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A rapid response time and highly sensitive amperometric glucose biosensor based on ZnO nanorod via citric acid-assisted annealing route
ARTICLE IN PRESS Physica E 42 (2010) 1830–1833
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Physica E journal homepage: www.elsevier.com/locate/physe
A rapid response time and highly sensitive amperometric glucose biosensor based on ZnO nanorod via citric acid-assisted annealing route Zao Yang a,n, Zhizhen Ye a,n, Binghui Zhao a, Xiaolin Zong b, Ping Wang b a
State Key Laboratory of Silicon Materials, Department of Materials Science and Engineering, Zhejiang University, Hangzhou 310027, China Biosensor National Special Laboratory, Key laboratory of Biomedical Engineering of National Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China b
a r t i c l e in f o
a b s t r a c t
Article history: Received 7 October 2009 Received in revised form 7 January 2010 Accepted 1 February 2010 Available online 10 February 2010
ZnO nanorods were synthesized by citric acid-assisted annealing route. In a phosphate buffer solution with a pH value of 7.4, glucose oxidase was immobilized on the surface of ZnO nanorod through chitosan-assisted cross-linking technique. The one-dimensional ZnO nanorods provide a large effective surface area with high surface-to-volume ratio and provide a favorable environment for the immobilization of GOx. The response time of this biosensor is less than 2 s. This biosensor has a very high sensitivity of 25.7 mA cm 2 mM 1. The low detection limit was estimated to be 0.01 mM. Two linear response ranges are 0.01–0.25 mM and 0.3–0.7 mM. The Michaelis–Menten constant is found to be 1.95 mM. These results demonstrate that zinc oxide nanorods have potential applications in biosensors. & 2010 Elsevier B.V. All rights reserved.
Keywords: Zinc oxide Nanorods Glucose biosensor
1. Introduction Glucose sensors, as one of the most popular biosensors, have been extensively investigated due to their important applications in biological and chemical analysis, clinical detection, and environmental monitoring. Among the numerous reports in glucose biosensors, the immobilization of enzymes on electrodes is generally the first step in fabrication, and thus, has attracted significant efforts, because enzymes are highly selective and quickly responsive to specific substrates [1]. Enzyme immobilization is considered as an important factor in biosensor technologies. The conventional techniques for enzyme immobilization include covalent attachment to the electrode surfaces [2], entrapment by ion-exchange polymers [3], nonconducting polymers [4], sol–gels [5], cross-linking in bovine serum albumin– glutaraldehyde [6]. On the other hand, nanomaterials play an important role in adsorption of biomolecules due to their unique properties of high specific surface area, good biological compatibility and stability. They can keep activity of enzyme due to the desirable microenviroment, and enhance the electron transfer between the enzyme’s active sites and the electrode [7]. The search for novel materials to design biosensors is of significance at present. Til date, many nanomaterials, such as gold nanoparticles and nanoclusters [8], Au/polyaniline nanocomposite [9], CNTs [10], nanocrystalline diamond [11], calcium carbonate nanoparticles [12], and Ag dendritic nanostructures [13] have been
n
Corresponding authors. Tel.: + 86 571 8795 3139; fax: +86 571 8795 2625. E-mail addresses: [email protected] (Z. Yang), [email protected] (Z. Ye).
1386-9477/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.physe.2010.02.001
studied as platforms for enzyme immobilization in glucose biosensors. ZnO is a biocompatible material with a high isoelectric point (IEP) of about 9.5, which makes it suitable for absorption of proteins with low IEPS, as the protein immobilization is primarily driven by electrostatic interaction [14]. Moreover, ZnO nanostructures have unique advantages including the high specific surface area, nontoxicity, chemical stability, electrochemical activity, and high electron communication features. Recently, GOx was immobilized on ZnO nanocombs and nanorods array to construct amperometric biosensors for glucose biosensing with sensitivities in the range of 15–23 mA/cm2 Mm [1,15]. The lower sensitivity and slower response of previously fabricated ZnO nanostructures based glucose biosensor have been demonstrated in the literature, hence more works are needed to obtain the higher sensitivity and faster response of the ZnO nanostructures based glucose biosensors. In this article, we shall report a biosensor for glucose detection using ZnO nanorods fabricated by citric acid-assisted annealing route. In a phosphate buffer solution with a pH value of 7.4, glucose oxidase was immobilized on the surface of ZnO nanorod through chitosan-assisted cross-linking technique. The response time of this biosensor is less than 2 s. This biosensor has a very high sensitivity of 25.7 mA cm 2 mM 1.
