Effects of the surface morphologies of ZnO nanotube arrays on the performance of amperometric glucose sensors

Materials Science in Semiconductor Processing 56 (2016) 137–144

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Effects of the surface morphologies of ZnO nanotube arrays on the performance of amperometric glucose sensors Fan Zhou, Weixuan Jing n, Qiong Wu, Weizhuo Gao, Zhuangde Jiang, Jiafan Shi, Qibing Cui State Key Laboratory for Manufacturing Systems Engineering, Xi’an Jiaotong University, Xi’an 710049, China

art ic l e i nf o

a b s t r a c t

Article history: Received 27 June 2016 Received in revised form 4 August 2016 Accepted 14 August 2016 Available online 24 August 2016

Amperometric glucose sensors have been fabricated with glucose oxidase (GOx) immobilized on ZnO nanotubes (ZnONTs) by physical adsorption. The ZnONTs were formed through selective dissolution of ZnO nanorods (ZnONRs) which were hydrothermally synthesized on Au cylindrical spiral (AuCS) electrodes. With the etching temperature regulated, the surface morphologies of the ZnONT arrays were tailored and their effects on the performance of the corresponding glucose sensors were investigated. It is found that at 65 °C the as-prepared ZnONT arrays show a Gaussian rough surface with larger surface area and better hydrophilicity, and have an effective solid–liquid interface with GOx solution, which further results in desirable GOx immobilization. Therefore favorable performance of the ZnONT-based glucose sensor was obtained, such as the sensitivity 2.63 μA/(mM cm2), linear range 0–6.5 mM, low detection limit 8 μM (S/N ¼3) and Michaelis-Menten constant 5.24 mM, which are superior to that of the ZnONR-based one. Moreover, the ZnONT-based glucose sensor exhibits good long-term stability, and excellent anti-interference ability to uric acid and ascorbic acid. The results can also be used for the performance optimization and the process standardization of other ZnONT-based amperometric biosensors. & 2016 Elsevier Ltd. All rights reserved.

Keywords: Glucose sensor ZnO nanotube Surface morphology Surface area Electrochemical measurement

1. Introduction The enzymatic electrochemical glucose sensors have been widely utilized for fast and accurate determination of glucose in clinical diagnosis [1], biological analysis [2], food processing [3] and environmental monitoring [4]. Efficient enzyme immobilization is a crucial issue for this type of glucose sensors and is determined by the surface morphology of the enzymatic electrode [5]. Therefore ZnO nanostructures including nanowires [6], nanorods [7], nanotubes [8], nanospheres [9], nanoflakes [10] and nanocombs [11] have drawn much more attention for enzyme immobilization owing to the large specific surface area, intrinsic hydrophilicity, good chemical stability, strong electron transfer capability and bio-compatibility [12]. What's more, the isoelectric point (IEP) of ZnO is about 9.5, making it easier to immobilize low IEP proteins or enzymes (e.g. GOx, IEP 4.2) by electrostatic adsorption [13]. Due to the hollow structure and large surface area, ZnONTs are favorable for enhancing the performance of glucose sensors. For instance, Kong immobilized GOx on ZnONTs that were formed through selective dissolution of electrodeposited ZnONRs in n

Corresponding author. E-mail address: [email protected] (W. Jing).

http://dx.doi.org/10.1016/j.mssp.2016.08.009 1369-8001/& 2016 Elsevier Ltd. All rights reserved.

0.125 M NaOH solution at 85 °C for 1.5 h, and demonstrated that the ZnONTs had better amperometric response to glucose than the ZnONRs did [8]. Ali employed ZnONTs to immobilize GOx which were etched from hydrothermally synthesized ZnONRs in 0.2 M KCl solution at 85 °C for 3 h, and thus constructed a potentiometric glucose sensor with a fast response to glucose [14]. As a matter of fact, these ZnONTs were formed under a certain etching parameter, which was not optimized. Moreover, the etching parameters determine the surface morphologies of ZnONT arrays [15], which then necessarily influence the actual surface area, the wettability, and further the degree of enzyme immobilization and the performance of glucose sensor [16]. However, related investigations on this topic have not been conducted to our knowledge. In most literatures, the surface morphology of ZnONT arrays is frequently characterized with diameter, length, wall thickness and density acquired from scanning electron microscopy (SEM). For instance, Yang and Ali obtained the diameter and length of ZnONTs from SEM micrographs, and analyzed their effects on the surface morphologies before and after GOx immobilization [14,17]. Similarly from SEM micrographs Gan determined the diameters, lengths, wall thicknesses and densities of the ZnONTs deposited at different synthesizing parameters, and concluded that the diameters of the ZnONRs influence significantly the formation of corresponding ZnONTs [18]. It is obvious that the employed

