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An Amperometric Biosensor of Determination H2O2 Based on Horseradish Peroxidase in Carbon Nanotubes/Ionic Liquid.

Byline: JUN WAN, WEINA WANG, GUANG YIN AND XIUJU MA

Summary: A novel amperometric biosensor for the determination of H2O2 based on horseradish peroxidase (HRP) in nanocomposite material of muti-walled carbon nanotubes/ionic liquid was explored. Cyclic voltammetry (CV) was used to characterize the performance of the biosensor. Under the optimized experimental conditions, H2O2 could be detected in a linear calibration range of 0.5 x 10-6 M ~ 6.0 x 10-6 M with a correlation coefficient of 0.9902 (n = 7), a detection limit of 1.5 x 10-7 M at 3s and the recovery ratio was of 96.2% ~ 110.8%, which indicated that the accuracy of this method is also satisfied. The modified electrodes display more excellent electrochemical performance, high sensitivity, good reproducibility, and long-term stability.

Keywords: Biosensor; Horseradish peroxidase; Muti-walled carbon nanotubes; Ionic liquid; Hydrogen peroxide.

Introduction

Hydrogen peroxide can exert detrimental effects on biological systems and appears to be involved in the neuropathology of central nervous system diseases. In addition, H2O2 has an impact on the forming of acid rain, owing to the importance of H2O2 in the oxidation of SO2 to H2SO4, which is harmful to the atmosphere aditionally. So the determination of H2O2 is of considerable importance in clinical and environmental applications. The methods of detecting H2O2 include spectrometry [1], chemiluminescence [2], and electrochemical analysis [3, 4]. Among these procedures, the electrochemical method offers high sensitivity, extended dynamic range and rapid response time. But the direct electrochemical detection of H2O2 often requires relatively high overpotentials. Hydrogen peroxide can be detected by employing peroxidase as bioelectrocatalysts to decrease the applied potentials. Among peroxidase, HRP has been most widely studied as enzyme-based amperometric biosensors because of its low cost.

The challenge of forming electrical contacts between redox proteins and electrode surfaces is crucial for the construction of electrochemical biosensor devices. The electrode surface modified with nanomaterials usually has good electron-transfer efficient between the matrix electrode and redox proteins such as enzymes. This modification is helpful in developing biosensors and the related apparatus. Among these applications, the amperometric enzyme-based electrode is quite unique, because it combines the enzyme specificity with the sensitivity and convenience of electroanalytical techniques in a compact form to facilitate analysis [5].

Carbon nanotubes (CNTs) as an important group of nanomaterials have grabbed considerable attention in recent years. Many studies have demonstrated that CNTs have excellent electronic, chemical, and mechanical properties. Their unique properties make them extremely attractive for fabricating electrochemical devices [6]. The high performance of the carbon nanotube electrodes is owing to the carbon nanotube dimensions, the electronic structure, and the topological defects present on the tube surface [7-13]. It can be coupled with enzymes to act as an electrical connector between the redox center of the enzyme and the electrode [14]. Cai reviewed the applications of carbon nanotubes in analytical ehemisny, and discussed the use of carbon nanotubes in scanning microscopy, gas sensors, modified electrode, gas chromatographic packing materials and detector of liquid chromatography [15].

Cai and co-worker [16] used a new type of nanocomposite of poly(nile blue A) with single-walled carbon nanotubes to modify GC electrode forming a new biosensor. This biosensor showed good electrocatalytic activity.

Nanomaterials and ionic liquids (ILs) have attracted researchers' interest recently [17-21]. Ionic liquids (ILs) are compounds consisting entirely of ions that exist in the liquid state at or near room temperature [22]. Due to their high intrinsic conductivity, wide electrochemical window, negligible vapour pressure, good chemical and thermal stability. ILs are also an excellent candidate for an electrolyte in electrochemical devices such as batteries, fuel cells, and dyesensitized solar cells [23, 24]. Researchers incorporate ionic liquids into carbon materials forming nanocomposite materials for their unique properties and potential applications in many areas [25]. Aida and co-workers [26] found that ILs are capable of dispersing carbon nanotubes. When a powder of ground single- walled carbon nanotubes was mixed with an excess amount of imidazolium-based ILs, the heavily entangled nanotube bundles were found to untangle to form much finer bundles.

