Hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase onto a poly(aniline-co-N-methylthionine) film

Synthetic Metals 212 (2016) 123–130

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Hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase onto a poly(aniline-co-N-methylthionine) film Chuanxiang Chen* , Xiaozhang Hong, Tingting Xu, Ankang Chen, Lin Lu, Yuhua Gao School of Environmental and Chemical Engineering, Jiangsu University of Science and Technology, 2 Mengxi Road, Zhenjiang 212003, People’s Republic of China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 August 2015 Received in revised form 3 December 2015 Accepted 11 December 2015 Available online xxx

In this present work, we developed a novel hydrogen peroxide (H2O2) biosensor fabricated using electrochemical doping to immobilize hydrogen peroxidase (HRP) in a new conducting polymer, poly (aniline-co-N-methylthionine) (PAN-PNMThH). Amperometric detection of H2O2 was evaluated by holding the PAN-PNMThH HRP electrode at 0.25 V (versus saturated calomel electrode (SCE)). PANPNMThH showed excellent redox activity and high porosity and acted as an electron transfer mediator. The biosensor had a wide linear response range from 5.0 mM to 60.0 mM H2O2 with a sensitivity of 35 mA M 1 cm 2, a detection limit of 3.2 mM (signal-to-noise ratio of 3) and an apparent Michaelis constant (KM) of 2.79 mM. The biosensor possessed good analytical performance and storage stability. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Hydrogen peroxide Hydrogen peroxidase Biosensor Poly(aniline-co-N-methylthionine)

1. Introduction Nowadays, some polymers are replacing traditional materials in many applications due to their low density, low cost, and specific properties [1–3]. Over the last few decades, conducting polymers, such as polyaniline and polypyrrole, have become of increasing interest for their novel optical, electrical, and electrochemical properties and promising applications in secondary batteries, supercapacitors, electrochromic devices, anticorrosive coatings, electrochemical sensors, and biosensors [4–6]. Advantages of using the conducting polymers in biosensors are acceleration of charge transfer, impressive signal amplification, and elimination of electrode fouling [7]. A conducting polymer film can be directly electrodeposited on a solid electrode surface. The film adheres strongly to the electrode surface, and its thickness and characteristics can be easily controlled from the charge consumed during the electropolymerization [8]. Moreover, the film offers an appropriate environment for immobilization of enzymes [9]. Thus, these polymers have been widely employed as enzyme immobilization materials in biosensors. Three methods for the enzyme immobilization with a conducting polymer are commonly used [10], namely, electrochemical entrapment, covalent bonding, and electrochemical doping. An important advantage of the electrochemical doping in a biosensor fabrication is that the enzyme immobilization can be

* Corresponding author. Fax: +86 511 85635850 E-mail address: [email protected] (C. Chen). http://dx.doi.org/10.1016/j.synthmet.2015.12.012 0379-6779/ ã 2015 Elsevier B.V. All rights reserved.

performed under a mild condition, which has no influence on the enzyme nature [11]. Fast and easy determination of hydrogen peroxide (H2O2) is of practical importance in clinical, pharmaceutical, biochemical, environmental and food analysis [12]. H2O2 has been determined by titrimetry, volumetry, colorimetry, chemiluminescence, and pectrophotometry. However, these methods generally suffer from various interferences and are complicated and time-consuming [8,13]. The sensitive determination of H2O2 can be achieved with the use of peroxidase enzyme-modified electrodes, since the enzymes possess excellent selectivity and high sensitivity [14]. Currently, numerous efforts have been made to develop the enzyme-based biosensors for the detection of H2O2, which is the basis of the measurement of many small biological molecules like glucose [15,16], choline [10,17], and cholesterol [18,19]. It is known that the optimum pH for most enzymes usually ranges from 4.0 to 10.0 [20]. However, this pH range is not suitable for polyaniline because it almost loses its electroactivity in solutions of pH greater than 4.0 [21–23]. Therefore, it is necessary to improve the electroactivity of polyaniline at high pH values through structural modifications. An effective approach to its structural modifications is the copolymerization of aniline with a suitable monomer. Methylene blue and other phenothiazine dyes have been commonly used as redox mediators in sensors and biosensors [24,25]. However, they can easily diffuse away from the electrode surface into the solution bulk during the continuous measurement, which would result in great signal loss and significantly affect the performance of the biosensor. The electropolymerized polymeric

