Direct electrochemistry of glucose oxidase based on its direct immobilization on carbon ionic liquid electrode and glucose sensing

Electrochemistry Communications 10 (2008) 1140–1143

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Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom

Direct electrochemistry of glucose oxidase based on its direct immobilization on carbon ionic liquid electrode and glucose sensing Xiaodong Shangguan, Hongfang Zhang, Jianbin Zheng * Institute of Analytical Science, Shaanxi Provincial Key Lab of Electroanalytical Chemistry, Northwest University, Xi’an, Shaanxi 710069, P.R. China

a r t i c l e

i n f o

Article history: Received 29 April 2008 Received in revised form 17 May 2008 Accepted 20 May 2008 Available online 25 May 2008 Keywords: Direct electrochemistry Glucose oxidase Carbon ionic liquid electrode Glucose sensor

a b s t r a c t Direct electrochemistry of glucose oxidase (GOx) has been achieved by its direct immobilization on carbon ionic liquid electrode (CILE) with a conductive hydrophobic ionic liquid, 1-butyl pyridinium hexafluophosphate ([BuPy][PF6]) as binder for the first time. A pair of reversible peaks is exhibited on GOx/CILE by cyclic voltammetry. The peak-to-peak potential separation (DEP) of immobilized GOx is 0.056 V in 0.067 M phosphate buffer solution (pH 6.98) with scan rate of 0.1 V/s. The average surface coverage and the apparent Michaelis–Menten constant are 6.69  1011 molcm2 and 2.47 lM. GOx/CILE shows excellent electrocatalytic activity towards glucose determination in the range of 0.1–800 lM with detection limit of 0.03 lM (S/N = 3). The biosensor has been successfully applied to the determination of glucose in human plasma with the average recoveries between 95.0% and 102.5% for three times determination. The direct electrochemistry of GOx on CILE is achieved without the help of any supporting film or any electron mediator. GOx/CILE is inexpensive, stable, repeatable and easy to be fabricated. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction Glucose oxidase (GOx), containing a flavin adenine dinucleotide (FAD) redox center that catalyzes the electron transfer from glucose to gluconolactone, has been extensively used to monitor the glucose in biochemistry, clinical chemistry, food processing and fermentation [1]. Many methods have been developed for the determination of glucose, especially amperometric biosensors [2]. It is well known that the active redox centre of GOD, FAD is deeply embedded in a protective protein shell, which makes the direct electron transfer with the conventional solid electrodes extremely difficult [3]. Hence, the different biocompatible immobilizing materials, including polymer [4], silica sol–gel film [5], poly acrylamide microgel matrix [6] and nano gold particles-ionic liquid-N, N-dimethylformamide etc. [7], are used to promote the electron transfer. Ionic liquids (ILs) are molten salts with the melting point close to or below room temperature, low flammability, high ionic conductivity, minimal vapour pressure, high thermal stability and a wide electrochemical window [8–11]. They are composed of two asymmetrical ions of opposite charges that only loosely fit together (usually bulky organic cations and smaller anions). In recent years, direct electrochemistry of biologically important enzymes has been studied with ILs in both theoretical and practical application because ILs are considered to be suitable

* Corresponding author. Tel.: +86 29 88302077; fax: +86 29 88303448. E-mail address: [email protected] (J. Zheng). 1388-2481/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2008.05.033

media for supporting biocatalytic processes with high polarity, non-coordination power, high selectivity, fast rate and great enzyme stability [12]. Enzymes are usually active and protein refolding is improved in ILs [13]. Zeng and co-workers constructed a modified glassy carbon electrode by entrapping GOx into nano gold particles-ionic liquid-N, N-dimethylformamide composite film [7]. Sun and co-workers constructed a modified imidazolium-based carbon ionic liquid electrode by entrapping Hb into sodium alginate hydrogel film [14]. Safavi and co-workers constructed a modified pyridinium-based carbon ionic liquid electrode by the use of [OcPy][Cl] to immobilize directly Hb on CILE [15]. In this paper, a new GOx/CILE has been constructed by directly immobilizing GOx on CILE, which comprises of graphite powder and ionic liquid ([BuPy][PF6]). The direct electrochemistry and electrocatalytic behaviour of GOx on CILE is investigated. Compared to our previous work [16], background current can be decreased highly on the proposed CILE. To our best knowledge, this is the first report on direct electrochemistry of GOx on CILE by direct immobilization of GOx without the need for modification of the electrode surface with a special film, nanoparticles or polymer as supporting materials.

