nano

4560

J. Phys. Chem. B 2007, 111, 4560-4567

Electrochemistry and Electrocatalysis of Hemoglobin in Nafion/nano-CaCO3 Film on a New Ionic Liquid BPPF6 Modified Carbon Paste Electrode Wei Sun,* Ruifang Gao, and Kui Jiao College of Chemistry and Molecular Engineering, Qingdao UniVersity of Science and Technology, Qingdao 266042, China ReceiVed: NoVember 29, 2006; In Final Form: March 4, 2007

Room temperature ionic liquid N-butylpyridinium hexafluorophosphate (BPPF6) was used as a binder to construct a new carbon ionic liquid electrode (CILE), which exhibited enhanced electrochemical behavior as compared with the traditional carbon paste electrode with paraffin. By using the CILE as the basal electrode, hemoglobin (Hb) was immobilized on the surface of the CILE with nano-CaCO3 and Nafion film step by step. The Hb molecule in the film kept its native structure and showed good electrochemical behavior. In pH 7.0 Britton-Robinson (B-R) buffer solution, a pair of well-defined, quasi-reversible cyclic voltammetric peaks appeared with cathodic and anodic peak potentials located at -0.444 and -0.285 V (vs SCE), respectively, and the formal potential (E°′) was at -0.365 V, which was the characteristic of Hb Fe(III)/Fe(II) redox couples. The formal potential of Hb shifted linearly to the increase of buffer pH with a slope of -50.6 mV pH-1, indicating that one electron transferred was accompanied with one proton transportation. Ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopy studies showed that Hb immobilized in the Nafion/nano-CaCO3 film still remained its native arrangement. The Hb modified electrode showed an excellent electrocatalytic behavior to the reduction of H2O2, trichloroacetic acid (TCA), and NaNO2.

1. Introduction Room temperature ionic liquids (RTILs) are ionic compounds consisting of ions that exist in the liquid state at room or related temperature. RTILs have received great attention in recent years due to their specific characteristics such as high chemical and thermal stability, relatively high ionic conductivity, negligible vapor pressure, and wide electrochemical windows. As a novel “green” solvent, RTILs have been used in organic synthesis, material science, catalysis, extraction, electrochemistry, and so forth.1-5 In the field of electrochemistry and electroanalysis, RTILs possess the characteristics of higher ionic conductivity and wider electrochemical windows, so they can be used as the supporting electrolyte or the modified material for electrochemical measurements. Endres et al.6 have reported the electropolymerization of benzene in 1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate. Sun et al.7 have investigated the electrochemical behavior of benzaldehyde using 1-ethyl-3-methylimidazolium bromide (EMIMBr) as the supporting electrolyte. Recently, some papers were reported by using RTILs to modify the electrode for bioelectrochemistry and electroanalysis. Moleki et al.8 used N-octylpyridinum hexafluorophosphate (OPFP) as a binder to make a high-performance carbon composite electrode; the resulting carbon ionic liquid electrode (CILE) showed excellent electrochemical behavior and provided a remarkable increase in the electron transfer rate of different organic and inorganic electroactive substances. Li et al.9 also fabricated an imidazolium-based ionic liquid modified carbon paste electrode, and the presence of ionic liquids caused an increase in the sensitivity of the response toward the detection of nitrite. Sun et al.10 also constructed an N-butylpyridinium hexafluorophos* Author to whom correspondence should be addressed. E-mail: [email protected]. Phone: 13156203237.

phate (BPPF6) modified carbon paste electrode and carefully investigated its electrochemical behavior. On the basis of its good ability to solve different kinds of compounds, RTILs are also used to construct a composite film to modify the traditional electrode. Dong et al.11 investigated the electrical-ionic properties of RTILs and carbon materials using multiwalled carbon nanotubes or mesocarbon microbeads. The hybrid carbon composite materials showed different conductivities and were further used as modifiers in the direct electrochemistry of protein. Li et al.12 used a novel chitosan/1-butyl-3-methylimidazolium hexafluorophosphate (BMIMBF6) composite material as a new immobilization matrix to entrap the proteins and studied the electrochemistry behavior of hemoglobin (Hb) on the glassy carbon electrode. Mao et al.13 described the formation of molecular film of water miscible imidazolium-based ionic liquids on glassy carbon electrodes, which possessed striking electrochemical properties to facilitate direct electron transfer of horseradish peroxidase (HRP). Such RTIL modified electrodes can also be applied to the electroactive organic substances. Zhao et al.14 applied RTIL modified electrodes for the voltammetric determination of dopamine in the presence of ascorbic acid and uric acid. Yan et al.15 studied the multiwalled carbon nanotube-ionic liquid paste coated glassy carbon electrode for the voltammetric determination of uric acid. The direct electrochemistry of proteins is an important foundation for biosensors and bioreactors.16 There are several ways for protein immobilization on the electrode surface including adsorption, covalent attachment, and film entrapment.17 Among them, film deposition is a popular technique for the various kinds of films available, and the ultrathin film can also provide a suitable microenvironment for redox proteins to transfer electrons with electrodes. Different approaches for casting films have been successfully proposed such as insoluble surfactants,18 hydrogel polymers,19 biopolymers,20 clay/surfac-