The use of a polymerizable ionic liquid as the gelling medium allows for the fabrication of highly electroconductive polymer/nanotube composite material, which showed a substantial enhancement in dynamic hardness. Youngseon Shim studied the single- and double-walled carbon nanotubes in the armchair configuration solvated in the room-temperature ionic liquid by molecular dynamics computer simulations method. The smallest nanotube that allows solvent ions inside the tunnel is with a diameter of 0.95 nm. Midazole rings of cations in the first internal and external solvation shells are mainly parallel to the nanotube surface, indicating p-stacking between the nanotubes and cations ions. Dong's group [27] immobilized glucose oxidase and laccase at mutiwalled carbon nanotubes-ionic liquid gel modified electrodes and used them as the catalysts of anode and cathode of biofuel cells (BFC), respectively.

The power output of the BFCs is ca. 4.1 (mu)W (power density ca. 10.0 (mu)W cm-2). Zhao et. al., [28] used a gel containing multiwalled carbon nanotubes (MWCNTs) and room-temperature ionic liquid of 1-octyl-3-methylimidazolium hexafluorophosphate (OMIMPF6) to modify a glass carbon electrode.

Under optimum conditions, linear calibration graphs were obtained over the DA concentration range of 1.0x10-6 to 1x10-4 M. The detection limit of the current technique was found to be 1.0x10-7 M based on the signal-to-noise ratio of 3. Our groups used a novel nanocomposite material of muti-walled carbon nanotubes and room- temperature ionic liquid of N-butylpyridinium hexafluorophosphate to construct a novel Microperoxidase-11 biosensor for the determination of H2O2. The detection limit of the current technique was of 3.8 x 10-9 M at 3s [29].

In this study, we explored a novel amperometric biosensor to determinate H2O2. Our approach is to assemble HRP onto a nanocomposite material of MWCNTs and 1-ethyl-3-methylimidazolium hexafluorophosphate (EMIMPF6) modified GC electrode by adsorption. The experiment result showed quite long-term stability (5 weeks) of the modified electrodes. This may be owe to the cooperativity of the MWCNTs and ionic liquids, and the high melt of solid room- temperature EMIMPF6. The prepared biosensor exhibited low detection limit about 1.5 x 10-7 M (3s). The biocompatible MWCNTs-IL composite may be widely used in chemical, biological, clinical, food, and environmental fields.

It was reported that the peroxidases can catalyze the oxidation reaction of phenols and aromatic amines by H2O2 [30]. A possible mechanism of H2O2 catalyzed by HRP/MWCNTs-EMIMPF6 -modified film is postulated as shown in Scheme 1.

The Cyclic voltammetry of various electrodes in 1.0 M KCl solution containing 5.0 x 10-3 M K3Fe(CN)6 at a scan rate of 20 mV s-1 was shown in Fig. 1. Bare GCE (Fig. 1a) gave a stable and well-defined redox peaks at 119 mV (Epc) and 184 mV (Epa), which was the characteristic of [Fe(CN)6]3-/4- redox couple in the solution. After coating with MWCNTs and MWCNTs-EMIMPF6 nanomaterials, the current was substantially increased and still displayed a steady-state diffusion plateau. So we choose the coupled MWCNTs and EMIMPF6 as modified material. The synergy effect of MWCNTs and EMIMPF6 bring about the excellent electron-transfer ability.

Influence of the Scan Rate

Typical CV curves of the HRP/Nafion /MWCNTs-EMIMPF6/GCE in 0.1 M phosphate buffer solutions (pH 6.5) at different scan rates are shown in Fig. 2a. It can be seen that a pair of roughly symmetric anodic and cathodic peaks appeared with almost equal peak currents in the scan rate range from 0.15 to 0.6 V s-1. The peak-to- peak separation also increased with the scan rate. A good linear relationship was found for the peak current and scan rate, with the results shown in Fig. 2b. The reduction and oxidation peak currents rise linearly with the linear regression equations as Ipc ((mu)A) = 2.8241n1/2 (V/s)1/2 + 0.2443 (n = 10, g = 0.9813),Ipa ((mu)A) = -5.5756n1/2 (V/s)1/2 + 1.1576 (n = 10, g = 0.9945), respectively, suggesting that the reaction is a quasi-reversible diffusion- controlled process.

