Direct electrochemistry of hemoglobin in gold nanowire array

Biosensors and Bioelectronics 23 (2007) 414–420

Direct electrochemistry of hemoglobin in gold nanowire array Minghui Yang, Fengli Qu, Yongjun Li, Yan He ∗ , Guoli Shen ∗∗ , Ruqin Yu State key laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering Center, Chemistry and Chemical Engineering College, Hunan University, Changsha 410082, PR China Received 2 February 2007; received in revised form 19 April 2007; accepted 9 May 2007 Available online 18 May 2007

Abstract Gold nanowire array has been proven to be efficient support matrixes for the immobilization of hemoglobin (Hb). The vertically oriented nanowire array provides an ordered well-defined 3D structure with nanowire density ∼5 × 108 cm2 . The adsorption of ferritin onto the nanowire surface was visualized by transmission electron microscopy. When Hb was adsorbed, UV–vis absorption and Fourier transform infrared (FT–IR) spectra show no obvious denaturation of Hb in the nanowire array. The Hb-modified nanowire array exerted direct electron transfer and gave a well-defined, nearly reversible redox couple with formal potential of −0.225 V. The quantity of electroactive Hb varied with the changing of the morphology of the electrode and found to increase with the increasing of the nanowire length. Comparisons of voltammetric and quartz crystal microbalance measurements show that 70% of the Hb molecules adsorbed are electroactive when the length of the nanowire was 2 ␮m. Both of the Hb-modified nanowire array and the unmodified nanowire array demonstrate good electrocatalytic reduction ability for hydrogen peroxide. With the adsorption of glucose oxidase onto the bare nanowire surface, sensitive and selective glucose biosensors can be fabricated. © 2007 Published by Elsevier B.V. Keywords: Direct electrochemistry; Gold nanowire array; Hemoglobin; Glucose

1. Introduction There has been increasing interest in the fabrication of biosensors based on direct electron transfer between metalloprotein and the electrode (Fan et al., 2001; Armstrong et al., 1988; Zhou et al., 2005). These studies may find applications in investigating the protein structure, mechanism of redox transformation of protein molecules, and mimicking catalytic roles in living systems (Beissenhirtz et al., 2004; Guan et al., 2004; Kluger and Zhang, 2003). However, many electron transfer metalloproteins are membrane-associated, the efficient electrical communication between redox proteins and solid electrode surfaces is hindered (Hamachi et al., 1997). To enhance electron transfer between proteins and electrode, various supporting films have been explored, such as organic films including insoluble surfactants (Bayachou et al., 1998), biopolymers (Panchagnula et al., 2002), Nafion and polyelectrolyte (Huang et al., 1996; ∗

Corresponding author. Tel.: +86 731 8821355; fax: +86 731 8821355. Corresponding author. E-mail addresses: [email protected] (Y. He), [email protected] (G. Shen). ∗∗

0956-5663/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.bios.2007.05.003

Yu et al., 2003). Recently, the adsorption of proteins on inorganic materials has been widely reported (Wang et al., 2004a,b; Topoglidis et al., 1998; Topoglidis et al., 2001). These materials are intrinsically more stable and provide the friendly microenvironment for protein loading and improve the stability of the immobilized biomolecules. However, to date, most studies on immobilization of proteins were focused on layered or mesopore-structured films. In most cases, the distribution of protein molecules is not uniform, sometimes spatially hindered, resulted in relatively low electron-transfer efficiency, that is only a very small percentage of the proteins immobilized are electroactive. Gold nanoparticles were among the most widely used inorganic materials for the immobilization of proteins (Lei et al., 2004; Lei et al., 2003; Hu et al., 2005). These gold nanoparticles can act as tiny conduction centers and can facilitate the transfer of electrons, the mechanism of which has been intensively investigated (Bharathi et al., 2001; Hicks et al., 2001). Further, many works have shown enzymes maintain their biocatalytic and electrochemical activity when immobilized on gold nanoparticles (Crumbliss et al., 1993; Zhao et al., 1996). However, rare attention has been paid in the use of gold nanowire