10.1021/jp067933n CCC: $37.00 © 2007 American Chemical Society Published on Web 04/11/2007

Hemoglobin in Nafion/nano-CaCO3 Film

J. Phys. Chem. B, Vol. 111, No. 17, 2007 4561

Figure 1. Molecular structure of BPPF6.

tant composites,21 nanoparticles,22 and layer-by-layer methods.23 These films can retain the native structure of protein and enhance the direct electron transfer between the protein and electrodes. Nafion films have been extensively used for electrode modification for their unique ion-exchange, discriminative, chemical resistance, and biocompatibility properties.24,25 As a perfluorosulfonate ionomer that contains less than 15% ionizable sulfonate groups per monomer unit, Nafion has a partly hydrophobic character leading to a very high affinity for hydrophobic cations but is almost impermeable to anions. Rusiling et al.26 have applied the composite films of surfactants, Nafion, and proteins on the edge plane pyrolytic graphite electrode and investigated the electrochemical behaviors of proteins. Recently, nanoparticles have been used in the fields of protein film voltammetry for their advantages of large surface area, thermal stability, good biocompatibility, and suitability for many surface immobilization mechanisms. Hu et al. have applied different nanoparticles such as SiO2,27 Fe3O4,28 Au,29 and carbon nanotubes30 in the layer-by-layer self-assembly of protein film. CaCO3 nanoparticles are biocompatible, spherical, cheap, nonaggregated material and can offer a large surface area for adsorption of substances of interest due to their porous structure. Sukhorukov et al.31 have used 30-50 nm porous CaCO3 as templates for the encapsulation of bioactive compounds. Hu et al.32 also constructed a core-shell nanocluster film with the layer-by-layer method containing heme proteins, nano-CaCO3, and polyelectrolytes. In this paper, RTILs of BPPF6 (the molecular structure is shown in Figure 1) were used as a binder to modify a carbon paste electrode and Hb was immobilized on the surface of the CILE by casting CaCO3 nanoparticles and Nafion film on the electrode surface step by step. The studies showed that the presence of nano-CaCO3 in the composite film can greatly enhance the electrochemical response of Hb, and Nafion was employed to fix the nano-CaCO3 and Hb tightly on the surface of the CILE. The electrochemical results indicated that Hb kept its native structure in the film and showed direct electron transfer behavior. The Hb modified electrode showed good electrocatalytic behavior to H2O2, trichloroacetic acid (TCA), and NaNO2. 2. Experimental Section 2.1. Chemicals. The bovine hemoglobin (Hb, MW 64 500) was from Tianjin Chuanye Biochemical Limited Company. The room temperature ionic liquid N-butylpyridinium hexafluorophosphate (BPPF6, g99%, melting point 65 °C) was purchased from Hangzhou Kemer Chemical Limited Company. nanoCaCO3 (average diameter 20.0 nm, Shandong Shengda Nanomaterial Limited Company). Nafion was obtained from Sigma, and 0.5% ethanol solution was prepared. 0.2 mol L-1 BrittonRobinson (B-R) buffers of various pH values were used as the supporting electrolytes. All other chemicals were of analytical reagent grade, and double-distilled water was used in all experiments. 2.2. Apparatus. A CHI 750B electrochemical workstation (Shanghai CH Instrument Company, China) was used for all of the electrochemical measurements such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). A traditional three-electrode cell was used with a Hb modified CILE as the working electrode, a platinum wire as the auxiliary

Figure 2. Cyclic voltammograms of (a) Nafion/nano-CaCO3/Hb/CILE, (b) bare CILE, (c) Nafion/CILE, (d) Nafion/nano-CaCO3/CILE, and (e) Nafion/Hb/CILE in pH 7.0 B-R buffer at a scan rate of 0.1 V s-1.