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characterization parameters are sparse, and the measurement data insufficient. In addition, the profiles of the ZnONT-based hierarchical structures are not extracted, so it is impossible to quantitatively describe the surface morphologies of the ZnONT arrays. These disadvantages further restrict the process regulation and performance optimization of the ZnONT-based glucose sensors. In this work, amperometric glucose sensors were fabricated with GOx immobilized on ZnONRs and related ZnONTs. The surface morphologies of the ZnONT arrays were tailored with the etching temperature regulated, thus the GOx immobilization was improved and the performance of the corresponding glucose sensors was optimized. Moreover the relationship between the etching temperatures, the surface morphologies and wettability of the ZnONT arrays, the degree of GOx immobilization and the performance of the glucose sensors was established.

contained 90 mM Zn(NO3)2  6H2O and 90 mM HMT. The solution was kept at 90 °C during the growth of ZnONRs and the growth time was 2.5 h. Subsequently the AuCS electrodes with ZnONRs were ultrasonically cleaned in de-ionized water and air-dried. Thus the ZnONRs modified AuCS (ZnONRs/AuCS) electrodes were acquired. 2.4. Selective dissolution of the ZnONRs to form ZnONTs

2. Experimental details

Three ZnONRs/AuCS electrodes were kept in 0.125 M NaOH solution for 1.5 h at 35, 65 and 95 °C respectively, and then rinsed with de-ionized water for several times. Thus ZnONT arrays with different surface morphologies were formed and the ZnONTs modified AuCS (ZnONTs/AuCS) electrodes were obtained. For convenience, the ZnONTs etched at 35, 65 and 95 °C are designated as ZnONTs-35, ZnONTs-65 and ZnONTs-95, whilst the corresponding ZnONTs/AuCS electrodes as ZnONTs-35/AuCS, ZnONTs65/AuCS and ZnONTs-95/AuCS electrodes in the following.

2.1. Materials and apparatus

2.5. Immobilization of GOx on the ZnONRs and ZnONTs

Optical fiber (125 mm in diameter) and Au fiber (30 mm in diameter) were got from Quanzhou Anpon Company and Beijing Doublink Solders Co., Ltd respectively. D-( þ)-glucose (99.7%), glucose oxidase (GOx, EC 1.1.3.4 from Aspergillus niger,  200 U/ mg), uric acid (Z99.0%) and ascorbic acid (499.0%) were purchased from Sigma-Aldrich. Acetone, absolute ethyl alcohol, zinc acetate dihydrate (Zn(CH3COO)2  2H2O, 99.9%), sodium hydrate (NaOH, 98.0%), zinc nitrate hexahydrate (Zn(NO3)2  6H2O, 99.9%) and hexamethylenetetramine (HMT, 99.0%) were bought from Tianjin Kemiou Chemical Reagent Co., Ltd. The phosphate buffer saline (PBS, 10 mM, pH 7.4) solution was prepared with Na2HPO4  12H2O and KH2PO4. GOx solution was prepared with 40 mg GOx dissolved in 1 mL PBS solution and stored in 4 °C when not in use. Glucose solution (0.25 M) was kept at least 24 h after preparation for mutarotation. All the chemicals were analytical reagent and used without further purification. Ultrasonic cleaner (KQ-100DE, Kunshan Ultrasonic Instruments Co. Ltd.), vacuum drier (DZF-6020, Shanghai Yiheng Technical Co. Ltd.), magnetic stirrer (BII-3, Shanghai Sile Automation Science & technology Co. Ltd.), water bath (DK-98-IIA, Tasite), field emission scanning electron microscope (FE-SEM, SU8010, Hitachi), X-ray diffractometer (XRD, Panak), Fourier Transform Infrared (FT-IR) Spectrometer (Nicolet iS10), contact angle meter (OCA20) and electrochemical workstation (CHI660D, Shanghai Chenhua Instrument Co., Ltd) were employed.

