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Direct electrochemistry of glucose oxidase immobilized on Au nanoparticles-functionalized 3D hierarchically ZnO nanostructures and its application to bioelectrochemical glucose sensor
Accepted Manuscript Title: Direct electrochemistry of glucose oxidase immobilized on Au nanoparticles-functionalized 3D hierarchically ZnO nanostructures and its application to bioelectrochemical glucose sensor Author: Linxia Fang Bing Liu Lulu Liu Yuehua Li Kejing Huang Qiuyu Zhang PII: DOI: Reference:
S0925-4005(15)30210-0 http://dx.doi.org/doi:10.1016/j.snb.2015.08.032 SNB 18887
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
21-4-2015 14-7-2015 8-8-2015
Please cite this article as: L. Fang, B. Liu, L. Liu, Y. Li, K. Huang, Q. Zhang, Direct electrochemistry of glucose oxidase immobilized on Au nanoparticlesfunctionalized 3D hierarchically ZnO nanostructures and its application to bioelectrochemical glucose sensor, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.08.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Direct electrochemistry of glucose oxidase immobilized on
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Au nanoparticles-functionalized 3D hierarchically ZnO
3
nanostructures and its application to bioelectrochemical
4
glucose sensor
5
Linxia Fanga,* , Bing Liua, Lulu Liua, Yuehua Lia, Kejing Huanga, Qiuyu Zhangb,∗
b
8 9
us
Xinyang ,China
Department of Applied Chemistry, School of Science, Northwestern Polytechnical University,
Xi’an, China
an
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College of Chemistry and Chemical Engineering, Xinyang Normal University,
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Abstract: Three-dimensional (3D) hierarchically ZnO nanoarchitecture with
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controlled morphology and dimensions was synthesized by trisodium citrate-assisted
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solution phase method and functionalized by Au nanoparticles (AuNPs) via in-situ
17
reduction of HAuCl4. The as-prepared AuNPs-functionalized 3D hierarchically ZnO
18
nanostructure (Au-ZnO nanocomposite) were used as a novel immobilization matrix
19
for glucose oxidase (GOD) and exhibited excellent direct electron transfer properties
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for GOD. The AuNPs-functionalized 3D hierarchically ZnO nanostructure (Au-ZnO
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nanocomposite) favored the immobilization of the glucose oxidase (GOD) and the
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penetration of water-soluble glucose molecules, which helped efficiently catalyze the
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Corresponding author E-mail address: [email protected]; [email protected]
*
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oxidation of glucose and facile direct electron transfer for GOD. The as-fabricated
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glucose biosensor exhibited satisfactory analytical performance with a low detection
25
limit (0.02 mM) and an acceptable linear range from 1 to 20 mM. These results
26
indicated that AuNPs-functionalized 3D hierarchically ZnO nanomaterial is a
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promising candidate material for high-performance glucose biosensors.
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Keywords: Au nanoparticle; 3D hierarchically ZnO nanostructure; glucose
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biosensor; direct electrochemistry
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1. Introduction
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Diabetes is a major public health problem in the world wide which classed as a
32
metabolism disorder and is one of the leading causes of death and disability [1-3].
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Accurate determination and regular monitoring of blood glucose concentration is very
34
important in the diagnosis and treatment of diabetes or metabolic disorders. Since the
35
initial development of glucose enzyme electrodes by Clark and Lyons in 1962,
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tremendous effort has been directed toward research into glucose enzyme biosensors
37
because of their great promise in a vast range of application fields such as medical
38
diagnosis, diabetes management, bioprocess monitoring, beverage industry, and
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environmental monitoring [4-6]. The electrochemical glucose biosensor based on the
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direct electrochemistry between an electrode and the immobilized glucose oxidase is
41
especially promising due to its high selectivity, sensitive glucose detection, and
42
relatively low-cost fabrication [7-9]. However, the lack of the direct electrical
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communication between redox proteins and electrode supports has been a key point
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that limits the development of this kind of biosensors. Thus, in order to promote the
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direct electron transfer between active site of GOD and electrode, many materials,
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including polymers, carbon nanotube, graphene, metal or metal oxide nanoparticles
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and their composites have been used to modify the electrode to immobilize GOD for
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improving the DET of GOD on the surface of electrode [10-13].
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The applications of biocompatible nanomaterials for enzymatic glucose sensors
50
were explored as they help the enzymes to retain its activity and to augment direct
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electron transfer between the active site of the enzyme and the electrode [14]. The
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advanced properties of nanostructures ZnO like non-toxicity, biocompatibility,
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chemical stability, good permeation and electrochemical activities have triggered a
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vast interest among the researchers to research and develop the applications especially
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in biomedical and sensor [15, 16]. Apart from these, ZnO has a high isoelectric point
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of about 9.5, which provide a positively charged substrate for immobilization of
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enzyme with low isoelectric point and serve as a potential biosensor electrode [17, 18].
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It has shown great promise in applications as electrode materials for immobilizing
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GOD and improving DET of GOD on electrode. It has been reported that gold
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nanoparticles (AuNPs) can greatly enhance the DET between some redox proteins
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and electrode due to their quantum characteristics and large specific surface area of
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small granule diameter as well as their ability to quickly transfer electrons at the
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surfaces of colloidal particles [19-21]. Recently, 3D nanostructures with complex
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morphology and high dimensionality have received great research interest due to the
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fact that their advanced geometric structure and atom arrangement on the specific
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facets of these nanostructures, which can offer novel properties [22-25]. Potentially,
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functionalizing 3D hierarchically ZnO nanomaterials with AuNPs, which combines
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the large surface area of 3D support and the unique property of AuNPs, will generate
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synergy effect and thus enhance their performance in applications of biosensor. In our
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previous works [26, 27], we fabricated biosensors based Au-ZnO nanocomposites to
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detect DNA arrays and dopamine respectively, and the good results were obtained.
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In the present study, 3D hierarchically ZnO nanoarchitecture was synthesized
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according to the reference [26]. High-density AuNPs were homogeneously loaded
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onto the porous matrix of ZnO to obtain AuNPs-functionalized 3D hierarchically ZnO
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nanomaterial which was used to modify glass carbon electrode (GCE). GOD was
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immobilized on the modified GCE and the DET between GOD and the modified GCE
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was studied. The electrochemical catalytic activity of the fabricated electrode in
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response to glucose was also investigated. Due to the synergy effect of 3D
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hierarchically ZnO nanoarchitecture and AuNPs, the fabricated electrode showed
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excellent direct electrochemical behavior and the DET between the GOD and the
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modified electrode was easily achieved, indicating that AuNPs-functionalized 3D
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hierarchically ZnO nanomaterial could be a good candidate material for immobilizing
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biomolecules and fabricating the third-generation biosensor.
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2. Experimental
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2.1. Apparatus
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The
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electrochemical
measurements
were
performed
on
a
CHI660D
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Electrochemical Workstation (Shanghai CH Instruments, China). All experiments
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were carried out by a conventional three-electrode system with a platinum wire 4
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electrode as the auxiliary electrode, a saturated calomel electrode (SCE) as the
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reference electrode and a modified glassy carbon electrode (GCE) (3.0 mm in
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diameter) as the working electrode. All of the potentials in this article were with
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respect to SCE. The pH measurements were made with a pH meter Leici Devices
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Factory of Shanghai, China. Electrochemical impedance spectroscopy (EIS)
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measurements were performed in 0.1 M KCl solution containing 5.0 mM
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K3Fe(CN)6/K4Fe(CN)6 (1:1) with a frequency ranging from 100 kHz to 0.1 Hz. The
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morphologies of the nanocomposite were recorded on a JEM 2100 transmission
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electron microscope (TEM) and a Hitachi S-4800 scanning electron microscope
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(SEM).
