pubs.acs.org/Langmuir © 2011 American Chemical Society
Direct Electrochemistry and Electrocatalysis of Myoglobin Immobilized on L-Cysteine Self-Assembled Gold Electrode Tercio de F. Paulo,†,‡ Izaura C. N. Diogenes,† and Hector D. Abru~na*,‡ †
Departamento de Quı´mica Org^ anica e Inorg^ anica, Universidade Federal do Cear a, Cx. Postal 6021, Fortaleza, Cear a, Brazil 60455-970, and ‡Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301, United States Received September 2, 2010. Revised Manuscript Received December 16, 2010
Myoglobin (Mb) has been successfully immobilized on a self-assembled monolayer (SAM) of L-cysteine (Cys) on a gold electrode, Au/Cys. The presence of a pair of well-defined and nearly reversible waves centered at ca. 0.086 V vs Ag/ AgCl (pH 6.5) suggests that the native character of Mb heme FeIII/II redox couple has been obtained. The formal potential of Mb on Cys SAM exhibited pH-dependent variation in the pH range of 5-9 with a slope of 55 mV/pH, indicating that the electron transfer is accompanied by a single proton exchange. Thermodynamic and kinetic aspects of Mb adsorption processes on Au/Cys were studied by using voltammetric and quartz-crystal microbalance methods. The Au/ Cys electrode with immobilized Mb exhibited electrocatalytic activity toward ascorbic acid (AA) oxidation with an overpotential decrease of over 400 mV and a linear dependence of current on the AA concentration from 0.5 to 5.0 mmol L-1.
Introduction Electron transfer reactions are an essential part of key biological processes including respiration, photosynthesis, and intermediary metabolism.1 Direct electrochemistry of redox proteins can provide a good model for mechanistic studies of their electron transfer activity in biological systems and is an important foundation for biosensors and bioreactors.2-5 Myoglobin (Mb) is a small heme protein found in muscle cells whose physiological function is to store and increase the diffusion rate of dioxygen. Although Mb does not act as an electron carrier, it undergoes a redox process in the respiratory system playing an essential role in biological processes. Therefore, Mb can be used as model for the study of electron transfer reactions of heme proteins, biosensing, and electrocatalysis. Moreover, the electroactive center of Mb allows its adsorption process to be monitored in real time with electrochemical techniques such as cyclic voltammetry. Nevertheless, heterogeneous electron transfer (hET) reactions of heme proteins are difficult to achieve at bare metal surfaces because adsorption generally leads to denaturation.2 Self-assembled monolayers (SAMs) of thiol species on gold have been successfully used *Corresponding author. E-mail:
[email protected]. (1) Moser, C. C.; Page, C. C.; Farid, R.; Dutton, P. L. J. Bioenerg. Biomembr. 1995, 27, 263–274. (2) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363–369. (3) Hu, S.; Lu, Q.; Xu, Y. Electrochemical Sensors, Biosensors and Their Biomedical Applications; Academic Press: New York, 2007. (4) Anderson, J. L.; Bowden, E. F.; Pickup, P. G. Anal. Chem. 1996, 68, 379R– 444R. (5) Arya, S. K.; Solanki, P. R.; Datta, M.; Malhotra, B. D. Biosens. Bioelectron. 2009, 24, 2810–2817. (6) Kumar, S. A.; Chen, S.-M. Biosens. Bioelectron. 2007, 22, 3042–3050. (7) Liu, Y.-C.; Cui, S.-Q.; Zhao, J.; Yang, Z.-S. Bioelectrochemistry 2007, 70, 416–420. (8) Zhang, H.-M.; Li, N.-Q. Bioelectrochemistry 2000, 53, 97–101. (9) Meng, X.; Wu, X.; Wang, Z.; Cao, X.; Zhang, Z. Bioelectrochemistry 2001, 54, 125–129. (10) Mozaffari, S. A.; Chang, T.; Park, S.-M. J. Phys. Chem. C 2009, 113, 12434–12442. (11) Nagaraju, D. H.; Pandey, R. K.; Lakshminarayanan, V. J. Electroanal. Chem. 2009, 627, 63–68. (12) Pinheiro, S. O.; Sousa, J. R. de; Santiago, M. O.; Carvalho, I. M. M.; Silva, A. L. R.; Batista, A. A.; Castellano, E. E.; Ellena, J.; Moreira, I. S.; Diogenes, I. C. N. Inorg. Chim. Acta 2006, 359, 391–400.
