Anal. Chem. 1993, 85, 1654-1657
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Mediated Electrochemical Reduction of Cytochrome c and Tyrosinase at Perfluorosulfonated Ionomer Coated Electrodes James W. Furbee, Jr., C. Robin Thomas, and Richard S. Kelly' Department of Chemistry, Lake Forest College, 555 North Sheridan Road, Lake Forest, Illinois 60045
Mitchell R. Malachowski Department of Chemistry, University of San Diego, San Diego, California 92110
Nafion-coatedglassy carbon electrodescontaining the copper(I1) complex prepared from the tripodal tris(pyrazo1-1-ylmethy1)amineligand are shown to effectively mediate electron transfer to cytochrome c and the copper-containing enzyme tyrosinase. For both species, a concentrationdependentincrease in cathodic current is observed at the peak potential for the mediator following addition of protein to the buffer solution containing the modified electrode. The average increase for a cytochrome c concentration of 0.19 mM is 11 f 3.2%) while that for 5.6 pM tyrosinase is 9.5 f 4.6%. The results are consistent with a secondorder EC catalytic scheme in which the reduction of protein is mediated by the copper complex in Nafion.
INTRODUCTION The modification of electrode surfaces to control their reactivity toward otherwise unreactive species has been a major area of electroanalytical research in recent years.13 Of specialinterest for such modified electrodesare redox proteins and enzymes because these molecules have historically exhibited irreversible electrochemical behavior and surface adsorption at untreated electrodes.4 The primary reason for irreversibility is the inability of the redox-active centers in these molecules to approach the electrode with a proper orientation for electrons to be rapidly transferred. Electron transfer from electrodes to biological molecules is important not only for fundamental reasons but in electrocatalytic and analytical applications, often as biosensors.6 For surface-modified electrodes, both direct unmediated electron transfer and indirect electron transfer involving an electroactive mediator have been reported. Notable examples have included the direct reduction of cytochrome c at noble metal electrodes after chemisorption of surfaceactive functionalgroupseand the oxidation of NADH at glassy carbon modified with ferrocenylmethanol and diaphorase.' Polymer coatings provide considerable versatility for surface-confined electron-transfer mediators. As evidenced by a growing number of reporta, mediators confined within
or formingthe polymer coating itself offer a favorablepathway for electron transfer to a solution species that does not otherwise undergo transfer at a bare electrode.8 The perfluorinated cation-exchange polymer Nafion is used in our work to confiie a copper-containing complex, which has an overall +2 charge, onto the surfaceof glassy carbon electrodes. The immobilized copper complex exhibits electrochemical characteristics similar to that in solution at a bare glassy carbon electrode. Electrodesmodified in this way are capable of mediating electron transfer to cytochrome c and to the copper-containing enzyme tyrosinase. Cytochrome c is a heme-containing protein that transfers electronsin the mitochondrialrespiratory chain. The protein has amolecular weight of 12 400, displays an overall +9 charge at pH 7,9 and has a reported standard electrode potential of +0.258 V vs the standard hydrogen electrode.10 Readily available in purified form, cytochrome c is often the protein used to demonstrate the effectivenessof mediator substances to enhance electron transfer. Tyrosinase is a monooxygenase found widely in nature which is involved in many reactions, including the conversion of monophenols to o-phenols and the oxidation of diphenols to quinones." Tyrosinase isolated from mushroom (Agaricus bisporus) has a molecular weight of 1.2 X 106 and contains two binuclear copper-containing active sites.'* The copper in the resting enzyme is thought to be in the oxidized antiferromagnetically coupled bicupric state.lS This enzyme carries a net negative charge in the pH range of 5.9-8.7." It has a reduction potential of +0.35 V vs the standard calomel reference.I3 Our interest in the enzyme stems from its ability to catalyze the air oxidation of catecholto quinone.'6 A tyrosinase-based sensor for phenols in which the enzyme was immobilized at carbon electrodes using dialysis membrane has been reported.16 It may be feasible to couple the enzyme with a reversible mediator to produce a more versatile sensor. In addition to containing the redox mediator, Nafion performs other functions important to mediating electron transfer to biological molecules. First, the film prevents depadative adsorption of the biomolecule to the electrode surface by electrostatic and/or size exclusion of proteins from the interior of the film. Second, it is capable of serving as an effective "docking" site for cytochrome c and tyrosinase.
