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Tailoring the surface area of ZnO nanorods for improved performance in glucose sensors
Sensors and Actuators B 192 (2014) 216–220
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Tailoring the surface area of ZnO nanorods for improved performance in glucose sensors Ji Yeong Kim, So-Yeon Jo, Gun-Joo Sun, Akash Katoch, Sun-Woo Choi, Sang Sub Kim ∗ Department of Materials Science and Engineering, Inha University, Inchoen 402-751, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 August 2013 Received in revised form 14 October 2013 Accepted 26 October 2013 Available online 1 November 2013 Keywords: ZnO nanorod Glucose sensor Hydrothermal growth Surface area Biosensor
a b s t r a c t Hydrothermally grown ZnO nanorods were used for enzyme immobilization in glucose sensors. In particular, the surface area of the ZnO nanorods was tailored by the use of a seed layer and/or by changing the concentration of the precursors. The glucose sensing capability was found to be strongly associated with the surface area of the nanorods. The results clearly demonstrated that hydrothermally grown ZnO nanorods be successfully applied to the electrode system for the detection of glucose. In addition, the growth conditions also need to be carefully optimized in order to grow ZnO nanorods that are as slim and long as possible in order to maximize the surface area. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the need for high-performance glucose sensors has been increasing because the fast and precise detection of glucose is a decisive means to diagnose diabetes or metabolic disorders that are currently widespread around the globe [1,2]. Accordingly, many efforts have been made to develop glucose sensors with excellent glucose-detection capability based on electrochemistry [3], chemoluminescence [4], etc. In particular, the enzyme-immobilized electrochemical glucose sensor is one of the most intensively investigated sensor types owing to its high selectivity, sensitive glucose detection, and relatively low-cost fabrication. An important issue with this type of glucose sensor is the choice of materials used for enzyme immobilization. The materials should possess efficient enzyme loading characteristics and good biocompatibility. Zinc oxide (ZnO) has been used extensively as a support material for enzyme immobilization. In the literature, nanorods [5], nanocombs [6], and nanowires [7] of ZnO have been successfully used as materials for enzyme immobilization. In particular, aligned ZnO nanorod films directly grown on the indium tin oxide layer were successfully employed for immobilization of glucose oxidase, demonstrating their potential for the use of amperometric glucose biosensors [8]. ZnO nanorods/Au hybrid nanocomposites were also used to entrap glucose oxidase, revealing reliable
∗ Corresponding author. Tel.: +82 32 860 7546; fax: +82 32 862 5546. E-mail address: [email protected] (S.S. Kim). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.10.113
glucose-sensing capability [9]. Two review papers well describe the state-of-the-art use of ZnO materials for enzyme immobilization in electrochemical-based biosensors [10] and the surface functionalization of ZnO materials for biosensing [11]. Because ZnO has demonstrated various useful properties that have led to its applications in many fields, many investigations have been carried out in efforts to synthesize various forms of ZnO nanomaterials. Among the various methods used to synthesize ZnO nanomaterials, the hydrothermal method has been regarded as one of the most promising owing to its cost-effectiveness in terms of production. A literature survey [12–15] shows that the precursors and preparation parameters used in the hydrothermal method, such as temperature and pH, determine the geometric shapes of ZnO nanomaterials, which in turn affect the electrical and optical properties of the ZnO nanomaterials. It is very likely that geometric factors such as the shape, size, and surface area of ZnO nanorods will greatly influence the glucose-sensing efficiency when ZnO nanorods are used for enzyme immobilization. However, in spite of its importance, since studies on the effect of the geometric factors are rare to our knowledge, it is urgent to confirm whether the geometric factors influence the glucose-sensing ability of ZnO nanorod-based sensors. In this work, ZnO nanorods were hydrothermally grown for enzyme immobilization in glucose sensors. Their glucose-sensing performance was investigated particularly as a function of the surface area of the ZnO nanorods, which was tailored by the use of a seed layer and/or by changing the concentration of the precursors in the hydrothermal growth solution. The glucose-sensing capabilities were found to be proportional to the surface area of
J.Y. Kim et al. / Sensors and Actuators B 192 (2014) 216–220
the nanorods, suggesting that densely packed, slim, and long ZnO nanorods are more favorable for obtaining superior ZnO nanorodbased glucose sensors. 2. Experimental details The following steps were applied to fabricate ZnO nanorodbased glucose sensors. The entire fabrication process is schematically shown in Fig. 1. All steps were performed in air at room temperature. For the growth of ZnO nanorods, a 90-nm-thick Au film was deposited on glass substrates by sputtering with a shadow mask whose opening size was 3 mm × 2 mm, resulting in a rectangular electrode area of 6 mm2 . The glass substrates were purchased from a company (Hansol Science, Korea) in the name of slide glass, whose dimensions were 76 mm × 52 mm in size and 1.2 mm in thickness. Before the sensor fabrication, they were ultrasonically cleaned using acetone and isopropanol in sequence for 10 min. To enhance the adhesion between the Au film and the glass substrate, a 20-nm-thick Cr interlayer was inserted between them. Then, the Au/Cr/glass wafers were sequentially rinsed with alcohol and deionized water three times. Three different shapes of