electrode, and a saturated calomel electrode (SCE) as the reference electrode. Voltammetry was carried out in B-R buffer containing no hemoglobin. The buffers were purged with highly purified nitrogen for about 30 min prior to a series of experiments, and a nitrogen environment was kept during the measurements. All of the electrochemical experiments were performed at an ambient temperature of 25 ( 2 °C. Ultravioletvisible (UV-vis) spectra were obtained on a Cary 50 probe UV-vis spectrophotometer (Varian Company, Australia). Fourier transform infrared (FT-IR) spectra were obtained on a Tensor 27 FT-IR spectrophotometer (Bruker, Germany). Scanning electron microscopy (SEM) measurements were carried out on a JSM-6700F scanning electron microscope (Japan Electron Company). 2.3. Procedures. 2.3.1. Construction of CILE. The CILE was fabricated with the following procedure: 3 g of graphite powder and 1 g of BPPF6 (mp 65 °C) were mixed thoroughly in a mortar and heated at a temperature of 80 °C to form a homogeneous carbon paste. A portion of the carbon paste was filled firmly into one end of a glass tube (A ) 0.12 cm2), and a copper wire was inserted through the opposite end to establish an electrical contact. The surface of the CILE had a metallic shine and showed good mechanical strength, which was smoothened on a piece of weighting paper. Such a CILE was used as the basic electrode for the preparation of the Nafion/nano-CaCO3/Hb film modified electrode. 2.3.2. Construction of the Nafion/nano-CaCO3/Hb Film Modified CILE. The 15 mg mL-1 Hb stock solution was prepared by dissolving 15 mg of Hb in 1.0 mL of pH 7.5 B-R buffer. The Hb modified electrode was prepared with the following procedure: A 5 µL volume of Hb solution was evenly pipetted onto the surface of the CILE and spread gently over the entire surface. The electrode was left in the air to dry under ambient conditions for about 6 h, and a small bottle was fit tightly over the electrode so that water was evaporated gradually. Then, a 5 µL volume of the 5 mg mL-1 nano-CaCO3 suspension solution was pipetted to cover the Hb modified CILE and dried at room temperature. Finally, a 5 µL volume of the 0.5% Nafion solution was spread onto the dry nano-CaCO3 film surface. Alternatively, different kinds of modified electrodes such as Nafion/CILE, Nafion/nano-CaCO3/CILE, and Nafion/Hb/CILE were also fabricated with a similar procedure.

4562 J. Phys. Chem. B, Vol. 111, No. 17, 2007

Sun et al.

3. Results and Discussion 3.1. Direct Electrochemistry of Hb. In this paper, Hb was fixed in the Nafion/nano-CaCO3 film on the surface of the CILE with electrostatic growth mode. Since BPPF6 is a pyridiniumbased ionic liquid, a layer of cationic BPPF6 film is formed on the surface of the CILE.8 Hb (isoelectric points at 7.0-7.4) has considerable negative surface charges at pH 7.5. The isoelectric point of nano-CaCO3 is at pH 8.5,33 and the nano-CaCO3 in the water solution is positively charged. Thus, it is possible for them to assemble on the electrode surface by electrostatic attraction between the oppositely charged species and form a stable composite film. Hb showed good direct electron transfer behavior on the CILE and the direct electrochemistry of Hb on the modified electrode was studied by cyclic voltammetry. Figure 2 showed cyclic voltammograms of different kinds of modified CILEs in pH 7.0 B-R buffer solution. A pair of welldefined, quasi-reversible cyclic voltammetric peaks (curve a) was found for Nafion/nano-CaCO3/Hb/CILE with Epa ) -0.285 V and Epc ) -0.444 V at a scan rate of 0.1 V s-1. The formal potential (E°′), which was calculated from the midpoint of the reduction and oxidation peak potentials, was -0.365 V (vs SCE), and the peak-to-peak potential difference (∆Ep) was 159 mV. The peaks were located at the potential characteristics of the heme Fe(III)/Fe(II) redox couples of proteins. However, no voltammetric peaks were observed at the bare CILE (curve b), Nafion/CILE (curve c), and Nafion/nano-CaCO3/CILE (curve d) in the same potential range. Curve e showed the cyclic voltammogram of Hb only immobilized on a bare CILE surface using Nafion. A small pair of redox peaks appeared, which suggested that the Hb could also have direct electron transfer on CILE but made little contribution to the observed redox peaks in curve a, so the presence of nano-CaCO3 can greatly enhance the electron transfer process of Hb. For Nafion/nano-CaCO3/ Hb films, the peak current reached the steady state after several cyclic voltammetric cycles in buffers. Thus, all of the voltammetric experiments with Nafion/nano-CaCO3/Hb film modified CILEs were conducted at their steady states. Figure 3A showed the cyclic voltammograms of Nafion/nanoCaCO3/Hb film in pH 7.0 B-R buffer solution with different scan rates. A pair of roughly symmetric anodic and cathodic peaks appeared with almost equal peak currents in the scan rate range from 0.06 to 0.50 V s-1. The peak-to-peak separation also increased with the scan rate. The results indicated that all of the electroactive Hb Fe(III) in the film was reduced to Hb Fe(II) on the forward scan and then reoxidized to Hb Fe(III) on the reverse scan. A good linear relationship was found for the peak current and scan rate, with the results shown in Figure 3B, which was characteristic of quasi-reversible surfacecontrolled thin-layer electrochemical behavior. The reduction peak currents increased with scan rate in the range 0.06-0.5 V s-1, and the integration of reduction peaks gave nearly constant charge (Q) values at different scan rates. According to the following equation, the surface concentration (Γ*) of electroactive Hb in the film was calculated.