(8) Ryan, M. D.; Chambers, J. Q. Anal. Chem. 1992,64,79R. (9)Bartlett, P.N.;Farington, J. J. Electroanal. Chem. 1989,261,471. (10)Hawkridge, F.M.; Kuwana, T.Anal. Chem. 1973,45,1021. (1)Abruna, H. D. Coord. Chen. Rev. 1988,86,135. (11)Sugumaran,M.Biochemistry 1986,25,4489. ( 2 ) Murray, R.W. In Electroanalytical Chemistry; Bard, A. J., Ed.; (12)Yong, G.;Leone, C.; Strothkamp, K. G. Biochemistry 1990,29, Marcel Dekker, Inc.: New York, 1984;VoL 13,p 191. 9684. (3)Merz, A. Top. Curr. Chem. 1990,152,49. (13)Makino, N.; McMahill, P.; Mason, H.S.; Moes, T.H. J. B i d . (4)Fultz, M. L.; Durst, R. A. Anal. Chim. Acta 1982,140, 1. Chem. 1974,249,6062. (5)Armstrong, F.A.; Hill, H. A. 0.;Walton, N. J. Acc. Chem. Res. (14)Bouchilloux, S.; McMahill, P.; Mason, H. 5.J. Biol. Chem. 1963, 1988,21,407. 238.1699. (6) Hill, H. A. 0.;Lawrance, G. A. J . Electroanal. Chem. 1989,270, - - , - - - -. (15)Malachowski,M. R.;Tomlineon, L. J.; Davidson, M. G.; Hall,M. 309. (7)Chang,H.-C.;Ueno,A.;Yamada,H.;Matsue,T.;Uchida,I.Analyst J. Inorg. Chim. Acta 1989,157,91. (16)Skladal, P. Collect. Czech. Chem. Commun. 1991,56,1427. 1991,116,793. 0003-2700/93/036C 1654$04.00/0
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993
Reversible binding of functional groups to the electrode surface is generally accepted as a necessary condition for mediated electron transfer to occur.17 The copper complex was prepared from the tris(pyrazo11-ylmethy1)amineligand.18 The copper(I1) complex derived from this tetradentate ligand exists in a trigonal bipyramidal environment, yet enough structural flexibility is present to allow the +1oxidation stateto also be accessible. The complex was observed to successfully act as an electron shuttle while immobilized within Ndion to facilitate electron transfer to cytochrome c and tyrosinase. It is interesting to note that the copper-tpzma complex acts as an efficient catalyst for the oxidation of catechol to quinone when the fifth donor dissociates to leave a vacant equatorial site.18 It is not clear whether this binding site can participate in catalysis when in Ndion. EXPERIMENTAL SECTION Reagents and Materials. [Cu(tpzma)CHsOHl(BF4)z(2) was prepared according to published procedures.18 Glassy carbon electrodes (3-mm diameter) were obtained from Bioanalytical Systems, Inc. (West Lafayette,IN). Ndion ( 5 % w/w in aliphatic alcohole/lO% water) was obtained from Aldrich Chemical Co. (Milwaukee,WI) and used as received. Cytochrome c (Type VI, 97%) was purchased from Sigma Chemical Co. (St. Louis, MO) and was either used as received or, in some experiments,purified prior to use on a CM-32 cation-exchange column (Whatman; Hillaboro,OR) as previously described.le Tyrosinase (mushroom, 2000-4000 units/mg) was obtained from Sigma and used as received. Apparatus. A three-electrode system was used in all the measurements, with potentialsreferred to the Ag/AgC1reference electrode. The glass cell consisted of a vial of ca. 5.0-mL volume with a Teflon cap. A platinum wire served as the auxiliary electrode. Cyclic voltammetry and chronoamperometry were performed using either an IBM EC/225 voltammetric analyzer and a Soltec Model VP-6415s X-Y recorder or an EG&G PAR Model 213 potentiostat/galvanostatcontrolled with a Proteus 286 IBM compatible computer running the EG&GPAR System 270 software package. All cyclic voltammetric sweeps, except where otherwise noted, were done at