ZnO nanorods were grown. First, ZnO nanorods were directly grown on the Au/Cr/glass wafer by the hydrothermal method. To accomplish this, a 0.1 M solution of equimolar zinc nitrate hexahydrate [Zn(NO3 )2 6H2 O] and hexamethylenetetramine (HMT) in deionized water was prepared and stirred for 2 h at room temperature. Then the solution was introduced into a home-made Teflon vessel in which the Au/Cr/glass wafer had been placed. The Teflon vessel was maintained at 95 ◦ C in an oven for 6 h. This process resulted in the growth of vertically aligned ZnO nanorods. Second, ZnO nanorods were grown on the Au/Cr/glass wafer with a ZnO seed layer by using the same hydrothermal process mentioned above. The 20nm-thick ZnO seed layer was deposited on the Au/Cr/glass wafer using the atomic layer deposition (ALD) technique. The ALD conditions used for growing the ZnO seed layer is described in detail in our earlier report [16]. Finally, ZnO nanorods were grown on the Au/Cr/glass wafer with the same ZnO seed layer but using another solution ratio: a 0.03 M solution of equimolar zinc nitrate hexahydrate [Zn(NO3 )2 6H2 O] and HMT in deionized water. The growth mechanism and the function for each component are discussed in our earlier report [15]. Hereafter, we respectively designate the three differently sized ZnO nanorods as ZNR-1, ZNR-2, and ZNR-3. To immobilize glucose oxidase (GOx ) on the ZnO nanorods, the ZnO nanorod-prepared wafers were immersed in a solution of 2 mg/mL GOx in phosphate-buffered saline (PBS) for 2 h and then rinsed with PBS three times. For the purpose of further stabilizing
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the immobilization of GOx , a 5% Nafion solution (Wako Pure Chemicals Industries) was dropped onto the GOx -immobilized ZnO nanorods and maintained at 4 ◦ C for 1 h. According to the specification provided by the company, the Nafion was dissolved in a mixed solvent of 45% water and 50% 1-rpopanol in addition to trace amounts of ethanol and ether. The microstructures of the ZnO nanorods were investigated by field-emission scanning electron microscopy (FE-SEM). Transmission electron microscopy (TEM) was used to further observe their microstructures. Cyclic voltammetry measurements were performed to investigate the electrochemical behavior of the fabricated electrode system to glucose. The measurements were based on a 3-electrode system consisting of an Ag/AgCl (3.0 M KCl) reference electrode, the fabricated electrode that served as the working electrode, and a platinum wire that was used as a counter electrode. To estimate the glucose-sensing capability of the fabricated electrode system, I–V curves were obtained while the electrodes were dipped in PBS (pH = 7.2) containing various concentrations of glucose. 3. Results and discussion Fig. 2(a) shows the microstructure of the ZnO nanorods, designated as ZNR-1, that were hydrothermally grown on the Au/Cr/glass substrates without the ZnO seed layer. It shows somewhat vertically aligned ZnO nanorods that are quite uniform in length and diameter; the length and diameter of ZNR-1 are approximately 7.7 and 1.5 m, respectively. One of the current authors has investigated the growth behavior (orientation and alignment) of vertically aligned ZnO nanorods as a function of the type of substrate used [17]. Based on the results from that paper, it is reasonable to assume that the alignment of the ZNR-1 nanorods grown on the Au/Cr/glass substrate is similar to what occurs with mismatched substrates such as Si. On mismatched Si substrates, ZnO nanorods usually grow with substantial vertical alignment but random in-plane alignment. Slim ZnO nanorods were grown using a ZnO seed layer under the same hydrothermal growth conditions, as shown in Fig. 2(b). These nanorods are designated as ZNR-2 and are 1.54 m in length and 0.105 m in diameter. In this case, ZnO nanorods were homoepitaxially grown on the ZnO seed layer. Based on the previous results [17], ZnO nanorods grown on well-matched substrates such as ZnO (0 0 0 1) are more likely to grow following the layer-by-layer growth. With the same ZnO seed layer but using a different solution concentration, much slimmer ZnO nanorods were obtained, as shown in Fig. 2(c). These nanorods are designated as ZNR-3 and are 1.4 m in length and 0.06 m in diameter. In this case,
Fig. 1. Fabrication of ZnO nanorod-based glucose sensors: (a) Au/Cr/glass substrate, (b) hydrothermally grown, vertically aligned ZnO nanorods on Au/Cr/glass substrate, (c) GO immobilization and subsequent stabilization by Nafion, (d) ALD-grown ZnO seed layer on the Au/Cr/glass substrate, (e) growth of ZnO nanorods on the ZnO/Au/Cr/glass substrate, and (f) GO immobilization and stabilization.
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Fig. 3. A low-magnification TEM image taken from an individual ZNR-1. The upper inset is a high-resolution TEM image. The lower inset is the corresponding selected area electron diffraction pattern.
selected area electron diffraction pattern is shown in the lower inset of Fig. 3, again showing the wurtzite ZnO structure. For avoiding redundancy, the TEM results taken from ZNR-2 and ZNR-3 samples were not presented here because they were basically similar to Fig. 3. The electrochemical behavior of the ZnO nanorod-based glucose sensors was investigated by cyclic voltammetry over a range of glucose concentrations in PBS. The results are shown in Fig. 4. It is apparent that the current increases gradually with the glucose concentration being increased. At the working electrode, the following electrochemical reactions are known to occur [18]: glucose + O2 + H2 O ↔ gluconicacid + H2 O2
Fig. 2. Microstructures of ZnO nanorods observed by FE-SEM: ZnO nanorods grown using the 0.1 M solution of equimolar zinc nitrate hexahydrate and HMT in deionized water on the Au/Cr/glass substrate (a) without the ZnO seed layer and (b) with the ZnO seed layer. (c) ZnO nanorods grown using the 0.03 M on the Au/Cr/glass substrate with the ZnO seed layer.
