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Platinum nanowire nanoelectrode array for the fabrication of biosensors
ARTICLE IN PRESS
Biomaterials 27 (2006) 5944–5950 www.elsevier.com/locate/biomaterials
Platinum nanowire nanoelectrode array for the fabrication of biosensors Minghui Yanga, Fengli Qua, Yashuang Lua, Yan Heb,, Guoli Shena,, Ruqin Yua a
Chemistry and Chemical Engineering College, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Hunan, Changsha 410082, PR China b State Key Laboratory of Chemo/BioSensing and Chemometrics, Biomedical Engineering Center, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410080, China Received 26 June 2006; accepted 7 August 2006
Abstract Platinum nanowire arrays can be grown by electrodeposition in polycarbonate membrane, with the average diameter of the nanowires about 250 nm and the height about 2 mm. The nanowire array prepared by the proposed method can be considered as nanoelectrode array (NEA) with nanoelectrode density of 5 108 cm2. While the NEA can improve the signal-to-noise ratio and decrease the detection limit, the high surface area of the platinum NEA circumvents the problem of conventional platinum electrodes associated with the limited electroactive site. The platinum NEA can direct response to hydrogen peroxide at low potential of 0 V with wide linear range (1 107–6 102 M) and sensitivity 50 times larger than that of the conventional platinum electrode. With the absorption of glucose oxidase onto the ordered NEA surface, the spatially patterned glucose oxidase improves greatly the resulting biosensor. The biosensor can achieve interference free determination of glucose with wide linear range (106–3 102 M). The sensitivity of the glucose biosensor is one-fifth of the sensitivity toward hydrogen peroxide, indicating high efficiency of signal transduction. The biosensor was used to determine glucose in real blood samples, and the glucose contents determined by the present biosensor were in agreement with the results of existing method. r 2006 Published by Elsevier Ltd. Keywords: Biosensor; Enzyme; Platinum nanowire; Polycarbonate membrane; Nanoelectrode array
1. Introduction Since the discovery of carbon nanotubes, there has been a series of rapidly expanding research for one-dimensional nanostructures as nanotubes and nanowires [1–3]. The interesting electrical, optical, and catalytic properties of the nanostructures have potential applications in nanoscale devices, sensors, and catalysts [4–6]. The adsorption of enzymes onto these nanostructures has been reported, as these materials provide large surface area for enzyme loading and friendly microenvironment to stabilize the immobilized enzymes, which enable further practical applications [7–9]. However, simply dropping the modified nanostructures onto electrode surface will result in a disarrayed and layered film, with the absorbed catalytic Corresponding authors. Tel./fax: +86 731 8821355.
E-mail addresses: [email protected] (Y. He), [email protected] (G. Shen). 0142-9612/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2006.08.014
enzyme sites partially blocked and the substrate transport to the enzymes hindered [10]. For an ideal biosensor fabrication method, besides large quantities of enzymes should be immobilized using mild chemical conditions and a favorable microenvironment to maintain the enzyme activity, large surface area for enzyme–substrate contact is also very important [11]. It would be a significant advancement if perpendicularly aligned nanotube or nanowire arrays can be formed as sensing devices [12]. Metal platinum is one of the mostly researched noble metals as its potential applications have been demonstrated in the fields of biosensors, catalysts and fuel cells [13–15]. Because of its nice performance toward the detection of hydrogen peroxide, a typical enzymatic product, platinum electrode, and platinum nanostructure modified electrode have been widely used to immobilize enzyme for the fabrication of biosensors [16–18]. Nanostructured platinum films were electrodeposited onto microelectrodes from hexagonal lyotropic liquid crystalline plating mixture and
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the films were confirmed to have high surface area with efficient mass transport characteristics [19]. Mesoporous platinum microelectrodes are shown to be excellent amperometric sensors for the detection of hydrogen peroxide over a wide range of concentration with good reproducibility and high precision [20]. Monitoring low levels of analytes is of great importance for modern medicine, environmental control, and food industry [21–23]. Recently, it has been reported that the use of microelectrode arrays instead of conventional ones could improve the signal-to-noise ratio (S/N) and decrease the detection limit [24–26]. For the microelectrode array to work efficiently, the spacing between microelectrodes needs to be sufficiently larger than the diameter of each microelectrode to prevent diffusion layer overlap with the neighboring electrodes [26]. Because the resulting current of the microelectrode array is proportional to the total number of individual electrodes, the size reduction of each individual electrode and the increased total number of electrodes are highly desirable. In this paper, considering the advantages of nanoelectrode array (NEA) and the properties of metal platinum, we synthesized platinum nanowire array in polycarbonate (PC) membranes by means of direct electrodeposition technique directly on glassy carbon (GC) electrode [27,28]. The diameter of each nanowire is about 250 nm with a length about 2 mm. The modified electrode can be considered consists of millions of nanoelectrodes with each nanowire acted as a single nanoelectrode. The electroactive surface area of the NEA is about six times larger than the conventional platinum electrode. Direct response of the NEA toward hydrogen peroxide can be achieved at low potential of 0 V with a sensitivity 50 times larger than that of the conventional platinum electrode. The high sensitivity of the NEA toward hydrogen peroxide and the large surface area make it ideal for the absorption of enzymes for the fabrication of biosensors. Glucose oxidase was selected and absorbed onto the NEA, and the resulting glucose biosensor enables selective determination of glucose with high sensitivity and wide linear range. 2. Experimental 2.1. Apparatus and reagents Track-etched porous PC (0.2 mm) membrane was provided by Whatman (Anodisc 47, 0.2 mm). Glucose oxidase (GOx, from Aspergillus niger; EC 1.1.3.4, type VII-S; 196,000 U g1) was purchased from Sigma. A 1/ 15 M phosphate buffer (PB, pH 6.98) solution was prepared using Na2HPO4 and KH2PO4. All other reagents were of analytical grade, and doubly distilled water was used throughout. Cyclic voltametric and amperometric measurements were carried out on CHI 760B electrochemical workstation (Shanghai, China). Scanning electron microscopy (SEM) analysis was performed using a JSM-5600LV microscope (JEO, Ltd., Japan). Transmission electron microscope (TEM) image was taken with a JEM-3010 TEM (JEOL Co. Ltd., Japan). A threeelectrode cell (10 mL) with the modified GC electrode as the working electrode, a saturated calomel electrode (SCE) as reference electrode and a platinum foil electrode as counter-electrode was used. All potentials were
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measured and reported versus the SCE and all experiments were carried out at room temperature.