2. Experimental The ZnO nanorods were prepared by citric acid-assisted annealing route [16]. All the chemicals used in this study were of analytical grade and used without further purification. Four chemicals were needed and they were analytically pure zinc
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acetate (Zn (CH3COO)2 2 H2O), citric acid (C6H8O7 H2O), pure distilled water (H2O) and absolute ethanol (CH3CH2OH). In a typical procedure, a solution of citric acid (0.19 M) in ethanol was added to Zn (CH3COO)2 solutions (2 M) in distilled water. After the addition was completed, the solution (pH value was equal to 6.2) was stirred at 80 1C for 8 h to get the precursor. The precursor was calcined in a muffle furnace for 2 h at 400 1C. Finally, the sample was obtained after cooling down at room temperature in air. Field-emission scanning electron microscopy (FE-SEM, HITACHI S-4800), X-ray diffraction (XRD, Rigaku, D/max-rA) with Cu Ka radiation, transmission electron microscopy (JEM-2010) were employed in characterization. To fabricate the glucose biosensor, the glassy carbon (GC) electrodes was polished by 0.3 and 0.05 mm aluminum slurries and then was cleaned by dipping into 1:1 (V/V) aqueous solution of HNO3, deionized water and ethanol with the assistance of ultrasonication prior to the experiment. The as-prepared ZnO nanorods were dispersed, in ethanol by ultrasonication for 1 h to 5 10 3 M suspension. Firstly, 5 ml of ZnO nanorod suspension was dropped onto the surface of GC electrode and dried at room temperature. Secondly, 5 ml of GOx solution with concentration of 1934 U/ml prepared in 0.01 M phosphate buffer solution (pH 7.4) was dropped onto the surface of GC electrode modified by ZnO nanorods. Finally, 5 ml of 0.5 wt% CHIT solution dissolved in acetic acid solution was dropped onto the surface of GOx/ZnO/GC electrode to avoid the leakage of the enzyme. The device was dried at 4 1C overnight in a refrigerator, followed by washing step to remove the unimmobilized GOx. The CHIT/GOx/ZnO/GC electrode was stored in PBS and kept at 4 1C in a refrigerator when not in use.
(SCE) were used as the counter and reference electrodes, respectively. Fig. 1(a) shows the XRD patterns of the sample. All the diffraction peaks are quite similar to that of a bulk ZnO, which ˚ C =5.206 A, ˚ has a hexagonal spiauterite structure (a =3.249 A, space group: P63mc (1 8 6)) and diffraction data are in agreement with JCPDS card of ZnO (JCPDS 36-1451). No other crystalline forms, such as Zn or other Zn compounds, were detected. Fig. 1(b) shows the typical FE-SEM image of the nanorods. As seen from the figure, the length of ZnO nanorods is 0.2–0.6 mm and the diameter is 30–100 nm. The detailed structure of ZnO nanorod is studied by TEM. Fig. 1(c) shows the TEM image of ZnO nanorod. It indicates that the ZnO nanorods have uniform diameters and no catalyst particle at the tip. The surface of the nanorods is very smooth. The EDS result (Fig. 1d) shows only Zn and O peaks for the nanorods. No other elements were detected from the nanorods. The enzyme electrode was characterized by cyclic voltammetry between the potentials of –0.4 and 0.8 V, as shown in Fig. 2. Curves (a) and (b) are the cyclic voltammetric responses of CHIT/ GOx/ZnO/GC electrode in stirred 0.01 M (pH 7.4) phosphate buffer solution in the absence and presence of 1 mM potassium ferricyanide. No electrochemical response is observed in the absence of potassium ferricyanide (curve a). The results indicate that potassium ferricyanide could be used as a diffusional electron-transferring mediator. In the presence of potassium ferricyanide, a pair of redox peaks with formal potential at 0.02 and 0.42 versus reference electrode was clearly observed, which can be assigned to reversible redox reaction of potassium ferricyanide. The typical enzyme-dependent catalytic process can be expressed as follows:
3. Results and discussion
Glucose+ GOD (FAD)-gluconolactone+ GOD (FADH2)
(1)
The electrochemical experiments were carried out with a CHI660A electrochemical workstation with a conventional threeelectrode cell. The glassy carbon (GC) electrode was used as the working electrode. The Pt wire and a saturated calomel electrode
GOD (FADH2)+ [Fe(CN)6]3 -GOD (FAD)+ [Fe(CN)6]4
(2)
where FAD is the oxidative (reductive) forms of flavin adenine dinucleotide and FADH2 is the active center of GOD.
Fig. 1. (a) XRD patterns of as-grown ZnO nanorods. (b) SEM images of the ZnO nanorods. (c) TEM image of as-grown ZnO nanorods. (d) EDX of ZnO nanorod.
