Direct Electron Transfer of Hemoglobin Founded on Electron

Aug 2, 2007 - College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China, State Key Laboratory of Transducer Technology,...
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J. Phys. Chem. B 2007, 111, 9808-9813

Direct Electron Transfer of Hemoglobin Founded on Electron Tunneling of CTAB Monolayer Qing Lu,†,‡ Chengguo Hu,† Ran Cui,† and Shengshui Hu*,†,‡ College of Chemistry and Molecular Sciences, Wuhan UniVersity, Wuhan 430072, P. R. China, State Key Laboratory of Transducer Technology, Chinese Academy of Sciences, Beijing 100080, P. R. China ReceiVed: February 12, 2007; In Final Form: April 26, 2007

Direct electron transfer and stable adsorption of hemoglobin (Hb) on a carbon paste (CP) electrode were achieved with the aid of a single-chain cationic surfactant, namely, cetyltrimethylammonium bromide (CTAB). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) indicated that CTAB could form a complete monolayer with a high density of positive charges on the surface of the CP electrode, which strongly adsorbed negatively charged protein molecules via electrostatic interactions. The surfactant molecules anchored the protein molecules to align in suitable orientations and acted as electron-tunneling pathways between the protein molecules and the CP electrode. The bioelectrocatalytic activity of the immobilized Hb was confirmed by RAIR and UV-vis spectroscopies, and rapid electrochemical responses to the reduction of oxygen (O2), hydrogen peroxide (H2O2), and nitrite (NO2-) were also obtained.

Introduction Surfactants are a type of amphiphilic molecule with a polar head at one end and a long hydrophobic tail at the other. They can spontaneously adsorb on the interfaces of two phases with different polarities or associate into micelles in solutions. These special structures and properties allow surfactants to have wide applications in electrochemistry and electroanalytical chemistry.1 For instance, Hu’s group has utilized surfactants in electroanalytical chemistry to improve the detection limits of several biomolecules.2-5 The presence of trace surfactants in electrolytes significantly improved the sensitivities of these substrates by at least a factor of 5. A “synergistic adsorption” mechanism was proposed to explain the enhancement effects of surfactants, i.e., surfactants might associate with the substrates in certain forms and strengthen their adsorption on the electrode surfaces through the adsorption of surfactants at the electrode surfaces. The adsorption of surfactants on the electrode surfaces might also alter the interface properties of the electrodes and promote the charge-transfer process of the electrode reaction, resulting in the reduction of the overpotentials.3,5 In addition to being added to the working solutions, surfactants could also be blended into the electrode matrixes or immobilized on the electrode surfaces to prepare chemically modified electrodes. Digua et al.6-8 mixed amphiphilic hexadecyl sulfonic acid into carbon paste to produce a chemically modified carbon paste electrode. This kind of electrode exhibited a strong cation-exchange property and an improved electrontransfer rate between the substrates and the electrode. Falaras et al.9-11 prepared an organoclay-modified glassy carbon electrode coated with a cationic surfactant bilayer. This electrode was found to exhibit an anion-exchange ability and to accumulate both negatively charged metal complexes and neutral redox-active reagents. Hu and Hu prepared a cetyltrimethylam* To whom correspondence should be addressed. E-mail: sshu@ whu.edu.cn. † Wuhan University. ‡ Chinese Academy of Sciences.