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D/Maxr-A
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X-ray
diffractometer
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X-ray powder diffraction (XRD) pattern was operated on a Japan Rigaku equipped
high-intensity Cu Ka radiation ( λ = 1.54178 Å).
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2.2 Reagents
graphite
monochromatized
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Zinc acetate dihydrate, hexamine (HMTA), absolute ethanol and sodium citrate
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were obtained from China National Pharmaceutical Industry Corporation Ltd. GOD
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and glucose was obtained from Sigma (Saint Louis, MO, USA). Phosphate-buffered
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saline (PBS, 0.01 M) at various pH values was prepared by mixing a stock standard
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solution of KH2PO4 and K2HPO4, which was used as the measuring buffer, and then
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adjusting the pH with 0.1 M KOH and H3PO4. All chemicals were of analytical grade
108
and used without further purification. Ultrapure water (18.2 MX) was obtained from a
109
Milli-Q water purification system and used throughout.
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2.3 Preparation of 3D hierarchically ZnO nanoarchitecture 5
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The preparation and the growth mechanism of 3D hierarchically ZnO
112
nanoarchitecture have been demonstrated in our previous work [26]. In a typical
113
synthesis, an equimolar ratio of zinc acetate dihydrate (25 mM）and HMTA (25 mM)
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was dissolved into 50 mL of deionized water with subsequent addition of trisodium
115
citrate (5 mM), followed by stirring at room temperature for 20 min. The final mixture
116
was transferred to a 100 mL Teflon-stainless beaker for hydrolysis reaction at 90 °C in
117
an oven for 6 h. After completion of the reaction, cooling to room temperature
118
naturally, the resulting white precipitate was collected by centrifugation and purified
119
by washing with deionized water and absolute ethanol several times and dried at 60
120
°C for 24 h.
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2.4 Preparation of AuNPs-functionalized 3D hierarchically ZnO nanomaterial
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The prepared 3D hierarchically ZnO nanomaterial were dispersed into 10mL
123
distilled water by ultrasonication. Then, 140 μL of freshly prepared HAuCl4 aqueous
124
solution (30 mM) was added into the dispersion by stirring. Subsequently, 0.25 mL of
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sodium borohydride (NaBH4) aqueous solution (0.2 M) was added drop by drop into
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the mixture solution with vigorous stirring at room temperature for 30 min. Finally,
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the products were collected by centrifugation and were washed with distilled water
128
and absolute ethanol several times to produce AuNPs-functionalized 3D hierarchically
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ZnO nanomaterial (Au-ZnO nanocomposite).
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2.5 Fabrication of AuNPs-functionalized 3D hierarchically ZnO nanomaterial
131
modified electrode
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Prior to electrode modification the GCE was polished with 0.05 µm alumina
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slurry and Buehler polishing cloth. It was then washed with deionized water and
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ultrasonicated for 3 min each in water and ethanol to remove any adsorbed alumina
135
particles or dirt from the electrode surface and finally dried in nitrogen airflow. 5 μL
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of Au-ZnO nanocomposite dispersion (2 mg mL-1 in PBS (pH7)) was drop casted onto
137
the pre-cleaned GCE and dried at room temperature. Then, 8 μL of GOD (10 mg mL-1
138
in PBS (pH7)) was drop casted onto the Au-ZnO modified GCE and dried at ambient
139
temperature. The GOD/Au-ZnO modified surface was smoothly washed with water to
140
remove loosely adsorbed enzyme. For comparison, Au-ZnO/GCE, ZnO/GCE,
141
GOD/ZnO/GCE and GOD/GCE were prepared by adopting the similar procedures.
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3. Results and discussions
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3.1 Characterization of the Au-ZnO nanocomposites
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The morphology of the as-prepared 3D hierarchically ZnO and the Au-ZnO
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nanocomposites were observed by SEM. Fig. 1a and b show the low magnification
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and high-magnification SEM images of the 3D hierarchically ZnO, respectively, from
147
which it can be clearly observed that the product has a spherical lamellar structure
148
with a diameter of 2-3µm, that is to say that the spheres are assembled by a large
149
amount of interconnected nanosheets. Plenty of voids and interspaces are present
150
among these nanosheets, which may increase the surface area of the materials and are
151
potentially useful for applications such as catalyst and sensor materials. Fig. 1c and d
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show the typical low magnification and high-magnification SEM images of the
153
Au-functionalized 3D hierarchically ZnO, respectively. An obvious change is that the
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Fig.1 (a) low magnification and (b) high-magnification SEM image of 3D
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hierarchically ZnO; (c) low magnification and (d) high-magnification SEM image of
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Au-functionalized 3D hierarchically ZnO
159
surface of microspheres become illegible and the porosity degrade, which is due to a
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high coverage of AuNPs deposited on the surface of ZnO nanosheet.
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The distribution of AuNPs on the 3D hierarchically ZnO support was further
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investigated by TEM. Typical TEM images of AuNPs-functionalized 3D
163
hierarchically ZnO with different magnification are displayed in Fig. 2(a-c). From the
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edges of ZnO microsphere, it can be seen clearly that a high density of AuNPs with
165
small sizes were uniformly anchored on the surface of ZnO nanosheets (Fig. 2a). In
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the higher resolution TEM picture of Fig. 2b, the AuNPs can be easily distinguished 8
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due to their darker contrast against the ZnO nanosheets. HRTEM image reveals
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clearly the dividing line of the two lattice stripes (Fig. 2c), and the lattice stripes
169
distance between adjacent lattice planes is 0.24 nm, corresponding to the interplanar
170
distance of the (111) plane of face centered cubic (fcc) Au, which means that AuNPs
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were espitaxially grown on the surface of ZnO nanosheets. The EDS of Au-ZnO
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hybrid nanostructure is shown in Fig. 2d which future confirms the presence of Au in
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the Au-ZnO nanocomposite. The phase compositions of the as-synthesized
174
productions were also determined by XRD. Fig. 3 shows the XRD patterns of ZnO
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(curve a) and Au-ZnO nanocomposite (curve b). The major diffraction peaks shown
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in curve a can be indexed to a phase from crystalline ZnO based on the data from the
177
JCPDS file (21-1486). The three additional peaks in curve b locating at 38.26°, 44.48°,
178
64.66°, and 77.64°are assigned to (111), (200), (220) and (311) planes reflection of
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AuNPs (JCPDS Card No. 65-2870), which proves the formation of crystalline Au on
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the ZnO nanosheets.
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hierarchically ZnO
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Fig. 2 TEM images (a-c) and (d) EDS spectra of AuNP-functionalized 3D
183
3.2 Electrochemical impedance spectroscopy of the modified electrodes
186
Electrochemical impedance spectroscopy (EIS) is a powerful tool to investigate
187
the impedance changes of electrode interface during the modification process. The
188
typical Nyquist impedance spectrum (presented in the form of the Nyquist plot)
189
includes a semicircle portion at high
190
electron-transfer-limited process and a linear part at low frequency range representing
191
the diffusion-limited process. The semicircle diameter observed at higher frequency
192
range is equaled to the electron transfer resistance (Ret).