the solution concentration was 0.03 M of equimolar zinc nitrate hexahydrate and HMT in deionized water. Table 1 summarizes the size and number density, and the corresponding surface area of ZnO nanorods prepared in this work. The detailed microstructure of individual ZnO nanorods was studied by TEM. Fig. 3 shows the low-magnification TEM image of an individual ZNR-1. The upper inset is a high-resolution TEM image taken from the individual ZNR-1 in which the spacing of the lattice plane 0.26 nm well corresponds the interplanar distance of the (0 0 0 2) plane in wurtzite ZnO. It demonstrates a good crystalline quality of ZNR-1 without showing considerable structural defects such as dislocations and staking faults. The corresponding
And at the platinum counter electrode, the following reaction occurs: H2 O2 ↔ 2H+ + O2 + 2e− The I–V measurements were based on the detection of the oxidation signal of hydrogen peroxide or the reduction signal of dissolved oxygen, which is produced or consumed in the oxidation process of glucose to gluconic acid catalyzed by glucose oxidase, respectively [8]. The cyclic voltammograms of the Nafion/ZnO nanorods/Au/glass electrode will more obviously reveal the effect of glucose oxidase in electrochemistry. Although we haven’t carried out the test, according to the reports [5,8], it is reasonable to assume that both ZnO nanorods and Nafion membrane are not electrochemically active in this potential window. In Fig. 4(b), (d) and (f), the obtained current densities measured at the working potential of 0.8 V are plotted as a function of the glucose concentration in order to evaluate the sensitivity of the glucose sensor fabricated from ZNR-1, ZNR-2 and ZNR-3, respectively. The current densities are proportional to the glucose concentration, exhibiting
Table 1 Summary of geometric details for ZnO nanorods grown in this work. Designation (substrate type)
ZNR-1 (Au/Cr/glass) ZNR-2 (ZnO seed layer) ZNR-3 (ZnO seed layer)
Ratio of zinc nitrate hexahydrate to hexamethylenetetramine
0.1 0.1 0.03
Geometric details of ZnO nanorods
Surface area of ZnO nanorods [m2 ]
Length [m]
Diameter [m]
# per electrode area [#/m2 ]
Unit area of electrode [m2 ]
7.7 1.54 1.4
1.5 0.105 0.06
0.375 57 170
13.6 28.9 44.8
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Fig. 4. Cyclic voltammograms of the ZnO nanorod-based glucose sensors: (a) ZNR-1, (c) ZNR-2 and (e) ZNR-3. The current density versus glucose concentration: (b) ZNR-1, (d) ZNR-2 and (f) ZNR-3. The slopes indicate the sensitivity of the fabricated sensors.
sensitivities of 16.5, 41.3, 69.8 nA/(M-cm2 ) for ZNR-1, ZNR-2 and ZNR-3, respectively. This proportionality has been often observed in various ZnO nanostructure-based glucose sensors [5,6,19,20], in which the current density tends to saturate for higher glucose concentrations. Fig. 5 shows the sensitivity as a function of the surface area of the ZnO nanorods, and a linear relationship is readily apparent. The larger the surface area is, the higher the glucose sensitivity is.
Fig. 5. Sensitivity versus surface area of ZnO nanorods per unit electrode area.
The strong interaction between glucose oxidase and ZnO nanorods was first confirmed by Liu et al. [8] on the basis of the infrared absorption spectra analysis. They found that glucose oxidase molecules are well anchored on the surface of ZnO nanorods. It is therefore reasonable to conclude that glucose oxidase molecules do not sit just at the tips of ZnO nanorods but cover the entire surface of ZnO nanorods. Although the ratio of the amount of glucose oxidase molecules that can be most fixed to the surface area of ZnO nanorods is not known yet, the larger surface area of ZnO nanorods like the ZNR-3 sample is likely to contain more amount of glucose oxidase molecules, thereby more pronounced oxidation and reduction signal in the above-mentioned electrochemical reaction. The response time is one of important parameters determining the sensing performance of ZnO nanostructure-based glucose sensors. According to the literature survey [5,6,8,19,21–25], despite a small variation in relation to the type of nanostructures, the response time is shorter than 10 s in various ZnO nanostructurebased glucose sensors. In this work, different surface areas were realized by growing the ZnO nanorods with different sizes, consequently leading to correspondingly different spaces between individual nanorods. This must affect the kinetics because different amounts of liquid are likely to be incorporated in the electrochemical reaction. The measurement of the response time will provide some idea to clarify this matter, which needs to be performed in the future.