monium bromide- (CTAB-) modified carbon paste (CP) electrode based on the surface modification method.12 This modified CP electrode could be applied to the immobilization of DNA, which was characterized by the adsorption isotherm of Co(phen)32+. Electron-transfer reactions are crucial in many natural energy conversion processes, from photosynthesis to respiration, most of which are mediated by proteins. When the proteins have redox-active centers, their direct electron-transfer reactions can be used to probe the nature of those energy conversion processes.13,14 Generally, a protein’s electroactive center is deeply embedded in the protein structure. An unfavorable orientation of protein molecules on the electrode surface can block electron transfer between the electrode and protein electroactive centers. Moreover, the adsorption of protein molecules onto the bare electrode surface would lead to their denaturation, which also decreases the direct electron-transfer rate.15,16 Because surfactants have an amphoteric structure similar to that of lipids and can form stable membranes similar to lipid membrane, several double-chain phosphate surfactants have been successfully utilized to immobilize proteins on electrode surfaces and shown to efficiently promote the direct electrochemistry of these proteins.17-19 In those earlier studies, it was proposed that protein molecules might be intercalated between multiple surfactant bilayers, just as they can be inserted into lipid membranes. That is, the multiple surfactant bilayers might provide microenvironments similar to those observed in real systems that are favorable for facilitating electron-transfer reactions of proteins. Unlike the method of protein immobilization with doublechain phosphate surfactants,17-19 a new approach was performed in this work to immobilize hemoglobin (Hb) on the surface of a CP electrode via electrostatic interactions. Because the singlechain surfactant, CTAB, can form a stable monolayer containing positive charges on the electrode surface based on the hydrophobic interactions between the hydrophobic long chain of the CTAB molecule and the paraffin oil in the carbon paste,12 the negatively charged Hb molecules can be adsorbed onto the

10.1021/jp071201t CCC: $37.00 © 2007 American Chemical Society Published on Web 08/02/2007

Direct Electron Transfer of Hemoglobin

J. Phys. Chem. B, Vol. 111, No. 33, 2007 9809

surface of CTAB/CP electrode stably. The surface immobilization method shows advantages over the embedding approaches, allowing for the convenient modulation of the desired properties of the immobilized proteins, such as density, orientation, and bioactivity, at the molecular scale. Furthermore, it is simple and inexpensive to prepare CTAB-modified CP electrodes and immobilize the protein. Here, a pair of well-defined and quasireversible cyclic voltammetric peaks were obtained at the Hb/CTAB/CP electrode, which suggests that CTAB could apparently enhance the direct electron-transfer interaction between Hb and the electrode. This is similar to the case of Hb entrapped in double-chain surfactant films, whereas the essential origin of the CTAB enhancement of the direct electron transfer of Hb is dissimilar. Substantively, CTAB could form monolayers of different types depending on its concentration on the electrode surface that would, in fact, influence the direct electron-transfer process of proteins. In addition, Hb at the modified electrode kept its catalytic activity toward oxygen (O2), hydrogen peroxide (H2O2), and nitrite (NO2-).

(Beijing Purkinje General Instrument, Beijing, China). Scanning electron microscopy (SEM) was performed with an X-650 scanning electron microanalyzer (Hitachi, Tokyo, Japan).