Prior to immobilizing GOx, the as-prepared ZnONRs/AuCS and ZnONTs/AuCS electrodes were rinsed with PBS solution to generate hydrophilic surfaces. Afterwards they were immersed in freshly prepared GOx solution overnight and dried in a refrigirator at 4 °C. Finally the electrodes were rinsed with PBS solution to remove the loosely adsorbed GOx and preserved in the refrigirator at 4 °C when not in use. Thus the GOx/ZnONRs/AuCS, GOx/ ZnONTs-35/AuCS, GOx/ZnONTs-65/AuCS and GOx/ZnONTs-95/ AuCS glucose sensors were constructed. Further, the maintenance of GOx activity after being immobilized on the ZnONTs was verified by FT-IR spectroscopy.

2.2. Preparation of AuCS electrode A clean Au fiber was manually spiraled around the core of an optical fiber, and thus an AuCS electrode was produced. The fabrication process of the AuCS electrode is described in detail in our previous work [19]. Four AuCS electrodes were ultrasonically washed with acetone, absolute ethyl alcohol and de-ionized water successively to remove the adsorbed residua from the electrode surfaces, and then dried in the air. 2.3. Hydrothermal synthesis of ZnONRs on the AuCS electrodes ZnO seed solution of 0.5 mM was prepared by dissolving Zn (CH3COO)2  2H2O and NaOH in absolute ethyl alcohol. The clean AuCS electrodes were dipped into the ZnO seed solution for 1 min, and then annealed at 120 °C for 10 min in the vacuum drier. This procedure was repeated twice to form homogeneous ZnO crystal nucleus. Afterwards the AuCS electrodes with the deposited crystal nucleus were put into ZnO growth solution which

2.6. Characterization of the ZnONRs and ZnONTs The microstructures of the ZnONRs and ZnONTs were characterized by FE-SEM. The profiles of the ZnONRs/AuCS and ZnONTs/AuCS electrodes were extracted with the use of FE-SEM images and Image Processing Toolbox of MATLAB. The least square circles were fitted and subtracted from the extracted profiles, thus the surface heights of the ZnONRs/AuCS and ZnONTs/AuCS electrodes were obtained. According to these surface heights, the characteristic parameters including roughness Ra, skewness Sk, kurtosis Ku, and correlation length ξ of the surface morphologies of the ZnONR and ZnONT arrays were determined. The detailed calculation of the characteristic parameters is available in the Supplementary material associated with this paper. The crystal structures of the ZnONRs and ZnONTs were examined with XRD using CuKα radiation generated at energy of 40 kV and current of 40 mA with the diffraction angle ranging from 20° to 80°. The contact angles (CAs) of the ZnONR and ZnONT arrays were measured in ambient condition. 2.7. Electrochemical measurements of the glucose sensors The electrochemical measurements were conducted at room temperature with the conventional three-electrode system, including the fabricated electrode as the working electrode, a Pt wire as the auxiliary electrode and an Ag/AgCl with saturated KCl solution as the reference electrode. The cyclic voltammetric measurements were performed in both PBS solution and 3 mM glucose solution to investigate the electrochemical behavior of the glucose sensors. The potentials are from  0.2 V to þ0.8 V and the scan rate is 50 mV/s. When the background current decayed to a steady state, the amperometric response was carried out with 50 μL glucose solution successively added in 25 mL PBS solution whilst

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being magnetic stirred at 400 rpm. Thus the performance of the glucose sensors was determined. All the potentials applied during the electrochemical measurements were with respect to the reference electrode.