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2. Experimental 2.1. Reagents and solutions N-Methylthionine and HRP (EC 1.11.1.7, type VI, 250–300 units mg 1) were bought from Sigma–Aldrich (Shanghai, China). H2O2 (30%, v/v) was purchased from Beijing Beihua Fine Chemicals (Beijing, China). Ascorbic acid, uric acid, ethanol, sodium chloride, hydrochloric acid, sodium hydroxide, sodium dihydrogen phosphate, and disodium phosphate were obtained from Sinopharm Chemical Reagent Corporation (Shanghai, China) and were of analytical grade. Aniline was distilled before use. All the solutions were prepared using deionized water. The phosphate buffer solution (PBS) was prepared using 0.20 M sodium dihydrogen phosphate, 0.20 M disodium phosphate and 0.20 M sodium chloride and adjusting the pH values with hydrochloric acid or sodium hydroxide, and fresh solutions of hydrogen peroxide were prepared daily. 2.2. Apparatus All electrochemical experiments were carried out using a CHI 660C electrochemical workstation (Chenhua, Shanghai, China). Cyclic voltammetry and chronoamperometry measurements were performed in a conventional three-electrode cell with a platinum foil and a saturated calomel electrode (SCE) as counter and reference electrodes, respectively. Another platinum foil was used as a working electrode. The area of each platinum foil was 4 mm  4 mm. All potentials were quoted relative to the SCE. The pH values of solutions were determined using a PHS-3C pH meter (Rex, Shanghai, China). 2.3. Electrosynthesis of PAN-PNMThH

Fig. 1. SEM images of PAN-PNMThH (A) and the copolymer HRP electrode polyaniline (B).

dye film is able to efficiently overcome these problems [26]. Furthermore, such a polymer film with a three-dimensional distribution of redox mediators is preferable to monolayer of the dye monomer because of the much larger catalytic response of the polymer due to the volume effect [27]. In recent years, considerable attention has been focused on tailoring the structure, morphology and properties of these polymers to achieve new improved biosensors based on polyphenothiazine dyes [25]. Recently, three-dimensional flower-like microparticles of a new conducting polymer, poly(aniline-co-N-methylthionine) (PANPNMThH), were electrosynthsized in our laboratory [28]. The copolymer, which contains phenothiazine units, exhibits a high electroactivity in acidic, neutral and basic solutions, which is favorable to enhancing the redox activity and charge transfer ability of the polymer at high pH. Taking into account that polythionine and other polyphenothiazine dyes can serve as redox mediators for the electrochemical sensors and biosensors [26–30], the use of the copolymer as a mediator would be of special interest for biosensing application. In this study, we used PAN-PNMThH to immobilize horseradish peroxidase (HRP) and studied effects of some experimental variables such as applied potential, pH, and temperature on the bioelectrochemical response of the hydrogen peroxide biosensor.