Experimental Section

Results and Discussion

Materials. Pig hemoglobin (MW 68000) was obtained from Sigma and used without further purification. CTAB, K3Fe(CN)6, K4Fe(CN)6, and NaNO2 (purchased from Shanghai Reagent Corporation, Shanghai, China) were dissolved in water to prepare 0.01, 0.1, 0.1, and 1 M stock solutions, respectively. Stock solutions of H2O2 were freshly diluted from 30% solution (purchased from Beijing Chemical Reagent Factory) and purged with high-purity nitrogen for 1 h. All other chemicals were of analytical grade and were used as obtained. All solutions were prepared with twice-distilled water. Preparations of the Modified Electrode. The working electrode was prepared as follows: One hundred milligrams of graphite powder and 16 µL of paraffin oil were mixed by hand to produce a homogeneous carbon paste. The carbon paste was packed into the cave of a homemade carbon paste electrode and then smoothed on a weighing paper. Subsequently, a 10µL drop of 1 × 10-4 M CTAB was dipped onto the surface of the bare electrode. Five minutes later, the electrode was completely rinsed with water to remove the unadsorbed modifier and air-dried. The effective surface area of the CTAB/CP electrode was determined by cyclic voltammmetry (CV) using the Randies-Sevcik equation for the reversible redox couple [Fe(CN)6]3-/[Fe(CN)6]4- with an average value of 0.19 cm2. Then, the electrode was immersed into the Hb solution (3 mg/ mL in pH 7.4 PBS) for 4 h. The final electrode was also rinsed thoroughly with water to remove the unadsorbed Hb molecules and dried in air. The Hb/CTAB/CP electrode was stored at 4 °C in a phosphate buffer (0.1 M, pH 7.4). Apparatus. Cyclic voltammetry (CV) was performed using a CHI660B electrochemical workstation (CH Instruments, Shanghai, China). A three-electrode system, including a working Hb/CTAB/CP electrode, a saturated calomel reference electrode (SCE), and a platinum wire counter electrode, was employed. Prior to each experiment, the buffer solutions were purged with high-purity nitrogen for at least 30 min, and a nitrogen environment was then kept over the solution in the cell. All experiments were carried out at room temperature (25 °C). Electrochemical impedance spectroscopy (EIS) was performed with a 283 potentiostat/galvanostat (EG&G Instruments, Oakridge, TN). Reflectance absorption infrared (RAIR) spectroscopy was collected using a Magna-IR Spectrometer 500 (Nicolet Instrument Corporation, Madison, WI). Ultraviolet visible (UV-vis) spectra were recorded on a TU-1901 UV-vis spectrophotometer

Direct Electron Transfer of Hb at a CTAB-Modified CP Electrode. The direct electron-transfer behavior of Hb at a CTAB/CP electrode was characterized by CV. To discern the roles of individual components of the modified electrode, the electrochemical behaviors of Hb/CTAB/CP, CTAB/CP, and bare CP electrodes in 0.1 M PBS (pH 7.4) were studied (Figure 1). Obviously, the Hb/CTAB/CP electrode yields a pair of welldefined and quasireversible redox peaks (curve d). However, there are no redox peaks at the CTAB/CP electrode (curve b) or the bare CP electrode (curve a). These results indicate that the pair of peaks is characteristic of the Fe(III)/Fe(II) redox couple for the heme protein. The anodic peak potential (Epa) and the cathodic peak potential (Epc) are located at -0.294 and -0.348 V, respectively, with a formal potential (E°′, defined as the average of Epa and Epc) at -0.321 V. Curve c shows the cyclic voltammogram of Hb adsorbed on a bare CP electrode. A couple of very small peaks are observed at the Hb/CP electrode with E°′ located at -0. 343 V, which differs from the results observed for Hb/CTAB/CPE. The CV behaviors of heme adsorbed on bare CP and CTAB-modified CP electrodes were also studied. A pair of nearly reversible redox peaks could be obtained at both the heme/CP and heme/CTAB/CP electrodes (not shown) with E°′ located at -0.343 and -0.357 V, respectively, also unlike the results for Hb/CTAB/CPE. Thus, it can be concluded that the redox peaks in curve d should profit from the electrochemical reaction of Hb at the CTAB-modified CP electrode. Furthermore, the ratio of the cathodic current to the anodic current for Hb at the CTAB/CP electrode is close to 1. The peak width at half-height goes well beyond the theoretical 90 mV for a one-electron surface reaction, and the separation of the peak potential (∆Ep), 54 mV, is in excess of the ideal thin-film value of 0 mV.20 These results coincide well with heme Fe(III)/Fe(II) couples of the proteins.21,22 The value of E°′, -0.321 V, is similar to that of Hb entrapping into surfactant films, polymer films, and composite films of surfactant and clay on pyrolytic graphite electrodes reported previously.18 It is also similar to those of other heme-containing proteins (enzymes), including myoglobin,23 horseradish peroxidase,24 and cytochrome P450cam.25 Figure 2 displays an overlap of cyclic voltammograms of Hb at scan rates (V) of 0.04-0.5 V s-1. The reduction and oxidation peak currents exhibit a linear relationship with the scan rate (shows in the inset with r ) 0.999), and the charge consumed,

Figure 1. Cyclic voltammograms at 0.1 V s-1 in pH 7.4 buffers for (a) bare CP, (b) CTAB/CP, (c) Hb/CP, and (d) Hb/CTAB/CP electrodes.