Q ) nFAΓ*

(1)

where Q is the charge passing through the electrode with full reduction of electroactive Hb in the film, A is the geometric area of the CILE, n and F have their usual meaning, and Γ* is the surface concentration of the electroactive substance. According to this method, the surface concentration of electroactive Hb in the film (Γ*) was calculated as 5.3 × 10-9 mol cm-2 by integration of the reduction peak in pH 7.0 B-R buffer solution. On the basis of the absolute concentration of

Figure 3. (A) Cyclic voltammograms of Nafion/nano-CaCO3/Hb film in pH 7.0 B-R buffer solution with different scan rates (a-i: 0.1, 0.15, 0.18, 0.2, 0.25, 0.3, 0.35, 0.4, and 0.5 V s-1). (B) Plots of cathodic (a) and anodic (b) peak current with the scan rate (V).

Hb on the geometric area of the electrode, the surface concentration of Hb on the electrode was estimated as 9.7 × 10-9 mol cm-2. Thus, the fraction of electroactive Hb was estimated as 54.6%, which indicated that most of the Hb molecules kept their bioactivity in the film. The apparent heterogeneous electron transfer rate constant (ks) for Nafion/nano-CaCO3/Hb/CILE was estimated by cyclic voltammetry with Laviron’s method34 of the following equations:

2.3RT log V RnF

(2)

2.3RT log V (1 - R)nF

(3)

Epc ) E°′ Epa ) E°′ +

RT nFV (1 - R)RF∆Ep (4) 2.3RT

log ks ) R log(1 - R) + (1 - R) log R - log

where R is the charge transfer coefficient and n is the number of electrons transferred. The relationships of peak potentials with log V were shown in Figure 4. According to eqs 2 and 3, the charge transfer



Figure 4. Relationship of the anodic (a) and cathodic (b) peak potential against log V.

coefficient (R) can be calculated and the result found was 0.58. The heterogeneous electron transfer rate constant (ks) was further calculated from the relationship of ∆Ep with log V on the basis of eq 4, and the result was 0.75 s-1. The pH effect on the electrochemistry of Hb was examined, and the results were shown in Figure 5A. Hb in the Nafion/ nano-CaCO3 film showed a stable and quasi-reversible voltammogram in the pH range from 3.5 to 9.0. The cyclic voltammetric peak potentials of the heme Fe(III)/Fe(II) redox couples shifted negatively with the increase of buffer pH. The changes in cyclic voltammetric peak potentials were reversible for Nafion/nano-CaCO3/Hb films. For example, the cyclic voltammogram of the Nafion/nano-CaCO3/Hb film at pH 4.0 was reproduced after immersing the electrode in pH 7.0 buffer and then putting it back into the pH 4.0 buffer. Figure 5B showed the relationship of the formal potentials (E°′) of Nafion/nano-CaCO3/Hb film with the pH of the buffer solution. It can be seen that a linear relationship was found in the pH range from 3.5 to 9.0 with a slope of -50.6 mV pH-1. The slope was slightly smaller than the theoretical value of -59.0 mV pH-1 at 25 °C for the reversible one-electron transfer coupled with single-proton transportation,35 which indicated that a single protonation accompanied one-electron transfer of Hb Fe(III) to the electrode. The electrochemical reduction of Hb can be simply expressed as follows:36

Hb heme Fe(III) + H+ + e- h Hb heme Fe(II)

(5)

The Nafion/nano-CaCO3/Hb film modified electrode was stored at 4 °C, and the stability was investigated by measuring the cyclic voltammogram periodically. The results indicated that the peak potentials and currents of Nafion/nano-CaCO3/Hb film were stable for 15 days and then decreased gradually. When the time was at 30 days, the cyclic voltammetric peak potentials appeared at the same position with the peak current decreased 10% compared with the initial response. 3.2. Spectroscopic Characterization. The structural information of Hb was investigated after it was immobilized on the surface of the electrode. Spectroscopic methods such as UVvis and FT-IR spectroscopy were used to compare the structural variations of native and immobilized Hb. FT-IR spectroscopy is a sensitive probe for the secondary structure of proteins. The shape and position of amide I (16001700 cm-1) and amide II (1500-1600 cm-1) of IR bands provide detailed information on the secondary structure of the polypeptide chain.37,38 The amide I band at 1700-1600 cm-1