193
electron-transfer kinetics of the redox probe at the electrode interface. Fig.4 presents
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the Nyquist plot of EIS for bare GCE (a), Au-ZnO/GCE (b), ZnO/GCE(c) and
195
GOD/Au-ZnO/GCE (d). As shown, the Ret of those modified electrodes is in the order
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of GOD/Au-ZnO/GCE > ZnO/GCE > Au-ZnO/GCE > GCE. The Ret of the ZnO/GCE
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is much higher than that of the bare GCE because of weak conductivity of ZnO,
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suggesting that a layer of ZnO film has formed on the surface of GCE and hindered
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frequencies
corresponding
to
the
This resistance controls the
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Fig. 3 (a) XRD pattern of 3D hierarchically ZnO nanostructure;
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(b) XRD pattern of AuNP-functionalized 3D hierarchically ZnO nanostructure
an
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the charge transfer from the redox probe of [Fe(CN)6]3-/4- to the GCE surface.
203
nanoparticles have excellent conductivity and this hetero-structure can extensively
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Obviously, the Au-ZnO/GCE shows comparatively low Ret value indicating that Au
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improves the electron transfer properties of the electrodes to a great extent. The
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GOD/Au-ZnO/GCE shows the largest Ret due to the blocking effects of high insulate
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GOD on the charge transfer, which also indicates that GOD has been successfully
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immobilized.
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3.3 Direct electrochemistry of GOD at Au-ZnO nanocomposites modified GCE
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The direct electrochemistry of GOD at Au-ZnO/GCE has been studied by cyclic
211
voltammetry. Fig. 5 shows the CVs acquired at ZnO/GCE, GOD/ZnO/GCE,
212
Au-ZnO/GCE and GOD/Au-ZnO/GCE in N2-saturated PBS (0.1M, pH 7.0) at a scan
213
rate of 100 mV s-1. No peaks were observed at the CVs of ZnO/GCE (curve a) and
214
Au-ZnO/ GCE (curve b), but a couple of well-defined redox peaks were obtained at 11
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GOD/ZnO/GCE (curve c) and GOD/Au-ZnO/GCE (d), which indicates the redox
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Fig. 4 The electrochemical impedance spectroscopy (EIS) of bare GCE (a),
218
Au-ZnO (b), ZnO (c), GOD/Au-ZnO (d) in 0.1 M KCl aqueous solution containing
219
1.0 mM [Fe(CN)6]3-/4-. The frequency range is from 0.1 to 104 Hz at the formal
220
potential of 0.2 V.
221
peaks of the GOD/Au-ZnO/GCE and the GOD/ZnO/GCE should be ascribed only to
222
GOD. The background current of the Au-ZnO/GCE is higher than that of ZnO/GCE.
223
This is ascribed to the improvement of electrical conductivity through
224
functionalization of ZnO by AuNPs. The peak currents of GOD/Au-ZnO/GCE are
225
higher than that of GOD/ZnO/GCE. This may be attributed to the good conductivity
226
and biocompatibility of Au-ZnO nanocomposite. The anodic peak potentials (Epa) and
227
cathodic peak potentials (Epc) of GOD/Au-ZnO/GCE were observed at -0.450 V and
228
-0.403 V, respectively. The peak-to-peak separation (△Ep) is about 47 mV, revealing a
229
fast electron transfer process. The formal potential (EcƟ´) calculated from the average
230
of cathodic and anodic peak potential is -0.426 V, which is close to the electrode
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potential of GOD in previous reports [28,29], indicating the direct electron transfer
232
from the redox site of the enzyme to the electrode. The outstanding direct electron
233
transfer ability of GOD at the Au-ZnO modified electrode surface is attributed to the
234
large surface area, favorable orientation of GOD, good biocompatibility, and high
235
electrical conductivity of Au-ZnO.
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Fig. 5 CVs of ZnO/GCE (a), Au-ZnO/GCE (b), GOD/ZnO/GCE (c),
237
GOD/Au-ZnO/GCE (d) in N2-saturated PBS solution (0.1 M, PH 7.0).
238
Scan rate: 0.1V s-1.
239 240 241
The isoelectric point (IEP) of ZnO is 9.5, whereas that of GOD is 4.2. At pH7.0, ZnO
242
surface is positively charged, while GOD surface is negatively charged. Therefore,
243
electrostatic interactions between positively charged ZnO nanostructures with
244
negatively charged surface of GOD lead to efficient immobilization of GOD.
245
However, GOD shows decreased direct electrochemistry at GOD-ZnO/ GCE due to 13
Page 13 of 26
the poor conductivity of ZnO film. The enhanced direct electron transfer of GOD at
247
the Au-ZnO support is due to the excellent electrical conductivity of AuNPs.
248
Therefore, we chose AuNPs as the functional material for ZnO to prepare Au-ZnO
249
composite for immobilizing GOD. The as-immobilized GOD exhibited promising
250
direct electrochemistry.
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3.4 Effect of scan rate
cr
251
The influence of the scan rates on the cyclic voltammetric performance of the
253
GOD/Au-ZnO/GCE was shown in Fig. 6. The scan rates from inner to outer curves
254
are 30, 50, 100, 120, 130, 160, 180, 200 and 300 mV s-1. As shown, the redox peak
255
currents linearly increased with the scan rates ranging from 30 to 300 mV s-1 (Fig. 6
256
inset). These characteristics indicate that the redox reaction of GOD on the surface of
257
Au-ZnO/GCE is a quasi-reversible surface-controlled electrochemical process. The
258
linear regression equations for the redox process are written as Ipa= 0.1084ｖ+10.75;
259
R2=0.9938 and Ipc= - 0.088ｖ+ 3.99; R2= 0.9956, where ｖ is the scan rate.
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3.5
260 261
Electrocatalytic
activity
of
GOD/Au-ZnO/GCE
toward
glucose
determination
262
Cyclic voltammetry was employed to measure the electrocatalytic activity of
263
glucose biosensor for Au-ZnO nanocomposite. This method is a fast and convenient
264
tool for characterizing glucose biosensor. Fig. 7 shows the cyclic voltammograms
265
obtained at the GOD/Au-ZnO modified electrode in the presence of different
266
concentrations of glucose in oxygen saturated PBS at the scan rate of 0.1V s−1.
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Fig. 6 CVs of GOD/Au-ZnO/GCE in N2 saturated PBS (0.1 M, PH 7.0) at various
269
scan rates: inner to outer are 30-300 mV s-1. Inset to (B) shows the linear dependence
270
of peak currents with scan rate.
271
The direct electrontransfer of GOD is a two-electron and two-proton coupled reaction.
272
The cathodic peak current (Ipc) is attributed to the reduction of GOD (FAD), while the
273
anodic peak current (Ipa) is attributed to the oxidation of GOD (FADH2). Upon
274
addition of glucose, the cathodic peak current decreased linearly, which could be
275
attributed to the enzyme-catalyzed glucose oxidation. As illustration by Liang et al.
276
[30], the oxidized form of GOD, GOD (FAD), is reduced by glucose, which restrains
277
the electrochemical reduction reaction of GOD (FAD) and decreases the reduction
278
current. The glucose biosensor is just built on the base of this characteristic. The
279
calibration curve (Fig. 7 inset) corresponding to cyclic voltammetry response is linear
280
against the glucose concentration ranging from 1 to 20 mM with a correlation
281
coefficient of 0.9962 and a sensitivity of 1.409 μA mM-1. The detection limit is
282
estimated to be 0.02 mM at a signal-to-noise ratio of 3. The linear range of the
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GOD/Au-ZnO/GCE is wider compared to other reported glucose biosensors based the
284
direct electron transfer for GOD [31-37].