PAN-PNMThH was synthesized using cyclic voltammetry in a potential range from 0.20 to 1.15 V at a scan rate of 60 mV s 1 for 100 cycles in a 0.20 M pH 2.0 PBS containing 0.10 M aniline and 2.5 mM N-methylthionine [28]. After electrodeposition, the PANPNMThH film was thoroughly rinsed with deionized water to remove unreacted monomer. 2.4. Fabrication of the PAN-PNMThH HRP electrode Enzyme immobilization on the surface of a conducting polymer modified electrode was performed using the electrochemical doping as described elsewhere [31–33]. The PAN-PNMThH modified electrode was first immersed in a 0.20 M pH 6.8 PBS and reduced at a potential of 0.60 V for 30 min until a steady state was achieved. Then, the reduced copolymer modified electrode was immediately moved into a solution of HRP at a potential of 0.60 V for 30 min. The enzyme solution is a 0.20 M pH 6.8 PBS containing 0.1 mg ml 1 HRP. Since the isoelectric point of HRP is 5.5 [32], the HRP carries a negative charge in the PBS. Under the electrostatic interaction, the negatively charged HRP was doped into the positively charged copolymer film to fabricate a conducting polymer enzyme electrode. The copolymer HRP electrode was rinsed thoroughly with de-ionized water to remove any enzyme not tightly bound to the copolymer, and used immediately or kept in 0.20 M phosphate buffer solution (pH 7.0) at 4  C if not in use. 2.5. UV–vis spectra The UV–vis spectra of HRP solutions were recorded on a U-3010 UV–vis spectrometer (Hitachi, Japan). The amount of HRP incorporated within the copolymer was determined by comparing

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0.3

125

2.7. Electrochemical measurements

A

1 The cell used for the electrocatalytic reduction of hydrogen peroxide consisted of the copolymer HRP electrode, platinum foil, and SCE as the working, counter, and reference electrodes, respectively. Cyclic voltammetry and chronoamperometry were used to measure the responses of the biosensor to H2O2. All experiments were performed at room temperature (unless stated otherwise) under aerobic conditions.

2 Abs

0.2

0.1

3. Results and discussion

0.0 300

3.1. Scanning electron microscopy

400

500

600 The surface morphologies of the PAN-PNMThH film and the copolymer HRP electrode were measured with SEM. The SEM micrograph of the PAN-PNMThH film shows complete grain coalescence with large micro-/nanoparticles (Fig. 1A). The thickness of the copolymer film is about 0.4 mm, as determined by SEM measurement (inset of Fig. 1A), After enzyme immobilization, the roughened surface changes to a image consisting of smaller nanoparticles (Fig. 1B). These differences indicates that HRP can be effectively immobilized on the copolymer film. Therefore, the HRP enzyme is located on the surface of the polymer film.

Wavelength / nm

0.3

B

Abs

0.2

0.1

0.0 0.00

0.02

0.04

0.06

0.08

0.10

HRP concentration / (mg ml-1)

A 0.0

0.2

I / mA

Fig. 2. UV–vis spectra of HRP solutions before (1) and after (2) incorporation into the copolymer (A) and calibration curve plotting the absorbance at 403 nm versus the HRP concentration (B).

1 2

0.0 3 I / mA

-0.1

1

-0.2

2 1

-0.2 -0.6

-0.4

-0.2

-0.4

0.0

0.2

0.4

E / V (vs. SCE)

-0.6 -0.6

-0.4

-0.2

0.0

0.2

0.4

0.00

B

E / V (vs. SCE)

-0.05

1

I / mA

Fig. 3. Cyclic voltammograms of the bare platinum foil (1), PAN-PNMThH film (1) and PAN-PNMThH HRP electrode (2) in a pH 6.0, 0.20 M PBS. Scan rate: 60 mV s 1.

-0.10 the UV absorbance at 403 nm for the HRP solution before and after immobilization. 2.6. Morphologies The scanning electron microscopy (SEM) images of the copolymer film and copolymer HRP electrode were characterized using an S4800 field-emission SEM (Hitachi, Japan). Both samples were sputter-coated with platinum prior to SEM observation.

2 -0.15 -0.6

-0.4

-0.2

0.0

0.2

0.4

E / V (vs. SCE) Fig. 4. Cyclic voltammograms of the PAN-PNMThH film (1) and PAN-PNMThH HRP electrode (2) in a pH 6.0, 0.20 M PBS containing 10.0 mM H2O2. Scan rates: 60 (A) and 10 mV s 1 (B).

