8100
J. Phys. Chem. 1992, 96, 8100-8105
Investigation of the Mass Transport Process In the Voitammetry of Cytochrome c at 4,4'-Bipyridyl Disulfide Modified Stationary and Rotated Macro- and Microdlsk Gold Electrodes Alan M. BOnd,*vl*-bH. Allen 0. Ha**1b bbojka Komorsky-LovriE,lCMilivoj LovriE,ld Mary E. McCartby,lbIoaMa S. M. Psalti,lb and Nicbolas J. Waltonlb Inorganic Chemistry Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, England OX1 3QR, Department of Chemistry, La Trobe University, Bundoora, 3083 Victoria, Australia, and Department of Chemical and Analytical Sciences, Deakin University, Geelong, 321 7 Victoria, Australia (Received: December 17, 1991; In Final Form: April 23, 1992)
The mass transport mechanism associated with the reduction of cytochrome c at gold electrodes modified by adsorption of 4,4'-bipyridyl disulfide (SS-bpy) has been investigated at a range of electrode configurations. Radial diffusion to the small electroactive sites on the electrode surface leads to the observation of sigmoidal shaped curves when the surface density of the modifier is low at a stationary conventionally sized gold electrode. High modifier coverage caw overlap of the diffusion layers which leaves linear diffusion as the dominant mode of mass transport resulting in peak-shaped cyclic voltammograms. In contrast, at a 5-pm radius gold disk microelectrode, radial diffusion is always the dominant mode of mam transport. However, the current is small or nonmeasurable when a microelectrode is modified ex situ at open circuit, whereas, if a suitable potential is applied to ensure a uniform distribution of SS-bpy over the entire surface of the microelectrode, then an easily measured reversible sigmoidal shaped voltammogram is observed by both in situ and ex situ methods of modification. In agreement with available spectroscopic evidence, data are consistent with electron transfer occurring at electroactive sites which are smaller than the 5-pm microelectrode radius and confm that SS-bpy and cytochrome c interactions on the electrode surface are dynamic rather than static. Studies on the time dependence of the voltammetry at a rotated gold disk electrode modified by adsorbed SS-bpy under conditions of low surface coverage give calculated values of the relative surface-interactionrate constants, Pf= kfI'(SS-bpy). For a 10" M SS-bpy solution k*fis 2.2 X lC3cm S-I for an accumulation time of 15 s and cm s-' for an accumulation time of 60 s. As predicted theoretically with this model, a linear relationship between 3.9 X k*f and the square root of the accumulation time is demonstrated for very short times.
Introduction Redox reactions involving proteins and enzymes are a research area which is currently of extremely high intere~t.~J In principle, electrochemical techniques should provide a powerful tool for studying their electron-transfer properties. However, direct electrochemistry at bare electrodes, such as gold and platinum, has been dificult to achieve,e7 although a few reports do exist8-I0 at unmodified gold, platinum, and silver electrodes. A major breakthrough in the field of protein electrochemistry came when Yeh and Kuwana" observed the reversible reduction of cytochrome c at tindoped indium oxide electrodes and Eddowes and Hill foundI2 a persistent and reversible response at a gold electrode modified by an adsorbed layer of 4,4'-bipyridyl. Since the work of Eddowes and Hill more than 10 years ago, numerous reportse7~10~13-20 on the use of modified electrodes to promote electron transfer of cytochrome c have appeared in the literature. The implication contained in the initial studies16,21-28 was that a binding interaction takes place between cytochrome c and the promoter at a surface-modified electrode in a manner similar to that which is known to take place between enzymes and proteins prior to electron transfer. Alternatively, the adsorbed surface modifier may simply prevent irreversible and degradative adsorption of cytochrome c or impurities on the electrode surface, thereby preventing the electrode surface from becoming completely blocked.zg Figure 1 provides a schematic illustration of the modified electrode surface as deduced from the various studies. Effectively, an array of adsorbed molecules provides electroactive sites at the electrode interface either by allowing intimate interaction with cytochrome c or by preventing blockage of the surface. Despite the postulate of an electrode-solution interface model of the kind given in Figure 1, until recently it has been commonly assumed that the mass transfer to and from the modified electrode surface of stationary electrodes occurs by linear diffusion of cytochrome c. This would be true if the sizes of the electroactive sites were large. However, in the case of the electrochemistry of proteins at graphite electrodes,z*z629 the response could best be explained by assuming radial, rather than linear, diffusion occurring at
microscopically small oxygen functionalized electroactive sites. Similarly, it has been postulatedzB that electron transfer at modified gold electrodes occurs predominantly by radial diffusion to microscopically small active sites with electron transfer not occurring at the unm&ied part of the gold electrode. If the gold electrode at which the reaction takes place is itself a microelectrode, then the appropriate model may well involve an array of ultramicroscopic electroactive sites contained within such a microsurface. In this paper, the reduction of cytochrome c has been re-examined in the presence of the modifier 4,4'-bipyridyl disulfide (SS-bpy) at a stationary gold disk macroelectrode as well as at a microdisk gold electrode. The experimentsat the microelectrode are used to provide a detailed test of the micrcwcopic model and the importance of radial diffusion which should be dominant, relative to linear diffusion, at such electrodes, even when the entire surface is electroacti~e.~~ Additional features introduced into the electrochemistry when the microelectrode is considered as an assembly of arrays of microscopic active sites are also discussed as is the situation when a gold electrode is rotated so that mass transport may be achieved by convection rather than diffusion.