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285
Fig. 7 CVs of GOD/Au-ZnO/GCE in the O2-saturated PBS (0.1 M, PH 7.0) solution
287
in various concentrations of glucose; Scan rate: 0.1V s-1. The inset shows the
288
calibration curve of the linear dependence of cathodic peak current on the glucose
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concentration.
289 290
The comparison of the analytical performance of the developed electrode with other
291
electrodes reported previously was given in Table1. It can be seen that the
292
GOD/Au-ZnO/GCE electrode exhibits a wider linear range, a lower detection limit
293
and a higher sensitivity. The as-fabricated glucose biosensor is suitable for detecting
294
human blood glucose concentration for the diagnosis of diabetes mellitus, since the
295
Table 1 Comparison of analytical performances between the proposed sensor and
296
other sensor for GOD direct electrochemistry Electrode
Linear range
Detection limit
Sensitivity
Ref
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(mM)
(μM)
(μA mM-1)
1-8
10
0.835
[31]
GOD/Au-OOPPye/GCE
1-8
70
0.851
[32]
GOD/Nff-GRg/GEh
2-14
40
1.547
[33]
GOD/ Agi- RGO/ GCE
0.5-12.5
160
0.27
[34]
GOD/GR-CNTj-ZnOk /GCE
0.01-6.5
4.5
GOD/MGFl/ GCE
1.0 - 12
250
0.2027
[36]
GOD/ GR-CdSm /GCE
2-16
700
0.124
[37]
GOD/Au-ZnO /GCE
1-20
20
1.409
This work
298
Au, gold nanoparticles.
299
c
RGO, reduced graphene oxide.
300
d
GCE, glassy carbon electrode.
301
e
cr
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OOPPy, overoxidized polypyrrole. f
302
M
GOD, glucose oxidase.
b
[35]
d
a
0.3865
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297
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GODa/Aub-RGOc/GCEd
Nf, nafion.
303
g
graphene.
304
h
GE, gold electrode.
305
i
Ag, silver nanoparticles.
306
j
CNT, carbon nanotube.
307
k
ZnO, zinc oxide.
308
l
MGF, mesocellular graphene foam.
309
m
310
normal range of blood glucose concentration is 4.4-6.6 mM. Moreover, the proposed
311
glucose biosensor was fabricated by a facile and low cost procedure without
312
expensive reagents and complicated experiments.
CdS, cadmium sulfide.
17
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313
3.6 Selectivity, stability, reproducibility and repeatability of the biosensor The selectivity of GOD/Au-ZnO film was evaluated in the presence of common
315
interfering species such as dopamine (DA), uric acid (UA), and ascorbic acid (AA) in
316
PBS. The influence of those interferents was examined by cyclic voltammograms of
317
the electrode with 1 mM DA, UA or AA added to 2 mM glucose solution in PBS. As
318
shown in Fig. 8, no noteworthy response was observed. In addition, the cathodic
319
peak currents and peak potentials are almost constant.
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Fig. 8 CVs of GOD/Au-ZnO/GCE 2 mM glucose solution, and glucose solution
321 322
containing 1 mM dopamine (DA), uric acid (UA) and ascorbic acid (AA) respectively
323
(0.1 M PBS, PH 7.4). Scan rate: 0.1 V s−1.
324
To evaluate the storage stability of the biosensor, GOD/Au-ZnO/GCE was stored in
325
PBS (PH 7) at 4 ℃ and the background current was recorded periodically by CV.
326
The response current of the biosensor was reduced by 5.6% of its initial response after
327
15 days, indicating its good stability. Such a high stability could be attributed to the 18
Page 18 of 26
effective immobilization of GOD and the good biocompatibility of the Au-ZnO film.
329
The relative standard deviation (RSD) of the current response to 5 mM glucose was
330
5.284% for five individual electrodes fabricated and measured under identical
331
conditions, which reveals that the fabrication method exhibits appreciable
332
reproducibility. Similarly, to evaluate the repeatability of the biosensors, the RSD for
333
six successive glucose determinations in different samples was obtained. The
334
biosensor displays good repeatability with an RSD of 4.163%.
335
3.7. Determination of glucose in serum samples
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To illustrate feasibility of the glucose sensor for real sample analysis, glucose
337
concentration in three different human serum samples were measured. The serums
338
were also analyzed with photometric kits in hospital. Five parallel determinations
339
were carried out. The comparative results are shown in Table 2.
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Table 2 Determination of glucose in serum samples with Au-ZnO/GCE
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340
Serum sample
Clinical assay (mM)
Proposed method (mM)
Relative error (%)
1
5.38
5.52
+2.60
2
5.62
5.41
-3.73
3
7.45
7.63
+2.42
341
It can be seen that the values measured by the glucose biosensor were very close to
342
the data provided by the hospital with relative error less than 4%, indicating the
343
suitability of the proposed glucose biosensor to practical applications.
344
4. Conclusions In summary, we proposed a simple and facile approach to fabricate a glucose
345
19
Page 19 of 26
biosensor with direct electron transfer based AuNPs-functionalized 3D hierarchically
347
ZnO nanostructure (Au-ZnO). 3D hierarchically ZnO nanostructure was prepared by
348
one-step solution route. AuNPs was deposited on the ZnO nanostructure through in
349
situ reduction of HAuCl4.2H2O. 3D hierarchically ZnO nanostructure provided a
350
wonderful platform to immobilize GOD and AuNPs because of its special surface
351
character. The introduction of AuNPs could not only improve conductivity but
352
enhance the immobilization of GOD by electrostatic interaction between AuNPs and
353
amidogen groups. The direct electrochemistry of GOD at the modified electrode has
354
been investigated. Cyclic voltammetric result showed a pair of well-defined redox
355
peaks corresponding to the electron transfer of GOD, which indicates that the Au-ZnO
356
nanocomposite can conduct electron transfer between GOD and the electrode. With
357
the wide linear range, fast electron transfer rate, high selectivity and the good stability,
358
this glucose sensor can be used for the detection of diabetic glucose concentrations.
359
The various characterization of GOD/Au-ZnO/GCE suggests that the fabrication
360
process proposed in this work will be useful for other analogous enzyme electrode
361
biosensors with direct electron transfer based on porous materials.
362
Acknowledgment
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The authors are grateful for the financial support provided by the National Natural
363 364
Science Foundation of China (U1304214, 21475115) and Program for University
365
Innovative Research Team of Henan (15IRTSTHN001) and the Natural Science
366
Foundation of Henan Province (nos. 132300410406).
367
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Author Biographies
478
Lin-Xia Fang received his Ph.D. degree in April 2015 from the Northwestern 25
Page 25 of 26
Polytechnical University. Presently, he is an Associate Professor at the Xinyang
480
Normal University. Her research interests are focusing on the novel nanomaterials and
481
electrochemical sensors.
482
Qu-Yu Zhang received her PhD degree in 1999 from the Northwestern Polytechnical
483
University. She is a Professor at the Northwestern Polytechnical University. Her
484
research
485
novelmicro/nano-materials.
486
Ke-Jing Huang received his PhD degree in 2006 from the Wuhan University.
487
Presently, he is an Associate Professor at the Xinyang Normal University. His
488
research interests include electrochemical analysis, electrochemical sensors and
489
biosensors.