2. Experimental 2.1. Reagents Glucose oxidase (E.C. 1.1.3.4, 182 U/mg, Aspergillus niger, Sigma, USA). b-D-glucose, butane chloride, ethylacetate, methanol,

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hexafluorophosphate acid and pyridinium were of analytical grades. [BuPy][PF6] was synthesized and purified following the similar procedures described in the literature [17]. High purity graphite powder (SP, China). All aqueous solutions were made with deionized water, which was further purified with a Milli-Q system (Millipore Co., USA).

A CHI660A electrochemical workstation (Shanghai Chenhua Co. Ltd., China) controlled by a microcomputer with CHI660A software was employed for all electrochemical measurements in the experiment. DL–180 ultrasonic cleaning machine (35 KHz, Zhejiang Haitian Electron Instrument Factory, China) was used to dissolve and form homogeneous solution. A three-electrode system, where a standard saturated calomel electrode (SCE) served as reference electrode, a platinum wire electrode as the auxiliary electrode and the prepared electrodes as the working electrode. All the electrochemical experiments were conducted at room temperature. All potentials reported were versus the SCE.

b

1

a 0

I/μA

2.2. Apparatus

2

-1 -2 -3 -4

-0.75

-0.60

-0.45

-0.30

-0.15

E/V(νs.SCE) Fig. 1. The CV curves of CILE (a) and GOx/CILE (b) in 0.067 M PBS (pH 6.98) at 100 mV/s.

2.3. Preparation of the CILE 3

8

1

Ip/μA

4

1

0 -1

I pc

-2 -3 -4

0.6

0

0

100

A 10 mL PBS (0.067 M, pH 6.98) with proper amount of glucose was transferred into a 10 mL cell and the three-electrode system was immersed into the solution. CV and DPV experiments were performed between 0.8 and 0.2 V at scan rate of 0.1 V/s. The peak currents and potentials of GOx were recorded by CHI660A workstation. The glucose content in the samples was obtained by using DPV. All experiments were performed at room temperature.

3. Results and discussion 3.1. Cyclic voltammetry of GOx/CILE Cyclic voltammograms of CILE and GOx/CILE are shown in Fig. 1. Under the same conditions and in the absence of GOx, bare CILE (Fig. 1a) does not show any peak in the working potential range mentioned above. While a pair of well-defined redox peaks is observed for GOx/CILE (Fig. 1b) with oxidation potential (Epa) of 0.445 V, reduction potential (Epc) of 0.501 V, DEP of 56 mV and Ipa/Ipc of 1. This indicates that the electrochemical reaction is almost reversible, which is better than that reported in literatures [7,19–21] and that [BuPy][PF6] is favorable to the immobilization of GOx and the direct electron transfer between GOx and CILE. It is thought that [BuPy][PF6] can improve the activity and stability of GOx.

200

log Ip

400

B

0.3

b

0.0

a

-0.3

-4

300

ν / (mV/s)

-0.6 -0.9

1.2

2.4. Experimental procedure

I pa

A

2

12

I/μA

CILE was prepared by hand-mixing of synthesized [BuPy][PF6] and graphite powder with a ratio of 50/50 (w/w) in the manner described in the literature [18]. A portion of the resulting paste was packed firmly into the cavity (2.0 mm i.d.) of a Teflon holder. In order to better homogeneity in the composite and lower background current, the electrode should be heated to a temperature above the melting point of IL (m.p. 65 °C) prior to its use [18]. A new surface was attained by polishing the electrode on fine grit sandpaper firstly and then smoothing on weighing paper. For preparing GOx/CILE, 4 lL 15 mg/mL GOx was immobilized on the surface of CILE by dropping method and dried in 4 °C for 6 h. After this process, the GOx/CILE was rinsed with water and then with 0.067 M phosphate buffer solution (PBS, pH 6.98). The GOx/CILE was stored at 4 °C in a refrigerator when not in use.