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to access hET reactions.6-15 For instance, SAMs of cysteine have been used to anchor the metalloprotein cytochrome c7 and the metalloenzyme bovine erythrocyte copper-zinc superoxide dismutase9 (SOD). Ascorbic acid (AA) is a water-soluble vitamin present in many biological systems being essential for cardiovascular function, immune cell development, connective tissue, and iron utilization in humans.16 Moreover, it is widely used in foods as an antioxidant for the stabilization of colors and flavors. Since there is a lack of the L-gulono-1,4-lactone oxidoreductase enzyme in the liver cells of humans, it is necessary to get AA from fruits, vegetables, and medicines to balance the amount of vitamin C. Taking these functionalities into account, the determination of AA has attracted a great deal of attention in food and pharmaceutical industries.17 Numerous methods have been developed for this purpose, including spectrophotometry,18 spectrophotofluorimetry,19 flame atomic absorption spectrometry,20 HPLC/MS,21 capillary electrophoresis (CE),22 and chemically modified electrodes (CMEs).23-25 The electrochemical detection is very attractive due to its high sensitivity, selectivity, and wide dynamic range. Furthermore, it is promising for the fabrication of simple and low-cost sensors. (13) Paulo, T. de F.; Silva, M. A. S. de; Pinheiro, S. O.; Meyer, E.; Pinheiro, L. S.; Freire, J. A.; Tanaka, A. A.; Lima-Neto, P. L.; Moreira, I. S.; Diogenes, I. C. N. J. Braz. Chem. Soc. 2008, 19, 711–719. (14) Paulo, T. de F.; Pinheiro, S. de O.; Silva, M. A. S. de; Lopes, L. G. F.; Pinheiro, L. S.; Aquino, G. A.; Temperini, M. L. A.; Lima-Neto, P. L.; Diogenes, I. C. N. Electroanalysis 2009, 21, 1081–1089. (15) Diogenes, I. C. N.; Nart, F. C.; Temperini, M. L. A.; Moreira, I. S. Inorg. Chem. 2001, 40, 4884–4889. (16) Koshiishi, I.; Imanari, T. Anal. Chem. 1997, 69, 216–220. (17) Bender, D. A. Nutritional Biochemistry of the Vitamins, 2nd ed.; Cambridge University Press: Cambridge, 2003. (18) Farajzadeh, M. A.; Nagizadeh, S. J. Anal. Chem. 2003, 58, 927–932. (19) Perez-Ruiz, T.; Martı´ nez-Lozano, C.; Tomas, V.; Fenol, J. Analyst 2001, 126, 1436–1439. (20) Yebra, M. C.; Cespon, R. M.; Moreno-Cid Anal. Chim. Acta 2001, 448, 157–164. (21) Frenich, A. G.; HernandezTorres, M. E.; Belmonte Vega, A.; Vidal, J. L. M.; Bola~nos, P. P. J. Agric. Food Chem. 2005, 53, 7371–7376. (22) Tang, Y.; Wu, M. Talanta 2005, 65, 794–798. (23) Khoo, S. B.; Chen, F. Anal. Chem. 2002, 74, 5734–5741. (24) Zhang, L.; Jiang, X. J. Electroanal. Chem. 2005, 583, 292–299. (25) Raoof, J.-B.; Ojani, R.; Rashid-Nadimi, S. Electrochim. Acta 2005, 50, 4694–4698.
Published on Web 01/18/2011
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Herein we report on the formation and characterization of myoglobin adsorption processes on an L-cysteine SAM on gold along with its electrocatalytic activity toward ascorbic acid oxidation.