490
Bing Liu is a graduate student at the Xinyang Normal University. Her current
491
researches include molecular electrochemistry and electrochemical materials.
concentrated
on
the
synthesis
and
application
of
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S0925-4005(15)30210-0 http://dx.doi.org/doi:10.1016/j.snb.2015.08.032 SNB 18887
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
21-4-2015 14-7-2015 8-8-2015
Please cite this article as: L. Fang, B. Liu, L. Liu, Y. Li, K. Huang, Q. Zhang, Direct electrochemistry of glucose oxidase immobilized on Au nanoparticlesfunctionalized 3D hierarchically ZnO nanostructures and its application to bioelectrochemical glucose sensor, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.08.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Direct electrochemistry of glucose oxidase immobilized on
2
Au nanoparticles-functionalized 3D hierarchically ZnO
3
nanostructures and its application to bioelectrochemical
4
glucose sensor
5
Linxia Fanga,* , Bing Liua, Lulu Liua, Yuehua Lia, Kejing Huanga, Qiuyu Zhangb,∗
b
8 9
us
Xinyang ,China
Department of Applied Chemistry, School of Science, Northwestern Polytechnical University,
Xi’an, China
an
7
College of Chemistry and Chemical Engineering, Xinyang Normal University,
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10
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Abstract: Three-dimensional (3D) hierarchically ZnO nanoarchitecture with
15
controlled morphology and dimensions was synthesized by trisodium citrate-assisted
16
solution phase method and functionalized by Au nanoparticles (AuNPs) via in-situ
17
reduction of HAuCl4. The as-prepared AuNPs-functionalized 3D hierarchically ZnO
18
nanostructure (Au-ZnO nanocomposite) were used as a novel immobilization matrix
19
for glucose oxidase (GOD) and exhibited excellent direct electron transfer properties
20
for GOD. The AuNPs-functionalized 3D hierarchically ZnO nanostructure (Au-ZnO
21
nanocomposite) favored the immobilization of the glucose oxidase (GOD) and the
22
penetration of water-soluble glucose molecules, which helped efficiently catalyze the
Ac ce pt e
14
Corresponding author E-mail address: [email protected]; [email protected]
*
1
Page 1 of 26
oxidation of glucose and facile direct electron transfer for GOD. The as-fabricated
24
glucose biosensor exhibited satisfactory analytical performance with a low detection
25
limit (0.02 mM) and an acceptable linear range from 1 to 20 mM. These results
26
indicated that AuNPs-functionalized 3D hierarchically ZnO nanomaterial is a
27
promising candidate material for high-performance glucose biosensors.
28
Keywords: Au nanoparticle; 3D hierarchically ZnO nanostructure; glucose
29
biosensor; direct electrochemistry
30
1. Introduction
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Diabetes is a major public health problem in the world wide which classed as a
32
metabolism disorder and is one of the leading causes of death and disability [1-3].
33
Accurate determination and regular monitoring of blood glucose concentration is very
34
important in the diagnosis and treatment of diabetes or metabolic disorders. Since the
35
initial development of glucose enzyme electrodes by Clark and Lyons in 1962,
36
tremendous effort has been directed toward research into glucose enzyme biosensors
37
because of their great promise in a vast range of application fields such as medical
38
diagnosis, diabetes management, bioprocess monitoring, beverage industry, and
39
environmental monitoring [4-6]. The electrochemical glucose biosensor based on the
40
direct electrochemistry between an electrode and the immobilized glucose oxidase is
41
especially promising due to its high selectivity, sensitive glucose detection, and
42
relatively low-cost fabrication [7-9]. However, the lack of the direct electrical
43
communication between redox proteins and electrode supports has been a key point
44
that limits the development of this kind of biosensors. Thus, in order to promote the
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direct electron transfer between active site of GOD and electrode, many materials,
46
including polymers, carbon nanotube, graphene, metal or metal oxide nanoparticles
47
and their composites have been used to modify the electrode to immobilize GOD for
48
improving the DET of GOD on the surface of electrode [10-13].
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The applications of biocompatible nanomaterials for enzymatic glucose sensors
50
were explored as they help the enzymes to retain its activity and to augment direct
51
electron transfer between the active site of the enzyme and the electrode [14]. The
52
advanced properties of nanostructures ZnO like non-toxicity, biocompatibility,
53
chemical stability, good permeation and electrochemical activities have triggered a
54
vast interest among the researchers to research and develop the applications especially
55
in biomedical and sensor [15, 16]. Apart from these, ZnO has a high isoelectric point
56
of about 9.5, which provide a positively charged substrate for immobilization of
57
enzyme with low isoelectric point and serve as a potential biosensor electrode [17, 18].
58
It has shown great promise in applications as electrode materials for immobilizing
59
GOD and improving DET of GOD on electrode. It has been reported that gold
60
nanoparticles (AuNPs) can greatly enhance the DET between some redox proteins
61
and electrode due to their quantum characteristics and large specific surface area of
62
small granule diameter as well as their ability to quickly transfer electrons at the
63
surfaces of colloidal particles [19-21]. Recently, 3D nanostructures with complex
64
morphology and high dimensionality have received great research interest due to the
65
fact that their advanced geometric structure and atom arrangement on the specific
66
facets of these nanostructures, which can offer novel properties [22-25]. Potentially,
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functionalizing 3D hierarchically ZnO nanomaterials with AuNPs, which combines
68
the large surface area of 3D support and the unique property of AuNPs, will generate
69
synergy effect and thus enhance their performance in applications of biosensor. In our
70
previous works [26, 27], we fabricated biosensors based Au-ZnO nanocomposites to
71
detect DNA arrays and dopamine respectively, and the good results were obtained.
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In the present study, 3D hierarchically ZnO nanoarchitecture was synthesized
73
according to the reference [26]. High-density AuNPs were homogeneously loaded
74
onto the porous matrix of ZnO to obtain AuNPs-functionalized 3D hierarchically ZnO
75
nanomaterial which was used to modify glass carbon electrode (GCE). GOD was
76
immobilized on the modified GCE and the DET between GOD and the modified GCE
77
was studied. The electrochemical catalytic activity of the fabricated electrode in
78
response to glucose was also investigated. Due to the synergy effect of 3D
79
hierarchically ZnO nanoarchitecture and AuNPs, the fabricated electrode showed
80
excellent direct electrochemical behavior and the DET between the GOD and the
81
modified electrode was easily achieved, indicating that AuNPs-functionalized 3D
82
hierarchically ZnO nanomaterial could be a good candidate material for immobilizing
83
biomolecules and fabricating the third-generation biosensor.
84
2. Experimental
85
2.1. Apparatus
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The
86
electrochemical
measurements
were
performed
on
a
CHI660D
87
Electrochemical Workstation (Shanghai CH Instruments, China). All experiments
88
were carried out by a conventional three-electrode system with a platinum wire 4
Page 4 of 26
electrode as the auxiliary electrode, a saturated calomel electrode (SCE) as the
90
reference electrode and a modified glassy carbon electrode (GCE) (3.0 mm in
91
diameter) as the working electrode. All of the potentials in this article were with
92
respect to SCE. The pH measurements were made with a pH meter Leici Devices
93
Factory of Shanghai, China. Electrochemical impedance spectroscopy (EIS)
94
measurements were performed in 0.1 M KCl solution containing 5.0 mM
95
K3Fe(CN)6/K4Fe(CN)6 (1:1) with a frequency ranging from 100 kHz to 0.1 Hz. The
96
morphologies of the nanocomposite were recorded on a JEM 2100 transmission
97
electron microscope (TEM) and a Hitachi S-4800 scanning electron microscope
98
(SEM).
99
D/Maxr-A
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X-ray
diffractometer
M
X-ray powder diffraction (XRD) pattern was operated on a Japan Rigaku equipped
high-intensity Cu Ka radiation ( λ = 1.54178 Å).