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Our results clearly demonstrate that not only can hydrothermally grown ZnO nanorods be successfully applied to highperformance glucose sensors but also that the growth conditions need to be carefully optimized to grow ZnO nanorods that are as slim and long as possible in order to attain maximum surface area. 4. Conclusions We have applied hydrothermally grown ZnO nanorods to the electrode system of glucose sensors. In particular, the ZnO nanorods played the role of enzyme immobilizers in the glucose sensors. Three different shapes of ZnO nanorods were vertically grown with the hydrothermal method by using an ALD-grown ZnO seed layer and/or another precursor solution concentration. Interestingly, the glucose sensitivity was strongly dependent on the surface area of the ZnO nanorods. Thus, ZnO nanorods with a larger surface area are more favorable for efficient glucose sensors. The results suggest that the growth conditions of ZnO nanorods needs to be fine-tuned in order to maximize the surface area for ZnO nanorod-based glucose sensors. Acknowledgment This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (No. 2012R1A2A2A01013899). In addition, this work was partially supported by Inha University Research Grant. References [1] N.S. Oliver, C. Toumazou, A.E.G. Cass, D.G. Johnston, Glucose sensors: a review of current and emerging technology, Diabetic Med. 26 (2009) 197–210. [2] E.H. Yoo, S.Y. Lee, Glucose biosensors: an overview of use in clinical practice, Sensors 10 (2010) 4558–4576. [3] X.-Y. Lang, H.-Y. Fu, C. Hou, G.-F. Han, P. Yang, Y.-B. Liu, Q. Jiang, Nanoporous gold supported cobalt oxide microelectrodes as high-performance electrochemical biosensors, Nat. Commun. 4 (2013) 1–8. [4] Y. Fan, Y. Huang, The effective peroxidase-like activity of chitosanfunctionalized CoFe2 O4 nanoparticles for chemiluminescence sensing of hydrogen peroxide and glucose, Analyst 137 (2012) 1225–1231. [5] A. Wei, X.W. Sun, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, W. Huang, Enzymatic glucose biosensor based on ZnO nanorod array grown by hydrothermal decomposition, Appl. Phys. Lett. 89 (2006) 123902. [6] J.X. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, Zinc oxide nanocomb biosensor for glucose detection, Appl. Phys. Lett. 88 (2006) 233106. [7] J. Zang, C.M. Li, X. Cui, J. Wang, X. Sun, H. Dong, C.Q. Sun, Tailoring zinc oxide nanowires for high performance amperometric glucose sensor, Electroanalysis 19 (2007) 1008–1014. [8] X. Liu, Q. Hu, Q. Wu, W. Zhang, Z. Fang, Q. Xie, Aligned ZnO nanorods: a useful film to fabricate amperometric glucose biosensor, Colloids Surf. B 74 (2009) 154–158. [9] Y. Wei, Y. Li, X. Liu, Y. Xian, G. Shi, L. Jin, ZnO nanorods/Au hybrid nanocomposites for glucose biosensor, Biosens. Bioelectron. 26 (2010) 275–278. [10] Z. Zhao, W. Lei, X. Zhang, B. Wang, H. Jiang, ZnO-based amperometric enzyme biosensors, Sensors 10 (2010) 1216–1231. [11] R. Yakimova, L. Selegård, V. Khranovskyy, R. Pearce, A.L. Spetz, K. Uvdal, ZnO materials and surface tailoring for biosensing, Front. Biosci. 4 (2012) 254–278. [12] S. Yamabi, H. Imai, Growth conditions for wurtzite zinc oxide films in aqueous solutions, J. Mater. Chem. 12 (2002) 3773–3778. [13] X. Liu, Z. Jin, S. Bu, J. Zhao, Z. Liu, Growth of ZnO films with controlled morphology by aqueous solution method, J. Am. Ceram. Soc. 89 (2006) 1226–1231.
[14] A.M. Peiro , P. Ravirajan, K. Govender, D.S. Boyle, P. O’Brien, D.D.C. Bradley, J. Nelson, J.R. Durrant, Hybrid polymer/metal oxide solar cells based on ZnO columnar structures, J. Mater. Chem. 16 (2006) 2088–2096. [15] J.Y. Park, S.-W. Choi, K. Asokan, S.S. Kim, Growth of ZnO nanobrushes using a two-step aqueous solution method, J. Am. Ceram. Soc. 93 (2010) 3190–3194. [16] J.Y. Park, S.-W. Choi, J.-W. Lee, C. Lee, S.S. Kim, Synthesis and gas sensing properties of TiO2 -ZnO core–shell nanofibers, J. Am. Ceram. Soc. 92 (2009) 2551–2554. [17] I.O. Jung, J.Y. Park, S.S. Kim, Substrate dependent growth modes of ZnO nanorods grown by metalorganic chemical vapor deposition, J. Cryst. Growth 355 (2012) 78–83. [18] M. Delvaux, A. Walcarius, S.D. Champagne, Bienzyme HRP–GOx-modified gold nanoelectrodes for the sensitive amperometric detection of glucose at low overpotentials, Biosens. Bioelectron. 20 (2005) 1587–1594. [19] Y. Lei, X. Yan, N. Luo, Y. Song, Y. Zhang, ZnO nanotetrapod network as the adsorption layer for the improvement of glucose detection via multiterminal electron-exchange, Colloids Surf., A 361 (2010) 169–173. [20] K. Yang, G.-W. She, H. Wang, X.-M. Ou, X.-H. Zhang, C.-S. Lee, S.-T. Lee, ZnO nanotube arrays as biosensors for glucose, J. Phys. Chem. C 113 (2009) 20169–20172. [21] T. Kong, Y. Chen, Y. Ye, K. Zhang, Z. Wang, X. Wang, An amperometric glucose biosensor based on the immobilization of glucose oxidase on the ZnO nanotubes, Sens. Actuators B 138 (2009) 344–350. [22] D. Pradhan, F. Niroui, K.T. Leung, High-performance, flexible enzymatic glucose biosensor based on ZnO nanowires supported on a gold-coated polyester substrate, ACS Appl. Mater. Interfaces 2 (2010) 2409–2412. [23] Y. Zhai, S. Zhai, G. Chen, K. Zhang, Q. Yue, L. Wang, J. Liu, Effects of morphology of nanostructured ZnO on direct electrochemistry and biosensing properties of glucose oxidase, J. Electroanal. Chem. 656 (2011) 198–205. [24] Y. Lei, X. Yan, J. Zhao, X. Liu, Y. Song, N. Luo, Y. Zhang, Improved glucose electrochemical biosensor by appropriate immobilization of nano-ZnO, Colloids Surf. B 82 (2011) 168–172. [25] N.K. Sarkar, S.K. Bhattacharyya, High electro-catalytic activities of glucose oxidase embedded one-dimensional ZnO nanostructures, Nanotechnology 24 (2013) 225502.