2.2. Synthesis of platinum nanowire arrays For the electrodeposition of platinum 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 GC (2 mm diameter) electrode surface and covered by a rubber O-ring. Electrodepositions were performed using chronoamperometry in 1% (w /w) H2PtCl4 solution containing 0.5 M per chloric acid at a potential of 0.35 V. After deposition, the PC template was dissolved by immersing the electrode in chloroform. A conventional platinum disk electrode (2 mm diameter) was used as a comparison reference.
2.3. Preparation of GOx modified platinum nanowire NEA Before modification, the NEA was washed successively using Piranha (3:1 v/v mixture of concentrated H4SO4 and H2O2), ethanol, and water. For the modification of the NEA with GOx, the NEA was immersed into GOx solution (5 mg/mL) overnight at 4 1C.
3. Results and discussion 3.1. Morphological characterization of the prepared platinum nanowire array During the electrolysis process, the color of the PC membrane changed from white to black indicating the electrodeposition of metal platinum in the pores of the membrane. After 400 s of deposition, the platinum nanowire array was obtained by etching away the template, and the morphology of the nanowire array was characterized by SEM. Fig. 1A shows SEM images of the platinum nanowire arrays, which confirmed the formation of nanowires. The nanowires are highly regular and uniform, and vertically oriented 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 400 s of deposition is about 2 mm. It can also be seen from the image that for most of the nanowires, the spacing between two nanowires are larger than the diameter of the nanowire, making each nanowire working as an individual nanoelectrode [26]. The nanoelectrode density is about 5 108 cm2 according to the SEM images. Fig. 1B shows TEM image of a single platinum nanowire. A straight nanowire can be seen with the diameter about 250 nm, which is in accordance with the SEM image. 3.2. Estimation of the electroactive surface area of the platinum NEA Fig. 2 represents cyclic voltammograms (CVs) of the conventional platinum electrode (a) and the platinum NEA in 20 mM K3Fe(CN)6 containing 0.2 M KCl at 0.1 V s1. The well-defined oxidation and reduction peaks due to the Fe3+/Fe2+ redox couple were observed at +0.30 and 0.085 V, respectively. According to the Randles–Sevcik
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800 600 b
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together with a signature for oxide formation (more anodic than 0.6 V) and stripping (centered at 0.4 V), exhibiting the characteristic features expected for polycrystalline platinum [30,31]. The stability of the NEA was also tested as shown in Fig. 3B. During 10 successive CV cycles in 0.5 M H4SO4 at scan rates of 0.02 V s1, no observable peak current change was found implying that the nanowire are stably immobilized onto the electrode.
3.4. Detection of hydrogen peroxide
Fig. 1. SEM image of the platinum nanowire array (A) and TEM image of a single platinum nanowire (B).
equation [29] I p ¼ 2:69 105 AD1=2 n3=2 g1=2 C, where D, n, g , and C are constant values, and the electroactive surface area (A) is a linear function of the peak current of the redox couple. The average value of the electroactive surface area for the platinum NEA was (0.1870.02) cm2 (n ¼ 6), which was about six times larger than the conventional platinum electrode. 3.3. Electrochemical characterization of the platinum NEA Fig. 3A shows CVs of the platinum NEA in 0.5 M H2SO4 in the potential range of 0.3 to 1.2 V at different scan rates. It is clear there are two pairs of hydrogen adsorption/ desorption peaks in the potential range 0 to 0.3 V,
Platinum is a widely used electrode material for the detection of hydrogen peroxide and for the fabrication of biosensors. However, for most biosensors based on platinum electrode, the detection of H2O2 was achieved at relatively high potential around 0.6 V, which made it liable to interferences from electroactive compounds. To solve this problem, additional permselective membranes are often needed. In this paper, the influence of potential on the response of the platinum NEA toward H2O2 was first investigated. Fig. 4 shows the current response of the platinum NEA toward 1 mM H2O2 at the potential range of 0.4–0.4 V in PB. As can be seen, during the potential range studied, high response current was observed and the potential of 0.3 V is a turning point. In the range of 0.3 to 0.4 V, with the negatively increasing potential, the current signal increased, and also started at 0.3 V, with the positively increasing potential, there is an increase of the current response. Compare the current response of the platinum NEA in the negative potential range with that in the positive potential range, it can be seen that in the negative potential range the response is much higher that in the positive range, indicating that the reduction of hydrogen peroxide is much
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Fig. 3. (A) CVs of the platinum NEA in 0.5 M H2SO4 at different scan rates. From inner to outsider, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 V s1. (B) Successive 10 CV cycles of the platinum NEA in 0.5 M H2SO4 at 0.02 V s1.
