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ZnO Nanotube Arrays as Biosensors for Glucose Kun Yang,†,§ Guang-Wei She,† Hui Wang,† Xue-Mei Ou,† Xiao-Hong Zhang,*,† Chun-Sing Lee,‡ and Shuit-Tong Lee‡ Nano-organic Photoelectronic Laboratory and Key Laboratory of Photochemical ConVersion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China, Center of Super-Diamond and AdVanced Film (COSDAF) and Department of Physics and Materials Science, City UniVersity of Hong Kong, Hong Kong SAR, People’s Republic of China, and Key Laboratory for Biomedical Informatics and Health Engineering, Institute of Biomedical and Health Engineering, Shenzhen, Institute of AdVanced Technology, Chinese Academy of Sciences, Shenzhen 518055, People’s Republic of China ReceiVed: March 1, 2009; ReVised Manuscript ReceiVed: September 30, 2009
Highly oriented single-crystal ZnO nanotube (ZNT) arrays were prepared by a two-step electrochemical/ chemical process on indium-doped tin oxide (ITO) coated glass in an aqueous solution. The prepared ZNT arrays were further used as a working electrode to fabricate an enzyme-based glucose biosensor through immobilizing glucose oxidase in conjunction with a Nafion coating. The present ZNT arrays-based biosensor exhibits high sensitivity of 30.85 µA cm-2 mM-1 at an applied potential of +0.8 V vs. SCE, wide linear calibration ranges from 10 µM to 4.2 mM, and a low limit of detection (LOD) at 10 µM (measured) for app sensing of glucose. The apparent Michaelis-Menten constant KM was calculated to be 2.59 mM, indicating a higher bioactivity for the biosensor. One-dimensional (1D) zinc oxide (ZnO) nanostructures can have significant applications in optics, optoelectronics, sensors, and actuators due to their semiconducting, piezoelectric, and pyroelectric properties.1-5 Extensive efforts have been made to fabricate various kinds of chemical and biochemical sensors based on ZnO nanostructures, such as fluorescent biosensors with nanoscale ZnO platforms,6,7 H2S gas sensor with single ZnO nanowire,8 intracellular pH sensor with ZnO nanorod,9 and ethanol sensor with flowerlike ZnO nanostructure.10 Due to their unique properties, these ZnO nanosensors show higher sensitivity and a lower limit of detection (LOD) as compared to those prepared from bulk ZnO devices. For enzyme-modified nanosensors, the 1D ZnO nanostructure is one of the most promising substrates for immobilizing enzyme because of their properties including biocompatibility, vast surface-to-bulk ratio, relative chemical stability in physiological environment, and electrochemical activity.11-13 Moreover, ZnO has a high isoelectric point (IEP) of about 9.5, which should provide a positively charged substrate for immobilization of low IEP proteins or enzyme such as GOx (IEP ≈ 4.2) at the physiological pH of 7.4. In this paper, we report sensing of glucose using the ZnO nanotube arrays based biosensor. The key difference between our approach and those reported previously14-16 is that in previous work the ZnO nanostructure was synthesized first and then assembled on the surface of the electrode resulting in a poor contact between the ZnO nanostructure and the electrode. In our work, the ZnO nanotubes have been grown directly on a conductive ITO glass to ensure excellent electrical contact between ZnO nanostructure and the electrode. In addition, due to the large surface-to-bulk ratio of * To whom correspondence should be addressed. E-mail: xhzhang@ mail.ipc.ac.cn.. † Chinese Academy of Sciences. § Also at the Graduate School of Chinese Academy of Sciences, Beijing, China. ‡ City University of Hong Kong.
the porous structures of the ZnO nanotubes, the ZnO nanotube arrays based electrode enhance the sensitivity for analytes as demonstrated by the detection of glucose without the presence of a mediator. The ZnO nanotube arrays were prepared by a two-step electrochemical/chemical process, as described in our previous work.17,18 Prior to fabricating the glucose biosensors, the asprepared ZnO nanotube arrays were washed with doubly distilled water several times and dried by high-purity nitrogen gas, and then a chip (0.2 cm × 1 cm) was split off, part of which was then encapsulated in wax, leaving an opening surface of 0.2 cm × 0.2 cm for use as a sensor. For immobilization of GOx (185 000 units/g, Type X-S: from Aspergillus niger) on the tailored ZnO nanotube arrays/ITO electrode, 5 µL of GOx solution with a concentration of 10 mg/mL prepared in 0.02 M phosphate buffer solution (PBS, pH 7.4) was deposited on the ZnO nanotube arrays/ITO electrode, and then dried at 4 °C overnight, from which the coverage of GOx on the ZnO nanotube arrays could be estimated to be 231 units/cm2. Then, the coated electrode was again rinsed carefully with doubly distilled water to remove any free GOx, 5 µL of 0.5% (weight) Nafion solution was then deposited onto the resulting surface and kept at 4 °C overnight. Then the electrode was washed carefully with doubly distilled water and the modified electrode was ready for use. All the electrochemical measurements were carried out on an electrochemical workstation in a conventional three-electrode configuration with a saturated calomel electrode (SCE) as the reference electrode, a Pt foil as the counter electrode, the modified ZnO nanotube arrays/ITO electrode as the working electrode, and 10 mL of PBS (0.02 M, pH 7.4, including 0.1 M KCl) as the supporting electrolyte medium. Amperometric detection proceeded with a single modified electrode in a stirred solution at an applied potential of +0.8 V, and the background current was first allowed to decay to a steady-state value. All
10.1021/jp901894j CCC: $40.75 2009 American Chemical Society Published on Web 11/02/2009
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Figure 1. (a) SEM image of the ZnO nanotube arrays; the EDS analysis (inset) indicating that the ZnO nanotubes consist of O and Zn. (b) SEM image of the surface modified ZNT arrays; the EDS analysis (inset) indicating that the modified ZnO nanotubes consist of O, Zn, C, and F.