Experimental Section Chemicals. Myoglobin from equine skeleton muscles, L-cysteine, and ascorbic acid were purchased from Aldrich and used as received. Aqueous solutions were prepared using Millipore water of at least 18 MΩ cm resistance. Phosphate buffer solution (PBS) used as supporting electrolyte at an ionic strength of μ=0.1 mol L-1 was prepared from K2HPO4 and KH2PO4. The pH was adjusted with either phosphoric acid or KOH and monitored with a pH meter. Mb concentration was estimated spectroscopically at pH 6.5 from its molar absorptivity26 of 188 L mmol-1 cm-1 at 409 nm. Apparatus. Cyclic voltammetric measurements were carried on a computer-controlled EC Epsilon potentiostat (Bioanalytical Systems, Inc., West Lafayette, IN). Electrochemical cells of conventional design were employed, and potentials were measured against a silver/silver chloride electrode (Ag/AgCl/3.5 mol L-1 KCl, BAS). Modified polycrystalline gold surfaces (BAS, A = 0.0314 cm2) and coiled platinum wires were used as working and auxiliary electrodes, respectively. A Pine Instruments rotating disk electrode system with a polycrystalline gold electrode (0.26 cm2 in area) was employed in rotating disk electrode experiments. The supporting electrolyte was purged with high-purity nitrogen for 20 min prior to experiments, and a nitrogen atmosphere was maintained during all the experiments. Spectroscopic measurements of a gold grid electrode (A=0.6 cm2) modified with L-cysteine and myoglobin were acquired in the ultraviolet and visible (UV-vis) regions on a Varian Cary 5000 spectrophotometer. AT-cut quartz crystals (5 MHz) of 24.5 mm diameter with Au electrodes deposited over a Ti adhesion layer (Maxtek Co.) were used for QCM measurements. An asymmetric keyhole electrode arrangement was used, in which the circular electrode geometrical areas were 1.370 cm2 (front side) and 0.317 cm2 (backside). The electrode surfaces were overtone polished. The quartz crystal resonator was set in a probe (TPS-550, Maxtek) made of Teflon, in which the oscillator circuit was included. The probe was immersed in various aqueous electrolytes solutions, which were thermostated at 25.0 °C by a water jacketed beaker connected to a thermostated bath (digital temperature controller 9101, Fisher Scientific). Nitrogen gas was used to degas the solutions before use and flowed over the solutions during experiments. The frequency was measured with a plating monitor (PM-740, Maxtek) that was interfaced to a desktop computer. Once the frequency reached a steady state, electrochemical experiments were performed. The frequency (ΔF) and mass (Δm) changes are related by a simple, linear equation27 (Sauerbrey equation): Δm ¼ - Cf ΔF -1
ð1Þ
-2
where Cf (17.7 ng Hz cm ) is the proportionality constant for the 5 MHz crystals used in this study. Quartz crystal resonators were also used as the working electrode in voltammetric experiments. The potential of the working electrode was controlled with a potentiostat (CV-27, BAS). Electrode Modification. Gold polycrystalline electrodes were mechanically polished on with a 0.05 μm alumina slurry, rinsed with water, sonicated for 10 min, immersed in fresh “piranha” solution (3H2SO4:1H2O2) for 2 min, rinsed with water, and sonicated again. (Caution: piranha solution is a strong oxidant solution that reacts violently with organic compounds.) Following this cleaning procedure, the electrode was pretreated by continuous cycling in 0.5 mol L-1 H2SO4 until the characteristic (26) Antonini, E.; Brunori, M. Hemoglobin and Myoglobin in Their Reactions with Ligands; North-Holland: Amsterdam, 1971. (27) Sauerbrey, G. Z. Phys. 1959, 155, 206–222.