Figure 5. (A) Influence of pH on cyclic voltammograms of Nafion/ nano-CaCO3/Hb/CILE (a-e: 4, 4.5, 5, 7, and 8, respectively). (B) pH effect on the formal potential (E°′) of Nafion/nano-CaCO3/Hb/CILE. Scan rate: 0.1 V s-1.

is caused by CdO stretching vibrations of the peptide linkage. The amide II band at 1600-1500 cm-1 results from a combination of NsH in-plane bending and CsN stretching of the peptide groups. As shown in Figure 6, the spectra of amide I and II bands of Hb in the Nafion/nano-CaCO3 film (1652.02 and 1532.05 cm-1) were nearly the same as those of natured Hb (1655.34 and 1535.80 cm-1). If Hb is denatured, the intensities of the amide I and II bands will significantly diminish or even disappear. Similarities of the spectra in parts A and B of Figure 6 suggested that Hb retained the essential features of its native structure on the CILE surface immobilized by Nafion/ nano-CaCO3 film. Positions of the Soret absorption band of heme may also provide information about possible denaturation of heme protein and particularly on conformational change in the heme group region.39 Therefore, UV-vis spectroscopy is a useful tool for conformational study of heme proteins. Figure 7 showed UVvis absorbance spectra of Hb in different pH buffer solutions. It can be seen that the Soret band of Hb in water solution (curve a) appeared at 405 nm. The spectra of the mixture of Hb with Nafion and nano-CaCO3 at the different pHs of external buffer solution were also tested. At pH values between 5.0 and 10.0,


Sun et al.

Figure 6. FT-IR spectra of the films of Hb (A) and Nafion/nano-CaCO3/Hb (B).

Figure 7. UV-vis absorption spectra of Hb in water solution (a) and Nafion/nano-CaCO3/Hb in pH (b) 2.0, (c) 5.0, (d) 6.0, (e) 7.0, and (f) 10.0 buffer solutions, respectively.

the Soret band appeared at 405 nm and at almost the same position as the native Hb solution (curves c-f), indicating that, in the medium pH range, Hb essentially retained its native conformation in nano-CaCO3 and Nafion solutions. When the pH shifted toward a more acidic or more basic direction, the Soret band became smaller and broader. At pH 2.0, for instance, the Soret band was located at 360 nm (curve b), which suggested that denaturation of Hb existed to a considerable extent. All of the UV-vis and FT-IR spectra suggested that the Hb in Nafion/ nano-CaCO3 films retained their near native states. 3.3. Morphology of Films. Scanning electron microscopy (SEM) was used to characterize and compare the morphologies of the different types of modified electrodes with the results shown in Figure 8. At the traditional carbon paste electrode (CPE), the surface was formed by isolated and irregularly shaped carbon flakes and each layer could be distinguished clearly (Figure 8A). At the surface of the CILE (Figure 8B), a more uniform surface was formed and no separated carbon layer could be observed. A mass of RTILs was embedded in carbon layers and bridged each layer of carbon flakes.40 At the Hb/CILE (Figure 8C), the aggregation of the immobilized Hb molecules was distributed regularly and showed a networklike structure.

Figure 8D presented the image of a nano-CaCO3/CILE. The nano-CaCO3 was discernible, which agglomerated in mass to form a spherical flowerlike structure with an average diameter of CaCO3 particles of about 20.0 nm. 3.4. Impedance Characterization. Electrochemical impedance spectroscopy (EIS) can provide information on the impedance changes of the electrode surface during the modification process. Figure 9 showed the typical results of AC impedance spectra of the bare CILE (curve a), Nafion/Hb/CILE (curve b), and Nafion/nano-CaCO3/Hb/CILE (curve c), respectively, which were obtained in a solution of 0.1 mol L-1 KCl containing 10 mmol L-1 Fe(CN)63- and 10 mmol L-1 Fe(CN)64with the frequencies ranging from 105 to 10-2 Hz. The electron transfer resistance (Ret) of a bare CILE was estimated to be 69 Ω, which was due to the presence of highly conductive RTILs in the mixture with carbon powder (curve a). When Nafion and Hb were assembled on the CILE without nano-CaCO3, the value of Ret was found to be 113 Ω (curve b), which was consistent with the fact that the presence of Nafion/Hb film on the surface of the CILE had more blocks to the approach of the redox probe Fe(CN)63-/4-. However, after the presence of nano-CaCO3 in the film, the Ret of Nafion/nano-CaCO3/Hb/CILE was 93 Ω (curve c), which located between that of the bare CILE and Nafion/Hb/CILE. This might be due to the presence of nanoparticles in the film playing an important role in enhancing the transfer of electrons and making it easier for the electron transfer to take place, thus decreasing the resistance of the Nafion/Hb film to Fe(CN)63-/4-. 3.5. Catalytic Reactivity. Electrochemical catalytic reduction of H2O2, trichloroacetic acid (TCA), and NaNO2 by the constructed Hb modified electrode was examined by cyclic voltammetry. The catalytic reduction of H2O2 on the Nafion/ nano-CaCO3/Hb film electrode was first examined. Formal studies showed that a pair of redox peaks of the Hb heme Fe(III)/Fe(II) redox couple can be obtained at -0.444 and -0.285 V in a pH 7.0 B-R buffer. After a certain volume of H2O2 was added to the pH 7.0 B-R buffer solution, a new and significant reduction peak appeared at about -0.320 V (Figure 10A). Meanwhile, the anodic peak wave decreased and disappeared after the addition of H2O2 into the buffer solution, which wascharacteristic of an electrochemically catalytic reaction.41