the measurements were performed at room temperature and all the potentials mentioned in this paper are relative to SCE. The surfaces of the ZnO array on ITO and the glucosemodified electrode were studied by field-emission scanningelectron microscopy (FE-SEM, Hitachi S-4300 FEG, equipped with energy dispersive spectroscopy (EDS)). Figure 1a shows the SEM image of the highly oriented ZNT arrays prepared on an ITO-coated glass. This shows that the tube-like structures have well-defined hexagonal cross sections with diameters of about 300 nm. Energy dispersive spectrometric (EDS) analysis in Figure 1a (inset) indicates that the ZnO nanotubes consist of O and Zn. After modification with GOx and Nafion, the SEM image (Figure 1b) of the electrode is more difficult to focus, which can be attributed to a layer of coated biochemical/polymer film. And the EDS analysis (Figure 1b, inset) also shows that the modified ZnO nanotubes consist of O, Zn, C, and F. The pattern of the modified ZnO nanotube arrays and the EDS analysis indicate that the Nafion and GOx have been distributed equally on the modified electrode surface. The sensor as constructed is sensitive to the concentration changes of glucose in PBS. Figure 2 shows the cyclic voltammetric curves of Nafion/GOx/ZnO nanotube arrays/ITO electrode in an unstirred 0.02 M PBS with 1 mM glucose (solid)
and without glucose (dashed) at the scan rate of 100 mV · s-1, respectively. It can be seen that the current begins to rise at 0.2 V in PBS with 1 mM glucose compared to that in PBS without glucose, indicating the good response to glucose by the modified electrode. The relatively minor anodic current increase could be attributed to the inherent electroconductivity of the ZnO nanostructure,19 which has been improved partly with the unique fabricating process of ZnO nanostructure ensuring excellent electrical contact between the ZnO nanotube arrays and substrate electrode. Figure 3 shows a typical amperometric response at 0.8 V of the modified electrode to successive addition of glucose to stirred PBS increasing the glucose concentration at 10 µM per step. The modified electrode responds favorably and very rapidly to the glucose concentration changes with the response time of less than 6 s, indicating a good catalytic property of the Nafion/GOx/ZnO nanotube arrays/ITO electrode. The glucose could be bioelectrocatalyzed by the immobilized GOx on ZnO nanotube arrays, and the mechanism of the process is schematically illustrated in Figure 4: glucose would be oxided by GOx(OX) to gluconolactone, while GOx(OX) is changed into GOx(R). The consumed GOx(OX) could be regenerated from GOx(R) through its reaction with the oxygen present in solution.
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Figure 4. The mechanism of the glucose sensing on the modified electrode. Figure 2. Cyclic voltammograms of Nafion/GOx/ZnO nanotube arrays/ ITO electrode in the same PBS (pH 7.4) in the absence (dashed) and the presence (solid) of 1 mM glucose.
Figure 3. Typical amperometric response curve of GOx/ZnO nanotube arrays/ITO electrodes for increasing glucose concentration in 10 µM per step with successive addition of glucose to the 0.02 M pH 7.4 PBS under stirring. The applied potential was +0.8 V vs. SCE.