easier than its oxidation. In this study, the potential of 0 V was selected. At such a low applied potential, the background current decreased, the responses of common interference species can be minimized, and the oxygen reduction current can be limited. Fig. 5A is CVs of the platinum NEA in PB in the absence (a) and in presence of 5 mM H2O2 (b) between 0.6 and 0.6 V at the scan rate of 100 mV s1. When H2O2 was added to the PB, an obvious increase of the cathodic and anodic current was observed, indicating the platinum NEA exhibited excellent electrocatalytic activity toward H2O2. Fig. 5B depicts the typical current–time curve at the platinum NEA (a) and conventional platinum electrode (b)
for successive addition of 1 mM H2O2 at 0 V and the inset (right) shows current–time curve at the platinum NEA for successive addition of 1 mM H2O2. Fast response can be observed at the two electrodes with steady-state current reached with 5 s. Noticeably, the sensitivity of the platinum NEA is about 50 times larger than that of the conventional electrode, indicating high catalytic activity of the platinum NEA. The linear range of the platinum NEA toward H2O2 is from 1 107 to 6 102 M, covering five orders of magnitudes with sensitivity of 0.54 A M1 cm2. Based on S=N ¼ 3, a detection limit of 5 108 M was obtained as shown in Fig. 5B (inset, left). The low detection limit of the sensor can be ascribed to the advantages of the NEA in improving the S/N ratio, while the perpendicularly orientation of the nanowire array provides large quantities of surface electroactive sites, circumventing the problems related to the limited number of active sites of conventional electrodes, which expanded the upper detection limit [32]. The well-defined surface area facilitated substrate–electrode contact, also contributed to the wide linear range. Repeated use of the electrode did not affect the longterm stability with good repeatability obtained. For example, 1 mM H2O2 was measured continuously for 10 times, and a relative standard deviation (RSD) of 4.1% was obtained. At relatively high H2O2 concentrations (410 mM), the electrode’s surface will be fouled, but a few CVs in PB solutions effectively released the blocked active sites. The H2O2 concentration of 15 mM was also measured for 10 times, between two successive measurements, the CV was performed, and a RSD of 6.5% was obtained. The satisfactory repeatability indicates the reliability of the NEA for the determination of H2O2 at both low and high concentrations. The overall performance of the platinum NEA for the detection of hydrogen peroxide is compared with different hydrogen peroxide sensors as shown in Table 1. A survey
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of the data reveals that this sensor displays the best combination of the low detection potential with wide linear range and high sensitivity.
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Fig. 5. (A) CVs of the platinum NEA in the absence (a) and presence (b) of 5 mM H2O2. (B) Current–time curve of the platinum NEA (a) and conventional platinum electrode (b) to the successive addition of 1 mM hydrogen peroxide at 0 V in PB. The inset (right) shows current–time curve of the platinum NEA to the successive addition of 1 mM hydrogen peroxide. The inset (left) shows detection limit of 50 nM with S=N ¼ 3.
The wide linear range and high sensitivity of the platinum NEA toward hydrogen peroxide detection made it an ideal biosensing platform for the fabrication of oxidase-based biosensors. In this study, glucose oxidase was selected and directly absorbed onto the nanowire surface. The large surface area of the nanowire array made it possible to absorb large amount of enzymes, while the ordered orientation of the NEA makes the enzymes spatially patterned, improving significantly the performance of the resulting biosensor. Fig. 6A is the typical current–time curve of the GOx modified platinum NEA for successive addition of 1 mM glucose at 0 V and the inset (right) shows current–time curve of the GOx modified platinum NEA for successive addition of 10 mM glucose. Fast and sensitive response of the biosensor toward glucose can be seen with steady-state current reached with 10 s. The linear calibration range is extended over four orders of magnitude of glucose concentration (106–3 102 M, Fig. 6B) and the detection limit of 5 107 M was obtained based on S=N ¼ 3 (Fig. 6A, inset, left). The wide linear range indicates large amount of enzymes was immobilized onto the electrode. The sensitivity of the biosensor to glucose is 0.11 A M1 cm2, one-fifth of the sensitivity to H2O2, which are higher than that of GOx immobilized on conventional platinum electrode surface (one seventh of the sensitivity to H2O2) [33], indicating high signal transduction efficiency. The apparent Michaelis–Menten constant (KMapp), which gives an indication of the enzyme–substrate kinetics, can be calculated from the linear part of the calibration curve, using the Lineweaver–Burk equation and a value of 3.08 mM for KMapp was obtained. The repeatability of the biosensor was also tested and found satisfactory. The glucose concentration of 1 mM was measured continuously for 10 times, and a RSD of 4.4%
Table 1 Comparison of the performance of different hydrogen peroxide sensors Type of H2O2 sensor
Detection potential (mV)
Sensitivity (A M1 cm2)
Linear range (M)
Detection limit (mM)
Reference
Pt NEA Mesoporous Pt electrodes Pt-NEGCF PB modified CP HRP/titania sol–gel HRP/CNT
0a 600a 600c 0c 150a 200c
0.54 2.8 0.055 0.427 0.49 0.07
1 107–6 102 2 105–4 102 5 107–2 103 5 107–5 103 8 105–5.6 104 1.67 105–7.4 104
0.05 4.5 0.007 0.5 1.5 13
b
[20] [34] [35] [36] [37]
Pt-NEGCF, Pt nanoparticles embedded in graphite-like carbon film; PB, Prussian blue; CP, carbon paste electrode; HRP, horseradish peroxidase; CNT, carbon nanotube. a Versus SCE. b Versus Ag/AgCl. c This paper.
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tested each week, after 1 month of storage, the response of the biosensor only decreased 10% compared to the initial response. The decreasing response may be due to the slight leaking of GOx. It indicates that the platinum nanowire array indeed provides a favorable microenvironment to maintain the activity of GOx.
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Fig. 6. (A) Current–time curve of the GOx modified platinum NEA to the successive addition of 1 mM glucose at 0 V in PB. The inset (right) shows current–time curve of the GOx modified platinum NEA to the successive addition of 10 mM glucose. The inset (left) shows detection limit of 0.5 mM with S=N ¼ 3. (B) Calibration curve of the GOx modified platinum NEA toward glucose.