Figure 8. SEM images of the different types of modified electrodes: (A) CPE; (B) CILE; (C) Hb/CILE; (D) nano-CaCO3/CILE.

catalyzed by Nafion/nano-CaCO3/Hb film is postulated as follows:

Hb Fe(III) + H2O2 f compound I + H2O

(6)

compound I + H2O2 f Hb Fe(III) + O2 + H2O

(7)

Hb Fe(III) + H+ + e- f Hb Fe(II) (at electrode) (8) Hb Fe(II) + O2 f Hb Fe(II)-O2 (fast)

(9)

Hb Fe(II)-O2 + 2H+ + 2e- f Hb Fe(II) + H2O2 (at electrode) (10) Figure 9. Electrochemical impedance spectroscopy for (a) bare CILE, (b) Nafion/Hb/CILE, and (c) Nafion/nano-CaCO3/Hb/CILE.

However, the direct reduction of H2O2 on the Nafion/nanoCaCO3 film electrode was not observed in the selected potential windows. The catalytic reduction peak current of Nafion/nanoCaCO3/Hb film increased linearly with the concentration of H2O2 in a linear range from 8.0 × 10-6 to 4.4 × 10-4 mol L-1 with a correlation coefficient of 0.997 (n ) 14). The linear regression equation was Iss (×10-5 A) ) 0.73 C (×10-5 mol L-1) + 9.49 × 10-5 (Figure 10B). When the H2O2 concentration was larger than 4.4 × 10-4 mol L-1, the CV response showed a level-off tendency and the curve tended to maximum at 5.0 × 10-4 mol L-1 H2O2, indicating a saturation of the enzymesubstrate reaction. A possible mechanism of reaction of H2O2

There are two catalytic cycles here: Hb Fe(II) reacts with O2 and forms Hb Fe(II)-O2 in eq 9, and the produced Hb Fe(II)-O2 will receive two electrons at electrodes and return to Hb Fe(II) again in eq 10; H2O2 produced in eq 10 will then induce or promote the catalytic cycle of eqs 9 and 10. The apparent Michaelis-Menten constant (Kapp M ) for Nafion/ nano-CaCO3/Hb film toward H2O2, which gave an indication of the enzyme-substrate kinetics, was estimated from the linear part of the calibration plot by using the electrochemical version of the Lineweaver-Burk equation.42

Kapp M 1 1 ) + Iss Imax ImaxC

(11)

where Iss is the steady-state current after the addition of the substrate, C is the bulk concentration of the substrate, and Imax


Figure 10. (A) Cyclic voltammograms obtained at Nafion/ nano-CaCO3/Hb/CILE in pH 7.0 B-R buffer before (a) and after the addition of 160.0, 240.0, 300.0, 360.0, and 440.0 µmol L-1 H2O2 (b-f), respectively. Scan rate: 0.1 V s-1. (B) Plot of the catalytic peak current versus the concentration of H2O2. Scan rate: 0.1 V s-1.

is the maximum current measured under saturation conditions. The value of Kapp M was determined according to the slope and the intercept of the plot of the reciprocals of the steady-state current and H2O2 concentration. The smaller Kapp M value means that the present electrode exhibits a higher affinity to H2O2. Accordingly, the Kapp M value for this Nafion/nano-CaCO3/Hb/ CILE was calculated as 0.12 mM, which was smaller than those of many horseradish peroxidase modified electrodes,43,44 and the results indicated that Hb in the Nafion/nano-CaCO3 film had a higher sensitivity to H2O2. Electrochemical catalytic reduction of TCA by the modified electrode was also investigated by cyclic voltammetry. When TCA was added to a pH 5.5 buffer, the Hb Fe(III) reduction peak of Nafion/nano-CaCO3/Hb film was observed at about -0.444 V (Figure 11A) and accompanied by a decrease of the Hb Fe(II) oxidation peak. The more TCA that was added, the greater the reduction peak current was increased. The results indicated that the Hb Fe(II) generated on the electrode was chemically oxidized by TCA and the produced Hb Fe(III) was reduced again at the electrode in a catalytic cycle. According

Sun et al.