3. Results and discussion 3.1. Characterization of the ZnONRs and ZnONTs Fig. 1(a) indicates that the high-density ZnONRs completely cover and grow perpendicularly to the surface of the AuCS electrode, and their cross-sections are typical hexagonal structures. The average diameter of the ZnONRs is about 395 715 nm and greater than 300 nm, thus the ZnONRs can be etched into nanotubes [18]. The microstructures of the ZnONTs-35, ZnONTs-65 and ZnONTs-95 are shown in Fig. 1(b)-(d). It is apparent that the center parts of the ZnONTs-35 are slightly dissolved and irregular pits of varied depths as well as rough bottoms are formed. The ZnONTs65 exhibits a perfect hexagonal tubular structure with greater etching depth, and both the internal and external surfaces are quite smooth. As for the ZnONTs-95, not only the center parts but also the lateral walls are dissolved owing to the over-etching. The aforementioned selective etching of the ZnONRs is ascribed to the following two reasons. On the one hand, the (001) plane of ZnO has high surface energy and is metastable, which leads to the preferential etching from the (001) plane and the fastest etching rate in the [001] direction. On the other hand, the defects (e.g. oxygen vacancies) in the ZnONRs are not balanced distribution,

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which gives rise to the preferential etching at the center parts of the ZnONRs [20]. Fig. 2 exhibits that all the diffraction peaks are indexed as the hexagonal wurtzite ZnO except for those of the Au fibers (space group: P63mc, PDF Card No.36-1451). This indicates that the ZnO is well crystallized and has high purity. The intensities of (002) diffraction peaks are much stronger than that of the other peaks. It suggests that the ZnO has a preferential growth orientation in the [001] direction, which is in agreement with the FE-SEM characterization in Fig. 1. Moreover, the intensities of (002) diffraction peaks in the ZnONTs are weaker than that of the ZnONRs, and also slightly decrease when the etching temperature increases. This is ascribed to the selective dissolution of the center parts of the ZnONRs. The XRD analysis also indicates that the ZnONRs are preferentially dissolved along the [001] direction in NaOH solution. 3.2. Characterization of GOx immobilized on the ZnONRs and ZnONTs Fig. 3(a) and (b) indicates that more amounts of GOx adsorbs on the top surfaces than the flanks of the ZnONRs and ZnONTs-35. It is ascribed to the air entrapped within the interstices of the adjacent ZnONRs and ZnONTs-35, which greatly prevents GOx solution from penetrating the interstices. Fig. 3(c) and (d) exhibits that GOx completely filled both the insides and adjacent clearances of the ZnONTs-65 and ZnONTs-95, and even accumulated on the surface of the ZnONT-95 arrays. This is due to the tubular structures of the ZnONTs, which remarkably push the entrapped

Fig. 1. The microstructures of the ZnONRs (a), ZnONTs-35 (b), ZnONTs-65 (c) and ZnONTs-95 (d). The ZnONRs were hydrothermally synthesized with 0.5 mM ZnO seed and 90 mM ZnO growth solution at 90 °C for 2.5 h. The ZnONTs were etched at temperatures of 35, 65 and 95 °C in 0.125 M NaOH solution for 90 min.

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Fig. 4. The surface heights of the ZnONRs/AuCS and ZnONTs/AuCS electrodes. Fig. 2. The XRD patterns of the ZnONRs and ZnONTs.

air out from the insides as well as the interstices and thus enlarge the solid–liquid interfaces and eventually improve the GOx immobilization. Apparently the FE-SEM images in Fig. 3 are a little blurry in comparison to those in Fig. 1. It is because GOx is such a nonconductive protein that electric charges could readily accumulate on the surfaces of the samples during the FE-SEM characterization. This also suggests the reliable immobilization of GOx on the ZnONRs and ZnONTs. The FT-IR spectroscopy in Fig. S2 of the Supplementary material shows that the secondary conformation of GOx is highly in

agreement with that of the free GOx, indicating the primary bioactivity maintenance of the immobilized GOx. 3.3. Surface morphologies and wettability of the ZnONR and ZnONT arrays Fig. 4 demonstrates that the surface heights of the ZnONTs/ AuCS electrodes fluctuate dramatically under higher etching temperatures. On the basis of the surface heights, the characteristic parameters are determined and listed in Table 1 to quantitatively describe the surface morphologies of the ZnONR and

Fig. 3. The microstructures of the ZnONRs (a), ZnONTs-35 (b), ZnONTs-65 (c) and ZnONTs-95 (d) with GOx immobilized on. The left upper corner insets are their corresponding cross-sections.

