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A Third-Generation Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Immobilized in Carbon Nanotubes/ SBA15 Film Zheng-Hui Fang ,a, b Li-Min Lu,b Xiao-Bing Zhang,*b Hong-Bo Li,b Bin Yang,b Guo-Li Shen,b Ru-Qin Yub a

Yueyang Vocational and Technical College, Yueyang 414000, P. R. China State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China Tel: + 86-731-88821960; *e-mail: [email protected] b

Received: April 12, 2011;& Accepted: July 12, 2011 Abstract A new third-generation biosensor for H2O2 assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of carbon nanotubes (CNTs)-SBA-15 modified gold electrode. The biological activity of HRP immobilizing in the composite film was characterized by UV-vis spectra. The HRP immobilized in the nanocomposite matrix displayed excellent electrocatalytic activity to the reduction of H2O2. The effects of the experimental variables such as solution pH and working potential were investigated using steady-state amperometry. Under the optimal conditions, the resulting biosensor showed a linear range from 1 mM to 7 mM and a detection limit of 0.5 mM (S/N = 3). Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results. Keywords: Horseradish Amperometry

peroxidase,

Carbon

nanotubes,

Third-generation

biosensor,

Hydrogen

peroxide,

DOI: 10.1002/elan.201100211

1 Introduction It has been well established that H2O2 has a direct relationship with numerous biological processes for the reason that H2O2 is a very important intermediate or product in biochemical reactions [1]. So the determination of H2O2 is practically important. Numerous techniques such as titrimetry [2], spectrometry [3], and electrochemistry [4] have been employed for this purpose. Among these techniques, the amperometric electrochemical biosensor based on electron transfer between an electrode and immobilized HRP is especially attractive because of its simplicity and high sensitivity [5–9]. For the fabrication of an efficient biosensor, the effective immobilization of enzymes on the electrode surface is a key step. It is highly desirable to use the substrates that would favor effective loading of enzymes to have high sensitivity towards the analyte [10–12]. Recent studies have demonstrated that CNTs are promising materials for electrochemical biosensors and show great potential for use in the next generation of biosensors [13–15]. For example, CNTs can enhance the electroactivity of biomolecules and promote the electron transfer reaction of proteins due to their electrocatalytic capabilities [16, 17]. Moreover, CNTs are robust and have mechanical strength. Wang et al. reported the direct electrochemistry Electroanalysis 2011, 23, No. 10, 2415 – 2420

of cytochrome c at electrochemically-activated SWCNT modified electrodes [17]. Gooding and co-authors also observed direct charge transfer between redox-active enzymes and the surface of aligned CNTs [18]. Willner et al. demonstrated that SWCNTs could plug into the active site of glucose oxidase, making feasible direct electron transfer from the redox center to electrodes through CNTs [16]. These studies demonstrated that direct electron transfer reactions of redox-active biomacromolecules can be much improved through the use of CNTs, which enhanced the sensitivity of given biosensors and offered an opportunity to develop reagentless biosensors. A series of organic compounds and inorganic materials such as polymers [19, 20], clay [21], montmorillonite [22– 24], porous alumina [25], and sol-gel matrix [26] etc. have been used as enzyme immobilization matrices. Mesoporous silicas (MPSs) have been proven to be promising as the immobilization matrix, due to their high mechanical, thermal, and chemical stability and good adsorption and penetrability. There have been a number of reports describing the use of MPSs as enzyme carriers [27–30]. Although MPSs have so many advantages, the poor conductivity of MPSs is also an important drawback of the materials, which might influence the performance of the biosensor in amperometric detection. To overcome the drawbacks, Bai et al. utilized Au nanoparticles-MPSs

2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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nanocomposites to develop glucose biosensor [31]. The introduction of Au nanoparticles to MPSs could enhance the conductivity of nanocomposites. We envisioned that the combination of CNTs with MPSs might also improve the conductivity of the naonocomposites, and therefore, improve the performance of the as-prepared amperometric biosensor. In this work, by combining the advantageous features of CNTs and SBA-15 (a kind of MPS with uniform tubular channels varying from 5 to 30 nm [32]), we developed a novel H2O2 biosensor based on immobilizing HRP in the CNTs-SBA-15 nanocomposite matrix. The large surface area and unique surface property of the biocompatible matrix resulted in a high enzyme adsorption capacity, and the enzyme adsorbed in this film retained its activity to a large extent. The proposed biosensor exhibited high sensitivity, fast response, good repeatability and longterm stability towards H2O2.