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Fig. 2. Cyclic voltammograms of the electrode in the absence (curve a) and presence (curve b) of potassium ferricyanide.
Fig. 3(a) is a typical amperometric response of the glucose biosensor for successive of 0.05 M glucose in 0.01 M PBS buffer (pH 7.4) at an applied potential of 0.8 V under stirring. It is found that the biosensor shows rapid and sensitive response to the change of glucose concentration, which is ascribed to the good electrocatalytic property of CHIT/GOx/ZnO/GC electrode. The sensor achieves 95% of steady-state current in less than 2 s indicating a fast electron exchange between GOx and ZnO nanorods. The corresponding calibration curve is shown in Fig. 3(b) and (c). Glucose biosensors have only one linear detection range in general, but the linear detection range of our ZnO nanorods-based glucose biosensor is divided into two parts. One part is 0.01–0.25 mM with a correction coefficient R= 0.9994. Another part is 0.3–0.7 mM with a correction coefficient R= 0.99976. The reason of two response ranges is as follows: when the substrate concentration is at low concentration range of 0.01–0.25 mM, the drop of the glucose solution has not been fully spread, then the sensitivity of the sensor is lower; when the substrate concentration is at high concentrations range of 0.3–0.7 mM, the drop of the glucose solution has been completely dispersed, then the sensitivity of the sensor is higher. This kind of biosensor has a low detection limit of 0.01 mM. The high affinity of GOx to glucose can be attributed to ZnO nanorods due to their biocompatibility, large surface area, and high electron communication capability. According to Hrapovic and Yany’s view [17], higher electroactive surface area of nanomaterials always means higher electrocatalytic activity resulting higher sensitivity. So, we select cyclic voltammograms (CVs) to estimate the electroactive surface area of the modified electrodes, Fig. 4 represents the CVs of ZnO nanorods modified GC electrode, which were recorded in a 5 mM [Fe(CN)6]3 /4 solution containing 0.1 M KCl at 50 m V s 1. As shown, well-defined oxidation and reduction peaks due to the Fe3 + /Fe2 + redox couple were observed. The average electroactive surface area could be calculated according to the Randles-Sevcik equation [18]: Ip ¼ 2:69 105 AD1=2 n3=2 g1=2 C
ð3Þ
where n is the number of electrons participating in the redox reaction, A is the area of the electroactive surface area (cm2), D is the diffusion coefficient of the molecule in solution (cm2 s 1) [19], C corresponds to the bulk concentration of the redox probe (mol cm 3), and g is the scan rate of the potential perturbation (V s 1). The [Fe(CN)6]3 /4 redox system used in this study is one
Fig. 3. (a) Amperometric responses of CHIT/GOx/ZnO/GC electrodes with the successive addition of 50 mM glucose to the 0.01 M, pH 7.4 PBS buffer under stirring. (b) One linear calibration curve of ZnO nanorod/GOx biosensor (0.01– 0.25 mM). The straight line is the linear fit to the calibration curve. A potential of +0.8 V was applied on the working electrode in the measurement. (c) The other linear calibration curve of ZnO nanorod/GOx biosensor (0.3–0.7 mM). The straight line is the linear fit to the calibration curve. A potential of + 0.8 V was applied on the working electrode in the measurement.
of the most extensively studied redox couples in electrochemistry and exhibits a heterogeneous one-electron transfer (n=1). C is equal to 5 mM, and the diffusion coefficient (D) is (6.770.02)
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to large surface-to-volume ratio and high conductivity of ZnO nanorod, and good biocompatibility of chitosan, which enhances the enzyme absorption and promotes electron transfer between redox enzymes and the surface of electrodes.
4. Conclusion
Fig. 4. Cyclic voltammograms of [Fe(CN)6]3 /4 containing 0.1 M KCl.
CHIT/GOx/ZnO/GC
electrodes
in
5 mM
Table 1 Performance parameter of glucose biosensors making use of various GOx-modified ZnO nanostructure as the working electrodes. Electrode material
Sensitivity (mA cm 2 mM 1)
LOD (mM)
Response time(s)
Ref.
