Journal of Analytical Sciences, Methods and Instrumentation, 2012, 2, 54-59
http://dx.doi.org/10.4236/jasmi.2012.22011 Published Online June 2012 (http://www.SciRP.org/journal/jasmi)
Simple Method for Preparing Glucose Biosensor Based on
Glucose Oxidase in Nanocomposite Material of Single-Wall
Carbon Nanotubes/Ionic Liquid
Weina Wang, Guang Yin, Xiuju Ma, Jun Wan*
College of Environment and Safety Engineering, Qingdao University of Science and Technology, Qingdao, China.
Received March 1st, 2012; revised March 21st, 2012; accepted April 9th, 2012
ABSTRACT
Based on electric conductivity and wide potential window of ionic liquid (IL) and electric property of single-wall car-
bon nanotubes (SWCNTs), composite material of IL-SWCNTs was prepared, glucose sensor was built with this mate-
rial for immobilizing glucose oxidase (GOx). It showed good response, sensitivity and stability for long time for glu-
cose detection. Linear range for the detection of glucose was from 0.5 × 10–6 M to 12 × 10–6 M while detection limit
was 6.26 × 10–8 M (S/N = 3).
Keywords: Biosensor; Glucose Oxidase; Single-Wall Carbon Nanotubes; Ionic Liquid; Glucose
1. Introduction
Single-walled carbon nanotubes (SWCNTs) and ionic
liquids (ILs) have attracted researchers’ interest recently.
SWCNTs are of significant interest due to their unique
properties and potential applications. The unique size
distribution and hollow geometry of SWCNTs bestow
upon them unique electronic, mechanical and chemical
properties and potentially wide application. It has been
reported that single-walled carbon nanotubes can be used
to promote electron transfer reactions when used as an
electrode material [1,2]. Ionic liquids (ILs) have good
chemical and physical properties, such as good chemical
and thermal stability, negligible vapour pressure, good
electrical conductivity and a wide electrochemical win-
dow [3]. The use of electrodes prepared from carbon
nanotubes and ILs have been reported in a number of
articles. One way is to modify the surface of a glassy
carbon electrode with a dispersion of CNTs/IL or CNTs
/IL/polymer in a suitable solvent [4-7]. This kind of
modified electrode provides a platform for fabrication of
biosensors, which shows promising application to detect
various biomacromolecules.
Glucose detection has important practical value in
analysis of food and fermentation, the textile industry,
environmental monitoring, medical diagnosis and other
areas [8,9]. People have done a lot of effort focused on
developing appropriate technology for accurate monitor-
ing of glucose in the view of high sensitivity, high stabil-
ity, high response rate, good selectivity and low produc-
tion cost detection requirements. Now the main detection
methods of glucose include surface plasmon resonance
biosensor [10], near-infrared light biosensors [11], ca-
pacitive sensing [12], electrochemical luminescence [13],
fluorescence detection [14], colorimetry [15], and current
biosensors [16]. Among them, the current biosensor is a
promising detection method because of its high sensitiv-
ity, miniaturization, compatibility, easy operation and
low cost. Usually, enzyme-based glucose sensor is the
use of glucose oxidase (GOx) to detect glucose.
In this paper, our approaches to development of new
kinds of enzyme biosensor are essentially based on assem-
bling glucose oxidase onto a SWCNTs-IL modified glassy
carbon (GC) electrode by adsorption. Cyclic voltammetry
and differential pulse voltammetry were used to investi-
gate the electrochemical behaviors of GOx/Nafion/
SWCNTs-IL/GC electrode. By this method, the compos-
ite modified electrode exhibited its better electrocatalysis
to glucose. The simple method provided an effective
means for fabricating the novel biosensors.
2. Experimental
2.1. Reagents and Apparatus
Glucose oxidase (GOx, EC1.1.3.4,118 U·mg–1, Type II)
purchased from Sigma and Aldrich. β-D(+)-glucose from
Tokyo Kasei Kogyo Co., Ltd.(TCI). N,N-dimethyl for-
*Corresponding autho
r
.

Simple Method for Preparing Glucose Biosensor Based on Glucose Oxidase in Nanocomposite
Material of Single-Wall Carbon Nanotubes/Ionic Liquid
55
mamide (DMF), from Tianjin Ruijinte Chemical Co., Ltd.
The ionic liquid 1-Ethyl-3-methylimidazolium Bromide,
from Songkong Chemical (HongKong) Co., Ltd. SWNTs
(2.73 wt%, -COOH), from Chengdu Organic Chemicals
Company Ltd. of Chinese Academy of Sciences. Nafion
(5 wt%) from Aldrich. 0.1 M PBS (containing 0.1 M KCl)
of various pH was prepared by mixing the stock solutions
of NaH2PO4 and Na2HPO4. The solutions were deaerated
with high purity nitrogen before the experiments and all
of the electrochemical experiments were performed at an
ambient temperature.