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voltammetric curve28 for a clean polycrystalline gold surface was obtained. From the integration of the area under the oxide formation wave, the electrochemically active area was determined using a conversion factor29 of 390 μC cm-2. The clean gold electrode was then immersed in an aqueous solution containing 2.0 mmol L-1 Cys for 12 h at 20 °C, rinsed with water, and dried under an argon flux. The Mb immobilization on this modified surface, thus forming the Au/Cys/Mb electrode, was made by immersion of the Au/Cys in a 0.1 mol L-1 PBS solution containing Mb (from 0.1 to 10 μmol L-1) at 20 °C. Successive cycles were performed over the potential range from 0.4 to -0.4 V until a stable voltammogram was obtained. The electrode was then rinsed with water and stored in 0.1 mol L-1 PBS (pH 6.5) at 20 °C. For the QCM measurements, the electrode was cleaned in a 0.5 mol L-1 H2SO4 solution and the electrode area determined as described above. Cys and Mb were dissolved in PBS and injected into the cell containing 0.1 mol L-1 PBS using a gastight syringe. The injected volumes were varied according to the desired final concentration. Although the injection sometimes caused a sudden frequency change, this parameter returned to its original value within a few minutes. Since the solution concentrations of the Mb were within the micromolar regime, their contribution to the measured voltammetric current was negligible, thus allowing the measured current to be assigned to the surface-confined species. For the acquisition of the UV-vis spectra, the cleanness procedure of the gold grid electrode was the same as that employed for the QCM electrodes. The spectra were acquired in 0.1 mol L-1 PBS.
Results and Discussion Direct Cyclic Voltammetry of Myoglobin at Cys/Au Electrodes. The cyclic voltammograms of the bare gold electrode in PBS containing Mb and Cys solutions are shown in Figure 1A (curves 1 and 2). Figure 1A (curve 3) shows the response obtained in PBS solution for the Au/Cys electrode. As can be ascertained, no voltammetric peaks were observed. On the other hand, well-defined redox waves centered at 0.086 V were observed in the cyclic voltammogram for the Au/Cys electrode in 10 μ mol L-1 Mb (Figure 1A, curve 4), indicating not only that is Mb immobilized but also that the FeIII in Mb can be effectively reduced to FeII on a L-cysteine-modified Au electrode. In addition, the observed half-wave potential was in good agreement with the formal potential of Mb reported by using DL-homocysteine SAM on a gold electrode.8 After 50 min of immersion in the Mb solution, the Au/Cys electrode was removed, thoroughly rinsed with water, and transferred to an electrochemical cell containing only the electrolyte solution (0.1 mol L-1 PBS, pH 6.5). The cyclic voltammogram thus obtained is presented in Figure 1A (curve 5). The voltammetric profile is very similar in shape to that acquired in PBS containing Mb (Figure 1A, curve 4). This result again indicates that Mb is adsorbed on the L-cysteine SAM. Moreover, a gold grid electrode was modified by following the same procedures, and UV-vis spectra were acquired. The Soret transition, which is characteristic of heme-containing proteins and indicative of its native state,30-32 is observed at 408 nm in the spectra of Mb in solution and adsorbed on Au/Cys electrode (see Figure S1 in the Supporting Information). Since a significant blue shift would be expected, had the Mb been denatured, this result indicates that the protein has retained its native state upon adsorption. (28) Sawyer, D. T.; Sobkowiak, A.; Roberts, J. L., Jr. Electrochemistry for Chemists, 2nd ed.; Wiley: New York, 1995. (29) Trasatti, S.; Petri, O. A. Pure Appl. Chem. 1991, 63, 711–734. (30) Schechter, A. N.; Epstein, C. J. J. Mol. Biol. 1968, 35, 567–589. (31) Brunori, M.; Giacometti, G. M.; Antonini, E.; Wyman, J. J. Mol. Biol. 1972, 63, 139–152. (32) Culbertson, D. S.; Olson, J. S. Biochemistry 2010, 49, 6052–6063.
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Figure 1. (A) Cyclic voltammograms at 0.10 V s-1 in 0.1 mol L-1 PBS, pH 6.5, of bare Au containing (1) 0.1 μmol L-1 Mb, (2) 2.0 mmol L-1
Cys, and of Au/Cys containing (3) 0.1 mol L-1 PBS, pH 6.5, and (4) 0.1 μmol L-1 Mb, and of Au/Cys/Mb containing (5) 0.1 mol L-1 PBS, pH 6.5. (B) Influence of scan rate on the peak current for the Au/Cys/Mb electrode in 0.1 mol L-1 PBS, pH 6.5. (C) Variation of peak potentials vs the logarithm of the scan rates.
