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Journal of Electroanalytical Chemistry Journal of Electroanalytical Chemistry 597 (2006) 51–59 www.elsevier.com/locate/jelechem Direct electrochemistry and electrocatalysis of hybrid ﬁlm assembled by polyelectrolyte–surfactant polymer, carbon nanotubes and hemoglobin Liang Chen a a,b , Gongxuan Lu a,* State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China b The Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100039, PR China Received 24 March 2006; received in revised form 24 July 2006; accepted 9 August 2006 Available online 22 September 2006 Abstract Functional hybrid ﬁlm based on the incorporation of multiwalled carbon nanotubes modiﬁed with hemoglobin into polyelectrolyte– surfactant polymer was fabricated. Such assembled ﬁlms are found to possess facilitated electron transfer of hemoglobin. Cyclic voltammetric results showed a pair of well-deﬁned redox peaks for the Hb heme Fe(III)/Fe(II) redox couple at about 0.273 (versus SCE) in a pH 7.0 phosphate buﬀer solution. The formal potential of the Hb heme Fe(III)/Fe(II) couple shifted linearly with pH with a slope of 52.8 mV pH1, indicating that an electron transfer is coupled with a one-proton transportation. An FT-IR and UV–vis spectroscopy study conﬁrms that the secondary structure of Hb entrapped in the hybrid ﬁlm still maintains the original arrangement. It is suggested that the achieved faradic response of the hemoglobin be due to the ability of carbon nanotubes to promote the electron transfer of enzymes and the biomembrane properties of the polyelectrolyte–surfactant polymer. And the entrapped Hb exhibits the features of a peroxidase and acts in an electrocatalytic manner in the reduction of hydrogen peroxide, oxygen, and nitrite. The properties of the functional hybrid ﬁlms, combined with the bioelectrochemical catalytic activity, could make them useful for the development of bioelectronic devices and investigation of protein electrochemistry at functional interface. 2006 Elsevier B.V. All rights reserved. Keywords: Hybrid ﬁlm; Polyelectrolyte–surfactant; Carbon nanotubes; Hemoglobin; Direct electrochemistry; Electrochemical catalysis 1. Introduction Among the best-known synthetic self-assembling polymeric systems, polyelectrolyte–surfactant polymer is a new kind of complexes of charged polymeric chain (polyelectrolytes) and appositely charged small amphiphilic molecules (surfactant), consisting of a polar ‘‘headgroup’’ and a nonpolar ‘‘tail’’ [1,2]. The complexation process is an ion-exchange reaction mainly driven by electrostatic attraction between the polymer chain units and ionic surfactants. Depending on the polyelectrolytes to surfactant * Corresponding author. Fax: +86 931 4968178. E-mail address: [email protected] (G. Lu). 0022-0728/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2006.08.002 ratio in aqueous solution, the complexes formed are as either stoichiometric or nonstoichiometric. Nonstoichiometric complexes containing an excess of either charged polymer chain units or surfactant molecules are generally soluble in water. If equimolar amounts of charged polymer chain units and surfactant molecules are mixed in water, stoichiometric complexes are formed. Stoichiometric polyelectrolyte–surfactant complexes can be viewed as a new type of comb-shaped polymer, in which every polymer chain unit has an electrostatically bound ‘‘side chain’’. Such complexes combine in unique ways the properties of polymers with those of low molecular weight amphiphiles. The polymeric components can provide, for instance, mechanical strength and thermal stability, while the surfactants retain their tendency to assemble in layered 52 L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59 structures. This may be particularly useful for fabrication of multifunctional materials for technological application, e.g., electronic devices, microsensors, separation membrane, and biomembranes, etc. [2–5]. Carbon nanotubes (CNTs) represents a new kind of carbon-based nanomaterials and is superior to other carbon material mainly in special structure feature, unique electronic properties and mechanical strength . The unique properties of carbon nanotubes make them extremely attractive for the construction of ultrasensitive electrochemical sensors . Recently, electrochemical studies have revealed that the ability of CNTs to promote electron transfer of some important molecules, such as catecholamine neurotransmitters , ascorbic acid , dopamine , nitric oxide  and enzymes (proteins) [12–16], etc. The striking properties of the CNTs coupled with the advantages of polyelectrolyte–surfactant polymer essentially suggest a new platform for facilitate the investigation of the protein electrochemistry. This article reports on a new and simple avenue for preparing bioelectrochemically hybrid ﬁlm based on polyelectrolyte–surfactant polymer, carbon nanotubes and hemoglobin. The direct electrochemistry of hemoglobin entrapped in the hybrid ﬁlm was studied and used to catalyze reduction of hydrogen peroxide, oxygen and nitrite. This demonstration may pave a new route to CNTs-based bioelectrochemical devices and would be useful for the investigation of protein electrochemistry at functional interface. 2. Experimental 2.1. Chemicals and solutions Hemoglobin (Hb, Mw 66,000) was purchased from Shanghai Lizhu Dong Feng Biotechnology Company (China). Poly(sodium-p-styrene-sulfonate) (PSS, Mw 70,000) was obtained from Acros Organics, USA. Cetyltrimethylammonium bromide (CTAB) was purchased from Shanghai Reagent Co. MWNTs (95%) purchased from Shenzhen Nanotech Port. Co., Ltd. (Shenzhen, China) were used as received. The 0.1 mol L1 phosphate buﬀers of various pHs were prepared by mixing the stock solutions of NaH2PO4 and Na2HPO4 and adjusted by 0.1 mM NaOH and 0.1 mM H3PO4 solutions. All solutions were made up with double-distilled water. All other chemicals were of analytical grade and used without further puriﬁcation. After the solution was centrifuged, washed, and dried, the dry yellow CTAB–PSS solids were obtained. The polymer can be dissolved in chloroform. Prior coating, the glassy carbon electrode (GCE, diameter 3 mm) was polished sequentially with slurries of 0.3 and 0.05 lm alumina to mirror ﬁnish. After rinsing with doubly distilled water, it was cleaned ultrasonically in water for 10 min. CNTs (10 mg) can be well dispersed in 10 ml aqueous CTAB solution (1% by weight) with aid of ultrasonication to give a 1 mg mL stable black suspension. CNTs suspensions (10 lL) were mixed well with 10 lL Hb solution (20 mg Hb dissolved in 1 mL pH 7.0 phosphate buﬀer) thoroughly. Then 6 lL of the mixture was deposited onto the surface of a GCE with a microsyringe and allowed to dry at ambient temperature. Finally, a total of 3 lL of 15 mg mL CTAB–PSS in chloroform was cast and used as binder to hold the Hb–MWNTs on the electrode surface stably. Then a small bottle was ﬁt tightly over the electrode to serve as a closed evaporation chamber so that chloroform was evaporated slowly to give uniform ﬁlm. 2.3. Instrumentation For all electrochemical experiments, a CHI660A electrochemical workstation (CH Instruments, Shanghai, China) was employed. The electrochemical cells consisted of a three-electrode arrangement with a Pt wire counter electrode, a saturated calomel electrode and a working electrode (hybrid ﬁlm modiﬁed GC electrode). The phosphate buﬀer solution (PBS) was purged with high-purity nitrogen for 15 min prior to experiments and a nitrogen environment was then kept over the solution in the cell. All experiments were performed at room temperature. Scanning electron microscopy (SEM) was carried out using a JSM-5600LV microscopy (Japan) at an acceleration voltage of 20 kV. MWNTs, Hb–MWNTs, polymer–Hb– MWNTs ﬁlms for SEM characterization were coated on copper slice and dried in the same way as the fabrication of the hybrid ﬁlm modiﬁed GC electrode. FT-IR spectra were obtained on an IFS120HRFTIR spectrometer (Bruker). Hb, Hb–MWNTs, surfactant polymer–Hb–MWNTs ﬁlms were coated on KBr wafer and dried for characterization. UV–vis spectra were obtained on a HP8453 UV–vis spectrometer (HP). 