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Colloids and Surfaces B: Biointerfaces 61 (2008) 93100

A high-sensitive amperometric hydrogen peroxide biosensor based on the immobilization of hemoglobin on gold colloid/l-cysteine/gold colloid/nanoparticles Ptchitosan composite lm-modied platinum disk electrode
Gan Yang, Ruo Yuan , Ya-Qin Chai
Chongqing Key Laboratory of Analytical Chemistry, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China Received 26 March 2007; received in revised form 9 July 2007; accepted 24 July 2007 Available online 9 August 2007

Abstract A hemoglobin (Hb)/gold colloid (nano-Au)/l-cysteine (l-cys)/nano-Au/nanoparticles Pt (nano-Pt)chitosan (CHIT) composite lm-modied platinum disk electrode (abbreviated to modied electrode) has been prepared to construct a biosensor for determination of H2 O2 . The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The modied process was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The morphologies of different composite lm were investigated with scanning electron microscopy (SEM) and the element of composite lm was investigated with X-ray photoelectron spectroscopy (XPS). Analytical parameters such as pH and temperature were also studied. The linear range for the determination of H2 O2 is 1.4 107 to 6.6 103 mol/L with a detection limit of 4.5 108 mol/L (S/N = 3). The sensor achieved 95% of the steady-state current within 10 s. The sensor exhibited high sensitivity (17.62 A/(mmol L)), selectivity and stability. The method is applied to the determination of H2 O2 with satisfactory results. 2007 Elsevier B.V. All rights reserved.
Keywords: Biosensor; Nanoparticles Pt; Chitosan; Gold colloid; l-Cysteine; Hb

1. Introduction Determination of hydrogen peroxide is of practical importance in clinical, environmental, and many other elds [1,2]. Hydrogen peroxide has been determined by titrimetry, spectrophotometry, and chemiluminescence methods that tend to be complex, time-consuming, and suffer from various interferences. The sensitive determination of hydrogen peroxide may be achieved by the use of Hb-modied electrodes [3], since Hb are known to show excellent selectivity for their substrates. The fabrication of H2 O2 sensors has attracted great interest as the measurement of H2 O2 is the basis of detecting many biologically active materials such as glucose and cholesterol. The performance of an electrode often depends on the method of Hb immobilization when fabricating biosensors [4]. There-

Corresponding author. Tel.: +86 23 68252277; fax: +86 23 68254000. E-mail address: yuanruo@swu.edu.cn (R. Yuan).

fore, the technique used to immobilize the Hb is one of the key factors in developing a reliable biosensor. Organicinorganic composite (or hybrid) materials have emerged in recent years [5]. It combines the physicochemical attributes of components and improves their features. Organic components benet the formation of defect-free inorganic membranes and make it less brittle. Organic membranes can have their chemical and thermal stability improved by an inorganic phase [6,7]. Because chitosan has excellent lm forming and adhesion ability, together with non-toxicity and biocompatibility, it has gained growing interest in using it to immobilize biomolecules in recent years [8]. Platinum plays an important role in many applications because of its unique optical, physical and chemical properties [9]. In particular, its stability in electrochemical reactions and its prominent catalytic activities are most attractive. Many areas use platinum in small dimensions, mostly in particles [10]. Electrochemical study of the third generation biosensor based on the direct electron transfer between the protein and the electrode [1114] has been paid more and more attention because

0927-7765/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2007.07.014

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Scheme 1. Preparation process of the Hb/nano-Au/l-cys/nano-Au/nano-PtCHIT composite lm-modied Pt electrode.