As shown in Figure 1B, the peak current (ip) was linearly dependent on scan rate (v), again consistent with a surfaceimmobilized redox couple.33 Neither the peak current nor formal potential changed after 100 continuous scans from 0.60 to -0.50 V in PBS solution. However, if the applied potential was higher than þ0.6 V, a degradation process appeared to take place. One can thus conclude that the Au/Cys/Mb electrode is quite stable as long as the applied potential does not exceed þ0.60 V. The surface coverage, Γ, of Mb was calculated according to eq 2: n2 F 2 νAΓ ð2Þ 4RT where n and A are respectively the number of electrons transferred and the electrode area, and R, T, and F have their usual meaning. A value of Γ = 6.10 10-11 mol cm-2 was calculated for Mb on the Au/Cys electrode under saturation conditions. In addition, as shown in Figure 1C, a shift to positive and negative potential directions is observed for the oxidation and reduction peak potentials (Ep), respectively, when the scan rate is increased. Based on Laviron’s theory34 a plot of Ep vs log v should yield two straight lines with slopes of -2.3RT/RnF and 2.3RT/(1 - R)nF for the cathodic and anodic peak potentials, respectively. From the intercept of these curves, the electron transfer coefficient, R, was calculated as 0.53. The electron transfer rate constant, ks = 1.66 s-1, was calculated based on eq 3:34 0 RT RnF RT Ep ¼ E 0 ln lnðνÞ ð3Þ RnF RTks RnF ip ¼
The calculated value is higher than the values reported for Mb adsorbed on graphite/nanoporous ZnO/Mb35 (1.0 s-1) and on a SAM formed with DL-homocysteine8 on gold (0.9 s-1); however, it is lower than those achieved by using multiwalled carbon
nanotubes, MWNTs36 (5.4 s-1), and a SAM of agarose37 (47 ( 3) and arylhydroxylamine6 on gold (51 ( 5 s-1). Influence of pH and Ionic Strength on Direct Electron Transfer of Mb. It is well established38 that the hET of Mb is affected by the solution’s ionic strength and pH. With regards to the pH dependence of the redox response, as can be seen in the Figure 2A, the formal potential was pH dependent. A plot of the formal potential vs pH was linear (Figure 2B) with a nearly Nernstian slope of 55 mV for a one-proton and one-electron process.39 Considering the denaturation of Mb,30-32,38 the redox reaction was not investigated above pH 9.0. In addition, for pH values below 5.0, the half-wave potential of Mb remained invariant. Figure 2C presents a plot of the formal potential as a function of μ-1/2. As can be ascertained, the plot is linear from 1.0 to 0.01 mol L-1, which corresponds to a Debye length of ca. 30 A˚, a value that is of the same order of magnitude as the modified layer thickness. For low ionic strength, i.e., 1.0 mmol L-1, the plot loses linearity, indicating that the screening length is much longer than the modifier layer. Thermodynamic and Kinetics Studies. The saturation surface coverage, Γs, and the adsorption coefficient, β, for the adsorption process of Mb on a Au/Cys electrode were determined based on the linearized Langmuir equation. Accordingly, a plot of [Mb]/Γ vs [Mb] yields 1/Γs and 1/Γsβ as slope and intercept, respectively, as illustrated in Figure 3 (inset). On the basis of this plot (Figure 3B), the values of 6.13 10-11 mol cm-2 and 1.06 107 L mol-1 were calculated for Γs and β, respectively. These data were used in the Frumkin isotherm (Figure 3A), and the best fit was obtained when the interaction parameter, g, was -0.15, indicating a weak repulsive interaction between the Mb adsorbed molecules. The β parameter was used to calculate the free energy of adsorption, ΔGad, according to eq 4: ΔGad ¼ - RT lnðas βÞ ¼ - RT lnð55:5βÞ
(33) Murray, R. W., Bard, A. J., Eds.; Marcel Dekker: New York, 1984; Vol. 13, pp 191-368. (34) Laviron, E. J. Electroanal. Chem. 1979, 101, 19–28. (35) Zhao, G.; Xu, J.-J.; Chen, H.-Y. Anal. Biochem. 2006, 350, 145–150. (36) Zhao, G. C.; Zhang, L.; Wei, Q. W.; Yang, Z. S. Electrochem. Commun. 2003, 5, 825–829. (37) Liu, H. H.; Tian, Z. Q.; Lu, Z. X.; Zhang, Z. L.; Zhang, M.; Pang, D. W. Biosens. Bioelectron. 2004, 20, 294–304.