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Figure 2. Cyclic voltammograms of Hb at a CTAB/CP electrode in a 0.1 M PB buffer (pH 7.4) at scan rates of (a) 0.5, (b) 0.4, (c) 0.3, (d) 0.2, (e) 0.1, (f) 0.08, (g) 0.06, and (h) 0.04 V s-1. The inset shows a plot of the reduction and oxidation peak currents i against the scan rate V.

Q (obtained from integrating the anodic or cathodic peak area in the cyclic voltammograms), was essentially constant under background correction. Furthermore, plots of log Ipc vs log V and log Ipa vs log V were linear and had a ratio of the slopes (SIpc/SIpa) of about 1. These results suggest that the electrochemical reaction of Hb immobilized at the surface of the CTAB/CP electrode is a surface-controlled behavior.26 In this process, all electroactive ferric proteins [protein Fe(III)], produced by the oxidation of ferrous proteins [protein Fe(II)] on the anodic scan, could be reduced to protein Fe(II) on the cathodic scan. In this system, the surface concentration of the electroactive Hb (Γ*) can be calculated by integrating the CV reduction peak and applying the formula of Q ) nFAΓ*, where Q is the integrated charge (C) of the reduction peak and the charge value (Q) is nearly constant at different scan rates, A is the effective area of the CP electrode (0.19 cm2), and the other symbols have their usual meanings. In the range of scan rate from 0.04 to 0.5 V s-1, the average Γ* value was 1.76 × 10-11 mol cm-2. This value is very similar to the theoretical monolayer coverage for Hb (i.e., 1.89 × 10-11 mol cm-2), when taking into account the crystallographic dimensions of 6.4 nm × 5.5 nm × 5.0 nm. This result reflects the fact that only the adsorbed Hb molecules of the innermost layer on the electrode surface could transfer electrons to and from the electrode to contribute to the observed redox peaks. The apparent heterogeneous electron-transfer rate constant, ks, of Hb at the CTAB/CP electrode was estimated by using the equation derived by Laviron for diffusionless CV.27 The average value of ks, 1.86 s-1, suggests reasonably fast electron transfer. It is well-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. Consequently, solution pH influences the redox potential of Hb. Figure 3 shows cyclic voltammograms of the Hb/CTAB/CP electrode in 0.1 M PBS at different pH values (curves d-k). As the inset shows, Epa (curve a), Epc (curve b), and E°′ (curve c) shift in the negative direction with increasing solution pH value. Within the pH range from 3.0 to 10.0, Epa, Epc, and E°′ are linearly dependent on the solution pH with slopes of -41, -38, and -39 mV/pH, respectively. These slope values are smaller than the theoretical value of -59 mV/pH for a single-proton coupled, reversible one-electron transfer. The reason for this deifference bears on the influence of the protonation of trans and residue ligands around the heme and the protonation of the water molecules coordinated with the central iron.28

Lu et al.

Figure 3. Cyclic voltammograms of Hb/CTAB/CP electrode in 0.1 M PBS at pH values of (d) 3.0, (e) 4.0, (f) 5.0, (g) 6.0, (h) 7.0, (i) 8.0, (j) 9.0, (k) 10.0 obtained at a scan rate of 0.1 V s-1. The inset shows the relationship between (a) Epa (9), (b) Epc (b), and (c) E°′ (2) and the solution pH value.

Figure 4. SEM images with the same magnification of (left) CTAB/ CPE and (right) Hb/CTAB/CPE.