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for the immobilization of proteins. It would be of significant advancement if perpendicularly aligned gold nanowire array could be used as immobilization matrix. The aligned nanowire array would provide a large well-defined surface area and the capacity for modifying the nanowire surface with proteins, so that the biomolecules would be spatially patterned, which would adopt a more flexible orientation and resulted in high amount of electroactive proteins. Gold nanowire ensembles have been prepared using electroless deposition method in porous template, but the electroless deposition method involves complex procedures, and especially, the lengths of the nanowires could not be controlled, so that nanowires are not aligned parallel, but bend toward each other, forming closely interacting bundles as the length of the nanowires is relatively long (Krishnamoorthy and Zoski, 2005; Menon and Martin, 1995). In this paper, considering the advantages of nanowire array and the biocompatibility of metal gold, we synthesized gold nanowire array in polycarbonate (PC) membranes by means of direct electrodeposition technique directly on glassy carbon (GC) electrode (Xue et al., 2005; Lux and Rodriguez, 2006). We take advantages of the large surface area of the 3D nanowire array to investigate the efficiency of biomolecule immobilization. Hemoglobin (Hb) is selected, as it is one of the most studied proteins in existence. Spectroscopy, electrochemistry and quartz crystal microbalance were used to characterize the Hb after it adsorbed onto nanowire surface. Keeping the amount of Hb on the electrode constant, we found that with increasing nanowire length, the electron-transfer efficiency increased; that is, more Hb molecules participated in the electron-transfer reaction. When the length of the nanowire was about 2 ␮m, the nanowire array was used to absorb Hb and 70% of the molecules are electroactive. Also during the experiment, we found the bare nanowire array has good electrocatalytic reduction ability toward hydrogen peroxide, and then glucose oxidase (GOx) was adsorbed onto the nanowire surface for the fabrication of glucose biosensor with high sensitivity and good selectivity. 2. Experimental

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urated calomel electrode (SCE) as reference electrode and a platinum foil electrode as counter electrode was used. All potentials were measured and reported versus the SCE. The piezoelectric quartz crystals (AT-cut, 9 MHz) were purchased from Chenxing Radio Equipments (Beijing, China). The frequency of the resonance crystal was recorded every 100 ms with data acquisition system connected to a computer system (QCA 922 Quartz Crystal Analyzer, SEIKO EG&G Co., Chiba, Japan). Scanning electron microscopy (SEM) analysis was performed using a JSM-5600LV microscope (JEOL, Ltd. Japan). Transmission electron microscope (TEM) image was taken with a JEM-3010 transmission electron microscope (JEOL Co. Ltd, Japan). Fourier transform infrared (FT–IR) spectra were obtained on a WQF-410 Fourier transform infrared spectrophotometer (Beijing Secondary Optical Instruments, China). UV–vis adsorption spectra were recorded on a Perkin–Elmer UV–vis-near-IR spectrometer, Lambda 900. All experiments were carried out at room temperature. 2.2. Synthesis of Au nanowire arrays For the electrodeposition of Au nanowire arrays, a thin film of Au (∼30 nm) was first sputtered onto one face of the PC template making the template conductive. In a typical experiment, the membrane was attached gold-side down on the glassy carbon (GC, 2 mm diameter) electrode surface and covered by a rubber O-ring. Electrodeposition was performed using chronoamperometry in 1% (w/w) HAuCl4 solution containing 0.5 M perchloric acid at potential of 0.18 V. After deposition, the PC template was dissolved by immersion the electrode in chloroform. 2.3. Modification of the nanowire with proteins ferritin In an attempt to immobilize ferritin molecules, the dispersed Au gold nanowire was simply stirred with diluted ferritin solution for 1 h. Then the samples were filtered, washed, and placed on copper grids for TEM characterization.