This process produces H2O2, which can be detected quantitatively on the modified electrode.20 The calibration curve of the glucose concentration versus anodic current at 0.8 V is shown in Figure 5 (solid square), which clearly reveals the linear relationship between the current and glucose concentrations at an applied potential until the glucose concentration reaches 4.2 mM. The linear regression equation is the following: J (µA · cm2) ) 1.967 + 30.85 C (mM), with a correlation coefficient R ) 0.9999. On the basis of the calibration curve, the linear range of the calibration curve is from 10 µM to 4.2 mM with a limit of detection (LOD) of 10 µM (measured). The present configuration of the biosensor shows a high sensitivity of 30.85 µA · cm-2 · mM-1, which is higher than all the nanomaterials-based biosensors for glucose sensing15,21-24 asshowninTable1.TheapparentMichaelis-Menten constant Kapp M is generally used to evaluate the biological activity of immobilized enzyme, which can be calculated according to app /imax)(1/C) + 1/imax the Lineweaver-Burk equation: iss ) (KM (where iss is the steady-state current after the addition of glucose, imax is the maximum current, and C is the concentration of
Figure 5. Calibration curve (solid square): anodic currents of the biosensor vs. solutions of different concentrations of glucose in 0.02 M PBS (pH 7.4) and the Lineweaver-Burk plot (open circle). The straight line is a linear fit to the squares.
glucose).25,26 According to the Lineweaver-Burk plot of 1/iss vs. 1/C as shown in Figure 5 (open circle), the Kapp M is calculated to be 2.59 mM, which is lower than previously reported,27-29 indicating that the ZnO nanotube arrays based electrode exhibits a higher affinity for glucose and the immobilized enzymess possess a higher bioactivity. The low value of LOD and the high sensitivity of the ZnObased biosensor can be attributed to two unique properties of our electrode: (1) the vast surface-to-bulk ratio and the porous structure of ZnO nanotube arrays, which can provide a favorable microenvironment for the immobilization of GOx and the enzyme catalysis of the glucose oxidation on electrode, and (2) excellent electrical contact between the ITO electrode and the ZnO nanotubes. Compared with the ZnO biosensors based on postassembly of ZnO on an electrode, our directly grown ZnO nanotube array on ITO should provide a better contact between the ZNT arrays and the ITO-coated glass. The present biosensor also has excellent selectivity for glucose sensing. The presence of ascorbic acid or uric acid (100 times that of glucose) had no influence on the response of the present
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TABLE 1: Summary of Sensitivities of Nanomaterials As Modified Electrochemical Sensors for Glucose electrode materials -2
-1
sensitivity (µA · cm · mM ) ref
ZnO nanocombs
TiO2 sol-gel
carbon nanotubes
TiO2 film
ZrO2/Chitson film
ZnO nanotubes
15.33 15
7.2 19
30.14 20
4.58 21
0.028 22
30.85 our work
biosensors to glucose (results not shown). We suggest that the good selectivity of the present biosensor can be attributed to the permselective (charge-exclusion) property30,31 of Nafion films coated on the electrode. In conclusion, ZnO nanotube arrays were directly grown on ITO glass through an electrodeposition process. GOx and Nafion were immobilized on the nanotube arrays to fabricate a glucose sensing electrode. Due to the vast surface-to-bulk ratio and the porous structure of the nanotubes, the good contact between the substrate electrode (ITO-coated glass) and ZnO nanotube arrays, and the permselective property of the Nafion film, the biosensor exhibited high sensitivity for glucose detection. The fabrication methodology of the present biosensor could be extended to other enzyme-based biosensors, such as those for detection of acetylcholine, cholesterol, phenol, etc. Acknowledgment. The work was partially supported by the National Basic Research Program of China (973 Program) (Grant Nos. 2006CB933000 and 2007CB936000), National Natural Science Foundation of China (Grant No. 50825304), and National High-tech R&D Program of China (863 Program) (Grant No. 2007AA03Z300). Dr. Jack Chang is gratefully acknowledged for his critical reading of the manuscript. References and Notes (1) Tseng, Y. K.; Huang, C. J.; Cheng, H. M.; Lin, I. N.; Liu, K. S.; Chen, I. C. AdV. Funct. Mater. 2003, 13, 811–814. (2) Bao, J. M.; Zimmler, M. A.; Capasso, F.; Wang, X. W.; Ren, Z. F. Nano Lett. 2006, 6, 1719–1722. (3) Li, Q. H.; Liang, Y. X.; Wan, Q.; Wang, T. H. Appl. Phys. Lett. 2004, 85, 6389–6391. (4) Wang, Z. L.; Song, J. H. Science 2006, 312, 242–246. (5) Johnson, J. C.; Knutsen, K. P.; Yan, H. Q.; Law, M.; Zhang, Y. F.; Yang, P. D.; Saykally, R. J. Nano Lett. 2004, 4, 197–204. (6) Dorfman, A.; Kumar, N.; Hahm, J. Langmuir 2006, 22, 4890–4895. (7) Dorfman, A.; Kumar, N.; Hahm, J. AdV. Mater. 2006, 18, 2685– 2690. (8) Liao, L.; Lu, H. B.; Li, J. C.; Liu, C.; Fu, D. J.; Liu, Y. L. Appl. Phys. Lett. 2007, 91, 173110/1–173110/3.
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