Figure 11. (A) Cyclic voltammograms in the absence of TCA (a) and the presence of 1.0 × 10-3, 2.0 × 10-3, 3.8 × 10-3, 4.8 × 10-3, 5.6 × 10-3, and 7.4 × 10-3 mol L-1 TCA (b-g) in pH 5.5 B-R buffer. (B) Plot of the cataclytic peak current versus the concentration of TCA. Scan rate: 0.1 V s-1.

to the literature,45 the catalytic reduction of TCA by Hb can be expressed by the following equations:

Hb Fe(III) + e- T Hb Fe(II) at electrode

(12)

Cl3CCOOH T Cl3CCOO- + H+ pKa ) 0.89 (13) 2[Hb Fe(II)] + Cl3CCOOH + H+ f 2[Hb Fe(III)] + Cl2HCCOOH + Cl- (14) The Hb Fe(III) reduction peak current was linearly proportional to the concentration of TCA. Figure 11B showed the linear dependence of the reduction peak currents on the TCA concentration. The linear regression equation was Iss(×10-4 A) ) 0.26 C (×10-3 mol L-1) + 0.61 × 10-4 in the range from 6.0 × 10-4 to 1.2 × 10-2 mol L-1 with a correlation coefficient of 0.996 (n ) 17). However, no corresponding electrochemical signal could be observed in the same potential window employing either a bare CILE or a Nafion/nano-CaCO3 film modified CILE in the same TCA solution. Therefore, the catalytic process came from the specific enzymatic catalytic reaction between Hb and TCA. The Kapp M value for the Nafion/


Figure 12. Cyclic voltammograms obtained at Nafion/nano-CaCO3/ Hb/CILE in pH 5.5 B-R buffer (from inner to outer: in the presence of 0, 1.0 × 10-5, 6.0 × 10-5, 1.2 × 10-4, and 2.0 × 10-4 mol L-1 NO2-, respectively). Scan rate: 0.1 V s-1.

nano-CaCO3/Hb film-based TCA sensor was calculated as 2.57 mM, lower than that of the previous report.46 Electrochemical catalytic reduction of nitrite was also observed at the Nafion/nano-CaCO3/Hb film electrode. After the addition of NO2- into a pH 5.5 B-R buffer, a new reduction peak at about -0.83 V appeared (Figure 12), while direct reduction of NO2- at Nafion/nano-CaCO3 film showed a peak potential at about -1.3 V. Thus, the presence of Hb in the film decreased the reduction overpotential of NO22- for at least 0.5 V. In addition, according to eq 11, the Kapp M value of NaNO2 was calculated as 0.22 mM. 4. Conclusion A room temperature ionic liquid BPPF6 modified carbon paste electrode was constructed, and direct electrochemistry of Hb was realized on it through the immobilization of Hb in a Nafion/ nano-CaCO3 film. The presence of BPPF6 in the CPE provided a highly conductive and biocompatible interface. Hb keeps its native structure in the film, and the electrochemical behaviors of Hb were carefully investigated. The immobilized Hb showed good electrochemical catalytical behaviors to different substances such as H2O2, TCA, and NaNO2. Acknowledgment. We are grateful to the financial support of the National Natural Science Foundation of China (nos. 20405008 and 20635020). References and Notes (1) Welton, T. Chem. ReV. 1999, 99, 2071-2083. (2) Wasserschied, P.; Welton, T. Ionic Liquids in Synthesis; WileyVCH: Weinheim, Germany, 2003. (3) Endres, F. ChemPhysChem 2002, 3, 144-154. (4) Buzzeo, M. C.; Evans, R. G.; Compton, R. C. ChemPhysChem 2004, 5, 1106-1120.