CTAB Enhancement for Direct Electron Transfer. Unlike common solid electrodes, such as glassy carbon electrodes and a variety of metal electrodes, the CP electrode is a combination electrode that is made up of adhesives (i.e., paraffin oil) and carbon particles. Generally, the adhesives in carbon paste electrodes are hydrophobic and can accumulate less soluble substrates from solutions on the electrode surfaces via hydrophobic interactions. Based on the hydrophobic interaction between the hydrophobic long chain of CTAB and the paraffin oil in carbon paste, CTAB could form a stable monolayer on the electrode surface.12 The CTAB/CP electrode has some unique properties, especially being covered with a high density of positive charges on the electrode surface. The isoelectric point of pig Hb is 6.9. Thus, Hb molecules are negatively charged when dissolved in pH 7.4 PBS. When the positively charged CTAB/CP electrode was immersed into the Hb solution, the negatively charged Hb molecules were adsorbed onto the surface through electrostatic attractions. Figure 4 shows SEM images of CTAB/CPE and Hb/CTAB/CPE. Because the CP electrode is made up of graphite power and paraffin oil, the surface of the CPE is quite rough. Compared to the rough surface of CTAB/CPE (left), the top view of Hb/CTAB/CPE (right) seems less rough. However, it is difficult to verify from the SEM images whether Hb molecules are adsorbed onto the surface of CTAB/CPE. To confirm the existence of electrostatic attractions between the Hb molecules and the CTAB/CP electrode, the electrochemical responses of 1.0 × 10-4 M K4Fe(CN)6 at CTAB/CP and Hb/CTAB/CP electrodes were examined (shown in Figure 5A). Because of the synergistic adsorption mechanism of CTAB, a pair of reversible redox peaks is achieved at the CTAB/CP electrode (curve a). However, the voltammetric response is apparently deteriorated at the Hb/CTAB/CP electrode (curve b). It is obvious that the positive charges of CTAB are

Direct Electron Transfer of Hemoglobin

Figure 5. (A) Electrochemical responses of 1.0 × 10-4 M K4Fe(CN)6 at (a) CTAB/CP and (b) Hb/CTAB/CP electrodes. (B) Impendence plane diagram (-Z′′ versus Z′) for the EIS measurements on (c) CTAB/ CP and (d) Hb/CTAB/CP electrodes in the presence of a 1.0 × 10-4 M K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture containing 0.1 M KCl at the formal potential of the system, 0.23 V.

neutralized by the negative charges of Hb. For this reason, the synergistic adsorption effect of CTAB for K4Fe(CN)6 is greatly decreased. Similar results were obtained by electrochemical impedance spectroscopy (EIS). Figure 5B shows the complex plane diagrams (-Z′′ versus Z′) of impedance spectroscopy on CTAB/CPE and Hb/CTAB/CPE for a 1.0 × 10-4 M K3[Fe(CN)6]/K4[Fe(CN)6] mixture in 0.1 M KCl. The EIS diagram of the CTAB/CP electrode (curve c) displays a nonlinear curve, very different from that of the bare CP electrode (not shown), suggesting that CTAB might form a monolayer on the surface of the CP electrode.29 In contrast, the EIS diagram of the Hb/ CTAB/CP electrode (curve d) includes a semicircular part and a linear part, of which the semicircular part at high frequencies corresponds to the electron-transfer-limited process and the linear part at low frequencies corresponds to the diffusionlimited process.30 We also investigated the Bode plots, log |Z| vs log f (frequency) and θ (phase angle) vs log f, of these two electrode systems (not shown). All of the EIS results were similar to the phenomena reported in Hu et al.’s work,29 indicating that the concentration of CTAB that could adsorb Fe(CN)63- and Fe(CN)64- was reduced at the Hb/CTAB/CP electrode. This decrease was attributed to the electrostatic attractions between Hb and CTAB, which lowered the positive charges on CTAB. When the CTAB concentration was changed from 1 × 10-6 to 7 × 10-4 M, similar differences between the Hb/CTAB/CPE and the CTAB/CPE were obtained by CV and EIS. The CTAB concentration of the CP electrode modification has a visible influence on the electrochemical response of Hb at the CTAB/CP electrode. When the CTAB concentration was 1 × 10-6 M, no redox peaks appeared. When 1 × 10-5 M CTAB was used, a small pair of redox peaks could be observed. With a further increase of the CTAB concentration, the currents of the redox peaks increased. When the concentration of CTAB reached 1 × 10-4 M, the redox peak currents attained a maximum. However, with a further increase of the CTAB concentration, the currents dropped slightly. It has been reported that, when the CTAB concentration is higher than 1 × 10-5 M, CTAB forms a complete monolayer on the electrode surface.29 With a further increase of the CTAB concentration, the density of the monolayer increases. Therefore, we can reasonably explain the experimental electrochemical results in view of the adsorption behaviors of CTAB at the CP electrode. The schematic representation is shown in Figure 6. Below 1 × 10-5 M (e.g., for 1 × 10-6 M), the adsorption of CTAB is as the monomer, and the CTAB monomer lies on the surface of the CP electrode (Figure 6A). The modified electrode at this phase adsorbs few Hb molecules irregularly. When the concentration reaches 1 × 10-5 M, CTAB forms a monolayer and is fixed