2 Experimental 2.1 Apparatus and Reagents The UV-vis spectra were recorded on Mutispec-1501 Shimadzu Hyper UV-vis spectrophotometer. Cyclic voltammetric and amperometric measurements were carried out on CHI 760B electrochemical workstation (Shanghai, China). A three-electrode cell (10 mL) was used with a modified Au electrode as working electrode, a saturated calomel electrode (SCE) as reference electrode and a platinum foil electrode as counter electrode. All potentials were measured and reported versus the SCE and all experiments were carried out at room temperature. HRP (from horseradish, 250 U mg1) was purchased from Shanghai Reagents (Shanghai, China) and chitosan (medium molecular weight) were purchased from Sigma (St. Louis, Mo, USA). Carbon nanotubes (CNTs) with approximately 95 % purity were obtained from Shenzhen Nanotech Port Co. Ltd. (Shenzhen, China). SBA-15 silicas (with a surface area of ~ 830 m2/g and pore sizes of ~ 6 nm) were purchased as commercial products from the Laboratory of Advanced Materials, Fudan University (Shanghai, China) and used without further purification. 30 % H2O2 solution was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd, and a fresh solution of H2O2 was prepared daily. All other chemicals were of analytical grade and used as received. The 0.1 M phosphate buffer solutions (PBS) at various pH values were prepared by mixing the stock solutions of 0.1 M KH2PO4 and 0.1 M Na2HPO4 with different proportion, and then adjusting the pH with 0.1 M H3PO4 or 0.1 M NaOH. Doubly distilled water was used throughout this work. 2.2 Electrode Modification Before each experiment, Au electrodes (polycrystalline gold rod, 99.99 %, 2 mm diameter) were polished using alumina slurries with particle diameters 0.3 mm and 2416

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0.05 mm, respectively, and subsequently cleaned through sonication in distilled water and ethanol, each for 5 min, Subsequently, the Au electrode was immersed in piranha solution (a bath of 7 parts H2SO4 to 3 parts 30 % H2O2) for 20 min. Finally, the electrode was rinsed with distilled water. A 1.0 wt.% chit solution was prepared by dissolving 1.0 g of chit flakes into 100 mL of 1.0 % acetic acid and stirred for 3 h at room temperature until complete dissolution. The chit solution was stored in refrigerator when not in use. The CNTs and SBA-15 were dispersed in PBS solutions respectively with the concentration of 2 mg mL1 by using a short 30-min sonication. HRP solution was prepared in PBS with the concentration of 10 mg mL1. Then, 10 mL 10 mg mL1 HRP was mixed with 5 mL 2 mg mL1 CNTs solution and 5 mL 2 mg mL1 SBA15 solution and the sonication was continued for 10 min so as to obtain a homogeneously CNTs-SBA-15-HRP solution. 5 mL of the mixture was dropped onto the Au electrodes and dried in air. Then, 2 mL chit was cast to form a protective film and dried similarly. The obtained electrode was CNTs-SBA-15-HRP/chit-modified Au electrode. As a comparison, SBA-15-HRP/chit and CNTsHRP/chit modified Au electrodes were prepared in a similar way.

3 Results and Discussion 3.1 Structural Characterization of HRP-CNTs-SBA-15 Hybrid Film Since the location of the UV-vis Soret absorption band of iron provides structural information about possible denaturation of heme proteins, especially about conformational change in the heme group region [33, 34], UV-vis was used to probe the structure change for heme proteins. As can be seen from Figure 1, the UV-vis spectra of HRP film (curve a) and HRP-CNTs-SBA-15-chit hybrid film (curve b) in 0.1 M PBS (pH 7.0) gave Soret bands at 403 nm. The HRP Soret absorption band position is not affected by the existence of CNTs-SBA-15-chit, both of which are the same as Sore absorption, which indicates that the HRP at CNTs-SBA-15-chit hybrid film remains a natural biological activity. The mesoporous CNTs-SBA15-chit should be an ideal matrix for immobilizing proteins and retaining its activity. 3.2 Electrochemical Characterization of the SelfAssembly Process Figure 2 shows cyclic voltammograms (CVs) of differently modified electrodes in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1 : 1) containing 0.1 M KCl. A well-defined CV was observed at the bare Au electrode (curve a). A further increase in peak current was observed (curve b) when CNTs was covered onto the electrode. In contrast, a peak current decrease was observed at the CNTs-SBA-15 composite film modified Au electrode due to the poor electri-


Electroanalysis 2011, 23, No. 10, 2415 – 2420

Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase

Fig. 1. UV-vis absorption spectra of HRP films (a) and HRPCNTs-SBA-15-chit hybrid film (b) in 0.1 M pH 7.0 PBS.