of its potential application in the study of the redox and electron transfer properties of biomolecules. Recently, We used nano-Au and multi-wall carbon nanotubes to immobilize protein to prepare biosensor for detection of H2 O2 , but the sensitivity needed to be provided [15,16]. In this work, we extended our methodology by using of the important material nano-PtCHIT as immobilization matrix and selecting Hb as a model simulation enzyme in the way of self-assembly technology to get high-sensitive biosensor for detection of H2 O2 . First, the nano-PtCHIT inorganicorganic composite lm containing NH2 group with positive charge were formed on Pt electrode. Secondly, negatively charged nanoAu were immobilized on the nano-PtCHIT composite lm based on electrostatic interaction between oppositely charged species. Then, l-cys was assembled on the surface of the nanoAu based on the strong afnity between the mercapto (-SH) and nano-Au and electrostatic adsorption. In the meantime, seconed nano-Au layer were immobilized on the surface of the electrode via NH2 groups of l-cys. Nano-Au has large specic surface area and good biocompatibility, furthermore, it can act as tiny conduction centers which facilitate the transfer of electrons [1719]. Finally, Hb was adsorbed onto the nano-Au layer to obtain hydrogen peroxide biosensor for the detection of H2 O2 (see Scheme 1). The biosensor obtained shows electrocatalytic activity, good accuracy, high sensitivity, good repeatability, long-term stability, and a low cost. 2. Experimental 2.1. Apparatus and chemicals Bovine (horse heart) hemoglobin, chitosan (MW1 106 ; 80% deacetylation), hydrogen hexachloroplatinate(IV) hydrate (H2 PtCl6 ), gold chloride tetrahydrate and sodium citrate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). l-cys were obtained from Shanghai Chemical Company, China. They were used directly without further purication. A stock solution of 10 mg/mL Hb was freshly prepared with 0.1 M phosphate buffer solutions (pH 6.0). Hydrogen peroxide (30%, w/v solution) were purchased from Chemical Reagent Company, Chongqing, China. The

concentration of the more diluted hydrogen peroxide solutions prepared from 30% hydrogen peroxide was determined by titration with potassium permanganate. The supporting electrolyte was a 0.1 M phosphate buffer solution (PBS) containing 0.1 M KCl, which was prepared with KH2 PO4 and Na2 HPO4 . All other chemicals employed were of analytical grade and used as received, doubly distilled water was used throughout the experiments. Gold nanoparticles with mean size of 16 nm were produced by reducing gold chloride tetrahydrate with citric acid at 100 C for half an hour [20] (the graph not shown). Electrochemical measurements were carried out on CHI 660A electrochemical workstation (CH Instruments, Chenhua Co., Shanghai, China). The impedance measurement unit (IM6e, ZAHNER elektrik, Germany) was equipped with THALES software v6.88. A conventional three-electrode system was employed with a modied platinum disk electrode (2.0 mm in diameter) as a working electrode, a saturated calomel electrode (SCE) as a reference electrode, and a platinum wire as an auxiliary electrode. All the potentials given in this paper were referred to the SCE. The size of nano-Pt and nano-Au was estimated from transmission electron microscopy (TEM, TECNAI 10, PHILIPS FEI Co., Holland). The morphologies of nano-PtCHIT composite lm, the nano-Au/nano-PtCHIT composite lm and the Hb/nano-Au/nano-PtCHIT composite lm were investigated with scanning electron microscopy (SEM, S4800, HITACHI Co., Japan). X-ray photoelectron spectroscopy (XPS) measurements were carried out using a VG Scientic ESCALAB 250 spectrometer, using Al K X-ray (1486.6 eV) as the light source. The experimental solutions were deaerated by highly pure nitrogen for 10 min, and a nitrogen atmosphere was kept over the solution during measurements. EIS measurements were carried out in a background solution of 5 mmol/L K3 Fe(CN)6 + K4 Fe(CN)6 phosphate buffer (pH 7.0) at a normal potential. The alternating voltage was 5 mV and the frequency range was 5.0 102 to 1.0 105 Hz. 2.2. Preparation of PtCHIT solution A 0.5 wt.% CHIT solution was prepared by dissolving 50 mg of CHIT akes into 10 mL of 1.0% acetic acid and stirred for 3 h at room temperature until complete dissolution. The pH was

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Fig. 1. TEM micrograph of the nano-PtCHIT sol.