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ð4Þ
where as is the activity of the solvent and/or ions in solution. The value of ΔGad, which gives a quantitative measure of the (38) Nassar, A. F.; Rusling, J. F.; Kumosinski, T. F. Biophys. Chem. 1997, 67, 107–116. (39) Bond, A. M. Modern Polarographic Methods in Analytical Chemistry; Marcel Dekker: New York, 1980.
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Figure 2. (A) Cyclic voltammograms of the Au/Cys/Mb electrode at 0.1 V s-1 in 0.1 mol L-1 PBS solution of different pH values. Plots of peak potentials vs pH (B) and inverse square root of PBS concentration, [PBS] (C).
Figure 3. (A) Adsorption isotherm for Mb on a Au/Cys electrode and fits to the Langmuir (solid curve) and Frumkin isotherms (dashed curve), with g = -0.15. (B) Linearized Langmuir isotherm.
adsorption strength, was calculated as -49.7 kJ mol-1, which is indicative of a chemisorptive interaction40 between Mb and the L-cysteine SAM. The adsorption kinetics of Mb were studied by following the surface coverage as a function of time, Γt, for different concentrations of Mb in solution. Figure 4 shows plots of Γt vs t and Γt/Γs vs t1/2 where the equilibrium surface coverage, Γe, and the adsorption rate constant, k0 , were used as adjustable parameters of the kinetic data according to eqs 5 and 6, which apply to transport and kinetically controlled process, respectively, and Γt =Γs ¼ KðC =Γe ÞðDtÞ1=2
ð5Þ
Γt ¼ Γe ð1 - expð- k C tÞÞ
ð6Þ
0
(40) Acevedo, D.; Bretz, R. L.; Tirado, J. D.; Abru~na, H. D. Langmuir 1994, 10, 1300–1305.
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where C* and D are the bulk concentration and diffusion coefficient of the adsorbate, respectively, and K is a constant equal to 2π1/2. As can be seen in Figure 4A, the agreement of the data to the fits suggests that a kinetically controlled model is applicable to the Mb adsorption process on a Au/Cys electrode. Moreover, the plots presented in Figure 4B are not linear, indicating that the process cannot be explained by means of a diffusion-controlled model. The dependence of the Mb solution concentration on the kinetics of adsorption was also evaluated. Table 1 shows k0 and Γe parameters obtained for the Mb adsorption on the Au/Cys electrode. The observation that Γe increases with an increase in the Mb concentration is consistent with the above. Quartz Crystal Microbalance. Prior to surface modification, a cyclic voltammogram of [Fe(CN)6]4- was acquired in order to study the electrochemical behavior of the bare gold crystal electrode. Results were as expected41 and are not shown here. After stabilization of the frequency, which takes ca. 300 s, the gold crystal was kept immersed in a 2.0 mmol L-1 L-cysteine aqueous solution for 12 h. A mass change of 87.8 ng cm-2 was observed for the overall adsorption process. This corresponds to a surface coverage of 7.2 10-10 mol cm-2. The Au/Cys electrode was then immersed in a 10 μmol L-1 PBS solution of Mb. A decrease in the frequency was observed during the first 15 min, followed by a much more gradual decrease that took about 90 min, after which a steady state was obtained. After 12 h of immersion, an overall frequency change of -94 Hz was observed indicating a mass change of 1.66 μg cm-2. The inset of Figure 5 shows the cyclic voltammogram obtained after 5000 s of immersion of the Au/Cys electrode in a 0.10 mol L-1 PBS aqueous solution containing 10.0 μmol L-1 Mb. Assuming that after this immersion time the saturation coverage, Γs, was reached, we calculated the surface coverage to be 6.53 10-11 mol cm-2, which would, in term, correspond to a mass variation of 1.1 μg cm-2. This value, however, is not in agreement with the experimental one of 1.66 μg cm-2. This discrepancy is illustrated in Figure 5. While somewhat speculative on our part, we believe that the change in the mass is likely due to hydration (41) Jiang, C.; Elliott, J. M.; Cardin, D. J.; Tsang, S. C. Langmuir 2008, 25, 534– 541.