Optimization Conditions for the Detection of H2O2 When the MAP concentration increased in the pH 6.5 phosphate buffer solutions containing 0.25 mM H2O2, the CV peak current response increased correspondingly and approached to a maximum value at the concentration of 5.0 mM (Fig. 3a). Thus, this concentration was chosen as the optimal MAP concentration.

The pH value of the substrate solution effect on the electrochemistry was examined, and the results were shown in Fig. 3b. The biosensor demonstrated the maximum response at the pH value of 6.5 in 0.1 M phosphate buffer solutions containing 5.0 mM MAP and 0.25 mM H2O2. Thus, the optional pH value of the enzymatic reaction was pH 6.5 and was chosen for all experiments.

Electrochemical Detection of H2O2

The modified electrode of HRP/Nafion

/MWCNTs-EMIMPF6/GCE showed good electro-

catalytic activity towards H2O2 reduction. The CV signals were obtained under the optimal conditions shown in Fig. 4. With the increasing H2O2 concentration the catalytic reduction peak current of HRP/Nafion /MWCNTs-EMIMPF6/GCE increased linearly. The linear response range of the biosensor to H2O2 concentration was from 1.0 (mu)M to 6.0 (mu)M and the linear regression equation was I (10-6 A) = 1.0320 C (10-6 M) - 0.1380 (n = 7, g = 0.9902) with a detection limit of 1.5 x 10-7 M at 3s. The results indicated that HRP in Nafion/MWCNTs-EMIMPF6/GCE had a higher sensitivity to H2O2. In addition, the long-term stability of the HRP/Nafion/MWCNTs- EMIMPF6/GCE was investigated over 40-day period. The catalytic current response could maintain about 90% of its original response in 40 days when the modified electrode was stored at 4 oC and measured intermittently.

The inset shows the plot of catalytic oxidation peak current versus the concentration of H2O2.

Table-1 shows the comparison of the linear range and detection limit obtained by several HRP enzyme-based electrodes for H2O2 detection. The detection limit of this developed H2O2 biosensor was lower than the biosensors based on HRP/MWCNTs/GCE [32], SA-HRP-CILE [33] and Th/HRP/TiO2 [34], and similar with HRP/Chitosan /IL/GCE [31] and HRP nanoparticles [35]. The result indicated that the modified electrodes provided a biocompatible microenvironment to keep the bioelectrocatalytic activity after enzyme adsorption on the surface of the modified electrodes.

Table-1: Comparison of the developed H2O2 biosensor with other HRP biosensors.

H2O2 biosensor###Linear range###Detection limit

Enzyme Electrode###((mu)M)###((mu)M)

HRP/Nafion/MWCNTs-

EMIMPF6/GCE (this work)###0.5 [?] 6###0.15

HRP/Chitosan/IL/GCE[31]###0.6 [?] 160###0.15

HRP/MWCNTs/GCE[32]###4 [?] 2000###1

SA-HRP-CILE[33]###1.0 [?] 6.0###0.5

Th/HRP/TiO2[34]###11 [?] 2000###1.2

HRP nanoparticles [35]###1 [?] 9###0.1

HRP: horseradish peroxidase; SA: sodium alginate; Th: Thionine.

Recovery

Experiments

The standard addition recovery experiment of simulated samples was also tested, the results shown in

Table-2. The recovery ratio was of 96.2% ~ 110.8%, which indicated that the accuracy of this method is also satisfied.

Table-2: Results of standard addition and recovery test.

Sample###Determined###Mean###Added###Found###Mean###Recovery

###((mu)g/L)###deviation###((mu)g/L)###((mu)g/L)###deviation###(%)

1###45.2###0.23###50###100.6###0.20###110.8

2###60.8###0.25###50###108.9###0.17###96.2

3###73.5###0.18###50###122.2###0.15###97.4

Experimental

Reagents

The following materials were obtained commercially and used as received: horseradish peroxidase (HRP) (500 U/mg, MW40 000, SERVA), H2O2 (analytical grade, Shanghai Chemical Plant, China), ionic liquid 1-ethyl-3-methylimidazolium hexafluorophosphate (EMIMPF6, 97%, melting point 58 ~ 62 oC, Guangzhou Weibo Chemical Limited Company, China), and m-aminophenol (MAP) (Beijing Hengyezhongyuan Limited Company, China). MWCNTs (with a diameter of about 10-20 nm and length of around 50 (mu)m) with carboxylic groups purchased from Shenzhen Nanotech. Port Ltd.co. (Shenzhen, China) were purified according to the reference [36]. The other chemicals were of analytical grade and used without further purification. The 0.1 M phosphate buffer solutions at various pH values were prepared by mixing the stock solutions of NaH2PO4 and Na2HPO4 and then adjusting the pH with 0.1 M NaOH and H3PO4.