J. Phys. Chem. B, Vol. 111, No. 33, 2007 9811 slantways (Figure 6B). In this way, a quantity of Hb molecules is adsorbed on account of the positively charged CTAB monolayer. The direct electron transfer of Hb at a CP electrode is difficult without CTAB because of the high anisotropy of the Hb R-helix. In the presence of a CTAB monolayer, CTAB can couple with the Hb molecules via electrostatic interactions and act as an electron-tunneling pathway between Hb and the electrode. Thus, the direct electrochemical response of Hb is obvious. With increasing CTAB concentration, the density of the monolayer is increased, and the CTAB molecules’ standing gradient is also increased. Increasing numbers of Hb molecules can be adsorbed because of the increase in the number of positive charges. On the other hand, the CTAB molecules’ standing gradient anchors the protein molecules to more perfectly align in a suitable orientation. Integrating these two aspects, the electrochemical response of Hb is notably enhanced (Figure 6C). When the CTAB concentration exceeds 1 × 10-4 M, even though the number of adsorbed Hb molecules keeps rising, the CTAB molecules’ standing gradients are not favorable to the direct electron transfer of Hb. As a result, the redox current drops slightly instead (Figure 6D). That is, the presence of CTAB (g1 × 10-5 M) can promote the electron transfer between the protein molecules and the electrode from several aspects. Biological Activity of Hb. Various other studies have shown that interactions between surfactants and myoglobin can induce the release of heme groups,31-33 resulting in the incorporation of the heme groups into micelles.32 In this work, CTAB forms not micelles but rather a monolayer on the surface of the CP electrode. It contacts Hb molecules only with its polar head through electrostatic interactions. The possibility for CTAB to release the heme groups from Hb molecules is very small. This can be validated by reflectance absorption infrared (RAIR) spectroscopy and ultraviolet visible (UV-vis) spectroscopy. RAIR spectroscopy is usually used to provide detailed information on the secondary structure of polypeptide chains and to detect conformational changes of proteins.34 Figure 7 shows the RAIR spectra of Hb/CPE and Hb/CTAB/CPE. We pay attention to the shapes of the amide I and amide II infrared absorbance bands. The amide I band at 1700-1600 cm-1 is caused by the CdO stretching vibrations of the peptide linkage. The amide II band at 1600-1500 cm-1 results from a combination of N-H in-plane bending and C-N stretching of the peptide groups. As can clearly be seen, the RAIR spectrum of Hb/CTAB/CPE (curve b) has shapes and positions of the amide I and amide II bands that are similar to those of Hb/CPE (curve a), which suggests that Hb adsorbed onto CTAB/CPE is not denatured. Furthermore, the RAIR data for the immobilized Hb after CV cycles (curve c) also have shapes and positions of the amide I and amide II bands similar to those of Hb/CTAB/CPE before the CV cycles, indicating that the protein molecules retain their natural structure during the CV cycles. The shape and position of the Soret absorption band of heme iron can also provide structural information about the heme pocket. Figure 8 displays UV-vis spectra of heme, Hb, and Hb/CTAB solutions, as well as dry Hb, CTAB, and Hb/CTAB films. For Hb/CTAB solution (curve a), the Soret band is located at 406 nm, the same as the solution of Hb alone (curve b). The UV-vis spectra of the dry Hb (curve c) and Hb/CTAB (curve d) films show Soret bands at 412 nm. A shift of only several nanometers in the Soret absorption band of the protein suggests that the microenvironment of the heme pocket is slightly changed when Hb is immobilized in dry film.35 Just because the quantity of Hb molecules in the films is much smaller than