Experimental Section Cyclic voltammograms were obtained with either an Ursar Scientific Instruments potentiostat or a Bioanalytical Systems Model 1OOA electrochemical analyzer. A homemade current amplifier was used for the microelectrode experiments. A 2-mm radius gold disk electrode was used for experiments in stationary solutions with a conventionallysized electrode. For rotating disk radius) electrode was used with experiments, a gold disk (1.5-mm a Metrohm rotating disk assembly having six discrete rotation rates between 500 and 3000 rpm. The gold microelectrode was constructed by sealing a 5.2-pm radius gold wire (Goodfellow Metals, Cambridge, England) into Pyrex glass in an analogous way to that reported for fabrication of platinum microdisk elect r o d e ~ . ~ 'Experiments .~~ with conventionally sized gold disk electrodes were carried out in a three-electrode cell. A saturated calomel or a Ag/AgCl (3 M KCl) reference electrode was used with a platinum gauze auxiliary electrode, and the three electrodes
0022-3654/92/2096-8 100$03.00/0 0 1992 American Chemical Society
Mass Transport Process of Cytochrome c
The Journal of Physical Chemistry, Vol. 96, No. 20, I992 8101
I red
(IJ A)
Part of madher moluule ccpable of adsorptlan
Part of malerule capable of interacting with protein
Electrode Monolayer of mod11ler
Proteln dlffuslon layer
Bulk solullon
Figure 1. Schematic reprcscntation of an array of adsorbed molecules on the surface of a gold electrode.
were mounted in a glass cell which contained approximately 0.5 mL of solution except for the rotating disk electrode experiments where the volume used was 50 mL. For the microelectrode experiments, a two-electrode system was used with a gold microdisk working electrode and either a platinum wire or Ag/AgCl reference electrode. Before each experiment, the gold working electrodes were polished with a 0.3-pm alumina/water slurry on cotton wool, or only on a damp Metron polishing cloth, rinsed with distilled water, sonicated for 30 s, and f d y again rinsed thoroughly with distilled water. Ex situ surface modification was achieved by dipping the freshly polished gold electrode into a solution of the modifier either at open circuit or with the electrode being poised at a chosen potential. In situ surface modification was achieved by adding the modifier to the bulk solution. Horse heart cytochromec, (Type VI, Sigma Chemical Co.) was purified on a CM-32 cellulose column (Whatman Biochemicals Ltd.) according to the method of Brautigan et al.33 The concentration of cytochromec was determined spectrophotometrically at 550 nm using an extinction coefficient of eSu) = 29.5 m-'M-'. 4,4'-Bipyridyl disulfide (SS-bpy) was obtained from Aldrich Chemical Co. under the trade name of Aldrithiol-4. The phosphate buffer was prepared from AnalaR grade potassium dihydrogen phosphate and sodium hydroxide. Sodium perchlorate was added to the buffer to control the ionic strength. All solutions were thoroughly degassed with humidified argon prior to undertaking the electrochemical experiments.