3. Results and discussion 3.1. FT-IR spectra of the polyelectrolyte–surfactant polymer 2.2. Film assembly on glassy carbon electrode Polyelectrolyte–surfactant polymer CTAB–PSS was prepared by a similar method described by Kunitake et al. . A total of 50 mL of 10 mg mL1 PSS solutions and 50 mL of 15 mg mL1 CTAB solutions were mixed well with stirring for 4 h at about 65 C until reaction was completed. A white precipitate of CTAB–PSS was formed. A comparison of CTAB, PSS and comb-shaped surfactant polymer CTAB–PSS FT-IR spectra is shown in Fig. 1. As shown in Fig. 1 (curve a), the symmetric and antisymmetric CH2 stretching vibrations modes of CTAB appear at 2849 and 2919 cm1, respectively, and the CH3–(N+) asymmetric deformation modes are at 1481 cm1. The characteristic peaks of PSS samples were found at about L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59 53 trolytes) and CTAB (surfactants) in the polymer. The details of peak-assignments of three samples are given in Table 1. Transmittance % a 3.2. Film characterization b c 3000 2500 2000 1500 1000 500 Wavenumber / cm -1 Fig. 1. FT-IR spectra of the three samples: CTAB (curve a), PSS (curve b) and polymer CTAB–PSS (curve c). 1195, 1128, 1039, 1011 and 836 cm1 . The SO 3 group antisymmetric and symmetric vibrational adsorption peaks can be assigned to the peaks at 1195 and 1039 cm1, respectively. Peaks at 1128 and 1011 cm1 can be attributed to the in-plane skeleton vibration of benzene ring and in-plane bending vibration of benzene ring. Peak at 835 cm1 is assigned to CH out-of-plane vibration for papa-disubstituted benzene. In the FT-IR spectrum of polymer, the CH3(N+) asymmetric deformation modes of CTA+ blue-shifted at 1486 cm1 , and the SO 3 group antisymmetric and symmetric vibration modes red-shifted at 1191 and 1037 cm1. These spectral changes clearly demonstrated their strong interaction between PSS (polyelecTable 1 Infrared frequencies and assignments of the three samples (CTAB, PSS and CTAB–PSS) CTAB (cm1) PSS (cm1) CTAB–PSS (cm1) 3063 3015 2919 2849 2923 2850 1603 1570 1497 1481 1469 1486 1473 1450 1195 1128 1191 1127 1039 1011 1037 1009 832 963 911 833 720 720 962 910 725 3017 2919 2850 1601 1571 Assignment Aromatic C–H stretch N+–CH3 C–H asym stretch CH2 C–H asym stretch CH2 C–H sym stretch Benzene ring [email protected] stretch Benzene ring [email protected] stretch Benzene ring [email protected] stretch N+–CH3 C–H sym bend CH2 scissoring Benzene ring [email protected] stretch SO 3 [email protected] asym stretch In-plane skeleton vibration of benzene ring SO 3 [email protected] sym stretch In-plane bending vibration of benzene ring C–N+ stretch C–N+ stretch Aromatic C–H out-of-plane bend CH2 rocking Macroscopic characterization of ﬁlms was studied using scanning electron microscopy. Fig. 2 illustrates the SEM photographs of the MWNTs ﬁlm (a), the Hb–MWNTs ﬁlm (b) and the polymer–Hb–MWNTs hybrid ﬁlm ((c) and (d)). As shown in photograph (a), many twisted MWNTs bundles can be observed. In the photograph (b), MWNTs bundles cannot be clearly observed. It could be due to the adsorption of hemoglobin molecules on the MWNTs. However, the surfactant ﬁlm shows signiﬁcant diﬀerences from above two ﬁlms. It is observed that this ﬁlm displays porous structure. The diameter of the holes varied from 100 nm to 1 lm. This unique porous structure could be propitious to the small molecule transfer between the electrode and the solution. Positions of the Soret absorption band of iron heme which is located at 406 nm may provide information about possible denaturation of heme proteins, especially that of conformational change in the heme group region. Previous studies showed that the band will diminish if the protein is fully denatured . Our experimental results revealed that the corresponding Soret