101
2.2 Reagents
graphite
monochromatized
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100
with
Zinc acetate dihydrate, hexamine (HMTA), absolute ethanol and sodium citrate
102 103
were obtained from China National Pharmaceutical Industry Corporation Ltd. GOD
104
and glucose was obtained from Sigma (Saint Louis, MO, USA). Phosphate-buffered
105
saline (PBS, 0.01 M) at various pH values was prepared by mixing a stock standard
106
solution of KH2PO4 and K2HPO4, which was used as the measuring buffer, and then
107
adjusting the pH with 0.1 M KOH and H3PO4. All chemicals were of analytical grade
108
and used without further purification. Ultrapure water (18.2 MX) was obtained from a
109
Milli-Q water purification system and used throughout.
110
2.3 Preparation of 3D hierarchically ZnO nanoarchitecture 5
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The preparation and the growth mechanism of 3D hierarchically ZnO
112
nanoarchitecture have been demonstrated in our previous work [26]. In a typical
113
synthesis, an equimolar ratio of zinc acetate dihydrate (25 mM）and HMTA (25 mM)
114
was dissolved into 50 mL of deionized water with subsequent addition of trisodium
115
citrate (5 mM), followed by stirring at room temperature for 20 min. The final mixture
116
was transferred to a 100 mL Teflon-stainless beaker for hydrolysis reaction at 90 °C in
117
an oven for 6 h. After completion of the reaction, cooling to room temperature
118
naturally, the resulting white precipitate was collected by centrifugation and purified
119
by washing with deionized water and absolute ethanol several times and dried at 60
120
°C for 24 h.
121
2.4 Preparation of AuNPs-functionalized 3D hierarchically ZnO nanomaterial
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The prepared 3D hierarchically ZnO nanomaterial were dispersed into 10mL
123
distilled water by ultrasonication. Then, 140 μL of freshly prepared HAuCl4 aqueous
124
solution (30 mM) was added into the dispersion by stirring. Subsequently, 0.25 mL of
125
sodium borohydride (NaBH4) aqueous solution (0.2 M) was added drop by drop into
126
the mixture solution with vigorous stirring at room temperature for 30 min. Finally,
127
the products were collected by centrifugation and were washed with distilled water
128
and absolute ethanol several times to produce AuNPs-functionalized 3D hierarchically
129
ZnO nanomaterial (Au-ZnO nanocomposite).
130
2.5 Fabrication of AuNPs-functionalized 3D hierarchically ZnO nanomaterial
131
modified electrode
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Prior to electrode modification the GCE was polished with 0.05 µm alumina
132
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slurry and Buehler polishing cloth. It was then washed with deionized water and
134
ultrasonicated for 3 min each in water and ethanol to remove any adsorbed alumina
135
particles or dirt from the electrode surface and finally dried in nitrogen airflow. 5 μL
136
of Au-ZnO nanocomposite dispersion (2 mg mL-1 in PBS (pH7)) was drop casted onto
137
the pre-cleaned GCE and dried at room temperature. Then, 8 μL of GOD (10 mg mL-1
138
in PBS (pH7)) was drop casted onto the Au-ZnO modified GCE and dried at ambient
139
temperature. The GOD/Au-ZnO modified surface was smoothly washed with water to
140
remove loosely adsorbed enzyme. For comparison, Au-ZnO/GCE, ZnO/GCE,
141
GOD/ZnO/GCE and GOD/GCE were prepared by adopting the similar procedures.
142
3. Results and discussions
143
3.1 Characterization of the Au-ZnO nanocomposites
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The morphology of the as-prepared 3D hierarchically ZnO and the Au-ZnO
145
nanocomposites were observed by SEM. Fig. 1a and b show the low magnification
146
and high-magnification SEM images of the 3D hierarchically ZnO, respectively, from
147
which it can be clearly observed that the product has a spherical lamellar structure
148
with a diameter of 2-3µm, that is to say that the spheres are assembled by a large
149
amount of interconnected nanosheets. Plenty of voids and interspaces are present
150
among these nanosheets, which may increase the surface area of the materials and are
151
potentially useful for applications such as catalyst and sensor materials. Fig. 1c and d
152
show the typical low magnification and high-magnification SEM images of the
153
Au-functionalized 3D hierarchically ZnO, respectively. An obvious change is that the
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Fig.1 (a) low magnification and (b) high-magnification SEM image of 3D
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157
hierarchically ZnO; (c) low magnification and (d) high-magnification SEM image of
158
Au-functionalized 3D hierarchically ZnO
159
surface of microspheres become illegible and the porosity degrade, which is due to a
160
high coverage of AuNPs deposited on the surface of ZnO nanosheet.
161
The distribution of AuNPs on the 3D hierarchically ZnO support was further
162
investigated by TEM. Typical TEM images of AuNPs-functionalized 3D
163
hierarchically ZnO with different magnification are displayed in Fig. 2(a-c). From the
164
edges of ZnO microsphere, it can be seen clearly that a high density of AuNPs with
165
small sizes were uniformly anchored on the surface of ZnO nanosheets (Fig. 2a). In
166
the higher resolution TEM picture of Fig. 2b, the AuNPs can be easily distinguished 8
Page 8 of 26
due to their darker contrast against the ZnO nanosheets. HRTEM image reveals
168
clearly the dividing line of the two lattice stripes (Fig. 2c), and the lattice stripes
169
distance between adjacent lattice planes is 0.24 nm, corresponding to the interplanar
170
distance of the (111) plane of face centered cubic (fcc) Au, which means that AuNPs
171
were espitaxially grown on the surface of ZnO nanosheets. The EDS of Au-ZnO
172
hybrid nanostructure is shown in Fig. 2d which future confirms the presence of Au in
173
the Au-ZnO nanocomposite. The phase compositions of the as-synthesized
174
productions were also determined by XRD. Fig. 3 shows the XRD patterns of ZnO
175
(curve a) and Au-ZnO nanocomposite (curve b). The major diffraction peaks shown
176
in curve a can be indexed to a phase from crystalline ZnO based on the data from the
177
JCPDS file (21-1486). The three additional peaks in curve b locating at 38.26°, 44.48°,
178
64.66°, and 77.64°are assigned to (111), (200), (220) and (311) planes reflection of
179
AuNPs (JCPDS Card No. 65-2870), which proves the formation of crystalline Au on
180
the ZnO nanosheets.
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hierarchically ZnO
184
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Fig. 2 TEM images (a-c) and (d) EDS spectra of AuNP-functionalized 3D
183
3.2 Electrochemical impedance spectroscopy of the modified electrodes
186
Electrochemical impedance spectroscopy (EIS) is a powerful tool to investigate
187
the impedance changes of electrode interface during the modification process. The
188
typical Nyquist impedance spectrum (presented in the form of the Nyquist plot)
189
includes a semicircle portion at high
190
electron-transfer-limited process and a linear part at low frequency range representing
191
the diffusion-limited process. The semicircle diameter observed at higher frequency
192
range is equaled to the electron transfer resistance (Ret).