R d t s d Discussion (a) Electrochemistry of cytochrome c at conveotionnllysued Electrodes. The electrochemistry of cytochrome c at a conventionally sized gold electrode modified ex situ with a high concentration of SS-bpy is shown in Figure 2a. The response corresponds to a reversible heme Fe(II1) to Fe(I1) one-electron reduction process under conditions of mass transport by linear diffusion as shown3'J5 by the separation of 65 f 5 mV for reduction and oxidation peak potentials and the linear dependence of peak height on the square root of the scan rate over the range 10-200 mV s-I. Furthermore, the average value of the oxidation and reduction peak potentials is 0.030 f 0.005 V vs SCE which corresponds closely to the standard redox potential, E", for cytochrome c as expected theoretically for a reversible process with linear d i f f u s i ~ n . The ~ * ~diffusion ~ coefficient calculated from the
Figwe 2. Voltammetry of 0.35mM cytochrome c at a 2-mm radius gold disk macroclcctrodeusing a scan rate of 20 mV s-l in 20 mM phosphate buffer/100 mM NaC10, (pH 7.0) with (a) a high surface coverage achieved by ex situ modification (insert is a plot of peak current for reduction versus the square root of the scan rate (v1I2), (b) moderate coverage achieved by in situ modification with 100 pM SS-bpy in the bulk solution (first three scans shown), and (c) low coverage achieved by in situ modification with 10 pM SS-bpy in the bulk solution (first two scans shown) with insert being a plot of E versus log [(il - i ) / i ] for the
s a n d scan.
slope of the insert in Figure 2a is 5 X IO-' cmz s-' using the Randles-Sevcik equation3' and assuming the entire surface is electroactive. At high concentrations of SS-bpy, it is postulated that the adsorbed molecules are close together and the surface density of electroactive sites is high. Under such conditions overlap of the individual diffusion layers occurs, leaving mass transport to be dominated by linear d i f f u s i ~ n . ~ - ~ ~ * ~ ~ According to the microscopic model, under conditions of submonolayer surface coverage, the electroactive sites will become well-separated and sigmoidal shaped voltammograms will result when no overlap of diffusion layers occurs. The predicted change in the shape and decrease in current is observed with in situ modification of the electrode and decreasing concentration of SS-bpy (lower surface coverages) and smaller electroactive electrode area as shown in Figure 2b,c. The shape becomes completely sigmoidal under the conditions of Figure 2c, with the half-wave potential being equal to the reversible potential F j l 2 (approximately E") and a plot of E vs log [(il- i)/i] is linear with a slope of (2.303RTlnF f 0.002) V over the temperature range of 15-25 "C (E = potential, i = current, il = the limiting current). The change from high to low concentration of surface modifier is assumed to represent a transition from approximately 100% linear diffusion (approaching monolayer coverage) is approximately 10096 radial diffusion (low surface coverage) as the spacing between electroactive sites increases. The microscopic model also provides an explanation of the time dependence of cyclic voltammograms obtained when the modified gold electrode surface is prepared by in situ exposure to SS-bpy. In Figure 2b,c there is virtually no response for initial scans commenced immediately after the electrode was in contact with the modifier. However, subsequent scans, which commence after significant time has e l a m for surface modification to occur,give the appropriate shaped wave predicted when the modifier merage
Bond et al.
8102 The Journal of Physical Chemistry, Vol. 96, No. 20, 1992 1
t (secs)
II
[modifier] ( p M 1
+/p= 2oo
TABLE I: Volt."ctric Parameters Obtained for Redoctioll of 0.35 &f ~ t O e h r O WC (h = u)mv f')8t 8 2 - W h d b cold Disk M.craketrode Modified in Situ awl ex Sits with Smb method of modification (W-bipyl (mM)) ex situ (2) ex situ (2) ex situ (2) ex situ (2) ex situ (2) ex situ (0.02) ex situ (0.2) ex situ (1.0) ex situ (2.0) ex situ (0.2) ex situ (1) in situ (0.02) in situ (0.2)
up or tmp
(SI
5 15
30 60 120 120
120 120 120 1800
300 3rd scan 3rd scan
slope (mV) 57 170 120 100
65 57 175
90 65 90 70
150 57
ipd. or ild. (PA)
0.09 0.14 0.60 0.60 0.65 0.45 0.44 0.60
0.65 0.60
0.60 0.30 0.60
shape sigmoidal
peaF peak peak peak sigmoidal pealr' peak peak peak peak
pcaF peak
"All solutions employed contained 20 mM phosphate buffer (pH 7) and 100 mM NaC10, as the electrolyte. bt, = time of exposure of electrode to SS-bpy solution at open circuit; AEp = peak-to-peak s e p aration of reduction and oxidation peak potentials from peak shape cyclic voltammogram; slope = slope of plots of E versus log [(II i ) / i ] obtained from a sigmoidal shaped curve at 20 O C ; ipd, = reduction peak current obtained from peak shaped curve; ild, = limiting current obtained from sigmoidal shaped curve. CAlmostsigmoidal, but small flat peak observed.