ZnO nanocombs ZnO nanorod array ZnO:Co nanoclusters ZnO nanorod
15.33 23.1 13.3 25.7
20 10 20 10
o 10 o5 8 o2
[15] [1] [19] This work
10 6cm2 s 1. According to the equation, the average value of the electroactive surface area for ZnO nanorods modified GC electrode is 0.03 cm2. Therefore, our glucose biosensors exhibited a high sensitivity of 25.7 mA cm 2 mM 1. The apparent Michaelis– app , which depicts the enzyme–substrate Menten constantKm kinetics of biosensor, can be calculated from the Lineweaver-Burk app =Imax Þð1=CÞ þ 1=Imax , where C is the conequation: 1=ISS ¼ ðKm centration of substrate, ISS is the steady-state current and Imax is the maximum current measured under substrate saturation [20]. app in this work can be calculated to be Therefore, the values of the Km app is lower than the recent report glucose 1.95 mM. The obtained Km biosensors base on ZnO:Co nanoclusters (21 mM) [20], ZnO nanorod array (2.9 mM) [1], and ZnO nanocombs (2.19 mM) [15]. app means the higher enzymatic activity of immobilized The lower Km GOx; thus, the above result further indicates that the ZnO nanorod modified possesses a high affinity to glucose. For comparison, Table 1 tabulates the key parameters of the glucose biosensors reported in this work and those previous papers making use of ZnO nanostruture as the working electrode. From Table 1, we can see that the ZnO nanorods biosensor show fast response and very high sensitivity. The excellent performance of the biosensor is attributed
In conclusion, our glucose biosensor based on ZnO nanorods by citric acid-assisted annealing route exhibits a low LOD of 0.01 mM, a high and reproducible sensitiving of 25.7 mA cm 2 mM 1 with a response time of less than 2 s. Glucose biosensors have only one linear detection range in general, but the linear detection range of our ZnO nanorods-based glucose biosensor is divided into two parts. One part is 0.01–0.25 mM with a correction coefficient R= 0.9994. Another part is 0.3–0.7 mM with app in this a correction coefficient R=0.99976. The values of the Km app work can be calculated to be 1.95 mM. The lower Km means the higher enzymatic activity of immobilized GOx. Thus, the above result further indicates that the ZnO nanorod modified possesses a high affinity to glucose. In comparison to the prior arts, the sensor reports in this work demonstrated superior performance in sensitivity, response time.
Acknowledgments This work was partly supported from ‘‘973’’ National Key Basic Research Program of China (Grant no. 2006CB604906). Postdoctoral fund of Zhe Jiang Province (2009-bsh-003) References [1] A. Wei, X.W. Sun, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, W. Huang, Appl. Phys. Lett. 89 (2006) 123902. [2] Y. Degani, A. Heller, J. Am. Chem. Soc. 110 (1988) 2615. [3] F. Mizutani, S. Yabuki, T. Katsura, Anal. Chim. Acta 274 (1993) 201. [4] M.M. Castillo-Ortega, D.E. Rodriguez, J.C. Encinas, M. Plascencia, F.A. MendezVelarde, R. Olayo, Sens. Actuators B 85 (2002) 19. [5] Z.J. Liu, B.H. Liu, J.L. Kong, J.Q. Deng, Anal. Chem. 72 (2000) 4707. [6] L.T. Jin, J.S. Ye, W. Tong, Y.Z. Fang, Mikrochim. Acta 112 (1993) 71. [7] J.B. Jia, B.Q. Wang, A.G. Wu, G.G. Cheng, Z. Li, S.J. Dong, Anal. Chem. 74 (2002) 2217. [8] S. Zhao, K. Zhang, Y. Bai, W.W. Yang, C. Sun, Bioelectrochemistry 69 (2006) 158163. [9] Y. Xian, Y. Hu, F. Liu, Y. Xian, H. Wang, L. Jin, Biosens. Bioelectron. 21 (2006) 1996. [10] G.D. Liu, Y.H. Lin, Electrochem. Commun. 8 (2006) 251. [11] W. Zhao, J.J. Xu, Q.Q. Qiu, H.Y. Chen, Biosens. Bioelectron. 22 (2006) 649. [12] D. Shan, M. Zhu, H. Xue, S. Cosnier, Biosens. Bioelectron. 22 (2006) 1612. [13] X. Wen, Y.J. Xie, M.W.C. Mak, K.Y. Cheung, X.Y. Li, R. Renneberg, S. Yang, Langmuir 22 (2006) 4836. [14] Z.M. Liu, Y.L. Liu, H.F. Yang, Y. Yang, G.L. Shen, R.Q. Yu, Electroanalysis 17 (2005) 1065. [15] J.X. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, Appl. Phys. Lett. 88 (2006) 233106. [16] Z. Yang, Z.Z. Ye, Z. Xu, B.H. Zhao, Physica E 42 (2009) 116. [17] M.H. Yang, Y.H. Yang, Y.L. Liu, G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 21 (2006) 1125. [18] A.J. Bard, L.R. Faulkner, Electrochemical Methods-Fundamentals and Applications, John Wiley and Sons, Beijing, 2002, p. 186. [19] S. Hrapovic, J.H. Luong, Anal. Chem. 75 (2003) 3308. [20] R.A. Kamin, G.S. Wilson, Anal. Chem. 52 (1980) 1198.