Fig. 3. Cyclic voltammograms of CNTs-SBA-15-HRP/chitmodified Au electrode in the absence (a), and presence (b) of 3.0 mM H2O2 in 0.1 M PBS (pH 7.0).

dation current decreased significantly at the CNTs-SBA15-HRP/chit-modified Au electrodes when 3 mM H2O2 was added (Figure 3b), indicating an obviously catalytic reduction of H2O2 at the biosensor. The mechanism for whole electrode response process could be expressed as the following reactions [35]: HRPðRedÞ þ H2 O2 ! HRPðOxÞ þ H2 O HRPðOxÞ þ 2e þ 2Hþ ! HRPðRedÞ þ H2 O Net reaction : H2 O2þ 2e þ 2Hþ ! 2H2 O Fig. 2. Cyclic voltammograms of (a) bare Au electrode; (b) CNTs-chit- modified Au electrode; (c) CNTs-SBA-15-chit-modified Au electrode; (d) CNTs-SBA-15-HRP/chit-modified Au electrode in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1 : 1) containing 0.1 M KCl.

cal conductivity of SBA-15(curve c). When HRP with the poor electrical conductivity was assembled onto the CNTs-SBA-15 composite film (curve d), the current decrease again. On the basis of cyclic voltammetry result, we can conclude that HRP is successfully immobilized on the CNTs-SBA-15 composite film. 3.3 Electrochemical Properties of CNTs-SBA-15-HRP/ chit-Modified Au Electrodes The bioelectrocatalytic activity of the CNTs-SBA-15HRP/chit-modified Au electrodes for the reduction of H2O2 was further investigated by CV in the absence and presence of 3 mM H2O2 in 0.1 M PBS (pH 7.0). As shown in Figure 3, the reduction current increased while the oxiElectroanalysis 2011, 23, No. 10, 2415 – 2420

Figure 4 shows the CVs of the CNTs-SBA-15-HRP/ chit-modified Au electrode recorded in 0.1 M PBS solution at different scan rates. It is found that both the anodic and cathodic peak current clearly increase with increasing potential scan rate. Moreover, the redox peak currents are proportional to the square root of scan rate, indicating a typical semi-infinite linear diffusion-controlled electrochemical behavior feature of the redox process. 3.4 Optimization of Experimental Parameters It is well known that the applied potential strongly affects the amperometric response of a biosensor. We have systemically investigated the impact of applied potentials on the amperometric response of the CNTs-SBA-15-HRP/ chit-modified Au electrode. Figure 5A shows the current response of the CNTs-SBA-15-HRP/chit-modified Au electrode toward 0.5 mM H2O2 in the potential range from 0 V to 0.4 V. As can be seen, the CNTs-SBA-15HRP/chit-modified Au electrode exhibits large reduction



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Fig. 4. Cyclic voltammograms of the CNTs-SBA-15-HRP/chit -modified Au electrode recorded in 1/15 M PBS (pH 7.0) at different scan rates (inner to outer): 0.02 V s1, 0.05 V s1, 0.1 V s1, 0.2 V s1, 0.3 V s1 and 0.5 V s1. Inset: dependence of the redox peak currents on the square root of scan rates; the cathodic and anodic peaks used to plot the current vs. scan-rate plot were at 0.35 V and 0.02 V, respectively.

currents in cases the applied potential is not larger than 0.35 V. The currents at 0.35 V, 0.40 V and 0.45 V are almost the same, but the background currents at 0.40 V and 0.45 V are larger than that at 0.35 V. Taking both the sensitivity and the signal/noise ratio into consideration, 0.35 V is chosen as the optimum applied potential. The pH dependence of the enzyme electrode over the pH range 4.0–9.0 is illustrated in Figure 5B. The amperometric response increased from pH 5.0 to 7.0 and then decreased above pH 7.0. The enzyme electrode had maximum sensitivity at pH 7.0, which was consistent with other results of HRP-based biosensors [36–38]. This indicated that the immobilization procedure did not alter the intrinsic characteristics of HRP. Therefore, pH 7.0 PBS was used as the electrolyte in subsequent experiments. 3.5 Amperometric Response and Enzymatic Kinetic Parameter Figure 6 shows amperometric responses for the SBA-15HRP/chit(a), CNTs- HRP/chit(b) and CNTs-SBA-15HRP/chit(c) modified Au electrode to subsequent additions of 10 mL of 1.0 M H2O2 in 10 mL PBS (the change of concentration for H2O2 being 1.0 mM) at 0.35 V. The CNTs-SBA-15-HRP/chit-modified Au electrode showed a rapid and sensitive bioelectrocatalytic response, reaching about 95 % of the steady-state current within 8 s after 2418


Fig. 5. A) Effect of working potential on the amperometric response of the biosensor in the presence of 0.5 mM H2O2 at different applied potentials measured in a pH 7.0 PBS. B) Influence of pH on the amperometric response of the biosensor in the presence of 0.5 mM H2O2 in PBS at a potential of 0.35 V.