a scan rate of 0.050 V/s. Electrode Preparation. The glassy carbon electrodes were first polished with. 0.3- and 0.05-pm alumina on microcloth polishing pads (all Buehler; Lake Bluff, IL). The electrodes were sonicated in distilled water after each polishing step to remove alumina particles. After the final sonication, the electrodes were dried with a Kimwipe and then 35 pL of Ndion solution was applied evenly to the surface from an Eppendorf micropipet. The electrode was dried at 100 O C for 30 min and allowed to sit in air overnight. The coated electrode was next soaked for 3 h in a 1mM solution of the copper complex in 0.1 M Tris buffer. Following a distilledwater rinse, the electrode was ready for use. For measurements at known concentration of complex incorporated in Nafion, a weighed amount of the complexwas dissolved in a fixed volume of the 5 % Ndion solution. The volume of the f i i layer formed after evaporation of the solvent was estimated from the known wet density of 1100EW Nd1on.P Film thickness, calculated in the same way, was ca. 30 wm for the 35-pL aliquot applied to the electrode surface. Electrochemistry of Cytochrome c. Cyclic voltammetry with the treated electrode was first performed in a solution of deoxygenated 0.1 M Tris buffer. A continuous triangular wave form was applied to the electrode until a steady-state response was obtained for the complex contained within the film. This steady-state response indicates that the complex was not lost from the film due to leaching. After a rest period of at least 1 min, a single cyclic voltammogram was recorded for the complex.
-2
-
-21""
W '
'
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I
400 200 0 -200 -400 600 400 200 0 -200-400 Potential vs Ag/AgCI, mV Potential vs AgIAgCI, mV Figure 1. Cyclic voltammograms recorded for [Cu(tpzme)CH,OH](BF& (2): (A) 1.0 mM solution in pH 7.4 Trls buffer (0.1 M); EP,* = +0.044 V, = 0.12 V. (B) Nafion-coatedelectrode soaked In 1.0 rnM 2 for 3 h; EPt2= +0.080 V, Up = 0.28 V. Scan rate for both scans was 0.050 V I S .
The cell lid containing all three electrodes was removed from the buffer, rinsed with deionized water, shaken lightly to remove excess water, and then placed in a cell containing cytochrome c dissolved in deoxygenated buffer. Single scans were recorded for cytochrome c at concentrations of 0.19,0.36, and 0.53 mM. In the case of the unpurified cytochrome c, these solutions were prepared by dilution of a 50 mg/mL stock solution prepared in buffer that had been previously deoxygenated. Experiments involving purified cytochrome c were also performed to m u r e that the results obtained for the unpurified substance were not due to electroactive impurities. In these cases, ca. 100mg of the unpurified substance was dissolved in 1.00 mL of the Tris buffer and passed through the CM-32 column using phosphate buffer at pH 7.0as the mobile phase. Only the center of the cytochrome c band was collected and used. Aliquots of the purified solution were added sequentiallyto a cell containing buffer alone. The concentration of cytochrome c in the cell was estimated from the volume collected for the fraction and the size of the aliquot. Electrochemistry of Tyrosinase. Cyclic voltammetry of tyrosinase at treated electrodes was obtained in the same way as for cytochromee, except that the solution concentrations for the enzyme were 0.0056,0.011, and 0.015 mM.