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Lu et al.

Figure 6. Schematic views of the interaction between Hb and CATB at different concentrations and the electron transfer between the Hb molecules and the electrode: (A) 1.0 × 10-6, (B) 1.0 × 10-5, (C) 1.0 × 10-4, and (D) 4.0 × 10-4 M.

Figure 7. Reflectance absorbance infrared (RAIR) spectra of (a) a Hb/CP electrode, (b) a Hb/CTAB/CP electrode before any CV cycles, and (c) a Hb/CTAB/CP electrode after CV cycles. The absorbance coordinate reflects only relative values.

Figure 8. UV-vis absorption spectra of (a) Hb solution, (b) Hb/CTAB solution, (c) dry Hb film, (d) dry Hb/CTAB film, and (e) dry CTAB film. The inset shows a UV-vis absorption spectrum of heme in solution.

that in solutions, the absorbance values of dry films are much lower than those of solutions. For comparison, the UV-vis spectrum of heme (inset) in solution was also investigated. The Soret band of heme is at 389 nm, very different from the values above. The electrocatalytic activity of a Hb/CTAB/CP electrode toward various substrates of biological or environmental significance, such as hydrogen peroxide (H2O2), oxygen (O2), and nitrite (NO2-), was examined and characterized by CV. A typical catalytic reduction peak of H2O2 at Hb/CTAB/CP electrode is shown in Figure 9. Upon addition of 84.6 µM H2O2 to the electrochemical cell, in comparison to the Hb/CTAB/ CPE in the absence of H2O2 (curve c), an increase of the reduction peak at about -0.33 V is observed, accompanied by a decrease of the oxidation peak (curve d), indicating a typical electrocatalytic reduction process of H2O2. The reduction peak current further increases upon the addition of H2O2 in buffer (curve e). For the CTAB/CPE, however, there is no difference between the absence (curve a) and presence (curve b) of H2O2.

Figure 9. Cyclic voltammograms at 0.1 V s-1 in pH 7.4 buffer for (a) CTAB/CPE and (c) Hb/CTAB/CPE without H2O2, (b) CTAB/CPE and (d) Hb/CTAB/CPE in buffer containing 84.6 µM H2O2, and (e) Hb/ CTAB/CPE in buffer containing 129.6 µM H2O2.

Figure 10. Amperometric responses at Hb/CTAB/CP electrode at the potential of -0.40 V in 5 mL of PB buffer (pH 7.4) with injection of 4.5 µM H2O2 every 30 s. The inset is a plot of the catalytic peak current against the concentration of H2O2.