2.1. Apparatus and reagents

2.4. Preparation of Hb-modified gold nanowire array

Track-etched porous polycarbonate (PC, 0.2 ␮m) membrane was provided by Whatman. Glucose oxidase (GOx, from Aspergillus niger; EC 1.1.3.4, type VII-S; 196,000 unit g−1 ), ferritin (from horse spleen, Type I) and hemoglobin (HB, MW 64,500) were purchased from Sigma. A 1/15 M phosphate buffer (PB, pH 6.98) solution was prepared with Na2 HPO4 and KH2 PO4 and the buffer was in thoroughly anaerobic conditions by bubbling with high purity nitrogen, except otherwise mentioned. All other reagents were of analytical grade, and doubly distilled water was used throughout. Cyclic voltammetric and amperometric measurements were carried out on CHI 760B electrochemical workstation (Shanghai, China). A three-electrode cell (10 mL) with the modified glassy carbon (GC) electrode as the working electrode, a sat-

Prior to modification, the nanowire array was washed successively using Piraha (3:1 v/v mixture of concentrated H4 SO4 and H2 O2 ), ethanol and water. A 2 ␮L aliquot of 0.1 mM Hb solution was then uniformly dropped onto the electrode surface. In a control experiment, Hb-modified conventional gold electrode (2 mm diameter) was prepared by dropping 2 ␮L of the Hb solution onto the electrode surface. These modified electrodes were dried at 4 ◦ C overnight before use. 2.5. Preparation of GOx modified gold nanowire array For the modification of the nanowire array with GOx, the nanowire array modified electrode was immersed into GOx solution (5 mg/mL) overnight at 4 ◦ C

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2.6. Quartz crystal microbalance (QCM) experiment The modification of the QCM with gold nanowire array was performed using the same method as the modification of GC electrode. After cleaning, the modified QCM was immersed into Hb solution (0.1 mM) for different times to absorb Hb molecules and then the resonance frequency change and cyclic voltammograms were recorded. 3. Results and discussion 3.1. Morphological characterization of the prepared gold nanowire array Recently, nanoelectrode-array based on nanowires and nanotubes have been widely used as composite electrode. For the preparation of nanoelectrode-array, the most often used method is to add an insulating material onto the electrode surface to insulate every nanoelectrode with other ones, so that the background current will decrease and the signal-to-noise ratio improve (Lin et al., 2004; Tu et al., 2005). But one problem of this method is that for biosensing application, the immobilized biomolecules will be restricted only to the nanoelectrode tip and the quantity will be greatly limited. The central theme of the present study is to take advantage of the large specific surface area of the 3D-gold nanowire array to improve biomolecule immobilization efficiency. During the electrolysis process, steady-state current was achieved within 10–20 s. After electrolysis, the color of the PC template changed from its pre-electrolysis white color to yellow, indicating the electrodeposition of metal gold within the pores of the template. After 300 s of deposition, the Au nanowire array was obtained by etching away the template, and the morphology of the nanowire array was characterized by SEM. Fig. 1 shows SEM images of the gold nanowire array, which confirmed the formation of nanowires. The nanowires are highly regular and uniform, with an average diameter of about 250 nm, which corresponds to the size of the nanopore in the template and the length