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0.0 1

I / mA

-0.1 2 -0.2

-0.3

3 -0.6

-0.4

-0.2

0.0

0.2

0.4

E / V (vs. SCE) Fig. 5. Cyclic voltammograms of the PAN-PNMThH HRP electrode in a pH 6.0, 0.20 M PBS with 0.0 (1), 10.0 (2), and 20.0 (3) mM H2O2. Scan rate: 60 mV s 1.

3.2. UV–vis spectra In order to the loading density of HRP incorporated within the PAN-PNMThH film, the HRP solution was studied using UV–vis spectrometer. As shown in curve 1 in Fig. 2A, an obvious absorption band occurs at 403 nm in a 0.20 M pH 6.8 PBS containing 0.1 mg ml 1 HRP, corresponding to the heme band of HRP. The absorption intensity of the HRP solution after enzymatic immobilization is lower than that before HRP immobilization. According to a calibration curve of different concentration HRP (Fig. 2B) and the absorption value in the UV–vis spectrum line of the HRP solution after enzymatic immobilization (curve 2 in Fig. 2A), the concentration of HRP incorporated within the copolymer film can be obtained, and then the mass weight of HRP is calculated to be 0.014 mg/mg (HRP wt./PAN-PNMThH wt.). 3.3. Cyclic voltammetric characterization of the biosensor Fig. 3 showed the cyclic voltammograms of bare platinum (1), PAN-PNMThH film (2) and PAN-PNMThH HRP electrode (3) in a pH 6.0, 0.20 M PBS with a scan rate of 60 mV s 1. The typical redox peaks were observed in the potential range from 0.60 to 0.50 V in curve 1, which was attributed to the hydrogen adsorption/ desorption on the platinum surface [34]. However, these redox peaks were completely diminished in curves 2 and 3, therefore the copolymer film could effectively reduce background interference from the platinum during the subsequent H2O2 measurement. Fig. 4 shows the cyclic voltammograms of the PAN-PNMThH film (1) and the PAN-PNMThH HRP electrode (2) in a pH 6.0, 0.20 M

PBS containing 10.0 mM H2O2. The oxidation peak current of curve 1 is a little higher than that of curve 2. This is due to the fact that the impedance of the copolymer enzyme electrode is higher than that of the copolymer film because of the HRP contained in the film. This result is consistent with those of the polypyrrole xanthine oxidase electrode [35] and the polyaniline glucose oxidase electrode [36]. More interestingly, the reduction current of H2O2 is started to increase at about 0.00 V and does not saturate even at 0.60 V in curve 1. However, a well-define reduction peak appears at 0.37 V in curve 2. This is caused by the catalytic effect of HRP toward the reduction of H2O2. It should be noted that the cyclic voltammogram of the PAN-PNMThH HRP electrode at a lower the scan rate (Fig. 4B) shows more clearly the catalytic wave with the limiting current than that at a scan rate of 60 mV s 1. To establish whether the copolymer HRP electrode can be used to detect H2O2, its cyclic voltammetry measurement was carried out in a pH 6.0, 0.20 M PBS with three different concentrations of H2O2. The resulting cyclic voltammograms are shown in Fig. 5. In the absence of H2O2, the cyclic voltammogram (curve 1) shows the reduction of the copolymer at approximately 0.30 V. When H2O2 was added to the buffer solution, an increase in the reduction peak current was observed with a decrease in the oxidation peak current (curve 2). Moreover, the reduction peak current increases with the concentration of H2O2 (curve 3). Such phenomenon reveals that the copolymer HRP electrode can electrocatalyze the reduction of H2O2 and be used as a biosensor to determine H2O2. During the electrocatalytic reduction of H2O2, the copolymer plays an important role in shuttling the electron transfer between the HRP and the base electrode. The reaction mechanism of the biosensor is illustrated in Scheme 1. At first, the oxidized state of HRP (compound I (Fe4+ = O)) is generated when the enzyme reacts with H2O2 diffusing from the buffer solution (Eq. (1)). Secondly, compound I (Fe4+ = O) gets one electron from the copolymer (PAN-PNMThH) to produce a new intermediate (compound II) and a copolymer free radical (PANPNMTh
) (Eq. (2)). Thirdly, compound II is subsequently reduced back to the nature HRP by capturing one electron from the copolymer free radical (Eq. (3)) [30,37]. Finally, the oxidized PANPNMTh+ formed in Eq. (3) is electrochemically reduced to PANPNMThH on the electrode, leading to an increase the reduction peak current of the biosensor as shown in Fig. 5. The net reaction is shown in Eq. (5). Therefore, the measurement of the response current of the biosensor is based on the reduction of H2O2 in the presence of HRP and the copolymer redox mediator. However, it should be noted that although the active center of HRP is hemin (Fe (III)-protoporphyrin IX), yet the responses observed are in fact not purely an effect of dissociated hemin. This is due to that the apoenzyme in HRP could fully enhance the intrinsic catalytic ability of the hemin cofactor [38,39].