bands for adsorbed Hb on MWNTs or entrapped Hb in the composite ﬁlm are located at 410 nm and have nice shapes. It is suggested that there exists slight structure variations in the vicinity of the heme group, and no signiﬁcant denaturation occurs (Fig. 3). FT-IR spectroscopy is a sensitive technique to probe into the secondary structure of proteins. Our FT-IR studies also show Hb is not denatured after incorporated into the hybrid ﬁlm. The proﬁles of the amide I and II infrared bands of Hb, especially, provide detailed information on the polypeptide chain . The amide I band (1700– 1600 cm1) is attributed to the [email protected] stretching vibration of peptide linkage in the backbone of protein, while the amide II band (1620–1500 cm1) is assignable to the combination of NAH bending and CAN stretching. Although the band of Hb from 1500 to 1600 cm1 overlaps with that of the polymer, the intensities of peaks at about 1653 and 1541 cm1 are mainly attributed to the absorbance of the amide I and II bands of Hb (see Fig. 4). It was shown that the amide I and II bands of Hb adsorbed on the MWNTs or entrapped in the hybrid ﬁlm (1653 and 1541 cm1), are very similar to those obtained in the protein spectrum (1658 and 1541 cm1). It is suggested that Hb retains the essential features of its original structure in the hybrid ﬁlm. Also ﬁve characteristic peaks of the surfactant polymer (1190, 1124, 1035, 1009 and 833 cm1) were observed in the spectrum of the hybrid ﬁlm. Our FT-IR spectroscopy study indicated that the functional hybrid ﬁlm could be successfully assembled by carbon nanotube, hemoglobin and polyelectrolyte–surfactant polymer. 54 L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59 Fig. 2. SEM images of the MWNTs ﬁlm (a), the Hb–MWNTs ﬁlm (b), the polymer–Hb–MWNTs hybrid ﬁlm 10,000· magniﬁcation (c) and 20,000· magniﬁcation (d). a a Hb Transmittance % Absorbance 1.0 b 0.5 c b Hb-MWNTs d c polymer-Hb-MWNTs 300 400 / nm 500 Fig. 3. UV–vis spectra of (a) 0.8 mg mL1 Hb solution, (b) 0.8 mg L1 Hb + 1 mg mL1 MWNT solution, (c) dry polymer–Hb–MWNTs hybrid ﬁlm, (d) 1 mg mL1 MWNT solution. 3.3. Direct electron transfer of Hb entrapped in the hybrid ﬁlm Fig. 5 depicts typical cyclic voltammograms obtained with the polymer–Hb-MWNTs hybrid ﬁlm modiﬁed electrode into pH 7.0 PBS buﬀer. For comparison, voltammograms of the Hb and Hb–MWNTs and polymer–Hb ﬁlms modiﬁed GC electrode were also given. As shown, no 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber / cm-1 Fig. 4. FT-IR spectra of the Hb ﬁlm (curve a), Hb–MWNTs ﬁlm (curve b) and polymer–Hb–MWNTs hybrid ﬁlm (curve c). redox peaks were observed at the above three ﬁlms in the potential range of interest, but a pair of well deﬁned and nearly symmetric redox peaks located at 0.213 V and 0.333 V was obtained at the polymer–Hb–MWNTs ﬁlm. And the potential diﬀerence between the two peak current, DEp, is 125 mV at a scan rate of 150 mV s1, which indicates a fast heterogeneous electron transfer process on the surface of the hybrid ﬁlm modiﬁed GC electrode. The 0 formal potential (E0 ) calculated by averaging the cathodic L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59 Current / 1e-6A 2 b a d c 0 -2 -0.8 -0.4 0.0 Potential / V vs SCE 0.4 Fig. 5. Typical cyclic voltammograms of Hb–MWNTs (curve a), polymer–Hb–MWNTs ﬁlm (curve b), Hb ﬁlm (curve c) and polymer–Hb ﬁlm (curve d) modiﬁed GC electrodes in 0.1 M phosphate buﬀer (pH 7.0) at a scan rate of 100 mV s1. and anodic peak potentials, is 0.273 V versus SCE, which agrees well with that obtained at a Hb ﬁlm at the CNT powder microelectrodes electrode . Table 2 lists the val0 ues of E0 of Hb at diﬀerent electrodes for comparison. According to those reported previously, the nearly revers- Table 2 0 Formal potentials ðE0 Þ and heterogeneous electron transfer rate constants (Ks) for hemoglobin on diﬀerent electrodes 0 Electrode pH E0 (V) versus SCE Ksa (s1) References Polymer–Hb–CNTs/GC Naﬁon–Hb–CNTs/GC Hb–AQ/PG Hb–PAM/PG Hb–DDAB/GC Hb–DDAB–PSS/PG