of the nanowire after 300 s of deposition is about 2 ␮m. The array has highly ordered 3D structure and the surface density of the nanowires is calculated to be ∼5 × 108 cm−2 . 3.2. Estimation of the electroactive surface area of the gold nanowire array Cyclic voltammograms (CVs) of the conventional gold electrode (a) and the gold nanowire array in 20 mM K3 Fe(CN)6 containing 0.2 M KCl at 0.1 V s−1 were performed (Supplemental information, Fig. S1). The well-defined oxidation and reduction peaks due to the Fe3+ /Fe2+ redox couple were observed at +0.085 and 0.30 V. The CV curve of the nanowire array is similar to the conventional gold electrode but not displaying a sigmoid shape, which may be due to the overlap of the diffusion layer from each nanowire (Krishnamoorthy and Zoski, 2005; Li et al., 2003). According to the Randles–Sevcik equation (Hrapovic et al., 2004): Ip = 2.69 × 105 AD1/2 n3/2 γ 1/2 C where D, n, γ and C are constant values, and the electroactive surface area (A) is linear to the peak current of the redox couple. The electroactive surface area of the gold nanowire array is about 6 times larger than the conventional gold electrode. From the CVs, one can see that the background current of the nanowire array increased about 3 times compared to the conventional gold electrode. 3.3. Characterization of ferritin molecules absorbed on gold nanowire Gold nanoparticles have been widely used for the adsorption of proteins due to their biocompatibility with biomolecules, and can also facilitate the transfer of electron between proteins and electrode surface. In this study, due to the vast surface area of the three-dimensional gold nanowire array, the adsorption ability of the nanowire toward proteins was tested. Ferritin molecules were selected as the iron core of ferritin molecules can be clearly seen under TEM. In an attempt to immobilize ferritin molecules, the dispersed gold nanowires were simply stirred with diluted protein solutions for 1 h. The gold nanowires before modification with proteins displays a smooth surface (Supplemental information, Fig. S2A). The successful immobilization of ferritin onto gold nanowire was visualized (Supplemental information, Fig. S2B). The periphery of the nanowire is surrounded with ferritin molecules and the higher magnification indicating the apoproteins appear amorphous around the core (Supplemental information, Fig. S2C) (Zhi et al., 2005). 3.4. Spectroscopic characterization of Hb absorbed onto gold nanowire

Fig. 1. SEM images of the gold nanowire array.

As protein molecules can be adsorbed directly onto nanowires, the bioactivity of proteins after absorption onto the nanowires and the possibility of direct electron transfer were investigated. The Soret band of Hb is sensitive to variations

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of the microenvironments around the heme site. The peak will be diminished if the protein is denatured (Nassar et al., 1995). In this experiment, the band for Hb after its absorption onto the nanowire is located at 406 nm, and there is no observable shift of the absorption peak compared with that of native Hb film (Supplemental information, Fig. S3A). This result suggests that there is no obvious denaturation for Hb after it adsorbed onto the nanowire. The weak and broad absorption peak at 550 nm is ascribed to the gold nanowire, as transforming the morphology of gold nanostructure from nanosphere to nanowire will decrease the absorption peak (Chen et al., 2006). Fourier transform infrared spectroscopy is sensitive to the secondary structure of the protein, as the shapes of the amide I and amide II infrared adsorption bands of Hb provide detailed information on the secondary structure of the polypeptide chain (Roach et al., 2006). The FT–IR spectroscopy was also used to characterize the structure of Hb after its absorption onto the Au nanowire, which shows the shapes of the amide I and amide II band of Hb on the nanowire located at 1652.49 and 1541.36 cm−1 , respectively, the locations are nearly the same as those (1653.74 and 1539.84 cm−1 ) from native proteins (Supplemental information, Fig. S3B), indicating that the absorbed Hb retains the essential features of its native structure. The spectroscopic data reveal that the bioactivity of the proteins is well preserved after adsorption. 3.5. Electrochemical characterization of Hb-modified nanowire array Fig. 2A shows CVs of Hb-modified gold electrode (a) and Hb-modified gold nanowire array (b) in PB at 0.2 V s−1 . There are no redox peaks for the Hb-modified gold electrode, indicating the electron transfer between the proteins and the electrode is hindered. However, a pair of well-defined, nearly reversible redox couple are observed for Hb-modified gold nanowire array at −0.19 and −0.26 V with formal potential (Eo = [(Ep, a + Ep,c)/2]) of −0.225 V, which is characteristic of direct electron transfer of Hb through the incorporation into the nanowire array. Fig. 2B shows CVs of the Hb-modified nanowire array in PB at different scan rates from 0.1 to 1.0 V s−1 . Both the cathodic and anodic peak currents increase linearly with the scan rate (the insert), which is characteristic of thin-layer electrochemical behavior, that is, almost all electroactive met-Hb in the nanowire array is converted to ferrous Hb on the forward CV scan and vice versa (Liu et al., 2005). By integration of CV peaks, the charges (Q) and thus the average surface concentration of electroactive species can be calculated according to the following equation (Laviron, 1979): Q = nFAΓ ∗ Here n is the number of electrons transferred, F is Faraday’s constant and A is the electrode area. The average surface concentration of electroactive Hb in the nanowire array was estimated to be 5.89 × 10−11 , which accounts for ∼30% of the total amount of Hb on the electrode surface. This may suggest