Contents lists available at ScienceDirect
Physica E journal homepage: www.elsevier.com/locate/physe
A rapid response time and highly sensitive amperometric glucose biosensor based on ZnO nanorod via citric acid-assisted annealing route Zao Yang a,n, Zhizhen Ye a,n, Binghui Zhao a, Xiaolin Zong b, Ping Wang b a
State Key Laboratory of Silicon Materials, Department of Materials Science and Engineering, Zhejiang University, Hangzhou 310027, China Biosensor National Special Laboratory, Key laboratory of Biomedical Engineering of National Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China b
a r t i c l e in f o
a b s t r a c t
Article history: Received 7 October 2009 Received in revised form 7 January 2010 Accepted 1 February 2010 Available online 10 February 2010
ZnO nanorods were synthesized by citric acid-assisted annealing route. In a phosphate buffer solution with a pH value of 7.4, glucose oxidase was immobilized on the surface of ZnO nanorod through chitosan-assisted cross-linking technique. The one-dimensional ZnO nanorods provide a large effective surface area with high surface-to-volume ratio and provide a favorable environment for the immobilization of GOx. The response time of this biosensor is less than 2 s. This biosensor has a very high sensitivity of 25.7 mA cm 2 mM 1. The low detection limit was estimated to be 0.01 mM. Two linear response ranges are 0.01–0.25 mM and 0.3–0.7 mM. The Michaelis–Menten constant is found to be 1.95 mM. These results demonstrate that zinc oxide nanorods have potential applications in biosensors. & 2010 Elsevier B.V. All rights reserved.
Keywords: Zinc oxide Nanorods Glucose biosensor
1. Introduction Glucose sensors, as one of the most popular biosensors, have been extensively investigated due to their important applications in biological and chemical analysis, clinical detection, and environmental monitoring. Among the numerous reports in glucose biosensors, the immobilization of enzymes on electrodes is generally the first step in fabrication, and thus, has attracted significant efforts, because enzymes are highly selective and quickly responsive to specific substrates [1]. Enzyme immobilization is considered as an important factor in biosensor technologies. The conventional techniques for enzyme immobilization include covalent attachment to the electrode surfaces [2], entrapment by ion-exchange polymers [3], nonconducting polymers [4], sol–gels [5], cross-linking in bovine serum albumin– glutaraldehyde [6]. On the other hand, nanomaterials play an important role in adsorption of biomolecules due to their unique properties of high specific surface area, good biological compatibility and stability. They can keep activity of enzyme due to the desirable microenviroment, and enhance the electron transfer between the enzyme’s active sites and the electrode [7]. The search for novel materials to design biosensors is of significance at present. Til date, many nanomaterials, such as gold nanoparticles and nanoclusters [8], Au/polyaniline nanocomposite [9], CNTs [10], nanocrystalline diamond [11], calcium carbonate nanoparticles [12], and Ag dendritic nanostructures [13] have been
n
Corresponding authors. Tel.: + 86 571 8795 3139; fax: +86 571 8795 2625. E-mail addresses: [email protected] (Z. Yang), [email protected] (Z. Ye).
1386-9477/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.physe.2010.02.001
studied as platforms for enzyme immobilization in glucose biosensors. ZnO is a biocompatible material with a high isoelectric point (IEP) of about 9.5, which makes it suitable for absorption of proteins with low IEPS, as the protein immobilization is primarily driven by electrostatic interaction [14]. Moreover, ZnO nanostructures have unique advantages including the high specific surface area, nontoxicity, chemical stability, electrochemical activity, and high electron communication features. Recently, GOx was immobilized on ZnO nanocombs and nanorods array to construct amperometric biosensors for glucose biosensing with sensitivities in the range of 15–23 mA/cm2 Mm [1,15]. The lower sensitivity and slower response of previously fabricated ZnO nanostructures based glucose biosensor have been demonstrated in the literature, hence more works are needed to obtain the higher sensitivity and faster response of the ZnO nanostructures based glucose biosensors. In this article, we shall report a biosensor for glucose detection using ZnO nanorods fabricated by citric acid-assisted annealing route. In a phosphate buffer solution with a pH value of 7.4, glucose oxidase was immobilized on the surface of ZnO nanorod through chitosan-assisted cross-linking technique. The response time of this biosensor is less than 2 s. This biosensor has a very high sensitivity of 25.7 mA cm 2 mM 1.