adjusted to 3.04.0 using a concentrated NaOH solution, followed by the addition of 15 mL of 0.01 M H2 PtCl6 , and the solution was then sonicated for 10 min. The Pt(IV) was then reduced by using 0.05 mL of freshly prepared 5% NaBH4 [21]. The TEM image in Fig. 1 depicts the production of high concentration Pt nanoparticles. The particles have an average diameter of 10 nm and are well distributed throughout the lm. 2.3. Preparation of hydrogen peroxide biosensor The bare platinum disk electrode was polished successively with 0.3 and 0.05 m Al2 O3 before modication, rinsed with double-distilled water, and sonicated in ethanol and doubledistilled water for 5 min, respectively. Then 2 l nano-PtCHIT solution was spread on the Pt electrode. After that, the electrode was dried in air, and immersed in a solution of gold colloid about 6 h. Following that, it was soaked in solution of l-cys (pH 2.3 PBS) for 6 h. Subsequently, the resulting electrode was immersed in gold colloid solution about 10 h. At last, the electrode was immersed in Hb solution (20 mg/ml, 0.1 mol/L pH 6.0 PBS) for 12 h to obtain the hydrogen peroxide biosensor, which was stored in a refrigerator (4 C) until further use. 3. Results and discussion 3.1. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) characterization of self-assembly process It is well known that electrochemical impedance spectroscopy is an effective tool for studying the interface properties of surface-modied electrodes. The typical impedance spectrum (presented in the form of the Nyquist plot) includes a semicircle portion at higher frequencies corresponding to the electrontransfer-limited process and a linear part at lower frequency range representing the diffusion limited process. The semicircle diameter in the impedance spectrum equals the electron-transfer resistance, Ret . This resistance controls the electron-transfer

kinetics of the redox probe at the electrode interface. Therefore, Ret can be used to describe the interface properties of the electrode. Its value varies when different substances are adsorbed on the electrode surface [22]. In this paper, the electrochemical impedance studies of the modied electrode were performed and the results are shown in Fig. 2. It can be seen that curve a have a small semicircle, implying very low Ret bare electrode to the redox probe dissolved in the electrolyte solution. After nano-PtCHIT composite lm was covered onto the Pt electrode, the diameter of the semicircle decreased (curve b), showing that the composite lm promoted the electron transfer of the electrochemical probe. This may be ascribed to the excellent conductivity of the nano-Pt and the electrostatic force between negatively charged Fe(CN)6 4/3 and the positively charged CHIT. When gold colloid was assembled on the lm (curve c), we found that the diameter of the semicircle increased. The reason may be that electrostatic force between negatively charged Fe(CN)6 4/3 and the negatively charged nano-Au. With the l-cys adsorption on nano-Au layer (curve d), the interfacial resistance decreased again, suggesting l-cys can promote the electrons transfer [23]. Moreover, after adsorption of Au colloid again (curve e), the interfacial resistance increased. At the last step of assembly, Hb was tied to the nano-Au (curve f), Ret increased again. This increase is attributed to the non-conductive properties of Hb, which would obstruct electron transfer of the electrochemical probe. The above results could clearly conrm the success of the assembly of the electrode. The cyclic voltammetry of ferricyanide is also a valuable and convenient tool to monitor the barrier of the modied electrode, because the electron transfer between the solution species and the electrode must occur by tunneling either through the barrier or through the defects in the barrier. Therefore, it was chosen as a marker to investigate the changes of electrode behav-

Fig. 2. EIS of (a) bare platinum disk electrode, (b) nano-PtCHIT composite lm-modied Pt electrode, (c) nano-Au/nano-PtCHIT composite lm-modied Pt electrode, (d) l-cys/nano-Au/nano-PtCHIT composite lmmodied Pt electrode, (e) nano-Au/l-cys/nano-Au/nano-PtCHIT composite lm-modied pt electrode, and inset: (f) modied electrode in a background solution of 5mM [Fe(CN)6 ]4/3 PBS (0.1 M, pH 7.0). The frequency range was 5.0 102 to 1.0 105 Hz at 25 C (Zre vs. Zim at 220 mV vs. SCE).

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ior after each assembly step. The experiment further conrmed that the Au colloid and Hb were successfully assembled on the platinum disk surface. Fig. 3 shows cyclic voltammograms (CVs) of differently modied electrodes in a 5 mM ferricyanide solution. Well-dened CVs, characteristic of a diffusion-limited redox process, are observed at the bare platinum disk electrode (curve a). A further increase in peak current is observed (curve b) after nanoparticles PtCHIT composite lm was covered onto the electrode. In contrast, a peak current decrease was observed at the nano-Au/nano-PtCHIT composite lmmodied Pt electrode (curve c). After l-cys were assembled on the nano-Au-modied electrode, the current increased, as shown in curve d. When nano-Au was adsorbed on the electrode surface again, a remarkable current decrease was observed as in curve e. With the Hb was assembled onto the nano-Au (curve f), current decrease again. There are reasons as same as the EIS. On the basis of the EIS and CV results, we can conclude that Hb is successfully immobilized on the self-assembled gold nanoparticles. 3.2. Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy The surface topology of the nano-PtCHIT composite lm, the nano-Au/nano-PtCHIT composite lm and the Hb/nanoAu/nano-PtCHIT composite lm were investigated using