Biographies Ji Yeong Kim received her M.S. degree from Inha University, Korea, in 2013. She works for a company that produces chemicals for surface treatments. So-Yeon Jo is a fourth-year undergraduate at Inha University. She has been working on synthesis and characterization of biosensors. Gun-Joo Sun received his B.S. degree from Inha University, Korea, in 2011. He is a doctoral student at Inha University, Korea, and has been working on oxide nanowires gas sensors. Akash Katoch received his B.S. degree from Himachal Pradesh University, India, in 2004. In 2009, he received his M.S. degree from Thapar University, India. From 2010, he has been a doctoral student at Inha University, Korea. He has been working on metal oxide gas sensors synthesized by the sol–gel process. Sun-Woo Choi received his B.S. and M.S. degrees from Inha University, Korea, in 2008 and 2010, respectively. He became a doctoral student at Inha University in 2010. He has been working on metal oxide gas sensors and on the synthesis of metal nanoparticles. Sang Sub Kim joined the School of Materials Science and Engineering, Inha University, in 2007 as a full professor. He received his B.S. degree from the Seoul National University and his M.S. and Ph.D. degrees from the Pohang University of Science and Technology (POSTECH) in Material Science and Engineering in 1987, 1990, and 1994, respectively. He was a visiting researcher at the National Research in Inorganic Materials (currently NIMS), Japan, for two years each in 1995 and in 2000. In 2006, he was a visiting professor at the Department of Chemistry, University of Alberta, Canada. In 2010, he also served as a cooperative professor at the Nagaoka University of Technology, Japan. His research interests include the synthesis and applications of nanomaterials such as nanowires and nanofibers, functional thin films, and surface and interfacial characterizations.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Tailoring the surface area of ZnO nanorods for improved performance in glucose sensors Ji Yeong Kim, So-Yeon Jo, Gun-Joo Sun, Akash Katoch, Sun-Woo Choi, Sang Sub Kim ∗ Department of Materials Science and Engineering, Inha University, Inchoen 402-751, Republic of Korea
a r t i c l e
i n f o
Article history: Received 9 August 2013 Received in revised form 14 October 2013 Accepted 26 October 2013 Available online 1 November 2013 Keywords: ZnO nanorod Glucose sensor Hydrothermal growth Surface area Biosensor
a b s t r a c t Hydrothermally grown ZnO nanorods were used for enzyme immobilization in glucose sensors. In particular, the surface area of the ZnO nanorods was tailored by the use of a seed layer and/or by changing the concentration of the precursors. The glucose sensing capability was found to be strongly associated with the surface area of the nanorods. The results clearly demonstrated that hydrothermally grown ZnO nanorods be successfully applied to the electrode system for the detection of glucose. In addition, the growth conditions also need to be carefully optimized in order to grow ZnO nanorods that are as slim and long as possible in order to maximize the surface area. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the need for high-performance glucose sensors has been increasing because the fast and precise detection of glucose is a decisive means to diagnose diabetes or metabolic disorders that are currently widespread around the globe [1,2]. Accordingly, many efforts have been made to develop glucose sensors with excellent glucose-detection capability based on electrochemistry [3], chemoluminescence [4], etc. In particular, the enzyme-immobilized electrochemical glucose sensor is one of the most intensively investigated sensor types owing to its high selectivity, sensitive glucose detection, and relatively low-cost fabrication. An important issue with this type of glucose sensor is the choice of materials used for enzyme immobilization. The materials should possess efficient enzyme loading characteristics and good biocompatibility. Zinc oxide (ZnO) has been used extensively as a support material for enzyme immobilization. In the literature, nanorods [5], nanocombs [6], and nanowires [7] of ZnO have been successfully used as materials for enzyme immobilization. In particular, aligned ZnO nanorod films directly grown on the indium tin oxide layer were successfully employed for immobilization of glucose oxidase, demonstrating their potential for the use of amperometric glucose biosensors [8]. ZnO nanorods/Au hybrid nanocomposites were also used to entrap glucose oxidase, revealing reliable
∗ Corresponding author. Tel.: +82 32 860 7546; fax: +82 32 862 5546. E-mail address: [email protected] (S.S. Kim). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.10.113
glucose-sensing capability [9]. Two review papers well describe the state-of-the-art use of ZnO materials for enzyme immobilization in electrochemical-based biosensors [10] and the surface functionalization of ZnO materials for biosensing [11]. Because ZnO has demonstrated various useful properties that have led to its applications in many fields, many investigations have been carried out in efforts to synthesize various forms of ZnO nanomaterials. Among the various methods used to synthesize ZnO nanomaterials, the hydrothermal method has been regarded as one of the most promising owing to its cost-effectiveness in terms of production. A literature survey [12–15] shows that the precursors and preparation parameters used in the hydrothermal method, such as temperature and pH, determine the geometric shapes of ZnO nanomaterials, which in turn affect the electrical and optical properties of the ZnO nanomaterials. It is very likely that geometric factors such as the shape, size, and surface area of ZnO nanorods will greatly influence the glucose-sensing efficiency when ZnO nanorods are used for enzyme immobilization. However, in spite of its importance, since studies on the effect of the geometric factors are rare to our knowledge, it is urgent to confirm whether the geometric factors influence the glucose-sensing ability of ZnO nanorod-based sensors. In this work, ZnO nanorods were hydrothermally grown for enzyme immobilization in glucose sensors. Their glucose-sensing performance was investigated particularly as a function of the surface area of the ZnO nanorods, which was tailored by the use of a seed layer and/or by changing the concentration of the precursors in the hydrothermal growth solution. The glucose-sensing capabilities were found to be proportional to the surface area of