2. Experimental The ZnO nanorods were prepared by citric acid-assisted annealing route [16]. All the chemicals used in this study were of analytical grade and used without further purification. Four chemicals were needed and they were analytically pure zinc
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acetate (Zn (CH3COO)2 2 H2O), citric acid (C6H8O7 H2O), pure distilled water (H2O) and absolute ethanol (CH3CH2OH). In a typical procedure, a solution of citric acid (0.19 M) in ethanol was added to Zn (CH3COO)2 solutions (2 M) in distilled water. After the addition was completed, the solution (pH value was equal to 6.2) was stirred at 80 1C for 8 h to get the precursor. The precursor was calcined in a muffle furnace for 2 h at 400 1C. Finally, the sample was obtained after cooling down at room temperature in air. Field-emission scanning electron microscopy (FE-SEM, HITACHI S-4800), X-ray diffraction (XRD, Rigaku, D/max-rA) with Cu Ka radiation, transmission electron microscopy (JEM-2010) were employed in characterization. To fabricate the glucose biosensor, the glassy carbon (GC) electrodes was polished by 0.3 and 0.05 mm aluminum slurries and then was cleaned by dipping into 1:1 (V/V) aqueous solution of HNO3, deionized water and ethanol with the assistance of ultrasonication prior to the experiment. The as-prepared ZnO nanorods were dispersed, in ethanol by ultrasonication for 1 h to 5 10 3 M suspension. Firstly, 5 ml of ZnO nanorod suspension was dropped onto the surface of GC electrode and dried at room temperature. Secondly, 5 ml of GOx solution with concentration of 1934 U/ml prepared in 0.01 M phosphate buffer solution (pH 7.4) was dropped onto the surface of GC electrode modified by ZnO nanorods. Finally, 5 ml of 0.5 wt% CHIT solution dissolved in acetic acid solution was dropped onto the surface of GOx/ZnO/GC electrode to avoid the leakage of the enzyme. The device was dried at 4 1C overnight in a refrigerator, followed by washing step to remove the unimmobilized GOx. The CHIT/GOx/ZnO/GC electrode was stored in PBS and kept at 4 1C in a refrigerator when not in use.
(SCE) were used as the counter and reference electrodes, respectively. Fig. 1(a) shows the XRD patterns of the sample. All the diffraction peaks are quite similar to that of a bulk ZnO, which ˚ C =5.206 A, ˚ has a hexagonal spiauterite structure (a =3.249 A, space group: P63mc (1 8 6)) and diffraction data are in agreement with JCPDS card of ZnO (JCPDS 36-1451). No other crystalline forms, such as Zn or other Zn compounds, were detected. Fig. 1(b) shows the typical FE-SEM image of the nanorods. As seen from the figure, the length of ZnO nanorods is 0.2–0.6 mm and the diameter is 30–100 nm. The detailed structure of ZnO nanorod is studied by TEM. Fig. 1(c) shows the TEM image of ZnO nanorod. It indicates that the ZnO nanorods have uniform diameters and no catalyst particle at the tip. The surface of the nanorods is very smooth. The EDS result (Fig. 1d) shows only Zn and O peaks for the nanorods. No other elements were detected from the nanorods. The enzyme electrode was characterized by cyclic voltammetry between the potentials of –0.4 and 0.8 V, as shown in Fig. 2. Curves (a) and (b) are the cyclic voltammetric responses of CHIT/ GOx/ZnO/GC electrode in stirred 0.01 M (pH 7.4) phosphate buffer solution in the absence and presence of 1 mM potassium ferricyanide. No electrochemical response is observed in the absence of potassium ferricyanide (curve a). The results indicate that potassium ferricyanide could be used as a diffusional electron-transferring mediator. In the presence of potassium ferricyanide, a pair of redox peaks with formal potential at 0.02 and 0.42 versus reference electrode was clearly observed, which can be assigned to reversible redox reaction of potassium ferricyanide. The typical enzyme-dependent catalytic process can be expressed as follows:
3. Results and discussion
Glucose+ GOD (FAD)-gluconolactone+ GOD (FADH2)
(1)
The electrochemical experiments were carried out with a CHI660A electrochemical workstation with a conventional threeelectrode cell. The glassy carbon (GC) electrode was used as the working electrode. The Pt wire and a saturated calomel electrode
GOD (FADH2)+ [Fe(CN)6]3 -GOD (FAD)+ [Fe(CN)6]4
(2)
where FAD is the oxidative (reductive) forms of flavin adenine dinucleotide and FADH2 is the active center of GOD.
Fig. 1. (a) XRD patterns of as-grown ZnO nanorods. (b) SEM images of the ZnO nanorods. (c) TEM image of as-grown ZnO nanorods. (d) EDX of ZnO nanorod.
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Fig. 2. Cyclic voltammograms of the electrode in the absence (curve a) and presence (curve b) of potassium ferricyanide.