The applicability of the biosensor was assessed by the determination of glucose concentration in real blood samples. Results were compared with those determined by Hunan University Hospital using colorimetric method with glucose kit (Shanghai Kehua-Dongling Diagnostic Products Co. Ltd.). A total of 10 serum samples were analyzed and the result is shown in Fig. 7. Glucose contents determined by the two methods agreed well, and a plot of the glucose concentration obtained by the two methods gave a straight line with a correlation coefficient of 0.989 (inset), indicating the biosensor can be used clinically for the detection of glucose. 4. Conclusions
was obtained. The low RSD means that the GOx are stably absorbed onto the electrode surface and can be used for practical application. The interferences from electroactive compounds commonly present in physiological samples used to cause problems in the accurate determination of glucose. In this study, the determination of glucose at the GOx modified platinum NEA is practically free of interferences. At the low detection potential of 0 V, the responses of 0.1 mM ascorbic acid, 0.1 mM uric acid, and 0.1 mM acetaminophen are negligible compared to the response of 1 mM glucose, indicating high selectivity of the biosensor. The storage stability of the biosensor was studied when it was stored in PB at 4 1C. The response to 1 mM glucose was
In this paper, we have demonstrated the preparation of platinum nanowire NEA. The nanostructuring improves analytical performances of the corresponding sensors compared to the conventional electrodes, specifically, the properties of the NEA improves the S/N ratio and decreases the detection limit, and the large surface area increases the number of electroactive sites and expanded the upper detection limit. The sensitivity of the NEA toward hydrogen peroxide is 50 times larger than that of conventional platinum electrode at detection potential of 0 V with wide linear range (1 107–6 102). The proposed modified electrode system is especially valuable when used in conjugation with an enzyme, which has been
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experimentally illustrated with glucose oxidase. Selective detection of glucose has been achieved, and the glucose oxidase was very stable after absorbed onto the electrode surface. The biosensor fabrication method is readily applicable to the fabrication of other biosensors based on oxidases, such as biosensors for choline, cholesterol, and alcohol. Acknowledgments This work was supported by the NNSF of China (No. 20435010, 20375012 and 20205005). References [1] Otten CJ, Lourie OR, Yu MF, Cowley JM, Dyer MJ, Ruoff RS, et al. Crystalline boron nanowires. J Am Chem Soc 2002;124:4564–5. [2] Li C, Curreli M, Lin H, Lei B, Ishikawa FN, Datar R, et al. Complementary detection of prostate-specific antigen using In2O3 nanowires and carbon nanotubes. J Am Chem Soc 2005;127:12484–5. [3] Barrelet CJ, Wu Y, Bell DC, Lieber CM. Synthesis of CdS and ZnS nanowires using single-source molecular precursors. J Am Chem Soc 2003;125:11498–9. [4] Mu YY, Liang HP, Hu JS, Jiang L, Wan LJ. Controllable Pt nanoparticle deposition on carbon nanotubes as an anode catalyst for direct methanol fuel cells. J Phys Chem B 2005;109:22212–6. [5] Lux KW, Rodriguez KJ. Template synthesis of arrays of nano fuel cells. Nano Lett 2006;6:288–95. [6] Yoon B, Wai M. Microemulsion-templated synthesis of carbon nanotube-supported Pd and Rh nanoparticles for catalytic applications. J Am Chem Soc 2005;127:17174–5. [7] Topoglidis E, Cass AEG, Gilardi G, Sadeghi S, Beaumont N, Durrant JR. Protein adsorption on nanocrystalline TiO2 films: an immobilization strategy for bioanalytical devices. Anal Chem 1998;70:5111–3. [8] Peng SG, Gao QM, Wang QG, Shi JL. Layered structural heme protein magadiite nanocomposites with high enzyme-like peroxidase activity. Chem Mater 2004;16:2675–84. [9] Kumar CV, Chaudhari A. Proteins immobilized at the galleries of layered a-zirconium phosphate: structure and activity studies. J Am Chem Soc 2000;122:830–7. [10] Wang J, Scampicchio M, Laocharoensuk R, Valentini F, Gonza´lezGarcy´a´ O, Burdick J. Magnetic tuning of the electrochemical reactivity through controlled surface orientation of catalytic nanowires. J Am Chem Soc 2006;128:4562–3. [11] Liu GD, Lin YH. Biosensor Based on self-assembling acetylcholinesterase on carbon nanotubes for flow injection/amperometric detection of organophosphate pesticides and nerve agents. Anal Chem 2006;78:835–43. [12] Wei C, Dai LM, Roy A, Tolle TB. Multifunctional chemical vapor sensors of aligned carbon nanotube and polymer composites. J Am Chem Soc 2006;128:1412–3. [13] Liu F, Lee JY, Zhou WJ. Template preparation of multisegment PtNi nanorods as methanol electro-oxidation catalysts with adjustable bimetallic pair sites. J Phys Chem B 2004;108:17959–63. [14] Sung WJ, Bae YH. A glucose oxidase electrode based on electropolymerized conducting polymer with polyanion-enzyme conjugated dopant. Anal Chem 2000;72:2177–81. [15] Niwa O, Horiuchi T, Morita M, Huang T, Kissinger PT. Determination of acetylcholine and choline with platinum-black ultramicroarray electrodes using liquid chromatography with a postcolumn enzyme reactor. Anal Chim Acta 1996;318:167–73. [16] Matsumoto N, Chen XH, Wilson GS. Fundamental studies of glucose oxidase deposition on a Pt electrode. Anal Chem 2002;74:362–7.