Fig. 2. (A) CVs of (a) Hb-gold electrode and (b) Hb-gold nanowire array in PB at 0.2 V s−1 . (B) CVs of Hb-gold nanowire array in PB at different scan rates. From inner to outside: 0.1 to 1 V s−1 with increasing the scan rate of 0.1 V s−1 each time. Insert: anodic and cathodic current plotted against the scan rate.

that only those Hb molecules adsorbed on the gold nanowire surface and with a suitable orientation can participate in the electron-transfer reaction. This estimated value is higher than the percentage of proteins immobilized in titanate nanotube film (15%) (Liu et al., 2005), that of the proteins immobilized in silica sol-gel film (11%) (Wang et al., 2004a,b), and that entrapped in agarose hydrogel film (1%) (Wang et al., 2005), indicating the advantages of the nanowire array in immobilization proteins. As the length of the nanowire could be altered with altering the deposition time, the effect of electrode morphology on the electron-transfer reaction of Hb was investigated. Five deposition times were selected as 30, 80, 160, 240 and 300 s. A 2 ␮L aliquot of 0.1 mM Hb solution was dropped onto the electrode to

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assure that the immobilized Hb is constant. The CVs of the five Hb-modified electrodes were shown in Fig. 3 and the insert is the percentage of electroactive Hb. It can be seen that the quantity of the electroactive Hb increased with increasing the deposition time. This might be due to that the length of the nanowire increased with increasing deposition time, so the quantity of Hb molecules adsorbed along the nanowire surface increased accordingly, thus resulting in the increase of the percentage of electroactive Hb. The above experiments are based on dropping the Hb solution on the electrode surface, and then in the following we further investigated the absorption ability of the nanowire array toward Hb molecules using QCM. The modification of the quartz crystal with gold nanowire array uses the same method as that for the modification of GC electrode. First, the relationship between the resonant frequency shift and the quantity of Hb was determined with a known amount of the proteins immobilized onto the nanowire array modified crystal surface, and a value of 3.2 ng/Hz was obtained. Then the crystal was immersed into Hb solution (0.1 mM) for different times, the resonant frequency shift and CV curves were measured. Fig. 4 depicts the CVs of the crystal after adsorption of Hb for different times and the insert is the frequency shift. As can be seen, with the increasing of the absorption time from 0.5 to 4 h, both of the cathodic and anodic peak currents in the CVs are increased, and also the frequency shift increased, indicating more Hb molecules are absorbed onto the nanowire surface. Further increasing the absorption time to 6 h, while still increasing the frequency shift, gives no obvious change on the CV curves, showing the Hb molecules further absorbed did not change into electroactive Hb. At the absorption time of 4 h, the amount of Hb immobilized onto the crystal surface calculated from the frequency shift is 4.98 × 10−11 mol, and according to the CV curve, the electroactive Hb is 3.42 × 10−11 mol, accounting for ∼70% of the total amount of Hb absorbed, which is much higher than simply dropping the Hb onto the electrode surface.

Fig. 3. CVs of the Hb-modified electrode with different deposition times. From inner to outside: 30,80,160,240,300 s. The insert is the percentage of the electroactive Hb.