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Table 1 The characteristic parameters of the surface morphologies of the ZnONR and ZnONT arrays. Etching temperature/°C / 35 65 95

ZnONRs ZnONTs-35 ZnONTs-65 ZnONTs-95

Ra/nm

ξ/μm

Sk

Ku

28.8 70.6 36.7 70.6 39.7 73.9 51.2 7 1.3

1.02 70.02 1.077 0.07 1.02 70.16 0.617 0.11

0.31 7 0.01 0.137 0.28  0.01 7 0.09  0.03 7 0.13

2.687 0.02 2.667 0.37 2.767 0.14 2.78 70.19

Fig. 5. The CAs of the ZnONR, ZnONT-35, ZnONT-65 and ZnONT-95 arrays.

ZnONT arrays. It demonstrates that Ra becomes larger and ξ smaller when the etching temperature increases. This indicates the rougher surfaces and thus the larger surface areas of the ZnONTs/AuCS electrodes. As for the ZnONTs-65 and ZnONTs-95 arrays, their Sk values go to 0 whilst the Ku values are close to 3, suggesting that Gaussian rough surfaces (Sk¼ 0, Ku¼ 3) are formed. Due to the good symmetry of surface height distribution, the Gaussian rough surface possesses effective pits of desirable sizes, which benefits efficient GOx immobilization. Fig. 5 shows the CAs of the ZnONR, ZnONT-35, ZnONT-65 and ZnONT-95 arrays are 48.5°, 38.2°, 25.2° and 14.1°. According to the Wenzel and Cassie-Baxter wetting models, the wettability of a hydrophilic surface is improved (namely the CA becomes smaller) when Ra enlarges [21]. The ZnO is intrinsically hydrophilic [22], thus the CAs decrease and the hydrophilicity of the fabricated ZnONT arrays enhanced with the etching temperature increased.

This provides effective solid–liquid interfaces for GOx solution sufficiently penetrating into the bottom of the ZnONTs as well as the interstices of the adjacent ZnONTs. To sum up, the favorable surface morphologies of the ZnONT arrays result in larger surface areas, better hydrophilicity and effective solid–liquid interfaces between the electrode surface and GOx solution. Therefore the amount of GOx immobilized on the ZnONTs-65 and ZnONTs-95 is the most. 3.4. Electrochemical measurements of the glucose sensors 3.4.1. Cyclic voltammetry The cyclic voltammograms in Fig. 6(a) indicate that the redox current increases apparently in 3 mM glucose solution compared with the case in PBS solution. This is attributed to the oxidation of glucose by GOx catalysis, and the direct electron transfer between

Fig. 6. (a) Cyclic voltammograms in PBS solution and 3 mM glucose solution; (b) Cyclic voltammograms of the four glucose sensors in 3 mM glucose solution. The potentials range from  0.2 V to þ 0.8 V, and the scan rate is 50 mV/s.