J.Y. Kim et al. / Sensors and Actuators B 192 (2014) 216–220
the nanorods, suggesting that densely packed, slim, and long ZnO nanorods are more favorable for obtaining superior ZnO nanorodbased glucose sensors. 2. Experimental details The following steps were applied to fabricate ZnO nanorodbased glucose sensors. The entire fabrication process is schematically shown in Fig. 1. All steps were performed in air at room temperature. For the growth of ZnO nanorods, a 90-nm-thick Au film was deposited on glass substrates by sputtering with a shadow mask whose opening size was 3 mm × 2 mm, resulting in a rectangular electrode area of 6 mm2 . The glass substrates were purchased from a company (Hansol Science, Korea) in the name of slide glass, whose dimensions were 76 mm × 52 mm in size and 1.2 mm in thickness. Before the sensor fabrication, they were ultrasonically cleaned using acetone and isopropanol in sequence for 10 min. To enhance the adhesion between the Au film and the glass substrate, a 20-nm-thick Cr interlayer was inserted between them. Then, the Au/Cr/glass wafers were sequentially rinsed with alcohol and deionized water three times. Three different shapes of ZnO nanorods were grown. First, ZnO nanorods were directly grown on the Au/Cr/glass wafer by the hydrothermal method. To accomplish this, a 0.1 M solution of equimolar zinc nitrate hexahydrate [Zn(NO3 )2 6H2 O] and hexamethylenetetramine (HMT) in deionized water was prepared and stirred for 2 h at room temperature. Then the solution was introduced into a home-made Teflon vessel in which the Au/Cr/glass wafer had been placed. The Teflon vessel was maintained at 95 ◦ C in an oven for 6 h. This process resulted in the growth of vertically aligned ZnO nanorods. Second, ZnO nanorods were grown on the Au/Cr/glass wafer with a ZnO seed layer by using the same hydrothermal process mentioned above. The 20nm-thick ZnO seed layer was deposited on the Au/Cr/glass wafer using the atomic layer deposition (ALD) technique. The ALD conditions used for growing the ZnO seed layer is described in detail in our earlier report [16]. Finally, ZnO nanorods were grown on the Au/Cr/glass wafer with the same ZnO seed layer but using another solution ratio: a 0.03 M solution of equimolar zinc nitrate hexahydrate [Zn(NO3 )2 6H2 O] and HMT in deionized water. The growth mechanism and the function for each component are discussed in our earlier report [15]. Hereafter, we respectively designate the three differently sized ZnO nanorods as ZNR-1, ZNR-2, and ZNR-3. To immobilize glucose oxidase (GOx ) on the ZnO nanorods, the ZnO nanorod-prepared wafers were immersed in a solution of 2 mg/mL GOx in phosphate-buffered saline (PBS) for 2 h and then rinsed with PBS three times. For the purpose of further stabilizing
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the immobilization of GOx , a 5% Nafion solution (Wako Pure Chemicals Industries) was dropped onto the GOx -immobilized ZnO nanorods and maintained at 4 ◦ C for 1 h. According to the specification provided by the company, the Nafion was dissolved in a mixed solvent of 45% water and 50% 1-rpopanol in addition to trace amounts of ethanol and ether. The microstructures of the ZnO nanorods were investigated by field-emission scanning electron microscopy (FE-SEM). Transmission electron microscopy (TEM) was used to further observe their microstructures. Cyclic voltammetry measurements were performed to investigate the electrochemical behavior of the fabricated electrode system to glucose. The measurements were based on a 3-electrode system consisting of an Ag/AgCl (3.0 M KCl) reference electrode, the fabricated electrode that served as the working electrode, and a platinum wire that was used as a counter electrode. To estimate the glucose-sensing capability of the fabricated electrode system, I–V curves were obtained while the electrodes were dipped in PBS (pH = 7.2) containing various concentrations of glucose. 3. Results and discussion Fig. 2(a) shows the microstructure of the ZnO nanorods, designated as ZNR-1, that were hydrothermally grown on the Au/Cr/glass substrates without the ZnO seed layer. It shows somewhat vertically aligned ZnO nanorods that are quite uniform in length and diameter; the length and diameter of ZNR-1 are approximately 7.7 and 1.5 m, respectively. One of the current authors has investigated the growth behavior (orientation and alignment) of vertically aligned ZnO nanorods as a function of the type of substrate used [17]. Based on the results from that paper, it is reasonable to assume that the alignment of the ZNR-1 nanorods grown on the Au/Cr/glass substrate is similar to what occurs with mismatched substrates such as Si. On mismatched Si substrates, ZnO nanorods usually grow with substantial vertical alignment but random in-plane alignment. Slim ZnO nanorods were grown using a ZnO seed layer under the same hydrothermal growth conditions, as shown in Fig. 2(b). These nanorods are designated as ZNR-2 and are 1.54 m in length and 0.105 m in diameter. In this case, ZnO nanorods were homoepitaxially grown on the ZnO seed layer. Based on the previous results [17], ZnO nanorods grown on well-matched substrates such as ZnO (0 0 0 1) are more likely to grow following the layer-by-layer growth. With the same ZnO seed layer but using a different solution concentration, much slimmer ZnO nanorods were obtained, as shown in Fig. 2(c). These nanorods are designated as ZNR-3 and are 1.4 m in length and 0.06 m in diameter. In this case,
Fig. 1. Fabrication of ZnO nanorod-based glucose sensors: (a) Au/Cr/glass substrate, (b) hydrothermally grown, vertically aligned ZnO nanorods on Au/Cr/glass substrate, (c) GO immobilization and subsequent stabilization by Nafion, (d) ALD-grown ZnO seed layer on the Au/Cr/glass substrate, (e) growth of ZnO nanorods on the ZnO/Au/Cr/glass substrate, and (f) GO immobilization and stabilization.