Fig. 3(a) is a typical amperometric response of the glucose biosensor for successive of 0.05 M glucose in 0.01 M PBS buffer (pH 7.4) at an applied potential of 0.8 V under stirring. It is found that the biosensor shows rapid and sensitive response to the change of glucose concentration, which is ascribed to the good electrocatalytic property of CHIT/GOx/ZnO/GC electrode. The sensor achieves 95% of steady-state current in less than 2 s indicating a fast electron exchange between GOx and ZnO nanorods. The corresponding calibration curve is shown in Fig. 3(b) and (c). Glucose biosensors have only one linear detection range in general, but the linear detection range of our ZnO nanorods-based glucose biosensor is divided into two parts. One part is 0.01–0.25 mM with a correction coefficient R= 0.9994. Another part is 0.3–0.7 mM with a correction coefficient R= 0.99976. The reason of two response ranges is as follows: when the substrate concentration is at low concentration range of 0.01–0.25 mM, the drop of the glucose solution has not been fully spread, then the sensitivity of the sensor is lower; when the substrate concentration is at high concentrations range of 0.3–0.7 mM, the drop of the glucose solution has been completely dispersed, then the sensitivity of the sensor is higher. This kind of biosensor has a low detection limit of 0.01 mM. The high affinity of GOx to glucose can be attributed to ZnO nanorods due to their biocompatibility, large surface area, and high electron communication capability. According to Hrapovic and Yany’s view [17], higher electroactive surface area of nanomaterials always means higher electrocatalytic activity resulting higher sensitivity. So, we select cyclic voltammograms (CVs) to estimate the electroactive surface area of the modified electrodes, Fig. 4 represents the CVs of ZnO nanorods modified GC electrode, which were recorded in a 5 mM [Fe(CN)6]3 /4 solution containing 0.1 M KCl at 50 m V s 1. As shown, well-defined oxidation and reduction peaks due to the Fe3 + /Fe2 + redox couple were observed. The average electroactive surface area could be calculated according to the Randles-Sevcik equation [18]: Ip ¼ 2:69 105 AD1=2 n3=2 g1=2 C
ð3Þ
where n is the number of electrons participating in the redox reaction, A is the area of the electroactive surface area (cm2), D is the diffusion coefficient of the molecule in solution (cm2 s 1) [19], C corresponds to the bulk concentration of the redox probe (mol cm 3), and g is the scan rate of the potential perturbation (V s 1). The [Fe(CN)6]3 /4 redox system used in this study is one
Fig. 3. (a) Amperometric responses of CHIT/GOx/ZnO/GC electrodes with the successive addition of 50 mM glucose to the 0.01 M, pH 7.4 PBS buffer under stirring. (b) One linear calibration curve of ZnO nanorod/GOx biosensor (0.01– 0.25 mM). The straight line is the linear fit to the calibration curve. A potential of +0.8 V was applied on the working electrode in the measurement. (c) The other linear calibration curve of ZnO nanorod/GOx biosensor (0.3–0.7 mM). The straight line is the linear fit to the calibration curve. A potential of + 0.8 V was applied on the working electrode in the measurement.
of the most extensively studied redox couples in electrochemistry and exhibits a heterogeneous one-electron transfer (n=1). C is equal to 5 mM, and the diffusion coefficient (D) is (6.770.02)
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to large surface-to-volume ratio and high conductivity of ZnO nanorod, and good biocompatibility of chitosan, which enhances the enzyme absorption and promotes electron transfer between redox enzymes and the surface of electrodes.
4. Conclusion
Fig. 4. Cyclic voltammograms of [Fe(CN)6]3 /4 containing 0.1 M KCl.
CHIT/GOx/ZnO/GC
electrodes
in
5 mM
Table 1 Performance parameter of glucose biosensors making use of various GOx-modified ZnO nanostructure as the working electrodes. Electrode material
Sensitivity (mA cm 2 mM 1)
LOD (mM)
Response time(s)
Ref.