J. Phys. Chem. B, Vol. 111, No. 17, 2007 4567 (5) Hiroyuki O. Electrochemical Aspects of Ionic Liquids; John Wiley & Sons, Inc.: New Jersey, 2005. (6) Zein, S.; Abedin, Z. E.; Borissenko, N.; Endres, F. Electrochem. Commun. 2004, 6, 422-426. (7) Sun, Q.; Zhao, P.; Lu, J. X. Chin. J. Chem. 2005, 23, 367-369. (8) Maleki, N.; Safavi, A.; Tajabadi, F. Anal. Chem. 2006, 78, 38203826. (9) Liu, H. T.; He, P.; Li, Z. Y.; Sun, C. Y.; Shi, L. H.; Liu, Y.; Zhu, G. Y.; Li, J. H. Electrochem. Commun. 2005, 7, 1357-1363. (10) Sun, W.; Gao, R. F.; Bi, R. F.; Jiao, K. Chin. J. Anal. Chem., in press. (11) Zhao, F.; Wu, X. E.; Wang, M. K.; Liu, Y.; Gao, L. X.; Dong, S. J. Anal. Chem. 2004, 76, 4960-4967. (12) Lu, X. B.; Hu, J. Q.; Yao, X.; Wang, Z. P.; Li, J. H. Biomacromolecules 2006, 7, 975-980. (13) Yu, P.; Lin, Y. Q.; Xiang, L.; Su, L.; Zhang, J.; Mao, L. Q. Langmuir 2005, 21, 9000-9006. (14) Zhao, Y. F.; Gao, Y. Q.; Zhan, D. P.; Liu, H.; Zhao, Q.; Kou, Y.; Shao, Y. H.; Li, M. X.; Zhuang, Q. K.; Zhu, Z. W. Talanta 2005, 66, 5157. (15) Yan, Q. P.; Zhao, F. Q.; Li, G. Z.; Zeng, B. Z. Electroanalysis 2006, 18, 1075-1080. (16) Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407-413. (17) Lojou, EÄ .; Bianco, P. Electroanalysis 2004, 16, 1113-1121. (18) Nassar, A.-E. F.; Zhang, Z.; Hu, N.; Rusling, J. F.; Kumosinski, T. F. J. Phys. Chem. B 1997, 101, 2224-2231. (19) Hu, N.; Rusling, J. F. Langmuir 1997, 13, 4119-4125. (20) Liu, H.; Hu, N. Anal. Chim. Acta 2003, 481, 91-99. (21) Wang, L.; Hu, N. J. Colloid. Interface Sci. 2001, 236, 166-172. (22) Li, Z.; Hu, N. J. Electroanal. Chem. 2003, 558, 155-165. (23) Lvov, Y. In Protein Architechture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mohwld, H., Eds.; Marcel Dekker: New York, 2000; pp 125-166. (24) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408-2409. (25) Fan, Z.; Harrison, D. J. Anal. Chem. 1992, 64, 1304-1311. (26) Huang, Q. D.; Lu, Z. Q.; Rusling, J. F. Langmuir 1996, 12, 54725480. (27) He, P. L.; Hu, N.; Rusling, J. F. Langmuir 2004, 20, 722-729. (28) Cao, D. F.; He, P. L.; Hu, N. Analyst 2003, 128, 1268-1274. (29) Zhang, H.; Lu, H. Y.; Hu, N. J. Phys. Chem. B 2006, 110, 21712179. (30) Zhao, L. Y.; Liu, H. Y.; Hu, N. Anal. Bioanal. Chem. 2006, 384, 414-422. (31) Sukhorukov, G. B.; Volodkin, D. V.; Gu¨nther, A. M.; Petrov, A. I.; Shenoy, D. B.; Mo¨hwald, H. J. Mater. Chem. 2004, 14, 20732081. (32) Liu, H. Y.; Hu, N. J. Phys. Chem. B 2005, 109, 10464-10973. (33) Salinas-Nolasco, M. F.; Mendze-Victor, J.; Lara, V. H.; Bosch, P. J. Colloid. Interface Sci. 2004, 274, 16-24. (34) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28. (35) Bond A. M. Modern Polarographic Methods in Analytical Chemistry; Marcel Dekker: New York, 1980. (36) Ma, H. Y.; Hu, N.; Rusling, J. F. Langmuir 2000, 16, 4969-4975. (37) Kauppinen, J. K.; Moffat, D. J.; Mantsch, H. H.; Cameron, D. G. Appl. Spectrosc. 1981, 35, 271-276. (38) Rusling, J. F.; Kumonsinski, T. F. Intell. Instrum. Comput. 1992, 10, 139-145. (39) Rusling, J. F.; Nassar, A. E. F. J. Am. Chem. Soc. 1993, 115, 11891-11897. (40) Sutto, T.; Trulove, E. P. C.; De Long, H. C. Electrochem. SolidState Lett. 2004, 6, A50-55. (41) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, Principle and Application; Wiley: New York, 1980; pp 459-462. (42) Kamin, R. A.; Wilson, G. S. Anal. Chem. 1980, 52, 1198-1205. (43) Liu, X. J.; Xu, Y.; Ma, X.; Li, G. X. Sens. Actuators, B 2005, 106, 284-288. (44) Li, J.; Tan, S. N.; Ge, H. Anal. Chim. Acta 1996, 335, 137143. (45) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369. (46) Wang, S. F.; Chen, T.; Zhang, Z. L.; Shen, X. C.; Lu, Z. X.; Pang, D. W.; Wong, K. Y. Langmuir 2005, 21, 9260-9266.

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