RESULTS AND DISCUSSION Electrochemistry of [Cu(tpzma)CH,OH](BF,)2 (2) in Solution. A cyclic voltammogram (CV) for a 1.0 mM solution of 2 in 0.1 M Tris buffer a t an unmodified glassy carbon (GC) electrode is shown in Figure 1A. The observed Eppfor the Cu(II)/Cu(I) couple is -0.18 V, with an observed Upof 115 mV a t 0.05 V/s (no iR compensation). The large value for AEpat this scan rate is symptomatic of a slow heterogeneous electron transfer with 2 at GC. Plots of both cathodic and anodic peak currents vs u1/2arelinear, indicative of a diffusioncontrolled reaction for both the oxidized and reduced forms of 2. Electrochemistry of 2 in Nafion. Figure 1Bshows a CV recorded at a Ndion-coated GC electrode which had been soaked in a 1.0 mM solution of the copper complex for 3 h. The incorporated complex has an Eppvalue of -0.13 V, which is only slighty shifted from the value observed in an aqueous solution. Significant negative shifts in the peak potentials of positively charged copper complexes after incorporation into Ndion have been reported.21 It is thought that these shifts are the result of a stabilizing effect on Cu2+over Cui+ in Ndion. The small shift observed for 2 incorporated in Ndion suggests that the environment for the complex remains similar to that in the aqueous solution.22
(17) Frew, J. E.; Hill, H. A. 0. Eur. J. Biochem. 1988,172, 261. (18) Malachoweki,M.R.;Davidmn,M.G.;Hoffmau,J.N.Znorg.Chim. Acta 1989,157, 91. (21) Bas& S.; Zachariae,P. S.; Rajeehwar, K. J.Electroanal. Chem. (19) Brautigan, D. L.; Fergueon-Miller, 5.; Margoliash, E. Methods 1991,319,111. Enzymol. 1978,53, 128. (22) White, H. 5.; Leddy, J.; Bard, A. J. J.Am. Chem. Sac. 1982,104, (20) Garcia, 0.;Kaifer, A. E. J. Electroanol. Chem. 1990,279, 79. 4811.
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0.0
0.5
1.o
1.5
2.0
(Ti rne)- I*, sec' l/* Flguro2. Cottrellplot for the chronoamperometric response observed at a Nafion-coatedelectrode containing 0.05 M 2. Potential step was from +0.60 to -0.30 V. Current was sampled between 0.4 and 5 s for thls plot.
Plots for the current vs the square root of scan rate of the cathodic and anodic peaks show a linear dependence, as was the case in solution. The separation between the cathodic and anodic peak potentials was observed to be ca. 270 mV for 2 in the film. AE, values this large can arise from a variety of sources, including kinetics of film reorganization and uncompensated resistance, as often found for cations bound to the interior of Nafion.21*22For example, the AE, value for Ru(NHg)63+in films of the same thickness was 145 mV at the scan rate without iR compensation. The apparent diffusion coefficient, D.,,, was estimated for 2 from chronoamperometric measurements in Ndion containing a fixed concentration of complex (0.050M). A Cottrell plot taking the average values for five electrodes is shown in Figure 2. A D,, of 3 X 1O-g cm2/s was calculated from the slope of this plot. It should be emphasized that the thickness of the film was not measured precisely, so that this value should be considered as an approximate one. An average value of 14.6 f 5.4 pA ( n = 28) was obtained for the cathodic peak current of Nafion-coated electrodes in Tris buffer after they had been soaked for 3 h in 0.10 M solutions of 2. This corresponds to a surface coverage, r, of approximately 1.8 X lo-' mol/cm2 and a film concentration of ca. 0.06M. For 30-pm-thick films, the t-l/2 dependence of the chronoamperometric current showed that a maximum concentration of the complex in the film was reached at a soak time of 3 h or more. The response was independent of time for long periods (days), indicating that the complex was not lost from the film. A steady-state response for the Cu(II)/Cu(I) couple was not observed in films thinner than 30 pm. Mediated Reduction of Cytochrome c. The cyclic voltammograms shown in Figure 3 illustrate the reduction of cytochrome c mediated by the copper complex 2 in the Nafion f i b . The CV of Figure 3A correspondsto a modified electrode run in 0.1 M Tris buffer. When the electrode was removed from the buffer and placed in solutions with increasing concentrations of cytochrome c, voltammograms B-D resulted. The increasesin the cathodic current for the reduction of the mediator are concentration dependent. The calibration plot for three concentrations of cytochrome c is shown in the inset of Figure 3. The average increase in the cathodic current for the treated electrode in going from buffer to a cytochrome c concentration of 0.19 mM was 11% (SD = 3.2,N = 12). Similar results were obtained for unpurified and purified cytochrome c, indicating that the observed current increases were not due to impurities.