Fig. 3. Cyclic voltammograms of the different electrodes in 5 mM [Fe(CN)6 ]4/3 solution (pH 7.0). (a) Bare platinum disk electrode, (b) nanoPtCHIT composite lm-modied Pt electrode, (c) nano-Au/nano-PtCHIT composite lm-modied Pt electrode, (d) l-cys/nano-Au/nano-PtCHIT composite lm-modied Pt electrode, (e) nano-Au/l-cys/nano-Au/nano-PtCHIT composite lm-modied pt electrode, and (f) modied electrode. The scan rate was 50 mV s1 .

Fig. 4. SEM images of (A) nano-PtCHIT composite lm, (B) nano-Au/nano-PtCHIT composite lm and (C) Hb/nano-Au/nano-PtCHIT composite lm.

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scanning electronic microscope (SEM) with images shown in Fig. 4. It can be seen in Fig. 4A that the particles of Pt have an average diameter of 10 nm and are well distributed. When gold colloid was assembled on the lm (Fig. 4B), the amount of nanoparticles increasal, implying the nano-Au were immobilized on the surface of the electrode. After Hb was assembled onto the nano-Au (Fig. 4C), a considerably change in morphology has been observed, which demonstrated a lot of Hb immobilized on the nano-Au/nano-PtCHIT composite lm surface. The Hb/nano-Au/nano-PtCHIT composite lm has been further investigated by XPS determination (Fig. 5). A characteristic Pt 4f peak was observed at about 74.3 eV, suggesting that Pt present in composite lm. The Au 4f spectrum consists of two peaks located at 83.7 and 87.4 eV, corresponding to the Au 4f7/2 and Au 4f5/2 , both distinctive for Au metal. Indicating that gold nanoparticle was adsorbed on the composite lm. We utilizing of XPS spectra to gain a ratio of weight of C to that of N is 5.62 that between the ratio of CHIT and Hb. Which demonstrate Hb immobilized on the composite lm surface. 3.3. Electrochemical characteristics of hydrogen peroxide biosensor The CVs of modied electrode measured in PBS (pH 7.0) at different scan rates show in Fig. 6. Here, cathodic peak currents increase linearly with square of root of scan rate in the range of 10250 mV/s, as shown in the inset of Fig. 6. Indicating the reaction is a diffusion controlled process. The dependence of the electrochemical behavior of modied electrode on the pH of the working solution was studied. The CVs of modied electrode in PBS at various pH values ranged from 5.0 to 9.0 are shown in Fig. 7. It is found that an increase of pH in solution leads to a negative shift in potential of cathodic peak for modied electrode. The cathodic peak potentials has a linear relationship with pH (from 5.0 to 9.0) with the slopes of 52.0 mV/pH is shown in Fig. 6A (inset in right of Fig. 7), this value is approximately close to the theoretical value of 57.6 mV/pH at 18 C for a reversible, one-proton-coupled single electron transfer.

Fig. 6. Cyclic voltammograms of the biosensor at different scan rates (from a to h): 20, 50, 80, 100,120, 150, 200, and 250 mV/s in 5 ml 0.1 mol/L PBS (pH 7.0) under room temperature. Inset: plots of cathodic peak currents vs. v1/2 .