each addition of H2O2. As can be seen, compared with the SBA-15-HRP/chit(a) and CNTs-HRP/chit(b) modified Au electrode, the CNTs-SBA-15-HRP/chit(c) modified Au electrode exhibits a much larger amperometric response, which could be attributed to the large surface area of the nanocomposite. The calibration curve of the CNTs-SBA-15-HRP/chit-modified Au electrode is shown in Figure 6 (left Inset). The reduction currents are linear within the concentration range from 1 mM to 7 mM with a relative coefficient 0.998, which is wider than that of molecular wires/polyethylene glycol-HRP-modified GCE [39] and Clay-HRP-Clay/AuCS-GCE [36]. The detection limit of the biosensor is 0.5 mM (S/N = 3), which is much lower than some other electrochemical biosensor systems using peroxidases [40, 41]. The relationship between the catalytic current and the concentration of H2O2 shows a Michaelis–Menten kinetic mechanism. The apparent MichaelisMenten constant (Kmapp ) could be calculated from the LineweaverBurk equation: 1=I ss ¼ 1=I max þ Km app =I max ½c ½42



Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase

Fig. 6. Amperometric responses of the SBA-15-HRP/chit (a); CNTs-HRP/chit (b); CNTs-SBA-15-HRP/chit (c) modified Au electrode to subsequent additions of 10 mL of 1.0 M H2O2 in 10 mL PBS at 0.35 V, and the calibration curve of the biosensor. Inset: relationship of the catalytic current with the concentration of H2O2.

where Iss, Imax and c represent the steady current, maximum current and H2O2 concentration, respectively. According to the intercept and slope of above regression equation, Kmapp can be estimated to be 1.61 mM. The value is smaller than 23.85 mM for HRP immobilized in sol-gel-derived ceramic-CNTs nanocomposite film [43] and 5.12 mM for SBP-sol-gel thin film modified electrode [44]. The small apparent Michaelisten constant shows a high affinity to H2O2 and good bioactivity of CNTs-SBA15-HRP/chit toward H2O2 reduction. 3.6 Reproducibility, Stability and Selectivity of the Biosensor The repeatability of the CNTs-SBA-15-HRP/chit-modified Au electrode was examined by the detection of 0.8 mM H2O2. A relative standard deviation (RSD) value of 2.5 % was obtained for 10 successive determinations, which indicated a good repeatability of the method. The fabrication reproducibility was also estimated with a series of 10 sensors constructed independently in the same way. The RSD was 4.3 % for the steady-state current to 0.8 mM H2O2, which demonstrated the reliability of the fabrication procedure. The long-term stability of the CNTs-SBA-15-HRP/chitmodified Au electrode was explored. It was investigated through the response to 0.8 mM H2O2 at 0.35 V in 0.1 M PBS. When not in use, electrode was stored at 4 8C in a refrigerator. The data shows that the sensitivity of Electroanalysis 2011, 23, No. 10, 2415 – 2420

the electrode remains relatively constant over the first 18 days, and decreases to about 83 % of the original value after 1 month (used more than 80 times). Good long-term stability seems to result from the favorable microenvironment that maintains the HRP activity and prevents the leakage of enzyme. The selectivity of this sensor was performed by comparing the amperometric response of 0.2 mM H2O2 before and after adding 2 mM some possible interferents into 0.1 M PBS (pH 7.0) and the results were given in Table 1. As shown, glucose, acetaminophen, ethanol, oxalic acid, ascorbic acid and uric acid did not interfere with the determination of H2O2. The good selectivity of this biosensor is largely attributed to the low working potential ( 0.35 V).

Table 1. Possible interferences tested with the CNTs-SBA-15HRP/chit-modified Au electrode Possible interference

Current ratio [a]

Glucose Acetaminophen Ethanol Oxalic acid Ascorbic acid Uric acid

1.00 1.02 1.00 1.02 0.93 0.98

[a] Ratio of currents for mixtures of 2 mM interfering substance and 0.2 mM H2O2 compared to that for 0.2 mM H2O2 alone.



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4 Conclusions In this paper, a novel electrochemical HRP biosensor based on a CNTs-SBA-15-chit composite was developed for the determination of H2O2. The bionanocomposite film provided a suitable microenvironment, which could effectively present a large loading amount of enzyme and enhance the direct electron transfer between the enzymes active sites and the electrode. The HRP immobilized in the nanocomposite matrix exhibited excellent electrocatalytic activity to the reduction of H2O2. Under optimal conditions, the designed biosensor exhibits fast amperometric response, excellent linear relationships, and a low detection limit. Moreover, the selectivity, stability and repeatability of the proposed biosensor were investigated with satisfactory results.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants 21177036, 20975034), The National Key Scientific Program of China (Grant 2011CB911000), “973” National Key Basic Research Program of China (Grant2007CB310500), Program for Changjiang Scholars and Innovative Research Team in University, and Ministry of Education of China (Grant NCET-07-0272).

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