Fe(CN)63-/4- solutions (5.0 mM, containing 0.1 M KCl) with equal concentration of Fe(CN)63- and Fe(CN)64- were prepared as electrochemical probes. All solutions were prepared with doubly distilled water.

Apparatus

Cyclic voltammetry (CV) and difference pulse

voltammetry (DPV) were performed on a CHI 832B electrochemical analyzer (Shanghai Chenhua Instrument, China) using a three-electrode system that consisted of a platinum wire as auxiliary electrode, a Ag/AgCl/KCl (sat) electrode as reference electrode, and a bare or modified GCE as working electrode. All the electrochemical experiments were performed at an ambient temperature of 20 +- 2 oC. All experimental solutions were deoxygenated by bubbling nitrogen for 30 min, and a nitrogen atmosphere was kept over the solutions during measurements.

Preparation of the MWCNTs-EMIMPF6/GCE

Prior to use, the glassy carbon electrode was carefully polished with polishing paper and 1.0, 0.3, 0.05 mm alumina slurry sequentially then washed ultrasonically in water and ethanol, finally dried in air. An amount of 2.0 mg of MWCNTs and 12.8 mg of EMIMPF6 that optimized were dissolved in 1 mL of N,N-Dimethylformamide (DMF) solution, as the stock solution. After about 40 min of sonication, uniformly dispersed MWCNTs and EMIMPF6 were formed. A 5 (mu)L of the prepared suspension was dipped onto GC electrodes to obtain the MWCNTs-EMIMPF6/GC modified electrodes.

Preparation of the HRP/Nafion/MWCNTs -EMIMPF6/GCE

After being air-dried, the MWCNTs- EMIMPF6 -modified electrodes were immersed into the aqueous solution of HRP (2.0 mg mL-1) for 6 h. The electrodes (denoted as HRP/MWCNTs- EMIMPF6 modified electrodes) were then thoroughly rinsed with distilled water to remove the nonadsorbed HRP. A 5 (mu)L of Nafion (1 wt%) solution was dipped onto modified electrodes and dried in air to obtain the HRP/Nafion/MWCNTs- EMIMPF6 modified electrodes. The modified electrode was stored at 4 oC in phosphate buffer solutions (pH 6.0) in a refrigerator when not in use. The fabrication procedure of the biosensor is shown in Fig. 5.

National Natural Science Foundation of China (No.21075073 and 21105053), and the Natural Science Foundation of Shandong Province (No. ZR2010BZ006 and ZR2010BM025).

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Electrochemical Detection

A 12.5 (mu)L of 0.1 M H2O2 were added to 5 mL of 5.0 mM MAP and 0.1 M phosphate buffer solutions (pH 6.5). The CV curves of the solution were recorded on a CHI 832B electrochemical analyzer with the three-electrode system. The scanning potential range from -0.8 to 0 V.

Conclusions

To conclude, the new HRP/Nafion/MWCNTs-EMIMPF6/GCE for quick determination H2O2 was studied. HRP was obtained at the MWCNTs-EMIMPF6 composite- film-modified glassy carbon (GC) electrode by direct electron transfer and a single electron transfer process between the enzyme and the GC electrode. H2O2 could be detected in a linear calibration range of 1.0 (mu)M to 6.0 (mu)M and a detection limit of 1.5 x 10-7 mol L-1 at 3s. The recovery ratio was of 96.2% ~ 110.8%, which indicated that the accuracy of this method is also satisfied. This new electrode presented very large current response from electroactive substrates due to its enhanced conductivity and biocompatible interface. The modified electrodes display more excellent electrochemical performance, high sensitivity, long-term stability and much lower detection limits.

Acknowledgments

This work was supported by the Nano Letters, 6, 1556 (2006).

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College of Environment and safety Engineering, Key Laboratory of Eco-chemical Engineering, Ministry of Education, Qingdao University of Science and Technology, Qingdao 266042, China wanjundz@sohu.com*
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Author:Jun Wan; Weina Wang; Guang Yin; Xiuju Ma
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9CHIN
Date:Dec 31, 2012
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