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Biomaterials 27 (2006) 5944–5950 www.elsevier.com/locate/biomaterials
Platinum nanowire nanoelectrode array for the fabrication of biosensors Minghui Yanga, Fengli Qua, Yashuang Lua, Yan Heb,, Guoli Shena,, Ruqin Yua a
Chemistry and Chemical Engineering College, State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Hunan, Changsha 410082, PR China b State Key Laboratory of Chemo/BioSensing and Chemometrics, Biomedical Engineering Center, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410080, China Received 26 June 2006; accepted 7 August 2006
Abstract Platinum nanowire arrays can be grown by electrodeposition in polycarbonate membrane, with the average diameter of the nanowires about 250 nm and the height about 2 mm. The nanowire array prepared by the proposed method can be considered as nanoelectrode array (NEA) with nanoelectrode density of 5 108 cm2. While the NEA can improve the signal-to-noise ratio and decrease the detection limit, the high surface area of the platinum NEA circumvents the problem of conventional platinum electrodes associated with the limited electroactive site. The platinum NEA can direct response to hydrogen peroxide at low potential of 0 V with wide linear range (1 107–6 102 M) and sensitivity 50 times larger than that of the conventional platinum electrode. With the absorption of glucose oxidase onto the ordered NEA surface, the spatially patterned glucose oxidase improves greatly the resulting biosensor. The biosensor can achieve interference free determination of glucose with wide linear range (106–3 102 M). The sensitivity of the glucose biosensor is one-fifth of the sensitivity toward hydrogen peroxide, indicating high efficiency of signal transduction. The biosensor was used to determine glucose in real blood samples, and the glucose contents determined by the present biosensor were in agreement with the results of existing method. r 2006 Published by Elsevier Ltd. Keywords: Biosensor; Enzyme; Platinum nanowire; Polycarbonate membrane; Nanoelectrode array
1. Introduction Since the discovery of carbon nanotubes, there has been a series of rapidly expanding research for one-dimensional nanostructures as nanotubes and nanowires [1–3]. The interesting electrical, optical, and catalytic properties of the nanostructures have potential applications in nanoscale devices, sensors, and catalysts [4–6]. The adsorption of enzymes onto these nanostructures has been reported, as these materials provide large surface area for enzyme loading and friendly microenvironment to stabilize the immobilized enzymes, which enable further practical applications [7–9]. However, simply dropping the modified nanostructures onto electrode surface will result in a disarrayed and layered film, with the absorbed catalytic Corresponding authors. Tel./fax: +86 731 8821355.
E-mail addresses: [email protected] (Y. He), [email protected] (G. Shen). 0142-9612/$ - see front matter r 2006 Published by Elsevier Ltd. doi:10.1016/j.biomaterials.2006.08.014
enzyme sites partially blocked and the substrate transport to the enzymes hindered [10]. For an ideal biosensor fabrication method, besides large quantities of enzymes should be immobilized using mild chemical conditions and a favorable microenvironment to maintain the enzyme activity, large surface area for enzyme–substrate contact is also very important [11]. It would be a significant advancement if perpendicularly aligned nanotube or nanowire arrays can be formed as sensing devices [12]. Metal platinum is one of the mostly researched noble metals as its potential applications have been demonstrated in the fields of biosensors, catalysts and fuel cells [13–15]. Because of its nice performance toward the detection of hydrogen peroxide, a typical enzymatic product, platinum electrode, and platinum nanostructure modified electrode have been widely used to immobilize enzyme for the fabrication of biosensors [16–18]. Nanostructured platinum films were electrodeposited onto microelectrodes from hexagonal lyotropic liquid crystalline plating mixture and
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the films were confirmed to have high surface area with efficient mass transport characteristics [19]. Mesoporous platinum microelectrodes are shown to be excellent amperometric sensors for the detection of hydrogen peroxide over a wide range of concentration with good reproducibility and high precision [20]. Monitoring low levels of analytes is of great importance for modern medicine, environmental control, and food industry [21–23]. Recently, it has been reported that the use of microelectrode arrays instead of conventional ones could improve the signal-to-noise ratio (S/N) and decrease the detection limit [24–26]. For the microelectrode array to work efficiently, the spacing between microelectrodes needs to be sufficiently larger than the diameter of each microelectrode to prevent diffusion layer overlap with the neighboring electrodes [26]. Because the resulting current of the microelectrode array is proportional to the total number of individual electrodes, the size reduction of each individual electrode and the increased total number of electrodes are highly desirable. In this paper, considering the advantages of nanoelectrode array (NEA) and the properties of metal platinum, we synthesized platinum nanowire array in polycarbonate (PC) membranes by means of direct electrodeposition technique directly on glassy carbon (GC) electrode [27,28]. The diameter of each nanowire is about 250 nm with a length about 2 mm. The modified electrode can be considered consists of millions of nanoelectrodes with each nanowire acted as a single nanoelectrode. The electroactive surface area of the NEA is about six times larger than the conventional platinum electrode. Direct response of the NEA toward hydrogen peroxide can be achieved at low potential of 0 V with a sensitivity 50 times larger than that of the conventional platinum electrode. The high sensitivity of the NEA toward hydrogen peroxide and the large surface area make it ideal for the absorption of enzymes for the fabrication of biosensors. Glucose oxidase was selected and absorbed onto the NEA, and the resulting glucose biosensor enables selective determination of glucose with high sensitivity and wide linear range. 2. Experimental 2.1. Apparatus and reagents Track-etched porous PC (0.2 mm) membrane was provided by Whatman (Anodisc 47, 0.2 mm). Glucose oxidase (GOx, from Aspergillus niger; EC 1.1.3.4, type VII-S; 196,000 U g1) was purchased from Sigma. A 1/ 15 M phosphate buffer (PB, pH 6.98) solution was prepared using Na2HPO4 and KH2PO4. All other reagents were of analytical grade, and doubly distilled water was used throughout. Cyclic voltametric and amperometric measurements were carried out on CHI 760B electrochemical workstation (Shanghai, China). Scanning electron microscopy (SEM) analysis was performed using a JSM-5600LV microscope (JEO, Ltd., Japan). Transmission electron microscope (TEM) image was taken with a JEM-3010 TEM (JEOL Co. Ltd., Japan). A threeelectrode cell (10 mL) with the modified GC electrode as the working electrode, a saturated calomel electrode (SCE) as reference electrode and a platinum foil electrode as counter-electrode was used. All potentials were
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measured and reported versus the SCE and all experiments were carried out at room temperature.