We compared the chronoamperometry response of differently modied electrodes with successive injections of 3.5 104 mol/L H2 O2 to the PBS (pH 7.0) at applied potential of 30 mV. The platinum disk electrode covered with only nano-PtCHIT composite lm exhibited a small amperometric response to hydrogen peroxide, as shown in Fig. 8a. According to Fig. 8b, for Hb/nano-PtCHIT composite lm-modied platinum disk electrode, a large response to H2 O2 was observed. Furthermore, nano-Au can act as tiny conduction centers which facilitate the transfer of electrons and nano-Au can be rmly tied to nano-PtCHIT composite lm-modied platinum disk electrode, so the current response increased in Fig. 8c. When Hb was chemisorbed on the nano-Au/nano-PtCHIT composite lm-modied electrode surface, current response to H2 O2 increased again, as shown in Fig. 8d. However, it has maximal response to H2 O2 when Hb was chemisorbed on the two layers nano-Au lm, as shown in Fig. 8f. This may be ascribed to

Fig. 5. XPS spectra of Hb/nano-Au/nano-PtCHIT composite lm. Inset: narrow scan spectrum of Au 4f (up) and Pt 4f (down) peaks.

Fig. 7. CVs of modied electrode in PBS at various pH values: (a) 5.0, (b) 6.0, (c) 7.0, (d) 8.0, and (e) 9.0 at scan rate of 50 mV/s. Inset in right: effect of pH on cathodic peak potential of composite-modied electrode in 0.1 M PBS. Inset in left: The inuence of pH on the response of the biosensor in 0.1 M PBS containing 3.5 104 M H2 O2 .

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3.4. Inuence of pH and temperature on biosensor response In order to obtain an efcient biosensor for H2 O2 , the inuence of pH on the response of modied electrode were investigated. The change of chronoamperometric current with the pH under constant hydrogen peroxide concentration (3.5 104 M) is shown in Fig. 7B (inset in left of Fig. 7). As can be seen, the current response increased from pH 5.0 to 9.0. But considered that Hb in strong acidic or alkaline solution may be denaturation, therefore, a pH 7.0 of the working buffer was selected. The effect of temperature on the performance of biosensor was investigated using constant concentration (3.5 104 mol/L) of H2 O2 in 0.1 mol/L PBS (pH 7.0) at the range from 15 to 60 C (gure not shown). It was observed that the cyclic voltammetry peak current increased with the temperature up to 40 C, the further increasing temperature led to a decrease of the peak current because of the partial denaturation of Hb. Considering both the lifetime and response characteristics of the biosensor, the room temperature (25 C) was employed as the optimum temperature in detection of hydrogen peroxide. 3.5. Electrochemical response to hydrogen peroxide Fig. 10 showed the cyclic voltammetric behavior of the hydrogen peroxide biosensor. An obvious increase of the cathodic peak current was observed when 4.3 106 mol/L H2 O2 was added to the PBS (curve b), indicating that a electron transfer reaction between the Hb and the electrode surface was achieved. With the addition of H2 O2 , cathodic peak current increased signicantly (curves bh), which indicated that the Hb kept its natural structure and eletrocatalytic property to H2 O2 after immobilized onto the electrode. We used the cathodic peak current to plot with the concentration of H2 O2 . There is a linear relation of the current with concentration of H2 O2 between 1.4 107 and 6.6 103 mol/L, as shown in the inset of Fig. 8. The linear regression equation is i( A) = 10.31

Fig. 8. The chronoamperometry response at applied potential of 30 mV with injection of 3.5 104 mol/L H2 O2 into 5 mL of stirring pH 7.0 PBS for (a) nano-PtCHIT composite lm-modied Pt electrode, (b) Hb/nano-PtCHIT composite lm-modied Pt electrode, (c) nano-Au/nano-PtCHIT composite lm-modied Pt electrode, (d) Hb/nano-Au/nano-PtCHIT composite lmmodied Pt electrode, (e) modied electrode, respectively.

the l-cys can promote the electrons transfer and more nano-Au can chemisorb more Hb. And the biosensor achieved 95% of response within 10s, the biosensor responded rapidly to the substrates due that nano-Au and l-cys can act as tiny conduction centers which facilitate the transfer of electrons. We also compared the cyclic voltammetry response of differently modied electrodes with injections of 3.5 104 mol/L H2 O2 to the PBS (pH 7.0) (Fig. 9). The Hb/nano-PtCHIT composite lm-modied platinum disk electrode response to hydrogen peroxide compared with Hb/nano-Au/nano-PtCHIT composite lm-modied platinum disk electrode response to hydrogen peroxide is smaller. When the lms contain l-cys response to hydrogen peroxide increased again. But our modied electrode response to hydrogen peroxide is maximal. There are reasons as same as the chronoamperometry.