Hb–DDAB–clay/PG Hb–Au colloid– cystamine/Au Hb–Au nanoparticle/GC 7.0 6.8 7.0 7.0 7.0 7.0 7.0 7.0 0.273 0.343 0.341 0.320 0.200 0.220 0.309 0.051 0.90 1.25 62 45 2.6 33 75 0.49 This work        7.0 0.257  Hb–sol–gel/PG (Hb–PDDA)n/PG Hb–DHP–PDDA/PG Hb/CNTPMEs Hb–SDS–CNTs/GC 7.0 7.0 7.0 7.0 7.0 0.312 0.340 0.340 0.278 0.300 Hb–DMPC/PG Hb–SP Sephadex/PG Hb–PHB/PG 7.0 7.0 5.0 0.329 0.290 0.254 Not shown 1.58 60 30 0.062 Not shown 70 102.12 10.33 55 ible CV peaks is a characteristic of the Hb heme Fe(III)/ Fe(II) redox couple. Hb is a kind of biological macromolecules containing four subunits. Several factors make it diﬃcult to achieve their direct electrochemistry at conventional electrodes such as GC, Au and Pt electrodes . These include (I) electroactive prosthetic group deep within the protein structure, (II) adsorptive denaturation of proteins onto electrodes, (III) unfavorable orientations at electrodes. In our experimental result, no redox peaks obtained at the Hb–MWNTs ﬁlm. It is probably ascribed to adsorbed Hb molecules in unfavorable orientations. The unique porous structure of surfactant polymer ﬁlm could yield biomembrane-like microenvironment for Hb. So Hb entrapped in the hybrid ﬁlm may has suitable orientations and exhibits well-behaved electrochemistry and good catalytic properties towards a variety of substrate. The CV reduction and oxidation peaks currents for entrapped Hb are found to increase linearly with potential scan rates from 20 to 400 mV s1 (see Figs. 6a and 6b). The linear regression equations are: y = 0.118 + 0.1260x, r = 0.9998 (anodic peak), and y = 0.3103 0.1240x, r = 0.9999 (cathodic peak). Fig. 7 shows the logarithm plot of anodic peak current versus logarithm of scan rate gives a linear relationship, with a correlation coeﬃcient of 0.9998 and a slope of 0.92, which is very close to the theoretical slope of 1 for thin layer voltammetry. These results suggest that all electroactive Hb Fe(III) in the ﬁlm has been converted to Hb Fe(II) on the reverse scan. Integration of reduction or oxidation peaks at diﬀerent scan rates gave nearly constant charge (Q) values. The surface concentration of the electroactive Hb entrapped in the hybrid ﬁlm, C (in mol/cm2), can be estimated using the Laviron’s equation :         GC, glassy carbon; PG, pyrolytic graphite; CNT, carbon nanotube; CNTPMEs, CNT powder microelectrode electrodes; AQ, Eastman AQ; PAM, polyacrylamide; DDAB, didodecyldimethylammonium bromide; PSS, poly(styrene sulfonate), PDDA, poly(diallyldimethylammonium); DHP, dihexadecylphosphate; SDS, sodium dodecylsulfate; DMPC, dimyristoyl phosphatidylcholine; PHB, poly-3-hydroxybutyrate. a Ks for hemoglobin on diﬀerent electrodes were estimated by diﬀerent methods. Fig. 6a. Cyclic voltammograms of the polymer–Hb–MWNTs hybrid ﬁlm modiﬁed GC electrode in a 0.1 MPBS buﬀer (pH 7.0) at various scan rates. Scan rate (from inner to outer): 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400 mV s1. 56 L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59 5 4 Peak current / 10-6A 3 2 1 0 -1 -2 -3 -4 -5 -6 0.0 0.1 0.2 0.3 0.4 V / vs -1 Fig. 6b. Plot of peak current versus scan rate in a 0.1 M pH 7.0 phosphate buﬀer. 0.8 log(peak current) 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 log(scan rate) Fig. 7. Linear logarithm plot of anodic peak current versus scan rate in 0.1 M phosphate buﬀer solution. Fig. 8. Cyclic voltammograms of the polymer–Hb–MWNTs ﬁlm modiﬁed GC electrode in 0.1 M phosphate buﬀer at diﬀerent pH values. Scan rate: 150 mV s1. that the electron transfer of Hb entrapped in the hybrid ﬁlm is a fast process. It is known that solution pH modulates the accessibility of water to the heme pocket of Hb and the protonation of the heme iron-bound proximal histidine and/or the distal histidine in the heme pocket and accordingly inﬂuences the redox potential of Hb . So the dependence of Epa, 0 Epc, and E0 of the hybrid ﬁlm modiﬁed electrode on the solution pH was examined (see Fig. 8). It is observed that the increase of the value of pH in buﬀers led to a negative 0 0 shift of Epa, Epc, and E0 . Epa, Epc, and E0 had linear relationships with solution pH with slopes of 45.8, 49.4, and 52.8 mV pH1 unit, respectively, in the range of 5–11. 