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the active sites of the immobilized GOx and the electrode surface. It suggests that GOx is well affined to the ZnONTs and still retains its intrinsic bioactivity. Fig. 6(b) shows that the redox currents and the peak areas (PAs) of the GOx/ZnONTs/AuCS glucose sensors are larger than that of the GOx/ZnONRs/AuCS one. In addition, the redox currents and the PAs of the GOx/ZnONTs/AuCS glucose sensors first increase when the etching temperature rises, then reach to the maximum value at 65 °C, and finally decrease at 95 °C because of GOx accumulation on the surface of the ZnONTs-95 arrays. It is ascribed to the different surface morphologies of the ZnONR and ZnONT arrays, which give rise to different contacting surface areas and further varied GOx immobilization. 3.4.2. Amperometric response Prior to conducting the amperometric response, the temperature, pH value and applied voltage were determined to optimize the test conditions. According to literatures [6,8], the catalytic activity of GOx increases with the test temperature varied from 20 °C to 50 °C, and then decreases after 50 °C due to the natural thermal degradation of the GOx. Nevertheless, the electrochemical experiment was carried out at 25 °C in order to prevent the analytical liquid from evaporation and also for easy operation. The glucose sensors showed optimal amperometric response at around pH 7.4 and the physiological pH value of human blood is about 7.4, thus PBS solution was adjusted to pH 7.4 [23]. The response current of the glucose sensors increase when the voltage is below 0.8 V and then almost saturated, hence the applied voltage to the working electrode was set as 0.8 V [8]. The amperometric responses in Fig. 7(a) suggest that the glucose sensors have fast and sensitive response to successive addition of glucose. From the calibration curves of the glucose concentrations versus the response currents in Fig. 7(b), the sensitivities of the glucose sensors are determined to be 0.60, 1.49, 2.63 and 1.25 μA/(mM cm2), higher than the graphite nanoparticles modified glassy carbon electrode (7.29  10  2 nA/(mM cm2)) [24] and NiO nanoparticles modified glassy carbon electrode (0.45 μA/ (mM cm2)) [25]. Based on the signal-to-noise of 3 (S/N ¼ 3), the low detection limits are estimated at 44, 15, 8 and 21 μM, lower than the multi-layered graphene modified electrode (154 μM) [26] and Au/ZnONRs modified ITO electrode (500 μM) [27]. The linear ranges are acquired to be 0.04–5.0, 0.01–7.0, 0–6.5 and 0.02– 6.5 mM, wider than the ZnONRs/ferrocenyl-alkanethiol modified Au electrode (0.05–1.0 mM) [28] and Ag nanowires modified glassy carbon electrode (0.01–0.8 mM) [29]. The better performance of our fabricated glucose sensors is mainly ascribed to two aspects, that is, the direct synthesis of ZnONRs and related ZnONTs on the AuCS electrodes as well as the favorable surface

morphologies of the ZnONR and ZnONT arrays. The former provides direct electron path from ZnONRs or ZnONTs to the AuCS electrodes, enabling the fast and sensitive response to glucose [5]. The latter ensures large surface areas of the working electrodes and effective solid–liquid interface between the ZnO nanostructures and GOx solution, more GOx immobilization and thus better performance of the glucose sensors. The Michaelis-Menten constant gives an indication of the enzyme substrate kinetics of an enzymatic sensor and is calculated from the Lineweaver-Burk equation: 1/Iss = (Kmapp/Imax )(1/C ) + 1/Imax , where Iss is the steady-state current, C the substrate concentration, Imax the maximum response current measured under the saturated app substrate solution, and Km the Michaelis-Menten constant. The Michaelis-Menten constants of the fabricated glucose sensors are obtained to be 2.45, 7.54, 5.24 and 5.57 mM, smaller than that of the GOx immobilized on polypyrrole films (37.6 mM) [30] and ZnO:Co nanoclusters (21 mM) [31]. The lower Michaelis-Menten constants reveal that the immobilized GOx exhibits better enzymatic activity and higher affinity to glucose due to the excellent biocompatibility of the ZnONRs and ZnONTs. Compared with the GOx/ZnONRs/AuCS glucose sensor, the performance of the GOx/ZnONTs-35/AuCS, GOx/ZnONTs-65/AuCS and GOx/ZnONTs-95/AuCS ones is better. It is attributed to the favorable surface morphologies and the large surface areas of the ZnONT arrays, which result in better hydrophilicity of the ZnONT arrays, larger solid–liquid interface between the ZnONTs and GOx solution, and more GOx immobilization. Nevertheless, it is the GOx/ZnONTs-65/AuCS glucose sensor instead of the GOx/ZnONTs95/AuCS one that exhibits the optimal performance because GOx accumulates on the ZnONTs-95 (referring to Fig. 2(d)) and hinders the generated electrons transferring from the redox centers of GOx to the electrode surface. 3.4.3. The anti-interference, long-term stability and real sample analysis Electroactive species such as uric acid (UA) and ascorbic acid (AA) are always present in human serum, and thus interfere with the accurate determination of glucose. Therefore amperometric response of the GOx/ZnONTs-65/AuCS glucose sensor was performed with successive addition of 0.5 mM glucose, 0.5 mM UA, 0.1 mM AA and 0.5 mM glucose into the PBS solution. Fig. 8 (a) shows that the response currents increase remarkably when glucose is added into PBS solution, but have little change when UA and AA are injected in the 0.5 mM glucose solution. More importantly, the relative fluctuation amounts of the response currents for UA and AA are estimated to be 1.5% and 1.9%. This indicates that the glucose sensor has excellent anti-interference