Fig. 4. CVs of Hb-gold nanowire array modified QCM in PB with different Hb adsorption times. From inner to outside: 0.5, 1, 2, 4, 6 h. The insert is the frequency shift of the QCM with increasing adsorption times.

3.6. Electrocatalytic reduction of hydrogen peroxide Electrocatalytic reduction of hydrogen peroxide by Hbmodified nanowire array was examined with CVs. Fig. 5A shows CVs of the Hb-modified nanowire array in PB before and after the addition of H2 O2 . With the addition of H2 O2 , the cathodic peak was greatly increased, while the corresponding anodic peak decreased, indicating an electrocatalytic reduction of H2 O2 had occurred. No peaks were observed at the nanowire array without Hb in the same H2 O2 solution, therefore, the catalytic process comes from the specific enzymatic catalytic reaction between Hb and H2 O2 . The electrocatalytic reaction mechanism is electrons shuttled directly between the electrode and Hb, leading to the catalytic reduction of H2 O2 . Amperometric method was also used to compare the response of the conventional gold electrode, gold nanowire array, and Hbmodified gold nanowire array toward H2 O2 . Fig. 5B displays typical current–time curve for successive addition of 10 ␮M H2 O2 at the three electrodes at −0.2 V. Fast response times can be obtained at the three electrodes with steady-state current reached within 5 s, but the response of the Hb-modified nanowire array is about three times larger than that of the nanowire array without Hb, again suggesting the participation of Hb in the H2 O2 catalytic reaction. Noticeably, the current of the bare nanowire array is more than 100 times higher than that of the conventional gold electrode, indicating high catalytic activity of the nanowire array. Based on S/N = 3, the detection limit was 5 × 10−8 M, and the linear range covers from 1 × 10−7 to 4 × 10−2 M with the linear regression coefficient of 0.997. From the amperometric measurement, the background current of the nanowire array increased about three times compared to the conventional gold electrode, while the response current of the nanowire array increased 100 times, resulting in the improved S/N ratio and

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Fig. 6. Current–time curve of GOx modified gold nanowire array for the successive addition of 1 mM glucose at −0.2 V. The insert is the calibration curve. Error bars = S.D. and n = 4.

Fig. 5. (A) CVs of Hb-modified gold nanowire array in the presence of (a) 0, (b) 100, (c) 200, and (d) 300 ␮M of H2 O2 . (B) Current–time curve of (a) Hbmodified gold nanowire array, (b) bare gold nanowire array, and (c) conventional gold electrode toward successive addition of 10 ␮M of H2 O2 at −0.2 V.

sensitivity. The high upper limit can be ascribed to the perpendicular orientation of the nanowire array that provides large quantities of surface electroactive sites and the well-defined surface area facilitated substrate-electrode contact. Further, 1 mM H2 O2 was measured continuously for six times, and a relative standard deviation (R.S.D.) of 3.2% was obtained, indicating the detection results were reliable. 3.7. Fabrication of glucose biosensor The bare gold nanowire array can directly respond to H2 O2 , so we also used the nanowire array for the fabrication of biosensors. Glucose oxidase was then absorbed onto the nanowire surface, and the amperometric responses of the GOx modified nanowire array for successive addition of 1 mM glucose at −0.2 V in PB (without bubbling with high purity nitrogen) are presented in Fig. 6, and the insert is the calibration curve. Sensitive response for glucose was obtained and response generated a steady-state current signal within 10 s. The fast response time and high sensitivity can also be ascribed to the well-defined surface area, which facilitated enzyme–substrate contact. The