HRP (Fe3+) + H2O2 ė compound I (Fe4+=O) + H2O

(1)

Compound I (Fe4+=O) + PAN-PNMThH ė compound II + PAN-PNMTh·

(2)

Compound II + PAN-PNMTh· + H+ ė HRP (Fe3+) + PAN-PNMTh+

(3)

PAN-PNMTh+ + H+ + 2e− ė PAN-PNMThH

(4)

H2O2 + 2H+ + 2e− ė 2H2O

(5)

Scheme 1. Possible reaction mechanisms of the PAN-PNMThH HRP electrode.

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Fig. 6. Effect of potential scan rate on the cyclic voltammograms of the PAN-PNMTH HRP electrode in a pH 6.0, 0.20 M PBS containing 10.0 mM H2O2. Curves: (1) 20, (2) 40, (3) 60, (4) 80, and (5) 100 mV s 1. The inset is a plot of the reduction peak current versus n1/2 based on the data of Fig. 6.

127

Fig. 8. The effect of pH on the amperometric response of the PAN-PNMTH HRP electrode toward 200.0 mM H2O2 at 0.25 V.

The different scan rate studies were performed at the copolymer HRP electrode in the presence of 10.0 mM H2O2. Fig. 6 shows the cyclic voltammograms of the copolymer HRP electrode at different scan rates from 20 to 100 mV s 1. The reduction peak current (Ipc) of H2O2 is linearly proportional to the square root of scan rate (inset of Fig. 6). This result indicates that the electrochemical reduction process is controlled by diffusion of H2O2 to the electrode and depends on the concentration of H2O2 in the solution, which is ideal for quantitative analysis in practical applications. In addition, it can be observed that the reduction peak potential (Epc) of H2O2 gradually shifts negatively with increasing scan rates, indicating that the electrochemical reduction of H2O2 at the HRP electrode is irreversible.

reactions of other electroactive species in the solution at a high negative applied potential, 0.25 V is selected as the applied potential for the amperometric determination of H2O2 in the following experiments.

1.2

0.6

1.0

0.4 I / mA

I / μA

Fig. 7 shows the effect of the applied potential on the steadystate current of the copolymer HRP electrode was studied between 0.15 and 0.35 V in a pH 6.8, 0.20 M PBS containing 200.0 mM H2O2. The electrochemical reduction of H2O2 can be observed at 0.15 V. Upon the applied potential shifting negatively from 0.15 to 0.35 V, the response current gradually increases, which is attributed to the increased driving force for the fast reduction of the copolymer at a lower potential. The reduced copolymer mediator can further reduce compound I to form HRP (Fe3+), which electrocatalyzes the reduction of H2O2. To minimize interfering

Fig. 9. Chronoamperometric responses of the PAN-PNMTH HRP electrode to the successive addition of H2O2 in a pH 6.0, 0.20 M PBS at 0.25 V. The inset is the current–time curve obtained upon the lower concentration of H2O2.