Electrochemical experiments were performed on a
CHI 832B electrochemical analyzer (Shanghai Chenhua
Instrument, China) with a three-electrode system com-
prising a modified GCE of diameter 4mm as working
electrode, a Ag/AgCl/ (sat KCl) electrode as reference
electrode, and a platinum wire as auxiliary electrode.
2.2. Preparation of the SWCNTs-IL/GCE
Prior to use, the glassy carbon electrode was carefully
polished with polishing paper and 1.0, 0.3 and 0.05 µm
alumina slurry, and sonicated respectively in water and
ethanol for 2 min. The electrode was rinsed in ethanol
again and allowed to dry under the infrared light. An
amount of 5.0 mg of SWCNTs and 0.1911 mg of ionic
liquid were dispersed in 2 mL of N,N-Dimethylforma-
mide (DMF) solution, then sonicated for 40 min with a
uniformly dispersed solution obtained. A 5 μL of the
prepared suspension was dipped onto GC electrodes.
After being dried under the infrared light, the SWCNTs-
IL modified GC electrodes were obtained.
2.3. Preparation of
GOx/Nafion/MWCNTs-IL/GCE
The 2 mg·mL–1 GOx solution was prepared by dissolving
GOx in 0.1 M PBS buffer (pH 7.0). The SWCNTs-IL-
modified electrodes were immersed into the GOx solu-
tion for 24 h at 4˚C. A 5 μL of Nafion (1 wt%) solution
was dipped onto modified electrodes and dried at 4˚C.
The modified electrode was stored at 4˚C when not in
use. Scheme 1 shows the preparation approach.
3. Results and Discussion
3.1. CV Behavior of the SWCNTs-IL/GCE
Figure 1 shows the CV of various electrodes in 2.5 mM
K3[Fe(CN)6] containing 0.1 M KCl at 50 mV·s–1. Bare
GCE after coating with SWCNTs-IL, the current was
substantially increased and still displayed a steady-state
diffusion plateau. The synergistic effects can be ascribed
to the excellent electron-transfer ability of SWCNTs and
the ionic liquid.
ionic liquid
GOx glucose
COOH
+ IL
SWCNTs
COOH
modification
GOx
inmersed into aqueous solution of GOx
GOx
GOx
GOx
Nafion solutio n
GOx
GOx
GOx Nafion
sing le-walled carbon nanotube
Scheme 1. Preparing schematic diagram of modified GOx/Nafion/SWCNTs–IL/GCE.

Simple Method for Preparing Glucose Biosensor Based on Glucose Oxidase in Nanocomposite
Material of Single-Wall Carbon Nanotubes/Ionic Liquid
56
3.2. Influence of the Scan Rate
Typical CV curves of GOx/Nafion/SWCNTs-IL/GCE
in 0.1 M PBS (pH 7.0) at different scan rates are shown
in Figure 2. A good linear relationship was found for the
peak current and scan rate, with the results shown in Fig-
ure 2. The reduction and oxidation peak currents rise
linearly with the linear regression equations as Ipc (µA)
= 9.624v1/2 (V/s)1/2 + 0.1005 (n = 8, r = 0.9986), Ipa (µA)
= –12.23v1/2 (V/s)1/2 – 0.4275 (n = 8, r = 0.9978), respec-
tively, suggesting that the reaction is a quasi-reversible
diffusion-controlled process.
3.3. Optimization Conditions for the Detection of
Glucose
Experiments were conducted to establish the effect of
various parameters such as the concentration of room
temperature ionic liquid, pH, the concentration of SWCNTs
on modified electrode performance.
In order to determine the optimal concentration of
room temperature ionic liquid in SWCNTs suspension,
we studied the influence of the temperature ionic liquid
concentration range form 0.01 M to 0.1 M on the elec-
trochemical characteristics of electrodes. Figure 3(a)
shows the modified electrode displayed the best electro-
chemical properties when the concentration of IL in
SWCNTs suspension is 0.05 M.
Figure 3(b) shows the i-pH curves obtained by the CV
curves of GOx/Nafion/SWCNTs-IL/GC electrode in 0.1
M PB solution with different pH at a scan rate of 50
mV· s –1 when the concentration of glucose oxidase was 1
mM. As shown in Figure 3(b), when the pH value of 0.1
M PB solution is 7.0, the redox peaks current ratio is
about 1, the peak potential diference is the smallest, re-
versibility is the best. Therefore, pH 7.0 was chosen as
the optimum value in the following experiments. All the
experiments were carried out at pH 7.0 unless specially
stated.
Figure 1. Cyclic voltammetry obtained with a GCE (a) SWCNTs/GCE (b) and SWCNTs-IL/GCE (c) in 0.1 M KCl solution
containing 2.5 mM K3Fe(CN)6 at a scan rate of 50 mV·s–1.