Fig. 7. (a) The amperometric responses of the glucose sensors; (b) The calibration curves of the glucose concentrations versus the response currents.

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Fig. 8. (a) Amperometric responses of the GOx/ZnONTs-65/AuCS glucose sensor with successive addition of 0.5 mM glucose, 0.5 mM UA, 0.1 mM AA and 0.5 mM glucose into PBS solution. (b) The long-term stability of the GOx/ZnONTs-65/AuCS glucose sensor.

ability due to the outstanding specificity of GOx to glucose. Fig. 8(b) exhibits the long-term stability of the GOx/ZnONTs65/AuCS glucose sensor, which is evaluated by measuring the response currents in 1 mM glucose solution intermittently after 5, 10, 15 and 20 days. Apparently the response currents of the glucose sensor still remain 97.3% and 90.7% of its initial value after being kept at 4 °C for 5 and 10 days, and decay to 82.5% and 80.8% after 15 and 20 days. The leakage of the weakly adsorbed GOx accounts for these changes. It concludes that the glucose sensor shows good long-term stability thanks to the friendly micro-environment that the ZnONTs provide for GOx to retain its bioactivity. The real sample analysis was performed with the usage of the GOx/ZnONTs-65/AuCS glucose sensors. The fasting blood sugar levels of two healthy persons were determined to be 5.38 mM and 5.61 mM, which are within the normal range of 3.89–6.1 mM. This implies that the glucose sensor has the ability to be used in real human serum analysis.

4. Conclusions In summary, two kinds of amperometric glucose sensors have been constructed with GOx immobilized on ZnONRs and corresponding ZnONTs. The surface morphologies of the ZnONT arrays were finely tailored with the etching temperature regulated, and then quantitatively characterized with the characteristic parameters Ra, Sk, Ku and ξ. The relationship between the etching temperatures, the surface morphologies and wettability of the ZnONT arrays, the degree of GOx immobilization and the performance of the glucose sensors was established. It is found that the ZnONTs-65 arrays have a Gaussian rough surface with Sk and Ku approximate to 0 and 3, Ra 39.77 3.9 nm and ξ 1.02 7 0.16 mm, larger surface area, better wettability and effective solid–liquid interface, which gives rise to desirable GOx immobilization. Thus the optimal performance of the related ZnONT-based glucose sensor was obtained, such as the sensitivity 2.63 μA/(mM cm2), the linear range 0–6.5 mM, the low detection limit 8 μM (S/N¼3) and the Michaelis-Menten constant 5.24 mM, which are better than that of the ZnONR-based one. The ZnONT-based glucose sensor also shows good long-term stability, and excellent antiinterference ability to uric acid and ascorbic acid. The results can also be used for the process regulation and performance optimization of other ZnONT-based biosensors for urea, uric acid, cholesterol, hydrogen peroxide and phenol.

Acknowledgments We would like to acknowledge the financial support from the NSFC Major Research Plan on Nanomanufacturing (Nos. 51075324 and 91323303), the Key Science and Technology Program of Shaanxi Province (No. 2015GY117), the National Key Scientific Instrument and Equipment Development Projects of China (No. 2012YQ03026101), the National Key Basic Research Program of China (No. 2015CB057400), and the 111 Project (No. B12016).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.mssp.2016.08.009.

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