Fig. 9. Cyclic voltammograms of the different electrodes in 0.1 M pH 7.0 PBS contain 3.5 104 M H2 O2 . (a) Hb/nano-PtCHIT composite lmmodied Pt electrode, (b) Hb/nano-Au/nano-PtCHIT composite lm-modied Pt electrode, (c) Hb/l-cys/nano-Au/nano-PtCHIT composite lm-modied Pt electrode, (d) modied electrode.

Fig. 10. Cyclic voltammograms of the biosensor in 0.1 M pH 7.0 PBS (curve a) and in the presence of 4.3 106 M H2 O2 (curve b), 6.3 104 M H2 O2 (curve c), 1.3 103 M H2 O2 (curve d), 2.4 103 M H2 O2 (curve e), 3.8 103 M H2 O2 (curve f), 5.2 103 M H2 O2 (curve g), and 6.6 103 M H2 O2 (curve h). Scan rate: 100 mV/s. Inset: the calibration curve of the biosensor.

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to 17.62[H2 O2 ] (mM) with a correlation coefcient of 0.999 (n = 24). From the slope of calibration curve, the detection limit of 4.5 108 mol/L is estimated at signal-to-noise ration of three. MichaelisMenten constant (K) give an indication of the protein-substrate kinetics, can be obtained from the electrochemical version of the LineweaverBurk equation [24]: 1 1 K = + ISS Imax Imax C where ISS is the steady-state current after the addition of substrate, which can be obtained from amperometric experiments (Fig. 8e), Imax the maximum current under saturated substrate condition and C is the bulk concentrate of the substrate. The value of the apparent MichaelisMenten constant (K) can be calculated from the slope (K/Imax ) and the intercept (1/Imax ) for the plot of the reciprocals of the steady-state current (1/ISS ) versus H2 O2 concentration (1/C).The K value for the modied electrode is estimated to be 0.12 mM. The low value of K in composite lm retains its bioactivity and has a high biological afnity to H2 O2 . 3.6. Repeatability and stability of the hydrogen peroxide biosensor The repeatability of the present biosensor was examined at a hydrogen peroxide concentration of 3.5 104 mol/L. The electrode showed an acceptable repeatability with a relative standard deviation (R.S.D.) of 2.5% for nine successive assays. The proposed biosensor shows high stability. When the 100 continuous cyclic scans was carried out in the potential range from 0.6 to 0.3 V with a 100 mV/s scan rate, only 6% decrease of the initial response is observed. On the other hand, the storage stability of the proposed biosensor was also studied. When not in use, the electrode was suspended above 0.1 M phosphate buffer at 4 C in a refrigerator. The response to 3.5 104 mol/L hydrogen peroxide was tested intermittently. The biosensor lost only 4.5% of the initial response after 2 weeks and maintained more than about 70% of the initial values after storage for more than 1 month. 3.7. Selectivity of the hydrogen peroxide biosensor Selectivity is also important in practical use of the biosensors. In the experiment, six interfering substances were used to evaluate the selectivity of the biosensor. The results are listed in Table 1. The current ratios were calculated by reading the current of the biosensor in the assay solution containing 3.5 104 mol/L H2 O2 and 7.0 104 mol/L interfering substance and comparing it with the current from the biosensor in the same assay solution containing only 3.5 104 mol/L H2 O2 . Accordingly, the degree of interference from the substances can be judged from the current ratios. In our experiments, the six tested substances did not interfere signicantly with the resulting biosensor. This was largely due to the low-working potential (about 30 mV), no electrochemical reactions occurred at this potential.

Table 1 Possible interferences tested with the biosensor Possible interferences Glucose Ethanol Acetic acid Ascorbic acid l-Tyrosine l-Cysteine Current ratio 0.99 0.98 1.03 1.04 0.97 0.96

Table 2 H2 O2 recoveries at various concentrations, determined with the biosensor Added (mM) 0.080 0.15 0.56 1.20 2.50 5.00
a

Founda (mM) 0.078 0.147 0.57 1.26 2.45 4.94

Recovery (%) 97.5 98.0 101.8 105.0 98.0 98.8

The average of three measurements.