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Figure 4. (A) Plots of Γt vs t and (B) Γ/Γs vs t1/2 for the adsorption of Mb on a Au/Cys electrode at different Mb concentrations. Table 1. k0 and Γe Parameters for Mb Adsorption on a Au/Cys Electrode [Mb]/μmol L-1
k0 /10-3 L mol -1 s-1
Γe/1011 mol cm-2
0.1 1.0 5.0
1.7 2.5 6.1
3.3 5.5 5.9
Figure 6. Cyclic voltammograms in 0.1 mol L-1 PBS (pH 6.5)
Figure 5. Observed (solid line) and calculated (open circles) plots of frequency shifts versus time for the adsorption of Mb on a Au/ Cys electrode. Inset: cyclic voltammogram at 100 mV s-1 of the Au/Cys electrode (gold crystal) in 0.10 mol L-1 PBS aqueous solution containing 10.0 μmol L-1 Mb after 5000 s of immersion.
and/or ions that are attached to the complex structure of the Mb. The numbers of water molecules of hydration (ca. 480 molecules per Mb) were calculated by dividing the measured excess mass by the molecular weight of water, and the number of adsorbed Mb molecules was estimated from the electrochemical measurements (Figure 5 inset). Electrocatalysis of Ascorbic Acid Oxidation. The electrocatalytic activity of the Au/Cys/Mb electrode toward ascorbic acid (AA) was evaluated. Cyclic voltammograms of this electrode as well as of the bare gold surface are presented in Figure 6. 2056 DOI: 10.1021/la103505x
containing 5.0 mmol L-1 AA of (A) Au/Cys/Mb (solid line) and Au (dotted line) electrodes at 100 mV s-1 and of (B) Au/Cys/Mb electrode at different sweep rates (a, 10; b, 25; c, 50; d, 75; e, 150; f, 250; g, 300; h, 400; and i, 500 mV s-1). (C) Plots of the catalytic current, i, vs the square root of sweep rate, v1/2 (O), and of the (b) sweep-rate-normalized current, i/v1/2, vs sweep rate, v.
For the Au/Cys/Mb electrode, the addition of AA to the PBS solution resulted in a dramatic change in the voltammetric shape with a large enhancement of the anodic current and virtually no current in the reverse (cathodic) sweep, as shown in Figure 6A. In addition, a decrease of more than 0.4 V in the anodic overpotential of AA can be clearly observed when the Au/Cys/Mb electrode is used, relative to the bare gold surface. Moreover, the oxidation of AA was evaluated by using the Au/Cys/Mb electrode after denaturation by immersion in 0.5 mol L-1 H2SO4 solution. Subsequently, a cyclic voltammogram similar to that obtained for a bare gold electrode was observed (see Figures S2 and S3 in the Supporting Information), indicating that the catalytic activity toward AA oxidation is directly related to the Mb protein in its native state. Langmuir 2011, 27(5), 2052–2057
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Figure 8. Cyclic voltammograms of a Au/Cyst/Mb electrode at 0.1 V s-1 in 0.1 mol L-1 PBS (pH 6.5) containing AA at different concentrations. Inset: j vs [AA]. Figure 7. (A) RDE voltammograms at 0.01 V s-1 of the Au/Cys/
Mb electrode in 0.1 mol L-1 PBS (pH 6.5) containing 5.0 mmol L-1 AA. Levich (B) and Koutecky-Levich (C) plots at 0.40 V for the AA oxidation reaction.