2.2. Synthesis of platinum nanowire arrays For the electrodeposition of platinum 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 GC (2 mm diameter) electrode surface and covered by a rubber O-ring. Electrodepositions were performed using chronoamperometry in 1% (w /w) H2PtCl4 solution containing 0.5 M per chloric acid at a potential of 0.35 V. After deposition, the PC template was dissolved by immersing the electrode in chloroform. A conventional platinum disk electrode (2 mm diameter) was used as a comparison reference.
2.3. Preparation of GOx modified platinum nanowire NEA Before modification, the NEA was washed successively using Piranha (3:1 v/v mixture of concentrated H4SO4 and H2O2), ethanol, and water. For the modification of the NEA with GOx, the NEA was immersed into GOx solution (5 mg/mL) overnight at 4 1C.
3. Results and discussion 3.1. Morphological characterization of the prepared platinum nanowire array During the electrolysis process, the color of the PC membrane changed from white to black indicating the electrodeposition of metal platinum in the pores of the membrane. After 400 s of deposition, the platinum nanowire array was obtained by etching away the template, and the morphology of the nanowire array was characterized by SEM. Fig. 1A shows SEM images of the platinum nanowire arrays, which confirmed the formation of nanowires. The nanowires are highly regular and uniform, and vertically oriented 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 400 s of deposition is about 2 mm. It can also be seen from the image that for most of the nanowires, the spacing between two nanowires are larger than the diameter of the nanowire, making each nanowire working as an individual nanoelectrode [26]. The nanoelectrode density is about 5 108 cm2 according to the SEM images. Fig. 1B shows TEM image of a single platinum nanowire. A straight nanowire can be seen with the diameter about 250 nm, which is in accordance with the SEM image. 3.2. Estimation of the electroactive surface area of the platinum NEA Fig. 2 represents cyclic voltammograms (CVs) of the conventional platinum electrode (a) and the platinum NEA in 20 mM K3Fe(CN)6 containing 0.2 M KCl at 0.1 V s1. The well-defined oxidation and reduction peaks due to the Fe3+/Fe2+ redox couple were observed at +0.30 and 0.085 V, respectively. According to the Randles–Sevcik
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800 600 b
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together with a signature for oxide formation (more anodic than 0.6 V) and stripping (centered at 0.4 V), exhibiting the characteristic features expected for polycrystalline platinum [30,31]. The stability of the NEA was also tested as shown in Fig. 3B. During 10 successive CV cycles in 0.5 M H4SO4 at scan rates of 0.02 V s1, no observable peak current change was found implying that the nanowire are stably immobilized onto the electrode.
3.4. Detection of hydrogen peroxide
Fig. 1. SEM image of the platinum nanowire array (A) and TEM image of a single platinum nanowire (B).
equation [29] I p ¼ 2:69 105 AD1=2 n3=2 g1=2 C, where D, n, g , and C are constant values, and the electroactive surface area (A) is a linear function of the peak current of the redox couple. The average value of the electroactive surface area for the platinum NEA was (0.1870.02) cm2 (n ¼ 6), which was about six times larger than the conventional platinum electrode. 3.3. Electrochemical characterization of the platinum NEA Fig. 3A shows CVs of the platinum NEA in 0.5 M H2SO4 in the potential range of 0.3 to 1.2 V at different scan rates. It is clear there are two pairs of hydrogen adsorption/ desorption peaks in the potential range 0 to 0.3 V,
Platinum is a widely used electrode material for the detection of hydrogen peroxide and for the fabrication of biosensors. However, for most biosensors based on platinum electrode, the detection of H2O2 was achieved at relatively high potential around 0.6 V, which made it liable to interferences from electroactive compounds. To solve this problem, additional permselective membranes are often needed. In this paper, the influence of potential on the response of the platinum NEA toward H2O2 was first investigated. Fig. 4 shows the current response of the platinum NEA toward 1 mM H2O2 at the potential range of 0.4–0.4 V in PB. As can be seen, during the potential range studied, high response current was observed and the potential of 0.3 V is a turning point. In the range of 0.3 to 0.4 V, with the negatively increasing potential, the current signal increased, and also started at 0.3 V, with the positively increasing potential, there is an increase of the current response. Compare the current response of the platinum NEA in the negative potential range with that in the positive potential range, it can be seen that in the negative potential range the response is much higher that in the positive range, indicating that the reduction of hydrogen peroxide is much
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Fig. 3. (A) CVs of the platinum NEA in 0.5 M H2SO4 at different scan rates. From inner to outsider, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 V s1. (B) Successive 10 CV cycles of the platinum NEA in 0.5 M H2SO4 at 0.02 V s1.