-8

-0.6

-0.3

1.6

0.0

2.0

logν

2.4

2.8

0.3

E/V (νs.SCE) Fig. 2. The CV curves of GOx in 0.067 M PBS (pH 6.98) at different scan rate on CILE. Scan rate 1–12: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400 mV/s. Inset A is linear relationship of Ip and v at GOx/CILE. Inset B is linear relationship of log Ip and log v at GOx/CILE (a log Ipa, b log Ipc).

3.2. Effect of scan rate The cyclic voltammograms for GOx/CILE are studied at different scan rates (v) in the range of 20–400 mV/s in Fig. 2. The results exhibit about symmetrical anodic and cathodic peaks of approximately equal heights for GOx at different scan rates. The peak currents (Ip) are linearly with v in range mentioned above in Fig. 2 inset A. The linear regression equation is Ipa (lA) = 4.904  102 + 7.440  103 v (mV/s), R = 0.9984; Ipc (lA) = 0.1420  8.370  103 v (mV/s), R = 0.9969, respectively. This indicates that the electron transfer process for GOx/CILE is a surface-confined mechanism in potential scope mentioned above. The logarithm of peak currents (log Ip) versus logarithm of scan rate (log v) shows linear relationships in (Fig. 2) inset B. The slope value of 1.001 for anodic currents and 0.9855 for cathodic currents is very close to the theoretical value of 1 expected for thin layer electrochemical behavior [22]. According to the equation of Ip = n2F2vAC*/4RT = nFQv/4RT [23], n is calculated as 1.72, meaning that 2e transfer is involved [24].

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At the same time, the Ipa/Ipc ratio and the formal potential E00 (E00 = (Epa + Epc)/2) keep almost unchanged, which are the typical characteristics of a reversible system. The average surface coverage (C*) of the electroactive GOx adsorbed on the CILE is calculated to be 6.69  1011 molcm2 from the slope of the Ip–v curve. This value is much larger than the literature value (2.86  1012 molcm2) [25], suggesting that CILE provides a large area for enzyme immobilization. 3.3. Influence of pH Cyclic voltammetry of GOx/CILE is studied in solutions with varying pH values. In the range of pH (5.29–7.38), GOx/CILE can exhibit a pair of stable and well-defined redox peaks. With rising of pH value, the cathodic peak currents increase and reach a maximum at around pH 6.98 and then decrease with further increasing pH value. The E0’ depend on pH value of the solution and shift to the negative potentials by increasing pH value of the solution, indicating that proton is present in the electron transfer process of GOx. The linear regression equation is E0’ = 0.058830.05589 pH, R = 0.9904. The slope is close to the theoretical value (58.6 mV/pH). This indicates that the numbers of electron and proton transferred in the electrochemical reaction are equal. 3.4. Biocatalytic activity of GOx When GOx/CILE is immersed in an air-saturated PBS (pH 6.98) containing glucose, the cathodic peak current of GOx decreases (Fig. 3). The reason is that the dissolved oxygen and GOx take part in the oxidation reaction of glucose [7]. The biocatalytical process for the oxidation of glucose in the presence of GOx can be summarized as following [26]

GOxðox:Þ þ glucose ! GOxðred:Þ þ gluconolactone þ 2Hþ

ð1Þ

GOxðred:Þ þ 2Hþ þ O2 ! GOxðox:Þ þ H2 O2

ð2Þ

Cathodic peak occurs at about 0.468 V in our experiments (Fig. 3) and differential pulse voltammetry (DPV) is used to determine glucose concentration in the range of 0.1–800 lM under corresponding optimized parameters. The detection limit estimated is 0.03 lM (S/N = 3). The optimum parameters are found to be amplitude of 0.05 V, pulse width of 0.05 s and pulse period of 0.2 s. The linear regression equation is Ipc (lA) = 2.986 + 3.070  103 Cglucose (lM), R = 0.9908.