-
120secs.2 mM
F m 3. Voltammetry of 0.35 mM cytochrome c at a 2-mm radius gold disk macroelectrode modified ex situ (I) from a 2 mM SS-bpy solution with indicated exposure times and (11) from indicated concentrations of SS-bpy after 2-min exposure. Other conditions the same as those for Figure 2. increases toward its equilibrium value. Data obtained for two situations relevant to the ex situ method of modification of macroelectrodes at open circuit provide further information on mechanistic aspects of the microscopic model. Firstly, different texpof the electrode to a constant concentration of modifier produces sigmoidal shaped voltammograms, at short periods of dipping and peak-shaped responses at longer exposure (Figure 31). Secondly, a constant exposure time, tcxp,of the electrode to an SS-bpy solution gives sigmoidal shaped voltammograms at low modifier concentrations and a peak shaped (linear diffusion) response at high concentrations (Figure 311). Additionally, the shape of the cyclic voltammograms obtained at ex situ modified electrodes varies considerably with scan duration, particularly for case I1 of Figure 3. This time dependence can be explained by postulating that open circuit dipping experiments at short tcxpproduce a non-uniform surface coverage of small aggregates of modifier which may congregate along cracks or other electrode imperfections. Redistribution of the modifier to give a more uniform coverage is then assumed to occur as the potential is scanned. This leads to an increase in the contribution of linear diffusion to the mass transport and implies that the modifier is mobile and able to move across the electrode surface in a time dependent manner. A summary of data obtained under various conditions of in situ and ex situ electrode preparation is contained in Table I. (b)ElectrocbemistryofQtocbnmecataColdMk"k. At a 5.2-pm radius gold microelectrode, even with a monolayer to coverage of modifier, radial diffusion would be be dominant with low scan rates. No response is observed at a bare gold microelectrode for reduction of cytochrome c (Figure 4a). However, Figure 4b demonstrates that a welldefined sigmoidal-shaped curve is observed at an in situ modified microelectrode. Furthermore, since the E,12-value of 0.030 i 0.005 V vs SCE equals the EO-value obtained at conventionally sized electrodes within experimental error and the plot of E vs log [(i,
(a)
obsence of SS-bipy
!/
presence of SS - bipy Figure 4. (a) Voltammetry of 0.35 mM cytochrome c in 20 mM phos-
phate buffer/lOo mM NaClO, (pH 7.0) in the absence (a) and presence (b) of 10 pM SS-bpy in solution at a 5.2-pm radius gold microdisk electrode using a scan rate of 20 mV s-'.
- i ) / i ] is linear with a slope of (2.303RTlnF f O.OO2)Vover the temperature range of 15-25 O C , the results are ~onsistent~~9~' with a very fast rate of electron transfer. At a microel-, even when all of the surface is electmactive, and steady-state conditions p r e ~ a i l , ' ~ . ~most ~ . ~ 'of the electrontransfer reaction occurs at the edge of the electrode and very little (only a few percent at most) m u r s in the center of the electrode. Apparently, this non-uniform concentration-current distribution has considerable implications for the voltammetry of cytochrome c at modified gold microelectrodes as ex situ modification carried out with microelectrodes at open circuit leads to no detectable response being observed for reduction of cytochrome c. In contrast, if the ex situ modification is carried out at potentials negative of then a reversible sigmoidal-shaped response is obtained, whereas if a potential positive of Ell2is used, no cytochrome c response is observed. It was deduced from data obtained at conventionally sized electrodes that ex situ preparation of modified electrodes leads to a non-uniform distribution of SS-bpy, which only becomes uniform on scanning of the applied potential. At a microelectrode at open circuit, it appears that minimal modification occurs at the edge of the microelectrode and that application of an a p propriate potential is necessary to achieve uniform surface modification with SS-bpy over the entire microdisk gold electrode surface, including the edge. Presumably, at open circuit, modification is dominant at the electrode center, so that currents are