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Fig. 3. A low-magnification TEM image taken from an individual ZNR-1. The upper inset is a high-resolution TEM image. The lower inset is the corresponding selected area electron diffraction pattern.
selected area electron diffraction pattern is shown in the lower inset of Fig. 3, again showing the wurtzite ZnO structure. For avoiding redundancy, the TEM results taken from ZNR-2 and ZNR-3 samples were not presented here because they were basically similar to Fig. 3. The electrochemical behavior of the ZnO nanorod-based glucose sensors was investigated by cyclic voltammetry over a range of glucose concentrations in PBS. The results are shown in Fig. 4. It is apparent that the current increases gradually with the glucose concentration being increased. At the working electrode, the following electrochemical reactions are known to occur [18]: glucose + O2 + H2 O ↔ gluconicacid + H2 O2
Fig. 2. Microstructures of ZnO nanorods observed by FE-SEM: ZnO nanorods grown using the 0.1 M solution of equimolar zinc nitrate hexahydrate and HMT in deionized water on the Au/Cr/glass substrate (a) without the ZnO seed layer and (b) with the ZnO seed layer. (c) ZnO nanorods grown using the 0.03 M on the Au/Cr/glass substrate with the ZnO seed layer.
the solution concentration was 0.03 M of equimolar zinc nitrate hexahydrate and HMT in deionized water. Table 1 summarizes the size and number density, and the corresponding surface area of ZnO nanorods prepared in this work. The detailed microstructure of individual ZnO nanorods was studied by TEM. Fig. 3 shows the low-magnification TEM image of an individual ZNR-1. The upper inset is a high-resolution TEM image taken from the individual ZNR-1 in which the spacing of the lattice plane 0.26 nm well corresponds the interplanar distance of the (0 0 0 2) plane in wurtzite ZnO. It demonstrates a good crystalline quality of ZNR-1 without showing considerable structural defects such as dislocations and staking faults. The corresponding
And at the platinum counter electrode, the following reaction occurs: H2 O2 ↔ 2H+ + O2 + 2e− The I–V measurements were based on the detection of the oxidation signal of hydrogen peroxide or the reduction signal of dissolved oxygen, which is produced or consumed in the oxidation process of glucose to gluconic acid catalyzed by glucose oxidase, respectively [8]. The cyclic voltammograms of the Nafion/ZnO nanorods/Au/glass electrode will more obviously reveal the effect of glucose oxidase in electrochemistry. Although we haven’t carried out the test, according to the reports [5,8], it is reasonable to assume that both ZnO nanorods and Nafion membrane are not electrochemically active in this potential window. In Fig. 4(b), (d) and (f), the obtained current densities measured at the working potential of 0.8 V are plotted as a function of the glucose concentration in order to evaluate the sensitivity of the glucose sensor fabricated from ZNR-1, ZNR-2 and ZNR-3, respectively. The current densities are proportional to the glucose concentration, exhibiting
Table 1 Summary of geometric details for ZnO nanorods grown in this work. Designation (substrate type)
ZNR-1 (Au/Cr/glass) ZNR-2 (ZnO seed layer) ZNR-3 (ZnO seed layer)
Ratio of zinc nitrate hexahydrate to hexamethylenetetramine
0.1 0.1 0.03
Geometric details of ZnO nanorods
Surface area of ZnO nanorods [m2 ]
Length [m]
Diameter [m]
# per electrode area [#/m2 ]
Unit area of electrode [m2 ]
7.7 1.54 1.4
1.5 0.105 0.06
0.375 57 170
13.6 28.9 44.8
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Fig. 4. Cyclic voltammograms of the ZnO nanorod-based glucose sensors: (a) ZNR-1, (c) ZNR-2 and (e) ZNR-3. The current density versus glucose concentration: (b) ZNR-1, (d) ZNR-2 and (f) ZNR-3. The slopes indicate the sensitivity of the fabricated sensors.
sensitivities of 16.5, 41.3, 69.8 nA/(M-cm2 ) for ZNR-1, ZNR-2 and ZNR-3, respectively. This proportionality has been often observed in various ZnO nanostructure-based glucose sensors [5,6,19,20], in which the current density tends to saturate for higher glucose concentrations. Fig. 5 shows the sensitivity as a function of the surface area of the ZnO nanorods, and a linear relationship is readily apparent. The larger the surface area is, the higher the glucose sensitivity is.
Fig. 5. Sensitivity versus surface area of ZnO nanorods per unit electrode area.
The strong interaction between glucose oxidase and ZnO nanorods was first confirmed by Liu et al. [8] on the basis of the infrared absorption spectra analysis. They found that glucose oxidase molecules are well anchored on the surface of ZnO nanorods. It is therefore reasonable to conclude that glucose oxidase molecules do not sit just at the tips of ZnO nanorods but cover the entire surface of ZnO nanorods. Although the ratio of the amount of glucose oxidase molecules that can be most fixed to the surface area of ZnO nanorods is not known yet, the larger surface area of ZnO nanorods like the ZNR-3 sample is likely to contain more amount of glucose oxidase molecules, thereby more pronounced oxidation and reduction signal in the above-mentioned electrochemical reaction. The response time is one of important parameters determining the sensing performance of ZnO nanostructure-based glucose sensors. According to the literature survey [5,6,8,19,21–25], despite a small variation in relation to the type of nanostructures, the response time is shorter than 10 s in various ZnO nanostructurebased glucose sensors. In this work, different surface areas were realized by growing the ZnO nanorods with different sizes, consequently leading to correspondingly different spaces between individual nanorods. This must affect the kinetics because different amounts of liquid are likely to be incorporated in the electrochemical reaction. The measurement of the response time will provide some idea to clarify this matter, which needs to be performed in the future.