linear calibration range is extended over four orders of magnitude of glucose concentration (10−6 −2 × 10−2 M with the linear regression coefficient of 0.997) and the detection limit of 5 × 10−7 M was obtained. The apparent Michaelis–Menten constant (Km app ) was estimated by using the Lineweaver-Burk plot of 1/I versus 1/[glucose] and a value of 2.05 mM for Km app was obtained. The repeatability of the glucose biosensor was also investigated with the detection of 1 mM glucose continuously for six times, the R.S.D. was 3.4%. The selectivity of the biosensor was determined. Electroactive compounds commonly present in physiological samples such as ascorbic acid, uric acid and acetaminophen used to interfere the accurate determination of glucose. In this study, these compounds cause interfering current at detection potential of −0.2 V, but the high sensitivity of the biosensor towards glucose makes these interferences negligible because the current due to the 0.1 mM electroactive compounds are less than 5% of the current due to 1 mM glucose. The performance of this biosensor was compared with literature report of glucose biosensors based on carbon nanotube array and Prussian blue array (Lin et al., 2004; Xian et al., 2007). The biosensor based on gold nanowire array has wider linear range than carbon nanotube array (0.08–30 mM) and Prussian blue array (0.005–8 mM). Further, the present biosensor has a higher sensitivity (58.91 ␮A mM−1 cm−2 ) than a biosensor based on carbon nanotube array (2.5 nA mM−1 cm−2 ) and Prussian blue array (14.18 ␮A mM−1 cm−2 ). 4. Conclusions We have demonstrated gold nanowire array as efficient support matrixes for the immobilization of hemoglobin. Protein molecules can be easily absorbed onto the nanowire surface, which has been demonstrated by absorption of ferritin molecules characterized by TEM. With the immobilization of Hb, UV–vis absorption and FT–IR spectra show there was no obvious denature of Hb after absorption. The Hb-modified gold nanowire array gave a well-defined redox couple with the apparent formal

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peak potential (Eo ) of −0.225 V, indicating direct electron transfer between Hb and the nanowire array was achieved, whereas no peak current was observed on Hb-modified conventional gold electrode. Noticeably, the bare gold nanowire array can direct reduce H2 O2 at −0.2 V with the current response 100 times larger than the conventional gold electrode and the linear range covers from 1 × 10−7 to 4 × 10−2 M. With the absorption of GOx, sensitive and selective glucose biosensor have been prepared. Acknowledgement This work was supported by the NNSF of China (No. 20675028, 20435010, 20375012 and 20205005). Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at doi:10.1016/j.bios.2007.05.003. References Armstrong, F.A., Hill, H.A.O., Walton, N.J., 1988. Acc. Chem. Res. 21, 407–413. Bayachou, M., Lin, R., Cho, W., Farmer, P.J., 1998. J. Am. Chem. Soc. 120, 9888–9893. Beissenhirtz, M.K., Scheller, F.W., Lisdat, F., 2004. Anal. Chem. 76, 4665–4671. Bharathi, S., Nogami, M., Ikeda, S., 2001. Langmuir 17, 1–4. Chen, C.C., Lin, Y.P., Wang, C.W., Tzeng, H.C., Wu, C.H., Chen, Y.C., Chen, C.P., Chen, L.C., Wu, Y.C., 2006. J. Am. Chem. Soc. 128, 3709–3715. Crumbliss, A.L., Stonehuerner, J.G., Henkens, R.W., Zhao, J., Odaly, J.P., 1993. Biosens. Bioelectron. 8, 331–337. Fan, C.H., Wang, H.Y., Sun, S., Zhu, D.X., Wagner, G., Li, G.L., 2001. Anal. Chem. 73, 2850–2854. Guan, F.Y., Uboh, C.E., Soma, L.R., Luo, Y., Jahr, J.S., Driessen, B., 2004. Anal. Chem. 76, 5127–5135. Hamachi, I., Fujita, A., Kunitake, T., 1997. J. Am. Chem. Soc. 119, 9096–9102. Hicks, J.F., Zamborini, F.P., Osisek, A.J., Murray, R.W., 2001. J. Am. Chem. Soc. 123, 7048–7053.