2

1

Sample

Found (lM)

Added (lM)

Found (lM)

Recovery (%)

1 2 3

11.6 11.6 11.6

2.0 4.0 6.0

13.5 15.7 17.5

95.0 102.5 98.3

The apparent Michaelis–Menten constant Km is generally used to evaluate the biological activity of GOx according to the Michaelis–Menten equation [27]. The corresponding plot yields Km of 2.47 lM. The value is smaller than those reported about enzymatic electrodes [28,29], suggesting that GOx immobilized on CILE has good affinity to glucose. The good microenvironment due to effect of [BuPy][PF6] might contribute to the improvement of the affinity and good performances of the biosensor. 3.5. Stability and repeatability of the GOx/CILE The stability of the GOx/CILE is evaluated by examining the cyclic voltammetric peak currents of GOx after continuously scanning for 30 cycles. No decrease of the voltammetric response is observed, indicating that the enzyme electrode is stable in PBS. The stability of GOx/CILE is also checked by measuring the current response over a period of 20 days. GOx/CILE is stable in a week and the electrochemical response of the GOx on the CILE decreases less than 4% after 20 days. To estimate the repeatability of the proposed method, the RSD of ten times successful measurement of peak current of 0.1 mM glucose is calculated to be 2.3%, which demonstrates the good repeatability of the method. The electrode-to-electrode reproducibility of DPV is examined on six GOx/CILEs constructed individually; the RSD of the six average peak current of 0.1 mM glucose is calculated to be 2.7%. 3.6. Application The GOx/CILE has been applied to the determination of blood sugar concentration in human plasma utilizing standard addition method. 20 lL plasma samples are transferred to the cell with 10 mL 0.067 M PBS (pH 6.98) for the determination. The results are listed in Table 1. The recoveries for the determination of glucose are between 95.0% and 102.5% for three times determination. It indicates that the detection of glucose using the enzyme biosensor is effective. The effects of interference on DPV responses of 0.1 mM glucose are also tested in presence of different concentrations of uric acid and ascorbic acid. The tested results show that no obvious current change occurs. 4. Conclusions

0

I/μA

Table 1 Determination of glucose in human plasma samples by GOx/CILE

5

-2

-4

-6 -0.8

-0.6

-0.4

-0.2

E/V(νs.SCE) Fig. 3. The CV curves of different concentration glucose in 0.067 M PBS (pH 6.98) at GOx/CILE with 100 mV/s. Concentration of glucose 1–5: 0, 100, 200, 400, 800 lM.

Carbon ionic liquid ([BuPy][PF6]) electrode can provide a unique microenvironment for the direct successful immobilization and electron transfer of GOx without the help of any supporting film or any electron mediator. The GOx/CILE possesses good electrochemical and electrocatalytic activities. The direct electron transfer between GOx and CILE is surface-confined reversible electrochemical process. GOx can exhibit a pair of well-defined reversible peaks at formal potential about 0.47 V (versus SCE) in 0.067 M PBS (pH 6.98) on the CILE. The GOx/CILE exhibits a high electrocatalytic effect towards glucose and determinates glucose in human plasma successfully. Meanwhile, GOx/CILE is quite stable for at least 20 days. The biosensor without using supporting films and redox mediators by direct immobilization and electron transfer of GOx on CILE can be expected to be used to construct other enzyme biosensor.

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Acknowledgments The authors appreciate the financial support from the National Natural Science Foundation of China (No. 20675062) and the Research Foundation for the Doctoral Program of Higher Education of China (No. 20060697013). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

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