2. SiO2 of same sizes with the different concentrationsIn order to testify the validity of the model and mechanism

further, we also did the following work. SiO2 of same sizes withdifferent concentrations on identical GC electrodes was illustratedin Fig. 6a. When the concentration of SiO2 is low, it is assumed thatthe ratio of the Cyt c amount adsorbed on the interface betweenSiO2 and GC electrode to those displaced per SiO2 grain on the GCelectrode is constant. As seen from the following equation:

practical value = Qa1�′1 − Qb1�1

Qb2�2 − Qa2�′2

(7)

number 1 and 2 represent peak 1 and peak 2, a and b represent thedifferent concentrations of SiO2, �1 and �2 represent the surfacecoverage of Cyt c adsorbed on the GC electrode and that on theinterface between SiO2 and GC electrode, respectively, �′

1 and �′2

correspondence to �1 and �2 in the case of low loading are alsoconsidered as constant. When the concentration

[

[[

[[[

[

ta 53 (2008) 7726–7729 7729

) the effect of the concentration on the ratio of charge quantities.

of SiO2 increases beyond a certain threshold, both the electroac-tive Cyt c (the red ones) and the Cyt c displaced by SiO2 on the GCelectrode (�′

1 and �′2) decrease due to the thicker casting of SiO2

which block the access of Cyt c to the GC electrode. The combinedresult is in a gradual increase in the practical value of the ratio incharge quantity (Fig. 6b) which can be deduced from Eq. (7). Theexperimental trends of peak 1 and peak 2 agree with those pre-dicted in theoretical model (as seen in Fig. 2), as is the ratio of chargequantity, in quantitative agreement with the proposal.

4. Conclusions

SiO2 can promote the direct electron transfer of Cyt c. The drivingforce of the direct electron transfer and the change of microen-vironment are related to the spatial geometrical configuration ofbifunctional structure. The present model qualitatively interpretedthe effect of nanoparticles size and concentration in direct elec-tron transfer of redox proteins, and opened a new way for theextensive applications of inorganic nanoparticles to promote theelectrochemistry of adsorbed.

Acknowledgement

This work was supported by the financial sponsorship of NatureScience Foundation of China (20433060).

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