3.8. Recovery experiment The analytical applicability of the biosensor was evaluated by determining the recoveries of six H2 O2 samples with different concentrations by standard addition method. The results were satisfactory with an average recovery of 99.8%, as listed in Table 2. 4. Conclusions In this paper, we have introduced a novel method for fabrication of an amperometric hydrogen peroxide biosensor. During fabrication of the biosensor, Hb, as a model protein, was adsorbed successfully on nano-Au/l-cys/nano-Au/nanoPtCHIT composite lm-modied platinum disk electrode. The biosensor showed an excellent sensitivity and fast response time (10 s) for detection of H2 O2 in 0.1 mol/L PBS (pH 7.0), with a wide linear response range from 1.4 107 to 6.6 103 mol/L. Acknowledgements Financial support for this work was provided by the National Natural Science Foundation of China (20675064), the Natural Science Foundation of Chongqing City (CSTC-2004BB4149, 2005BB4100) and High Technology Project Foundation of Southwest University (XSGX 02), China. References
[1] J. Wang, Y. Lin, L. Chen, Analyst 118 (1993) 277. [2] J. Kulys, L. Wang, A. Maksimoviene, Anal. Chim. Acta 274 (1993) 53. [3] J. Yang, N.F. Hu, Bioelectrochem. Bioenerg. 48 (1999) 117. [4] Y. Liu, T. Yu, Chem. Phys. C37 (1997) 459. [5] X. Chen, S.J. Dong, Biosens. Bioelectron 18 (2003) 999. [6] M.Ah. Kim, W.Y. Lee, Anal. Chim. Acta 479 (2003) 143.

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G. Yang et al. / Colloids and Surfaces B: Biointerfaces 61 (2008) 93100 [16] Sh.H. Chen, R. Yuan, Y.Q. Chai, L.Y. Zhang, N. Wang, X.L. Li, Biosens. Bioelectron. 22 (2007) 1268. [17] L. Wang, E. Wang, Electrochem. Commun. 6 (2004) 225. [18] D.P. Tang, R. Yuan, Y.Q. Chai, X. Zhong, Y. Liu, J.Y. Dai, Biochem. Eng. J. 22 (2004) 43. [19] S.Y. Xu, X.Z. Han, Biosens. Bioelectron 19 (2004) 1117. [20] B.V. En st n, J. Turkevich, J. Am. Chem. Soc. 85 (1963) 3317. u u [21] M.H. Yang, Y. Yang, H.F. Yang, G.L. Shen, R.Q. Yu, Biomaterials 27 (2006) 246. [22] Y.J. Liu, F. Yin, Y.M. Long, Z.H. Zhang, S.Z. Yao, Colloid Interface Sci. 258 (2003) 75. [23] E. Ferapontova, K. Schmengler, T. B rchers, T. Ruzgas, L. Gorton, Biosens. o Bioelectron. 17 (2002) 953. [24] R.A. Kamin, G.S. Wilson, Anal. Chem. 52 (1980) 1198.

[7] G. Wang, J.J. Xu, H.Y. Chen, Z.H. Lu, Bioelectronics 18 (2003) 335. [8] J.M.C.S. Magalhaes, A.A.S.C. Machado, Talanta 47 (1998) 183. [9] M.H. Yang, F.L. Qu, Y.Sh. Lu, Y. He, G.L. Shen, R.Q. Yu, Biomaterials 27 (2006) 5944. [10] M. Sasaki, M. Osada, N. Higashimoto, T. Yamamoto, A. Fukuoka, M. Ichikawa, J. Mol. Catal. A: Chem. 141 (1999) 223. [11] A.L. Ghindilis, P. Atanasov, E. Wilkins, Electroanalysis 9 (1997) 661. [12] L. Gorton, A. Lindgren, T. Larsson, F.D. Munteanu, T. Ruzgas, I. Gazaryan, Anal. Chim. Acta 400 (1999) 91. [13] F.A. Armstrong, G.S. Wilson, Electrochim. Acta 45 (2000) 2623. [14] J.F. Rusling, Acc. Chem. Res. 31 (1998) 363. [15] Y. Liu, R. Yuan, Y.Q. Chai, D.P. Tang, J.Y. Dai, X. Zhong, Sens. Actuators B 115 (2006) 109.