The Journal of Physical Chemistry, Vol. 96, No. 20, 1992 8103
Mass Transport Process of Cytochrome c small until after application of an applied potential which produces a uniform modifier coverage. (c) Time Dependeace of Adsorption at a Rotating Disk Electrode. A rotating disk electrode can be used to change the mode of mass transport from diffusion to convection (assuming the rotation rate, o,is fast enough) so that the time dependence of the surface process can be calculated in a relatively simple way. If the SS-bpy modifier simply provides a suitable site for electron transfer by removing a blocked part of the surface, without specific interaction with cytochrome c, then effectively, a time-dependent electroactive area should be created as SS-bpy is adsorbed. Under these conditions, and with a sufficiently fast rotation rate so that mass transport occurs solely by convection, the theory for a reversible process would predict E l l 2 = E O . If a specific interaction does occur at an electrode surface, then El12may or may not equal E O . In this case, the experiment may be modeled by assuming that the redox reaction is preceded by the formation of a surface complex which is then followed by the dissociation of the complex20as in eqs 1-3. Here CytFe"' and
a difference between the observed Ell2and the standard potential Eos might be expected, if the surface complex formation mechanism is valid. In this sense, the fact that Ell2= Eosmay favor the concept of the unblocking mechanism, but only, of course, by inference. If the formation of the surface complex in eq 1 is slow (forward reaction in eq 1 is rate determining) so that the following applies, d( r (Cyt Fe" '-SS-bpy)) / dt = kfI'(SS-bpy) [CytFelll]x,o - kbI'(CytFellLSS-bpy) (12) then a combination of eqs 5-9 and 12 results in i(kin.) = (1
+ 2 + exp(4*))-li(lim,masstrans.)
i(lim,kin.) = (1
+ Z)-'i(lim,mass trans.)
(13) (14)
+ (SS-bpy),d, (CytFe"'-SS-bpy),d, (CytFe"'-SS-bpyJad, + e- e (CytFe"-SS-bpy),d,
(1)
where Z = D(6k*f)-I and k*f = kfI'(SS-bpy). The kinetic parameter 2 can be calculated from the ratio between the convection or mass transport controlled and kinetically controlled limiting currents: 2 = (i(lim,masstrans.)/i(lim,kin.)) - 1 (15)
(2)
Thus, under these conditions, both Z and i(lim,masstrans.) are linear functions of w1I2:
(CytFeILSS-bpylad,e {SS-bpy),d, + CytFe"
(3)
Z = (X/k*f)o"2
k
CytFe"'
CytFe" denote the ferric and ferrous forms of cytochrome c respectively, {SS-bpy),, stands for the adsorbed surface modifier and (CytFe"'-SS-bpy),, designates the adsorbed adduct which undergoes the electron-transfer reaction. The reactions given in eqs 1-3 are characterized by eqs 4-6, where 4 = F(E-Eos)/RT, K1 = r(CytFellLSS-bpy)([CytFelll]x=oI'(SS-bpy))-l (4)
r(CytFeIIl-SS-bpy) = exp(4) I'(CytFeILSS-bpy) K2 = r(CytFeII-SS-bpy)( [CytFe"] x*or( SS-bpy))-l
(5) (6)
Eosis a standard potential of the surface confined redox reaction 2, K I and K2 are stability constants of the adsorbed adducts, kf and kb are the forward- and back-reaction rate constants associated r(z)is a surface concentration of a species with eq 1 (kf/kb = K1), z,and [Y]xsois the concentration of species Y near the electrode surface. Under steady-state conditions, at a rotating disk electrode where mass transport occurs solely by convection, the reduction current is independent of time and the following equations apply: i = FSD([CytFe"'*] - [CytFe111],,o)/6 (7) i = FSD[CytFe11],,o/6
(8)
d(r(CytFe1I1-SS-bpy))/dt = i/FS
(9)
where i is the current, S is the electrode surface area, D is a common diffusion coefficient, [CytFe"'*] is the bulk concentration of the oxidized form of cytochrome c, 6 is the diffusion layer thickness (6 = 01/3u1/6w-1/2[0.62(2a/60)1~2]-1), u is the kinematic viscosity, and w is the rotation rate of the electrode in revolutions per minute. If reaction 1 is reversible, the current is equal to i = (1 exp(4*))-li(lim,mass trans.) (10)
+
where i(lim,mass trans.) = FSD[CytFe"'*]/6 and exp(4*) = ( K 2 / K I ) exp(4). Under these conditions, a rotating disk steady-state voltammogram has a reversible half-wave potential Fl12equal to
+ (RT/F!
(1 1) In (Kl/K2) F I t 2is obviously identical to Eos and therefore to EO, as is observed experimentally at a gold electrode if K I = K2. This requires that both adsorbed adducts are either equally unstable (k,