0.8

0.3

0.2

I / mA

3.4. Effect of the applied potential on the biosensor response

0.6

0.1 0.0 0

0.0 0.4

R2=0.9986

0.2

0 -0.15

-0.20

-0.25

-0.30

-0.35

E / V(vs.SCE) Fig. 7. The effect of applied potential on the amperometric response of the PANPNMThH HRP electrode toward 200.0 mM H2O2 in a pH 6.0, 0.20 M PBS.

40

80

20

[H2O2]

120

40 / mM

160

60

200

[H2O2] / mM Fig. 10. The relationship between the response current of the PAN-PNMTH HRP electrode at 0.25 V and the concentration of H2O2 in a pH 6.0, 0.20 M PBS, based on the data of Fig. 9.

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R2=0.9997

I-1 / mA-1

24

16

8

0 0

50

100 150 -1 -1 [H2O2] / mM

200

Fig. 11. Determination of the apparent Michaelis–Menten constant K PAN-PNMTH HRP electrode, based on the data of Fig. 10.

M

app

for the

3.5. Effect of pH on the biosensor response The pH dependence of the response current of the biosensor was evaluated in the range from 4.0 to 8.0 in 0.20 M PBS containing 200.0 mM H2O2 at the applied potential of 0.25 V. As shown in Fig. 8, the response current increases with pH until it reaches a maximum at pH 6.0 and then decreases. This indicates that the response current of the biosensor is controlled by the activity of the immobilized HRP in this pH range. The decrease of the response current at low and high pH is attributed to the decrease of the enzymatic activity, leading to the decrease of the response current. Therefore, pH 6.0 is the optimal pH for the immobilized HRP in this work, which is very close to other reported values [40,41]. Since the activity of the enzyme often varies with its surrounding microenvironment, the optimal pH of HRP changes from 5.5 to 7.4 in different studies [26,41–43]. 3.6. Effect of the concentration of H2O2 on the biosensor response Fig. 9 shows the typical amperometric response of the copolymer HRP electrode on successive injection of different concentrations of H2O2 into a pH 6.0, 0.20 M PBS under the applied potential of 0.25 V. Upon addition of H2O2, the amperometric response increases and achieves 95% of the maximum value within 2 s. This indicates the amperometric response of the copolymer HRP electrode is fast. The fast response can be mainly due to the enhanced electron transfer ability of the copolymer. The

Fig. 12. The relationship between the temperature and the response current of the PAN-PNMTH HRP electrode at 0.25 V in a pH 6.0, 0.20 M PBS containing 200.0 mM H2O2.

Fig. 13. Plot of log I versus T

1

in the temperature range from 5.0 to 35.0  C.

amperometric response of the biosensor increases linearly with the increasing H2O2 concentration from 5.0 mM to 60.0 mM (R2 = 0.9986) (shown in Fig. 10). The linear range of the biosensor is wider than those of the HRP biosensors based on tetraethoxysilicone sol-gel film and multi-wall carbon nanotubes (70 mM– 3 mM) [42], laponite/chit film (29 mM–1.4 mM) [43], and oleylamine-stabilized gold nanowires and nanoparticles (20–500 mM) [44]. The sensitivity is 0.035 A M 1 cm 2, which is higher than 0.031 and 0.027 A M 1 cm 2 of oleylamine-stabilized gold nanowires and particles based HRP biosensors [44]. The detection limit of the biosensor is 3.2 mM, estimated at a signal-to-noise ratio of 3 (S/N = 3), which is better than those of the above-mentioned HRP biosensors (14 [42], 5 [43], 5 (gold nanowires), and 8 mM (gold nanoparticles) [44]). The apparent Michaelis–Menten constant (KMapp), which gives an indication of the enzyme-substrate kinetics, can be evaluated from the electrochemical version of the Lineweaver–Burk equation: 1/Iss = 1/Imax + KMapp/(Imax[H2O2]). The KMapp value for the biosensor was determined to be 2.79 mM from the Lineweaver– Burk plot (Fig. 11). This value is lower than those of 17.89 mM for HRP immobilized in carbon nanotubes-polyethyleneimine nanocomposite film [45], 11.94 mM for HRP entrapped in graphene and dsDNA composite modified carbon ionic liquid electrode [46], 5.19 and 4.42 mM for HRP immobilized in supramolecular hydrogel composite without and with 0.548% PANI nanoparticles [47], and close to those of 2.3 mM for HRP based on ferrocene-bovine serum albumin and multiwall carbon nanotube modified ormosil composite [37] and 2.14 mM for HRP immobilized in laponite/ Chit film self-assembled modified GCE [43]. The low KM value