I a -200
-8 400
-400
200
Potential vs Ag/AgCI, mV Flguro 3. Mediated reductionof cytochrome c: (A) modified electrode In pH 7.4 Trls buffer (0.1 M); ( E D ) modified electrode In Trls buffer (pH7.4)contalnIng0.19,0.36,and0.53mMcytochr~nec,respectively. Inset shows the dependence of observed cathodic current on cytochrome c concentration. All scans done at 0.050 V/s.
The results are consistent with a second-order EC catalytic scheme= in which the reduction of cytochrome c is mediated by the copper complex in Ndion. This process can be represented as Cu(II)
+ e-
Cu(I)
t -
CU(I) +
cyt,
Cytrd + CU(II)
where Cu(I1) and Cu(1) represent the oxidized and reduced forms of the immobilized complex, respectively, and Cy&,= and C h are the two states for the cytochrome c molecule. As shown by Dimarco et al.,u the reverse mediator wave is not observed for a pseudo-first-order catalytic scheme. It is thought that the mediated electron transfer occurs at the surface of the Nafion film, with the current being limited either by the electron-transfer rate at the film/solution boundary or by the rate of electron transport through the film. Hahn et al. have shown that cytochrome c entrapped within a Nafion film does not show an electrochemical response, while cytochrome c ~ 1 cytochrome , bs, and azurin display quasi-reversible electron transfer.% This behavior is thought to be the result of strong binding of the protein by the polymer, preventing its interaction with the electrode. The demonstration here of mediated electron transfer to cytochrome c at the surface of a Nafion film is made all the more interesting in light of these results. A size-exclusion argument can also be made to support the assertion that electron transfer is localized at the polymer/ solution interface. Andrieux and Saveant have dealt with (23) Zak, J.; Kuwana, T. J. Electroanal. Chem. 1983, 150, 645. (24)Dimarco, D. M.; Forshey, P. A.; Kuwana, T. In Chemically Modified Surfaces in Catalyeis and Electrocatdyeis; Miller, J. S., Ed.; ACS Symposium Series 192; American Chemical Society: Washington, DC, 1982; p 71. (25) Hahn,C.E. W.;Hill,H.A.O.;Ritchie,M.D.;Sear,J. W. J.Chem. Soc., Chem. Commun. 1990, (2), 125.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 13, JULY 1, 1993
the diffusion of Substrate in polymer filmsin terms of pinholes of radius R,through which penetration of molecules into the film from the bathing solution would occur.% It seems reasonable to assume that if the average size of the pinholes were known, it would be predicted whether a molecule of certain size would be able to diffuse into the polymer."' The cluster/channel model for the structure of Ndion has gained acceptance, with the channels connecting the hydrophilic regions of polymer being on the order of 10-15 A in diameter.28129 Cytochrome c is roughly spherical, with a molecular diameter of 30-34 A.* This size should prevent the molecule from entering the film. The lack of diminished currents from surface adsorption at higher concentrations of protein also supports its exclusion from the film interior. Mediated Reduction of Tyrosinase. The cyclic voltammograms in Figure 4 show the mediated reduction of tyrosinase at electrodes treated in the same way as for cytochrome c reduction. The CV of Figure 4A shows the response of a treated electrode in buffer before immersion in solutions of tyrosinase. The responses shown in Figure 4B-D