Simple Method for Preparing Glucose Biosensor Based on Glucose Oxidase in Nanocomposite Material of Single-Wall Carbon Nanotubes/Ionic Liquid

doi:10.4236/jasmi.2012.22011

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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.

Email: *[email protected]

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

Simple Method for Preparing Glucose Biosensor Based on Glucose Oxidase in Nanocomposite

Material of Single-Wall Carbon Nanotubes/Ionic Liquid

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

Nafion solutio n

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

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

(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

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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