Fig. 14. The influence of 0.20 mM electroactive interferences (ascorbic acid, uric acid and ethanol) in 0.20 mM H2O2 at 0.25 V.

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also provided a biocompatible platform for the immobilization and bioactivity maintenance of HRP. The resultant biosensor exhibited excellent electrocatalytic activity toward H2O2 reduction. This was proved by cyclic voltammetry result and low activation energy (50.64 kJ mol 1). The experimental variables such as applied potential, solution pH and temperature were optimized. The biosensor exhibited linear response to H2O2 from 5.0 mM to 60.0 mM with a detection limit of 3.2 mM (S/N = 3). In addition, the biosensor showed satisfactory stability and anti-interferent ability. Thus, the copolymer HRP electrode can be used to determine the concentration of H2O2. Acknowledgements

Fig. 15. Stability of the copolymer HRP electrode.

indicates that the enzyme immobilized into the copolymer film retains its activity with high affinity to H2O2. 3.7. Effect of the temperature on the biosensor response

Financial supports from the Social Development Foundation of Zhenjiang (SH2011016), the Natural Science Foundation of Jiangsu Province (BK2012699), the National Natural Science Foundation of China (51208233) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. References [1] [2] [3] [4] [5] [6] [7] [8] [9]

The temperature dependence of the response current of the biosensor was investigated in the range from 5.0 to 40.0  C in a pH 6.0, 0.20 M PBS containing 200.0 mM H2O2 at the applied potential of 0.25 V. As shown in Fig. 12, the response current increases with the temperature between 5.0 and 35  C and then decreases. The maximum response current appears at 35  C, which is the optimum temperature for HRP immobilized in the copolymer film. It is known that an enzyme-catalyzed reaction rate generally increases with temperature until it reaches a maximal value, and then decreases due to the thermal denaturalization of the enzyme [11]. In the temperature range from 5.0 and 35.0  C, the response current versus temperature therefore follows the electrochemical version of the Arrhenius equation: IT = I0exp( Ea/RT). On the basis of the data shown in Fig. 12, the log I versus T 1 relationship is a straight line (Fig. 13). The apparent activation energy (Ea) was calculated to be 50.64 kJ mol 1 from the slope of the straight line, which is bigger than that of the native HRP (16.8 kJ mol 1) [48] and that (39.1 kJ mol 1) reported by Wang et al. [49] for HRP immobilized in polyaniline films. However, the activation energy of the copolymer HRP electrode is close to that for most enzymecatalyzed reactions (20–85 kJ mol 1) [11], which is additional evidence that the electrocatalytic reduction of H2O2 occurred at the copolymer HRP electrode.

[20] [21] [22] [23] [24] [25] [26]

3.8. Selectivity and stability of the biosensor

[27]

Interference experiments were performed to evaluate the selectivity of the biosensor, in which ascorbic acid, uric acid, and ethanol were chosen as the interferents. As seen in Fig. 14, ethanol and uric acid do not have observable interference while ascorbic acid has slight interference (the current ratio is less than 0.05). Fig. 15 shows the long-term stability of the biosensor stored at 4  C was studied by measuring its response current to 200.0 mM H2O2. The response current of the biosensor is nearly unchanged during the initial first 7 days. It still retains 86.6% of its original response current after 30 days of storage. 4. Conclusions In this work, we have demonstrated that the PAN-PNMThH film is highly useful for the biosensing applications. This copolymer not only acted as an efficient redox mediator in the H2O2 biosensor, but

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