ZnO nanocombs ZnO nanorod array ZnO:Co nanoclusters ZnO nanorod
15.33 23.1 13.3 25.7
20 10 20 10
o 10 o5 8 o2
[15] [1] [19] This work
10 6cm2 s 1. According to the equation, the average value of the electroactive surface area for ZnO nanorods modified GC electrode is 0.03 cm2. Therefore, our glucose biosensors exhibited a high sensitivity of 25.7 mA cm 2 mM 1. The apparent Michaelis– app , which depicts the enzyme–substrate Menten constantKm kinetics of biosensor, can be calculated from the Lineweaver-Burk app =Imax Þð1=CÞ þ 1=Imax , where C is the conequation: 1=ISS ¼ ðKm centration of substrate, ISS is the steady-state current and Imax is the maximum current measured under substrate saturation [20]. app in this work can be calculated to be Therefore, the values of the Km app is lower than the recent report glucose 1.95 mM. The obtained Km biosensors base on ZnO:Co nanoclusters (21 mM) [20], ZnO nanorod array (2.9 mM) [1], and ZnO nanocombs (2.19 mM) [15]. app means the higher enzymatic activity of immobilized The lower Km GOx; thus, the above result further indicates that the ZnO nanorod modified possesses a high affinity to glucose. For comparison, Table 1 tabulates the key parameters of the glucose biosensors reported in this work and those previous papers making use of ZnO nanostruture as the working electrode. From Table 1, we can see that the ZnO nanorods biosensor show fast response and very high sensitivity. The excellent performance of the biosensor is attributed
In conclusion, our glucose biosensor based on ZnO nanorods by citric acid-assisted annealing route exhibits a low LOD of 0.01 mM, a high and reproducible sensitiving of 25.7 mA cm 2 mM 1 with a response time of less than 2 s. Glucose biosensors have only one linear detection range in general, but the linear detection range of our ZnO nanorods-based glucose biosensor is divided into two parts. One part is 0.01–0.25 mM with a correction coefficient R= 0.9994. Another part is 0.3–0.7 mM with app in this a correction coefficient R=0.99976. The values of the Km app work can be calculated to be 1.95 mM. The lower Km means the higher enzymatic activity of immobilized GOx. Thus, the above result further indicates that the ZnO nanorod modified possesses a high affinity to glucose. In comparison to the prior arts, the sensor reports in this work demonstrated superior performance in sensitivity, response time.
Acknowledgments This work was partly supported from ‘‘973’’ National Key Basic Research Program of China (Grant no. 2006CB604906). Postdoctoral fund of Zhe Jiang Province (2009-bsh-003) References [1] A. Wei, X.W. Sun, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, W. Huang, Appl. Phys. Lett. 89 (2006) 123902. [2] Y. Degani, A. Heller, J. Am. Chem. Soc. 110 (1988) 2615. [3] F. Mizutani, S. Yabuki, T. Katsura, Anal. Chim. Acta 274 (1993) 201. [4] M.M. Castillo-Ortega, D.E. Rodriguez, J.C. Encinas, M. Plascencia, F.A. MendezVelarde, R. Olayo, Sens. Actuators B 85 (2002) 19. [5] Z.J. Liu, B.H. Liu, J.L. Kong, J.Q. Deng, Anal. Chem. 72 (2000) 4707. [6] L.T. Jin, J.S. Ye, W. Tong, Y.Z. Fang, Mikrochim. Acta 112 (1993) 71. [7] J.B. Jia, B.Q. Wang, A.G. Wu, G.G. Cheng, Z. Li, S.J. Dong, Anal. Chem. 74 (2002) 2217. [8] S. Zhao, K. Zhang, Y. Bai, W.W. Yang, C. Sun, Bioelectrochemistry 69 (2006) 158163. [9] Y. Xian, Y. Hu, F. Liu, Y. Xian, H. Wang, L. Jin, Biosens. Bioelectron. 21 (2006) 1996. [10] G.D. Liu, Y.H. Lin, Electrochem. Commun. 8 (2006) 251. [11] W. Zhao, J.J. Xu, Q.Q. Qiu, H.Y. Chen, Biosens. Bioelectron. 22 (2006) 649. [12] D. Shan, M. Zhu, H. Xue, S. Cosnier, Biosens. Bioelectron. 22 (2006) 1612. [13] X. Wen, Y.J. Xie, M.W.C. Mak, K.Y. Cheung, X.Y. Li, R. Renneberg, S. Yang, Langmuir 22 (2006) 4836. [14] Z.M. Liu, Y.L. Liu, H.F. Yang, Y. Yang, G.L. Shen, R.Q. Yu, Electroanalysis 17 (2005) 1065. [15] J.X. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, Appl. Phys. Lett. 88 (2006) 233106. [16] Z. Yang, Z.Z. Ye, Z. Xu, B.H. Zhao, Physica E 42 (2009) 116. [17] M.H. Yang, Y.H. Yang, Y.L. Liu, G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 21 (2006) 1125. [18] A.J. Bard, L.R. Faulkner, Electrochemical Methods-Fundamentals and Applications, John Wiley and Sons, Beijing, 2002, p. 186. [19] S. Hrapovic, J.H. Luong, Anal. Chem. 75 (2003) 3308. [20] R.A. Kamin, G.S. Wilson, Anal. Chem. 52 (1980) 1198.