193
electron-transfer kinetics of the redox probe at the electrode interface. Fig.4 presents
194
the Nyquist plot of EIS for bare GCE (a), Au-ZnO/GCE (b), ZnO/GCE(c) and
195
GOD/Au-ZnO/GCE (d). As shown, the Ret of those modified electrodes is in the order
196
of GOD/Au-ZnO/GCE > ZnO/GCE > Au-ZnO/GCE > GCE. The Ret of the ZnO/GCE
197
is much higher than that of the bare GCE because of weak conductivity of ZnO,
198
suggesting that a layer of ZnO film has formed on the surface of GCE and hindered
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frequencies
corresponding
to
the
This resistance controls the
10
Page 10 of 26
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199
Fig. 3 (a) XRD pattern of 3D hierarchically ZnO nanostructure;
201
(b) XRD pattern of AuNP-functionalized 3D hierarchically ZnO nanostructure
an
200
the charge transfer from the redox probe of [Fe(CN)6]3-/4- to the GCE surface.
203
nanoparticles have excellent conductivity and this hetero-structure can extensively
204
Obviously, the Au-ZnO/GCE shows comparatively low Ret value indicating that Au
205
improves the electron transfer properties of the electrodes to a great extent. The
206
GOD/Au-ZnO/GCE shows the largest Ret due to the blocking effects of high insulate
207
GOD on the charge transfer, which also indicates that GOD has been successfully
208
immobilized.
209
3.3 Direct electrochemistry of GOD at Au-ZnO nanocomposites modified GCE
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202
210
The direct electrochemistry of GOD at Au-ZnO/GCE has been studied by cyclic
211
voltammetry. Fig. 5 shows the CVs acquired at ZnO/GCE, GOD/ZnO/GCE,
212
Au-ZnO/GCE and GOD/Au-ZnO/GCE in N2-saturated PBS (0.1M, pH 7.0) at a scan
213
rate of 100 mV s-1. No peaks were observed at the CVs of ZnO/GCE (curve a) and
214
Au-ZnO/ GCE (curve b), but a couple of well-defined redox peaks were obtained at 11
Page 11 of 26
GOD/ZnO/GCE (curve c) and GOD/Au-ZnO/GCE (d), which indicates the redox
us
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215
216
Fig. 4 The electrochemical impedance spectroscopy (EIS) of bare GCE (a),
218
Au-ZnO (b), ZnO (c), GOD/Au-ZnO (d) in 0.1 M KCl aqueous solution containing
219
1.0 mM [Fe(CN)6]3-/4-. The frequency range is from 0.1 to 104 Hz at the formal
220
potential of 0.2 V.
221
peaks of the GOD/Au-ZnO/GCE and the GOD/ZnO/GCE should be ascribed only to
222
GOD. The background current of the Au-ZnO/GCE is higher than that of ZnO/GCE.
223
This is ascribed to the improvement of electrical conductivity through
224
functionalization of ZnO by AuNPs. The peak currents of GOD/Au-ZnO/GCE are
225
higher than that of GOD/ZnO/GCE. This may be attributed to the good conductivity
226
and biocompatibility of Au-ZnO nanocomposite. The anodic peak potentials (Epa) and
227
cathodic peak potentials (Epc) of GOD/Au-ZnO/GCE were observed at -0.450 V and
228
-0.403 V, respectively. The peak-to-peak separation (△Ep) is about 47 mV, revealing a
229
fast electron transfer process. The formal potential (EcƟ´) calculated from the average
230
of cathodic and anodic peak potential is -0.426 V, which is close to the electrode
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Page 12 of 26
potential of GOD in previous reports [28,29], indicating the direct electron transfer
232
from the redox site of the enzyme to the electrode. The outstanding direct electron
233
transfer ability of GOD at the Au-ZnO modified electrode surface is attributed to the
234
large surface area, favorable orientation of GOD, good biocompatibility, and high
235
electrical conductivity of Au-ZnO.
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236
Fig. 5 CVs of ZnO/GCE (a), Au-ZnO/GCE (b), GOD/ZnO/GCE (c),
237
GOD/Au-ZnO/GCE (d) in N2-saturated PBS solution (0.1 M, PH 7.0).
238
Scan rate: 0.1V s-1.
239 240 241
The isoelectric point (IEP) of ZnO is 9.5, whereas that of GOD is 4.2. At pH7.0, ZnO
242
surface is positively charged, while GOD surface is negatively charged. Therefore,
243
electrostatic interactions between positively charged ZnO nanostructures with
244
negatively charged surface of GOD lead to efficient immobilization of GOD.
245
However, GOD shows decreased direct electrochemistry at GOD-ZnO/ GCE due to 13
Page 13 of 26
the poor conductivity of ZnO film. The enhanced direct electron transfer of GOD at
247
the Au-ZnO support is due to the excellent electrical conductivity of AuNPs.
248
Therefore, we chose AuNPs as the functional material for ZnO to prepare Au-ZnO
249
composite for immobilizing GOD. The as-immobilized GOD exhibited promising
250
direct electrochemistry.
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246
3.4 Effect of scan rate
cr
251
The influence of the scan rates on the cyclic voltammetric performance of the
253
GOD/Au-ZnO/GCE was shown in Fig. 6. The scan rates from inner to outer curves
254
are 30, 50, 100, 120, 130, 160, 180, 200 and 300 mV s-1. As shown, the redox peak
255
currents linearly increased with the scan rates ranging from 30 to 300 mV s-1 (Fig. 6
256
inset). These characteristics indicate that the redox reaction of GOD on the surface of
257
Au-ZnO/GCE is a quasi-reversible surface-controlled electrochemical process. The
258
linear regression equations for the redox process are written as Ipa= 0.1084ｖ+10.75;
259
R2=0.9938 and Ipc= - 0.088ｖ+ 3.99; R2= 0.9956, where ｖ is the scan rate.
Ac ce pt e
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3.5
260 261
Electrocatalytic
activity
of
GOD/Au-ZnO/GCE
toward
glucose
determination
262
Cyclic voltammetry was employed to measure the electrocatalytic activity of
263
glucose biosensor for Au-ZnO nanocomposite. This method is a fast and convenient
264
tool for characterizing glucose biosensor. Fig. 7 shows the cyclic voltammograms
265
obtained at the GOD/Au-ZnO modified electrode in the presence of different
266
concentrations of glucose in oxygen saturated PBS at the scan rate of 0.1V s−1.
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Page 14 of 26
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Fig. 6 CVs of GOD/Au-ZnO/GCE in N2 saturated PBS (0.1 M, PH 7.0) at various
269
scan rates: inner to outer are 30-300 mV s-1. Inset to (B) shows the linear dependence
270
of peak currents with scan rate.
271
The direct electrontransfer of GOD is a two-electron and two-proton coupled reaction.
272
The cathodic peak current (Ipc) is attributed to the reduction of GOD (FAD), while the
273
anodic peak current (Ipa) is attributed to the oxidation of GOD (FADH2). Upon
274
addition of glucose, the cathodic peak current decreased linearly, which could be
275
attributed to the enzyme-catalyzed glucose oxidation. As illustration by Liang et al.
276
[30], the oxidized form of GOD, GOD (FAD), is reduced by glucose, which restrains
277
the electrochemical reduction reaction of GOD (FAD) and decreases the reduction
278
current. The glucose biosensor is just built on the base of this characteristic. The
279
calibration curve (Fig. 7 inset) corresponding to cyclic voltammetry response is linear
280
against the glucose concentration ranging from 1 to 20 mM with a correlation
281
coefficient of 0.9962 and a sensitivity of 1.409 μA mM-1. The detection limit is
282
estimated to be 0.02 mM at a signal-to-noise ratio of 3. The linear range of the
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Page 15 of 26
GOD/Au-ZnO/GCE is wider compared to other reported glucose biosensors based the
284
direct electron transfer for GOD [31-37].
an
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283
285
Fig. 7 CVs of GOD/Au-ZnO/GCE in the O2-saturated PBS (0.1 M, PH 7.0) solution
287
in various concentrations of glucose; Scan rate: 0.1V s-1. The inset shows the
288
calibration curve of the linear dependence of cathodic peak current on the glucose
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M
286
concentration.