represent increasing concentrations of the enzyme. The results are similar to those observed for mediated reduction of cytochrome c, with electron transfer to the enzymeoccurring at the reduction potential of the mediating copper complex. The average current increase at a treated electrode on going from a buffer to a tyrosinase concentration of 5.6 pM was 9.5% (SD = 4.6, N = 8). The inset of Figure 4 shows a calibration plot for three concentrations of tyrosinase. The increasedsensitivity of the electrode toward tyrosinase may be explained partially by the existence of tetramers, each of which contains an electroactive copper center and which may be experiencing electron transfer individually. Tyrosinase, being negatively charged at pH 7.0, is also prohibited from entering the film due to Donnan exclusion. Thus, the substrate fii-diffusion current density is zero, and the electron-transfer process occurs exclusively at the film/solution interface. These results are analogousto those observed by Sharp31 for the oxidation of ferrocytochrome c at quaternary poly(viny1pyridine) (QPVP)coated electrodes mediated by Fe(CN)e9-. In that case, Donnan exclusion of the positively charged protein limited the mediated oxidation to the solution/polymer interface. A report by Kaiferm
600
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0
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Potential vs AglAgCI, mV Figure 4. Mediated reduction of tyrosinase: (A) modlfled electrode In pH 7.4 Trls buffer (0.1 M); (ED) modlfled electrode In Tris buffer (pH 7.4) containing 5.6, 11, and 15 pM tyroslnase, respectlvely. Inset shows the dependence of observed cathodic current on tyroslnase concentration. All scans done at 0.050 VIS.
includes similar results for the oxidation of ferrocyanide mediated by ferrocene derivatives immobilized in Ndion.
CONCLUSION The results presented here indicate that the copper complex 2 is capable of mediating electron transfer to both negatively
and positively charged proteins of varying molecular weight and differing redox-active centers. Work is underway to investigate further the structure/activity relationships of Ndion-bound copper complexes similar to 2. The applicability of such electrodes as chromatographic detectors and biological sensors will be explored.
ACKNOWLEDGMENT (26) Andriem, C. P.;Saveant,J.-M.lnMolecularDesign ofElectrode Surfaces; Murray, R. W., Ed.;Techniques of Chemistry XXII;Wiley: New York, 1992; p 207. (27) Shimazu, K.; Kuwana, T. J. Electrochem. SOC.1988, 135, 1603. (28) Gierke,T.D.;Hsu, W. Y. InPerfluorinatedZonomerMembranes; Eieenberg, A., Yeager,H. L., Ede.;ACS SymposiumSeries 180; American Chemical Society: Washington, DC, 1982; p 283. (29) Schmidt, M. H. Doctoral Dieeertation, Stanford University; University Microfilms International, Ann Arbor, MI, 1989. (30)Sun,S.;Reed, D.E.;Hawkridge, F. M. In Redor Chemistry and Znterjacial Behavior of BiologicalMolecules; Dryhmt, G., Kataumi, N., Eda., Plenum Press: New York, 1988, p 47. (31) Sharp, M. In ref 30, p 499.
Grant-in-aid support of this project by Lake Forest College is gratefdy acknowledged. We thankTed Kuwana for helpful comments during the preparation of the manuscript. A preliminary report of this work was presented at the 25th Great Lakes Regional Meeting of the American Chemical Society, Milwaukee, WI, June 1992.
RECEIVED for review January 14, 1993. Accepted March 12, 1993.