By using the Au/Cys/Mb electrode, one can observe that the oxidation peak potential of AA presents a dependence on the sweep rate, shifting to positive values with an increase in the scan rate, as illustrated in Figure 6B. This behavior is indicative of a kinetic limitation in the reaction between the Au/Cys/Mb electrode and AA. A similar suggestion was proposed for the catalytic oxidation of AA by using Mb in solution42,43 and adsorbed on a modified glassy carbon electrode.44 However, the plot of the catalytic current vs the square root of sweep rate is linear as shown in Figure 6C (O), suggesting that, at sufficiently high overpotentials, the reaction is transport limited. A plot of the sweep-rate-normalized current (i/v1/2) vs sweep rate (Figure 6C (b)) exhibits the characteristic shape of an ECcat process.39 Andrieux and Saveant40 proposed a theoretical model for such a mechanism in which there was a correlation of the peak current and the concentration of the substrate (ascorbic acid in our case). Taking into account the surface coverage allows one to calculate the pseudo-first-order rate constant, kf, for the process. On the basis of this approach and using Figure 1 of the theoretical paper of Andrieux and Saveant,40 a value of kf=2.40 104 M-1 s-1 was calculated for the AA f AA2þ at a Mb surface coverage of 6.1 10-11 mol cm-2 in the presence of 5.0 mmol L-1 AA. The electrocatalytic activity of the Au/Cys/Mb electrode toward AA oxidation was also investigated by means of rotating disk electrode (RDE) measurements. Figure 7 shows the RDE responses of 5.0 mmol L-1 AA in which a Au/Cys/Mb electrode with a surface coverage of 6.1 10-11 mol cm-2 was used as the working electrode. The RDE voltammograms exhibited an increase in current with increasing of the rate of rotation, ω, up to about 150 rpm and (42) Giulivi, C.; Cadenas, E. FEBS Lett. 1993, 332, 287–290. (43) Galaris, D.; Cadenas, E. Arch. Biochem. Biophys. 1989, 273, 497–504. (44) Chen, S.-M.; Tseng, C.-C. J. Electroanal. Chem. 2005, 575, 147–160. (45) Pariente, F.; Lorenzo, E.; Tobalina, F.; Abru~na, H. D. Anal. Chem. 1995, 67, 3936–3944. (46) Andrieux, C. P.; Saveant, J.-M. J. Electroanal. Chem. 1978, 93, 163–168. (47) Koutecky, J.; Levich, V. G. Zh. Fiz. Khim. 1958, 32, 1565–1575.
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then leveled off. A plot of the limiting current, iL, versus the square root of ω, Levich plot, was clearly nonlinear suggesting kinetic limitations, as illustrated in Figure 7B. Considering that the oxidation of the AA substrate is the rate-determining step, the Koutecky-Levich41 relation was used to construct the plot displayed in Figure 7C from which the rate constant, kf, was calculated as 1.9 104 mol-l L-l s-l. This value is in excellent agreement with that calculated from cyclic voltammetric experiments (Figure 6). The analytical performance of the Au/Cys/Mb electrode toward AA determination was evaluated based on the cyclic voltammograms obtained for different AA concentrations in solution (Figure 8). The Au/Cyst/Mb electrode showed a linear response for AA from 0.5 to 5.0 mmol L-1 with a linear regression equation of ip(A)=4.43 10-3[AA] (mol L-1) and correlation coefficient (R2) of 0.9970.
Conclusions The direct electron transfer of myoglobin on L-cysteine-modified gold electrode has been achieved. The immobilized material retains the redox reactivity of the heme group, and the formal potential exhibits a variation of 55 mV/pH unit, which is close to the anticipated Nernstian value of 59 mV. Using cyclic voltammetry, we have studied the thermodynamics of adsorption of Mb on an L-cysteine SAM-modified gold electrode. A fit of the experimental adsorption isotherms to theoretical models indicated the presence of slight repulsive interactions (molecule/molecule and electrode/molecule) and a large free energy of adsorption (-49.7 kJ/mol). The kinetics of adsorption of Mb on L-cysteine SAM can be represented by a model in which the process is kinetically controlled rather than mass transport controlled. The Au/Cyst/Mb electrode exhibited excellent electrocatalytic activity toward the oxidation of ascorbic acid with a rate constant of kf = 2.40 104 M-1 s-1. Acknowledgment. T.d.F.P. thanks CAPES for a PDEE grant, and I.C.N.D. is thankful to CNPq (303530/2008-1) for the grants. Supporting Information Available: Figures S1-S3. This material is available free of charge via the Internet at http:// pubs.acs.org.
DOI: 10.1021/la103505x
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