Hrapovic, S., Liu, Y.L., Male, K.B., Luong, J.H.T., 2004. Anal. Chem. 76, 1083–1088. Hu, S.Q., Xie, Z.M., Lei, C.X., Shen, G.L., Yu, R.Q., 2005. Sens. Actuators, B 106, 641–647. Huang, Q.D., Lu, Z.Q., Rusling, J.F., 1996. Langmuir 12, 5472–5480. Kluger, R., Zhang, J., 2003. J. Am. Chem. Soc. 125, 6070–6071. Krishnamoorthy, K., Zoski, C.Z., 2005. Anal. Chem. 77, 5068–5071. Laviron, E., 1979. J. Electroanal. Chem. 101, 19–28. Lei, C.X., Hu, S.Q., Shen, G.L., Yu, R.Q., 2003. Talanta 59, 981–988. Lei, C.X., Yang, Y., Wang, H., Shen, G.L., Yu, R.Q., 2004. Anal. Chim. Acta. 513, 379–384. Li, J., Ng, H.T., Cassell, A., Fan, W., Chen, H., Ye, Q., Koehne, J., Han, J., Meyyappan, M., 2003. Nano. Lett. 3, 597–602. Lin, Y.H., Lu, F., Tu, Y., Ren, Z.F., 2004. Nano. Lett. 4, 191–195. Liu, A.H., Wei, M.D., Honma, I., Zhou, H.S., 2005. Anal. Chem. 77, 8068–8074. Lux, K.W., Rodriguez, K.J., 2006. Nano. Lett. 6, 288–295. Menon, V.P., Martin, C.R., 1995. Anal. Chem. 67, 1920–1928. Nassar, A.E.F., Willis, W.S., Rusling, J.F., 1995. Anal. Chem. 67, 2386–2392. Panchagnula, V., Kumar, C.V., Rusling, J.F., 2002. J. Am. Chem. Soc. 124, 12515–12521. Roach, P., Farrar, D., Perry, C.C., 2006. J. Am. Chem. Soc. 128, 3939–3945. Topoglidis, E., Cass, A.E.G., Gilardi, G., Sadeghi, S., Beaumont, N., Durrant, J.R., 1998. Anal. Chem. 70, 5111–5113. Topoglidis, E., Campbell, C.J., Cass, A.E.G., Durrant, J.R., 2001. Langmuir 17, 7899–7906. Tu, Y., Lin, Y.H., Yantasee, W., Ren, Z.F., 2005. Electroanalysis 17, 79–84. Wang, Q., Lu, G., Yang, B., 2004a. Biosens. Bioelectron. 20, 1342–1347. Wang, Q.L., Lu, G.X., Yang, B.J., 2004b. Biosens. Bioelectron. 19, 1269–1275. Wang, S.F., Chen, T., Zhang, Z.L., Shen, X.C., Lu, Z.X., Pang, D.W., Wong, K.Y., 2005. Langmuir 21, 9260–9266. Xian, Y.Z., Hu, Y., Liu, F., Xian, Y., Feng, L.J., Jin, L.T., 2007. Template synthesis of highly ordered Prussian blue array and its application to the glucose biosensing. Biosens. Bioelectron. 22, 2827–2833. Xue, F.H., Fei, G.T., Wu, B., Cui, P., Zhang, L.D., 2005. J. Am. Chem. Soc. 127, 15348–15349. Yu, X., Sotzing, G.A., Papadimitrakopoulos, F., Rusling, J.F., 2003. Anal. Chem. 75, 4565–4571. Zhao, J.G., Odaly, J.P., Henkens, R.W., Stonehuerner, J., Crumbliss, A.L., 1996. Biosens. Bioelectron. 11, 493–502. Zhi, C.Y., Bando, Y., Tang, C.C., Golberg, D., 2005. J. Am. Chem. Soc. 127, 17144–17145. Zhou, H., Gan, X., Wang, J., Zhu, X.L., Li, G.X., 2005. Anal. Chem. 77, 6102–6104.