The amperometric response of the Hb/CTAB/CP electrode upon successive additions of 4.5 µM H2O2 (PBS, pH 7.4) at an applied potential of -0.40 V is illustrated in Figure 10. Upon addition of an aliquot of H2O2 to the electrochemical cell, the reduction current increases rapidly to reach a stable value. The modified electrode reaches 95% of the steady-state current in less than 2 s, which indicates that the electrocatalytic response is very fast. The current response and the concentration of H2O2 have a linear relationship in the concentration range from 4.5 × 10-6 to 5.85 × 10-5 M with a correlation coefficient of 0.998 (inset). The detection limit was estimated to be 1.8 × 10-6 M at a signalto-noise ratio of 3. When the concentration of H2O2 was higher than 2.2 × 10-4 M, the calibration curve tended to a plateau and then decreased upon addition of H2O2, implying progressive enzyme inactivation in the presence of higher concentrations of H2O2. The similar catalytic results were observed for O2 in the same type of experiments on a Hb/CTAB/CP electrode (not shown). The reduction peak current increased linearly with the concentration of O2 in the solution in the range from 1.23 ×

Direct Electron Transfer of Hemoglobin

J. Phys. Chem. B, Vol. 111, No. 33, 2007 9813 which was confirmed by RAIR spectroscopy, UV-vis spectroscopy, and catalysis of the reductions of H2O2, O2, and NO2-. The system can also be used to achieve direct electron transfer of other heme proteins, such as myoglobin, cytochrome C, and even redox enzymes. It will provide a new aspect toward understanding the kinetics and thermodynamics of biological redox processes. Acknowledgment. Financial support from the National Nature Science Foundation of China (Nos. 30370397 and 60571042) is gratefully acknowledged. References and Notes -1

Figure 11. Cyclic voltammograms at 0.1 V s in pH 5.0 buffer for (a) Hb/CPE and (c) Hb/CTAB/CPE without NO2-, (b) Hb/CPE and (d) Hb/CTAB/CPE in buffer containing 85.8 µM NO2-, and (e) Hb/ CTAB/CPE in buffer containing 214.5 µM NO2-. The inset is a plot of the reduction peak current against the concentration of NO2-.

10-7 to 4.02 × 10-5 M. The linear regression equation is I/µA ) 0.313 + 0.0544C/µM, with a correlation coefficient of 0.998 (n ) 20). Catalytic reduction of nitrite was also studied at a Hb/CTAB/ CP electrode, and the results are shown in Figure 11. A new reduction peak located at -0.72 V is observed at the Hb/CTAB/ CP electrode upon the addition of NO2- to a pH 5.0 buffer (curve d). The further addition of NO2- causes an increase of the peak current (curve e). Meanwhile, the reduction and oxidation peak currents of the heme Fe(III)/Fe(II) redox couple for Hb decrease. In control experiments, no reduction peak appeared at a Hb/CP electrode (curve b) with the same concentration of NO2-. The reduction product at -0.72 V is most likely N2O, which was detected previously by mass spectroscopy with Mb-DDAB films upon electrolysis at -0.895 V in pH 7.0 buffers.36 In addition, the new reduction peak is related to the [HbFe(II)-NO]+ nitrosyl adduct.37 The mechanism of the electrocatalytic reduction of nitrite at Hb/CTAB/ CPE starting from HbFe(III) can be proposed as follows36-38

HbFe(III) + e- f HbFe(II) HbFe(II) + HONO + H+ f [HbFe(II)-NO]+ + H2O [HbFe(II)-NO]+ + e- f [HbFe(II)-NO]• [HbFe(II)-NO]• + e- f [HbFe(II)-NO][HbFe(II)-NO]- + H+ f HbFe(II) + HNO 2HNO f N2O + H2O The reduction current has a linear relationship with the concentration of NO2- as shown in the inset. The linear regression equation is I/µA ) 0.558 + 0.555C/µM (R ) 0.997, n ) 10), in the range 0.286-4.576 mM. Conclusions In this article, the single-chain cationic surfactant CTAB was successfully utilized to immobilize Hb onto a CP electrode. The surfactant molecules could interact with the electrode surface in a specific manner and anchor the protein molecules to align in a suitable orientation, which, in turn, promoted electron transfer between the protein molecules and the CP electrode. Moreover, Hb could retain its bioelectrocatalytic activity after adsorption on the surface of the CTAB-modified CP electrode,

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