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Our results clearly demonstrate that not only can hydrothermally grown ZnO nanorods be successfully applied to highperformance glucose sensors but also that the growth conditions need to be carefully optimized to grow ZnO nanorods that are as slim and long as possible in order to attain maximum surface area. 4. Conclusions We have applied hydrothermally grown ZnO nanorods to the electrode system of glucose sensors. In particular, the ZnO nanorods played the role of enzyme immobilizers in the glucose sensors. Three different shapes of ZnO nanorods were vertically grown with the hydrothermal method by using an ALD-grown ZnO seed layer and/or another precursor solution concentration. Interestingly, the glucose sensitivity was strongly dependent on the surface area of the ZnO nanorods. Thus, ZnO nanorods with a larger surface area are more favorable for efficient glucose sensors. The results suggest that the growth conditions of ZnO nanorods needs to be fine-tuned in order to maximize the surface area for ZnO nanorod-based glucose sensors. Acknowledgment This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (No. 2012R1A2A2A01013899). In addition, this work was partially supported by Inha University Research Grant. References [1] N.S. Oliver, C. Toumazou, A.E.G. Cass, D.G. Johnston, Glucose sensors: a review of current and emerging technology, Diabetic Med. 26 (2009) 197–210. [2] E.H. Yoo, S.Y. Lee, Glucose biosensors: an overview of use in clinical practice, Sensors 10 (2010) 4558–4576. [3] X.-Y. Lang, H.-Y. Fu, C. Hou, G.-F. Han, P. Yang, Y.-B. Liu, Q. Jiang, Nanoporous gold supported cobalt oxide microelectrodes as high-performance electrochemical biosensors, Nat. Commun. 4 (2013) 1–8. [4] Y. Fan, Y. Huang, The effective peroxidase-like activity of chitosanfunctionalized CoFe2 O4 nanoparticles for chemiluminescence sensing of hydrogen peroxide and glucose, Analyst 137 (2012) 1225–1231. [5] A. Wei, X.W. Sun, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, W. Huang, Enzymatic glucose biosensor based on ZnO nanorod array grown by hydrothermal decomposition, Appl. Phys. Lett. 89 (2006) 123902. [6] J.X. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, Zinc oxide nanocomb biosensor for glucose detection, Appl. Phys. Lett. 88 (2006) 233106. [7] J. Zang, C.M. Li, X. Cui, J. Wang, X. Sun, H. Dong, C.Q. Sun, Tailoring zinc oxide nanowires for high performance amperometric glucose sensor, Electroanalysis 19 (2007) 1008–1014. [8] X. Liu, Q. Hu, Q. Wu, W. Zhang, Z. Fang, Q. Xie, Aligned ZnO nanorods: a useful film to fabricate amperometric glucose biosensor, Colloids Surf. B 74 (2009) 154–158. [9] Y. Wei, Y. Li, X. Liu, Y. Xian, G. Shi, L. Jin, ZnO nanorods/Au hybrid nanocomposites for glucose biosensor, Biosens. Bioelectron. 26 (2010) 275–278. [10] Z. Zhao, W. Lei, X. Zhang, B. Wang, H. Jiang, ZnO-based amperometric enzyme biosensors, Sensors 10 (2010) 1216–1231. [11] R. Yakimova, L. Selegård, V. Khranovskyy, R. Pearce, A.L. Spetz, K. Uvdal, ZnO materials and surface tailoring for biosensing, Front. Biosci. 4 (2012) 254–278. [12] S. Yamabi, H. Imai, Growth conditions for wurtzite zinc oxide films in aqueous solutions, J. Mater. Chem. 12 (2002) 3773–3778. [13] X. Liu, Z. Jin, S. Bu, J. Zhao, Z. Liu, Growth of ZnO films with controlled morphology by aqueous solution method, J. Am. Ceram. Soc. 89 (2006) 1226–1231.
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Biographies Ji Yeong Kim received her M.S. degree from Inha University, Korea, in 2013. She works for a company that produces chemicals for surface treatments. So-Yeon Jo is a fourth-year undergraduate at Inha University. She has been working on synthesis and characterization of biosensors. Gun-Joo Sun received his B.S. degree from Inha University, Korea, in 2011. He is a doctoral student at Inha University, Korea, and has been working on oxide nanowires gas sensors. Akash Katoch received his B.S. degree from Himachal Pradesh University, India, in 2004. In 2009, he received his M.S. degree from Thapar University, India. From 2010, he has been a doctoral student at Inha University, Korea. He has been working on metal oxide gas sensors synthesized by the sol–gel process. Sun-Woo Choi received his B.S. and M.S. degrees from Inha University, Korea, in 2008 and 2010, respectively. He became a doctoral student at Inha University in 2010. He has been working on metal oxide gas sensors and on the synthesis of metal nanoparticles. Sang Sub Kim joined the School of Materials Science and Engineering, Inha University, in 2007 as a full professor. He received his B.S. degree from the Seoul National University and his M.S. and Ph.D. degrees from the Pohang University of Science and Technology (POSTECH) in Material Science and Engineering in 1987, 1990, and 1994, respectively. He was a visiting researcher at the National Research in Inorganic Materials (currently NIMS), Japan, for two years each in 1995 and in 2000. In 2006, he was a visiting professor at the Department of Chemistry, University of Alberta, Canada. In 2010, he also served as a cooperative professor at the Nagaoka University of Technology, Japan. His research interests include the synthesis and applications of nanomaterials such as nanowires and nanofibers, functional thin films, and surface and interfacial characterizations.