0 The E0 values is close to the 58 mV pH1 unit expected for a reversible, one-electron coupled one-proton reaction process at 20 C, which indicates that a single protonation accompanies a single electron transfer between the electrode and those of the four heme Fe(III) of Hb. I p ¼ n2 F 2 ACv=4RT ¼ nFQv=4RT 3.4. Catalytic reduction of H2O2 Here, n is the number of electron transferred, F, Faraday’s constant, and A, the electrode area. According to the C Q relationship, the average surface concentration of electroactive Hb was estimated to be 1.1 · 1010 mol cm2, which accounted for only about 2.5% of the total amount of Hb deposited on the electrode. This may suggest that only those Hb molecules in the inner layers of the ﬁlms closest to the electrodes and with a suitable orientation can exchange electrons with the electrode and contribute to the observed redox reaction. From the dependence of DEp on the various scan rates, the apparent heterogeneous electron transfer rate constant, Ks, can be estimated to be 0.9 s1 using the method developed by Laviron  for a surface-controlled electrochemical system. It was indicated Having demonstrated the redox properties of the hybrid ﬁlm, we next investigated its bioelectrochemically catalytic activity. It has been reported that proteins containing the heme group had the ability to reduce H2O2 electrocatalytically. To check the bioelectrocatalytic activity of the polymer–Hb–MWNTs hybrid ﬁlm modiﬁed electrode, cyclic voltammetric experiments were performed (Fig. 9). When H2O2 was added to a pH 7.0 buﬀer, an increase in reduction peak at about 0.34 V was seen with the disappearance of the oxidation peak for HbFe(II) . Controlled experimental results indicated that no catalytic current was observed at the bare GC electrode and only a very small cathodic current was observed at the MWNT/GC electrode in the presence of H2O2, which was also observed in previous publication . Fig. 10 showed the steady- L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59 57 Here, Iss is the steady-state current after the addition of substrate, c is the bulk concentration of the substrate and Imax is the maximum current measured under saturated substrate conditions. The Km value of the hybrid ﬁlm was found to be 1.4 · 104 M. The low value of Km implied that entrapped Hb exhibits a high biological aﬃnity for H2O2. 3.5. Catalytic reduction of O2 and NO 2 Fig. 9. Cyclic voltammograms obtained at the hybrid ﬁlm modiﬁed GC electrode in 0.1 M phosphate buﬀer with pH 7.0 before (curve a) and after the addition of 0.06 (curve b), 0.21 mM (curve c) H2O2. Scan rate: 150 mV s1. Catalytic CV behavior was also observed for oxygen at the hybrid ﬁlm modiﬁed electrodes (Fig. 11), which is very similar to that of a hydrogen peroxide system. When a certain volume of air was injected into a previously deaerated buﬀer at pH 7.0 in a sealed cell by a syringe, a signiﬁcant increase in reduction peak at 0.25 V was observed, accompanied by the disappearance of the oxidation peak for HbFe(II), the reduction peak current increased with the amount of oxygen in solution. Compared with MWNT ﬁlm modiﬁed electrode, the hybrid ﬁlm modiﬁed electrode decreased the reduction overpotential of oxygen about 200 mV and the peak current for the oxygen reduction is larger (see Fig. 12). So it was concluded that the active Hb entrapped in the hybrid ﬁlm play an important role in the electroreduction of oxygen. The catalytic eﬃciency expressed as the ratio of the reduction peak current of HbFe(II) in the presence (Ic) and absence of oxygen (Id), i.e., Ic/Id, decreases within the increase of scan rate, which is also a characteristic of electrochemical catalytic reduction of oxygen by Hb entrapped in the composite ﬁlm. The potential of