289 290
The comparison of the analytical performance of the developed electrode with other
291
electrodes reported previously was given in Table1. It can be seen that the
292
GOD/Au-ZnO/GCE electrode exhibits a wider linear range, a lower detection limit
293
and a higher sensitivity. The as-fabricated glucose biosensor is suitable for detecting
294
human blood glucose concentration for the diagnosis of diabetes mellitus, since the
295
Table 1 Comparison of analytical performances between the proposed sensor and
296
other sensor for GOD direct electrochemistry Electrode
Linear range
Detection limit
Sensitivity
Ref
16
Page 16 of 26
(mM)
(μM)
(μA mM-1)
1-8
10
0.835
[31]
GOD/Au-OOPPye/GCE
1-8
70
0.851
[32]
GOD/Nff-GRg/GEh
2-14
40
1.547
[33]
GOD/ Agi- RGO/ GCE
0.5-12.5
160
0.27
[34]
GOD/GR-CNTj-ZnOk /GCE
0.01-6.5
4.5
GOD/MGFl/ GCE
1.0 - 12
250
0.2027
[36]
GOD/ GR-CdSm /GCE
2-16
700
0.124
[37]
GOD/Au-ZnO /GCE
1-20
20
1.409
This work
298
Au, gold nanoparticles.
299
c
RGO, reduced graphene oxide.
300
d
GCE, glassy carbon electrode.
301
e
cr
us
an
OOPPy, overoxidized polypyrrole. f
302
M
GOD, glucose oxidase.
b
[35]
d
a
0.3865
Ac ce pt e
297
ip t
GODa/Aub-RGOc/GCEd
Nf, nafion.
303
g
graphene.
304
h
GE, gold electrode.
305
i
Ag, silver nanoparticles.
306
j
CNT, carbon nanotube.
307
k
ZnO, zinc oxide.
308
l
MGF, mesocellular graphene foam.
309
m
310
normal range of blood glucose concentration is 4.4-6.6 mM. Moreover, the proposed
311
glucose biosensor was fabricated by a facile and low cost procedure without
312
expensive reagents and complicated experiments.
CdS, cadmium sulfide.
17
Page 17 of 26
313
3.6 Selectivity, stability, reproducibility and repeatability of the biosensor The selectivity of GOD/Au-ZnO film was evaluated in the presence of common
315
interfering species such as dopamine (DA), uric acid (UA), and ascorbic acid (AA) in
316
PBS. The influence of those interferents was examined by cyclic voltammograms of
317
the electrode with 1 mM DA, UA or AA added to 2 mM glucose solution in PBS. As
318
shown in Fig. 8, no noteworthy response was observed. In addition, the cathodic
319
peak currents and peak potentials are almost constant.
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314
320
Fig. 8 CVs of GOD/Au-ZnO/GCE 2 mM glucose solution, and glucose solution
321 322
containing 1 mM dopamine (DA), uric acid (UA) and ascorbic acid (AA) respectively
323
(0.1 M PBS, PH 7.4). Scan rate: 0.1 V s−1.
324
To evaluate the storage stability of the biosensor, GOD/Au-ZnO/GCE was stored in
325
PBS (PH 7) at 4 ℃ and the background current was recorded periodically by CV.
326
The response current of the biosensor was reduced by 5.6% of its initial response after
327
15 days, indicating its good stability. Such a high stability could be attributed to the 18
Page 18 of 26
effective immobilization of GOD and the good biocompatibility of the Au-ZnO film.
329
The relative standard deviation (RSD) of the current response to 5 mM glucose was
330
5.284% for five individual electrodes fabricated and measured under identical
331
conditions, which reveals that the fabrication method exhibits appreciable
332
reproducibility. Similarly, to evaluate the repeatability of the biosensors, the RSD for
333
six successive glucose determinations in different samples was obtained. The
334
biosensor displays good repeatability with an RSD of 4.163%.
335
3.7. Determination of glucose in serum samples
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To illustrate feasibility of the glucose sensor for real sample analysis, glucose
337
concentration in three different human serum samples were measured. The serums
338
were also analyzed with photometric kits in hospital. Five parallel determinations
339
were carried out. The comparative results are shown in Table 2.
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Table 2 Determination of glucose in serum samples with Au-ZnO/GCE
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340
Serum sample
Clinical assay (mM)
Proposed method (mM)
Relative error (%)
1
5.38
5.52
+2.60
2
5.62
5.41
-3.73
3
7.45
7.63
+2.42
341
It can be seen that the values measured by the glucose biosensor were very close to
342
the data provided by the hospital with relative error less than 4%, indicating the
343
suitability of the proposed glucose biosensor to practical applications.
344
4. Conclusions In summary, we proposed a simple and facile approach to fabricate a glucose
345
19
Page 19 of 26
biosensor with direct electron transfer based AuNPs-functionalized 3D hierarchically
347
ZnO nanostructure (Au-ZnO). 3D hierarchically ZnO nanostructure was prepared by
348
one-step solution route. AuNPs was deposited on the ZnO nanostructure through in
349
situ reduction of HAuCl4.2H2O. 3D hierarchically ZnO nanostructure provided a
350
wonderful platform to immobilize GOD and AuNPs because of its special surface
351
character. The introduction of AuNPs could not only improve conductivity but
352
enhance the immobilization of GOD by electrostatic interaction between AuNPs and
353
amidogen groups. The direct electrochemistry of GOD at the modified electrode has
354
been investigated. Cyclic voltammetric result showed a pair of well-defined redox
355
peaks corresponding to the electron transfer of GOD, which indicates that the Au-ZnO
356
nanocomposite can conduct electron transfer between GOD and the electrode. With
357
the wide linear range, fast electron transfer rate, high selectivity and the good stability,
358
this glucose sensor can be used for the detection of diabetic glucose concentrations.
359
The various characterization of GOD/Au-ZnO/GCE suggests that the fabrication
360
process proposed in this work will be useful for other analogous enzyme electrode
361
biosensors with direct electron transfer based on porous materials.
362
Acknowledgment
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The authors are grateful for the financial support provided by the National Natural
363 364
Science Foundation of China (U1304214, 21475115) and Program for University
365
Innovative Research Team of Henan (15IRTSTHN001) and the Natural Science
366
Foundation of Henan Province (nos. 132300410406).
367
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Author Biographies
478
Lin-Xia Fang received his Ph.D. degree in April 2015 from the Northwestern 25
Page 25 of 26
Polytechnical University. Presently, he is an Associate Professor at the Xinyang
480
Normal University. Her research interests are focusing on the novel nanomaterials and
481
electrochemical sensors.
482
Qu-Yu Zhang received her PhD degree in 1999 from the Northwestern Polytechnical
483
University. She is a Professor at the Northwestern Polytechnical University. Her
484
research
485
novelmicro/nano-materials.
486
Ke-Jing Huang received his PhD degree in 2006 from the Wuhan University.
487
Presently, he is an Associate Professor at the Xinyang Normal University. His
488
research interests include electrochemical analysis, electrochemical sensors and
489
biosensors.
490
Bing Liu is a graduate student at the Xinyang Normal University. Her current
491
researches include molecular electrochemistry and electrochemical materials.
concentrated
on
the
synthesis
and
application
of
cr
is
Ac ce pt e
d
M
an
us
work
ip t
479
492
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