easier than its oxidation. In this study, the potential of 0 V was selected. At such a low applied potential, the background current decreased, the responses of common interference species can be minimized, and the oxygen reduction current can be limited. Fig. 5A is CVs of the platinum NEA in PB in the absence (a) and in presence of 5 mM H2O2 (b) between 0.6 and 0.6 V at the scan rate of 100 mV s1. When H2O2 was added to the PB, an obvious increase of the cathodic and anodic current was observed, indicating the platinum NEA exhibited excellent electrocatalytic activity toward H2O2. Fig. 5B depicts the typical current–time curve at the platinum NEA (a) and conventional platinum electrode (b)
for successive addition of 1 mM H2O2 at 0 V and the inset (right) shows current–time curve at the platinum NEA for successive addition of 1 mM H2O2. Fast response can be observed at the two electrodes with steady-state current reached with 5 s. Noticeably, the sensitivity of the platinum NEA is about 50 times larger than that of the conventional electrode, indicating high catalytic activity of the platinum NEA. The linear range of the platinum NEA toward H2O2 is from 1 107 to 6 102 M, covering five orders of magnitudes with sensitivity of 0.54 A M1 cm2. Based on S=N ¼ 3, a detection limit of 5 108 M was obtained as shown in Fig. 5B (inset, left). The low detection limit of the sensor can be ascribed to the advantages of the NEA in improving the S/N ratio, while the perpendicularly orientation of the nanowire array provides large quantities of surface electroactive sites, circumventing the problems related to the limited number of active sites of conventional electrodes, which expanded the upper detection limit [32]. The well-defined surface area facilitated substrate–electrode contact, also contributed to the wide linear range. Repeated use of the electrode did not affect the longterm stability with good repeatability obtained. For example, 1 mM H2O2 was measured continuously for 10 times, and a relative standard deviation (RSD) of 4.1% was obtained. At relatively high H2O2 concentrations (410 mM), the electrode’s surface will be fouled, but a few CVs in PB solutions effectively released the blocked active sites. The H2O2 concentration of 15 mM was also measured for 10 times, between two successive measurements, the CV was performed, and a RSD of 6.5% was obtained. The satisfactory repeatability indicates the reliability of the NEA for the determination of H2O2 at both low and high concentrations. The overall performance of the platinum NEA for the detection of hydrogen peroxide is compared with different hydrogen peroxide sensors as shown in Table 1. A survey
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of the data reveals that this sensor displays the best combination of the low detection potential with wide linear range and high sensitivity.
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Fig. 5. (A) CVs of the platinum NEA in the absence (a) and presence (b) of 5 mM H2O2. (B) Current–time curve of the platinum NEA (a) and conventional platinum electrode (b) to the successive addition of 1 mM hydrogen peroxide at 0 V in PB. The inset (right) shows current–time curve of the platinum NEA to the successive addition of 1 mM hydrogen peroxide. The inset (left) shows detection limit of 50 nM with S=N ¼ 3.
The wide linear range and high sensitivity of the platinum NEA toward hydrogen peroxide detection made it an ideal biosensing platform for the fabrication of oxidase-based biosensors. In this study, glucose oxidase was selected and directly absorbed onto the nanowire surface. The large surface area of the nanowire array made it possible to absorb large amount of enzymes, while the ordered orientation of the NEA makes the enzymes spatially patterned, improving significantly the performance of the resulting biosensor. Fig. 6A is the typical current–time curve of the GOx modified platinum NEA for successive addition of 1 mM glucose at 0 V and the inset (right) shows current–time curve of the GOx modified platinum NEA for successive addition of 10 mM glucose. Fast and sensitive response of the biosensor toward glucose can be seen with steady-state current reached with 10 s. The linear calibration range is extended over four orders of magnitude of glucose concentration (106–3 102 M, Fig. 6B) and the detection limit of 5 107 M was obtained based on S=N ¼ 3 (Fig. 6A, inset, left). The wide linear range indicates large amount of enzymes was immobilized onto the electrode. The sensitivity of the biosensor to glucose is 0.11 A M1 cm2, one-fifth of the sensitivity to H2O2, which are higher than that of GOx immobilized on conventional platinum electrode surface (one seventh of the sensitivity to H2O2) [33], indicating high signal transduction efficiency. The apparent Michaelis–Menten constant (KMapp), which gives an indication of the enzyme–substrate kinetics, can be calculated from the linear part of the calibration curve, using the Lineweaver–Burk equation and a value of 3.08 mM for KMapp was obtained. The repeatability of the biosensor was also tested and found satisfactory. The glucose concentration of 1 mM was measured continuously for 10 times, and a RSD of 4.4%
Table 1 Comparison of the performance of different hydrogen peroxide sensors Type of H2O2 sensor
Detection potential (mV)
Sensitivity (A M1 cm2)
Linear range (M)
Detection limit (mM)
Reference
Pt NEA Mesoporous Pt electrodes Pt-NEGCF PB modified CP HRP/titania sol–gel HRP/CNT
0a 600a 600c 0c 150a 200c
0.54 2.8 0.055 0.427 0.49 0.07
1 107–6 102 2 105–4 102 5 107–2 103 5 107–5 103 8 105–5.6 104 1.67 105–7.4 104
0.05 4.5 0.007 0.5 1.5 13
b
[20] [34] [35] [36] [37]
Pt-NEGCF, Pt nanoparticles embedded in graphite-like carbon film; PB, Prussian blue; CP, carbon paste electrode; HRP, horseradish peroxidase; CNT, carbon nanotube. a Versus SCE. b Versus Ag/AgCl. c This paper.
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tested each week, after 1 month of storage, the response of the biosensor only decreased 10% compared to the initial response. The decreasing response may be due to the slight leaking of GOx. It indicates that the platinum nanowire array indeed provides a favorable microenvironment to maintain the activity of GOx.
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Fig. 6. (A) Current–time curve of the GOx modified platinum NEA to the successive addition of 1 mM glucose at 0 V in PB. The inset (right) shows current–time curve of the GOx modified platinum NEA to the successive addition of 10 mM glucose. The inset (left) shows detection limit of 0.5 mM with S=N ¼ 3. (B) Calibration curve of the GOx modified platinum NEA toward glucose.