the reduction peak is about 100 mV more 0 positive than the E0 of the Hb heme Fe(III)/Fe(II) redox couple, which could be explained by the fact that the reduction peak belonged to the reduction of HbFe(II)–O2, rather than reduction of HbFe(III). When the solution was deoxygenated and oxygenated in turn, a reproducible reduction Fig. 10. Current–time response curve of the hybrid ﬁlms with successive additions of 0.06 mM H2O2, in a 0.1 M phosphate buﬀer (pH 7.0) at the applied potential 0.2 V. Inset is the calibration curve. state current response of H2O2. The response was linearly within the concentration range from 8 · 106 M to 2.4 · 104 M, the linearly regression equation was I (lA) = 0.3752 + 2.236 · 103[H2O2] (M), with a correlation coeﬃcient of 0.997. The detection limit was 4 · 106 M (three times the ratio of signal to noise). When the concentration of H2O2 was higher than 3 · 104 M, a response plateau is observed, showing the characteristic of the Michaelis–Menten kinetic mechanism. The apparent Michaelis–Menten constant (Km), which is an indication of the enzyme–substrate kinetics, can be calculated using the Lineweaver–Burk equation : 1=I ss ¼ 1=I max þ K m =I max c Fig. 11. Cyclic voltammograms of the hybrid ﬁlm modiﬁed GC electrode in 15 mL 0.1 MPBS (pH 7.0): (a) with no oxygen present; (b) after injection of 6 mL air; (c) after injection of 12 mL air; (d) after injection of 18 mL air; (e) after injection of 24 mL air. Scan rate: 100 mV s1. 58 L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59 0.8 Current/1e-5A 0.4 0.0 -0.4 -0.8 a b c -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 Potential / V vs SCE Fig. 12. Cyclic voltammograms of the MWNTs ﬁlm modiﬁed GC electrode in 15 mL 0.1 MPBS (pH 7.0): (a) with no oxygen present; (b) after injection of 12 mL air; (c) after injection of 24 mL air. Scan rate: 100 mV s1. peak current could be obtained. In other words, the reduction activity of Hb to oxygen did not decrease in the processes. The exact mechanism of catalytic reduction of oxygen on the hybrid ﬁlm is not yet clear but probably similar to that reported by Hu  and Rusling . For example, direct electrochemical reduction of HbFe(III) to HbFe(III) + e- HbFe(II) at electrode HbFe(II) + O 2 → HbFe(II)-O2 fast HbFe(II)-O2 + 2e + 2H → HbFe(II) + H2O2 at electrode - + Scheme 1. HbFe(II) occurred at the electrode, followed by a fast reduction of HbFe(II) with oxygen. The product of HbFe(II)–O2 could then undergo electrochemical reduction at the potential of HbFe(II) reduction, producing hydrogen peroxide and HbFe(II) again. Thus, the simpliﬁed mechanism for the electrochemical catalytic reduction of O2 on the hybrids modiﬁed electrode maybe expressed as Scheme 1. The hybrid ﬁlm modiﬁed electrode can be also used to catalyze reduction of nitrite (see Fig. 13). When a modiﬁed electrode was placed in pH 6.0 PBS buﬀer containing NO 2, a new reduction peak appeared at about 0.82 V. The direct reduction of NO 2 at blank MWNTs ﬁlm electrode was hardly observed in the studied potential window. The reduction peak currents of NO 2 at the hybrid ﬁlm varied approximately linearly with the concentration of NO 2. Although the mechanism of NO 2 reduction at on the polymer–Hb–MWNTs hybrid ﬁlm is yet not clear, probably it is similar to that on Mb–DDAB ﬁlm reported by Farmer et al. . On Mb–DDAB ﬁlm modiﬁed electrodes, the conformed catalytic reduction product was N2O, which was detected by mass spectroscopy on electrocatalysis at 0.895 V in pH 7.0 buﬀer. 4. Conclusions The properties of Hb entrapped in the hybrid ﬁlm were characterized by both spectroscopic and electrochemical methods. It was observed that Hb could facilely exchange electrons with the electrode at the same time and achieve a high peroxidase activity. It was also demonstrated that carbon nanotubes combined with polyelectrolyte–surfactant polymer could yield a new platform for facilitate direct electron transfer of proteins. 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