Figure 2. Typical CV curves of GOx/Nafion/SWCNTs-IL/GCE in 0.1 M PB solution (pH 7.0) at different scan rates (V·s–1): (a)
0.05 V·s–1; (b) 0.1 V·s–1; (c) 0.15 V·s–1; (d) 0.2 V·s–1; (e) 0.25 V·s–1; (f) 0.3 V·s–1; (g) 0.35 V·s–1; (h) 0.4 V·s–1. Inset shows a cali-
bration plot of peak current versus scan rate.

Simple Method for Preparing Glucose Biosensor Based on Glucose Oxidase in Nanocomposite
Material of Single-Wall Carbon Nanotubes/Ionic Liquid
57
(a) (b)
(c)
Figure 3. (a) Different concentration of room temperature ionic liquid in 0.1 M KCl solution containing 2.5 mM K3Fe(CN)6.
Error bars represent device-to-device standard deviation (n = 3); (b) I-pH curves obtained by the CV curves of GOx/
Nafion/SWCNTs-IL/GC electrode in 0.1 M PB solution with different pH at a scan rate of 50 mV·s–1 when the concentration
of glucose oxidase was 1 mM. Error bars represent device-to-device standard deviation (n = 3); (c) Different concentration of
SWCNTs in 0.1 M KCl solution containing 2.5 mM K3Fe(CN)6. Error bars represent device-to-d evice standard d eviation (n = 3).
The concentration of SWCNTs impacts the concentra-
tion of glucose oxidase’s adsorption. In this paper, we
studied the influence of the SWCNTs concentration
range form 1.0 mg·mL–1 to 3.0 mg·mL–1 on the electro-
chemical characteristics of electrodes when the concen-
tration of IL in suspension was 0.05 M. As shown in Fig-
ure 3(c), with the increase of the concentration of
SWCNTs, the content of glucose oxidase’s adsorption
increased. When the concentration of SWCNTs was 2.5
mg· mL –1, increased the concentration, the curve tended
to a horizontal line. The concentration of 2.5 mg·mL–1
SWCNTs was chosen as the optimal concentration for all
experiments.
3.4. Stability Investigation of
GOx/Nafion/SWCNTs-IL/GCE
As shown in Figure 4, the long-term storage stability of
GOx/Nafion/SWCNTs-IL/GC electrode was studied over
a certain period of time by monitoring the CV currents in
0.1 M PB solution (pH 7.0). When the modified elec-
trode was stored at 4˚C for several days, the electrode
sensitivity to glucose decreased gradually, but the reduc-
tion rate is relatively small, the modified electrode shows
good stability. The better long-term stability could be
attributed to SWCNTs and IL entrapped in the film pro-
viding desirable microenvironment for glucose oxidase
immobilization. All the results indicated that the com-
posite electrode had better stability.
Figure 4. Typical CV curves of GOx/Nafion/ SWCNTs-IL/
GCE in 0.1 M PB solution (pH 7.0) at different time: (a)
initialization; (b) 7 d; (c) 14 d.

Simple Method for Preparing Glucose Biosensor Based on Glucose Oxidase in Nanocomposite
Material of Single-Wall Carbon Nanotubes/Ionic Liquid
58
Figure 5. Differential pulse voltammetry curves obtained
with different concentration of glucose in 0.1 M PB solution
of GOx/Nafion/SWCNTs-IL/GCE: (a) 0.5 × 10–7 M; (b) 1.0
× 10–6 M; (c) 2.0 × 10–6 M; (d) 4.0 × 10–6 M; (e) 6.0 × 10–6 M;
(f) 8.0 × 10–6 M; (g) 1.0 × 10–5 M; (h) 1.2 × 10–5 M. The inset
shows the plot of catalytic oxidation peak current versus the
concentration of glucose.
3.5. Electrochemical Detection of Glucose
In order to examine the applicability of GOx/Nafion
/SWCNTs-IL/GCE, differential pulse voltammetry method
was used for glucose detection. The differential pulse
voltammetry curves were obtained under the optimal
conditions shown in Figure 5. With the increasing glu-
cose concentration, the peak current of the modified
electrode increased linearly. The linear response range to
glucose concentration was from 0.5 × 10–6 M to 12 × 10–6
M and linear regression equation was I (10–5 A) =
–0.05279 C (10–7 M ) – 3.8333 (n = 8, r = 0.995) with a
detection limit of 6.26 × 10–8 M at 3σ. The results indi-
cated that glucose oxidase in Nafion/SWCNTs-IR/GCE
had a higher sensitivity to glucose.
4. Conclusion
With immobilization of glucose oxidase at the surface of
SWCNTs/IL modified GC electrode a simple and prom-
ising sensor for glucose detection was demonstrated. The
modified electrodes display more excellent electro-
chemical characteristics, long-term stability, high sensi-
tivity and much lower detection limits. The composite
material based on single-walled carbon nanotubes and
ionic liquid can find wide potential applications in direct
electrochemistry, biosensors and biocatalysts.
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