The applicability of the biosensor was assessed by the determination of glucose concentration in real blood samples. Results were compared with those determined by Hunan University Hospital using colorimetric method with glucose kit (Shanghai Kehua-Dongling Diagnostic Products Co. Ltd.). A total of 10 serum samples were analyzed and the result is shown in Fig. 7. Glucose contents determined by the two methods agreed well, and a plot of the glucose concentration obtained by the two methods gave a straight line with a correlation coefficient of 0.989 (inset), indicating the biosensor can be used clinically for the detection of glucose. 4. Conclusions
was obtained. The low RSD means that the GOx are stably absorbed onto the electrode surface and can be used for practical application. The interferences from electroactive compounds commonly present in physiological samples used to cause problems in the accurate determination of glucose. In this study, the determination of glucose at the GOx modified platinum NEA is practically free of interferences. At the low detection potential of 0 V, the responses of 0.1 mM ascorbic acid, 0.1 mM uric acid, and 0.1 mM acetaminophen are negligible compared to the response of 1 mM glucose, indicating high selectivity of the biosensor. The storage stability of the biosensor was studied when it was stored in PB at 4 1C. The response to 1 mM glucose was
In this paper, we have demonstrated the preparation of platinum nanowire NEA. The nanostructuring improves analytical performances of the corresponding sensors compared to the conventional electrodes, specifically, the properties of the NEA improves the S/N ratio and decreases the detection limit, and the large surface area increases the number of electroactive sites and expanded the upper detection limit. The sensitivity of the NEA toward hydrogen peroxide is 50 times larger than that of conventional platinum electrode at detection potential of 0 V with wide linear range (1 107–6 102). The proposed modified electrode system is especially valuable when used in conjugation with an enzyme, which has been
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experimentally illustrated with glucose oxidase. Selective detection of glucose has been achieved, and the glucose oxidase was very stable after absorbed onto the electrode surface. The biosensor fabrication method is readily applicable to the fabrication of other biosensors based on oxidases, such as biosensors for choline, cholesterol, and alcohol. Acknowledgments This work was supported by the NNSF of China (No. 20435010, 20375012 and 20205005). References [1] Otten CJ, Lourie OR, Yu MF, Cowley JM, Dyer MJ, Ruoff RS, et al. Crystalline boron nanowires. J Am Chem Soc 2002;124:4564–5. [2] Li C, Curreli M, Lin H, Lei B, Ishikawa FN, Datar R, et al. Complementary detection of prostate-specific antigen using In2O3 nanowires and carbon nanotubes. J Am Chem Soc 2005;127:12484–5. [3] Barrelet CJ, Wu Y, Bell DC, Lieber CM. Synthesis of CdS and ZnS nanowires using single-source molecular precursors. J Am Chem Soc 2003;125:11498–9. [4] Mu YY, Liang HP, Hu JS, Jiang L, Wan LJ. Controllable Pt nanoparticle deposition on carbon nanotubes as an anode catalyst for direct methanol fuel cells. J Phys Chem B 2005;109:22212–6. [5] Lux KW, Rodriguez KJ. Template synthesis of arrays of nano fuel cells. Nano Lett 2006;6:288–95. [6] Yoon B, Wai M. Microemulsion-templated synthesis of carbon nanotube-supported Pd and Rh nanoparticles for catalytic applications. J Am Chem Soc 2005;127:17174–5. [7] Topoglidis E, Cass AEG, Gilardi G, Sadeghi S, Beaumont N, Durrant JR. Protein adsorption on nanocrystalline TiO2 films: an immobilization strategy for bioanalytical devices. Anal Chem 1998;70:5111–3. [8] Peng SG, Gao QM, Wang QG, Shi JL. Layered structural heme protein magadiite nanocomposites with high enzyme-like peroxidase activity. Chem Mater 2004;16:2675–84. [9] Kumar CV, Chaudhari A. Proteins immobilized at the galleries of layered a-zirconium phosphate: structure and activity studies. J Am Chem Soc 2000;122:830–7. [10] Wang J, Scampicchio M, Laocharoensuk R, Valentini F, Gonza´lezGarcy´a´ O, Burdick J. Magnetic tuning of the electrochemical reactivity through controlled surface orientation of catalytic nanowires. J Am Chem Soc 2006;128:4562–3. [11] Liu GD, Lin YH. Biosensor Based on self-assembling acetylcholinesterase on carbon nanotubes for flow injection/amperometric detection of organophosphate pesticides and nerve agents. Anal Chem 2006;78:835–43. [12] Wei C, Dai LM, Roy A, Tolle TB. Multifunctional chemical vapor sensors of aligned carbon nanotube and polymer composites. J Am Chem Soc 2006;128:1412–3. [13] Liu F, Lee JY, Zhou WJ. Template preparation of multisegment PtNi nanorods as methanol electro-oxidation catalysts with adjustable bimetallic pair sites. J Phys Chem B 2004;108:17959–63. [14] Sung WJ, Bae YH. A glucose oxidase electrode based on electropolymerized conducting polymer with polyanion-enzyme conjugated dopant. Anal Chem 2000;72:2177–81. [15] Niwa O, Horiuchi T, Morita M, Huang T, Kissinger PT. Determination of acetylcholine and choline with platinum-black ultramicroarray electrodes using liquid chromatography with a postcolumn enzyme reactor. Anal Chim Acta 1996;318:167–73. [16] Matsumoto N, Chen XH, Wilson GS. Fundamental studies of glucose oxidase deposition on a Pt electrode. Anal Chem 2002;74:362–7.
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