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Enhancing Electron Transfer at a Cytochrome c-Immobilized Microelectrode and Macroelectrode Ela Strauss,† Bill Thomas,‡,§ and Siu-Tung Yau*,† Department of Physics and Astronomy, Hunter College of the City Universty of New York (CUNY), New York, New York 10021, and BAE Systems, Marshall Space Flight Center, Huntsville, Alabama 35806 Received January 7, 2004. In Final Form: June 28, 2004 The redox reaction of cytochrome c immobilized on the bare surfaces of microelectrodes and macroscopic electrodes (macroelectrodes) composed of different planes of highly oriented pyrolytic graphite has been investigated using cyclic voltammetry. The protein-immobilized microelectrodes were fabricated using a simple masking method. For both macroelectrodes and microelectrodes, the redox reaction of immobilized cytochrome c needs to be activated by increasing the electrochemical potential maximum of cyclic voltammetry to a high positive value. The redox currents of this protein-electrode system can be enhanced using two approaches. The oxidation and reduction currents of cytochrome c adsorbed on microelectrodes that are composed of the edge plane show an anomalous enhancement compared to those for macroelectrodes composed of the basal plane. The difference in the surface chemical properties of the two kinds of electrodes results in the current anomaly. The oxidation current of the macroelectrode can be selectively enhanced by decreasing the potential minimum.
Introduction The search for an interface between an electrode and immobilized proteins/enzymes that gives rise to enhanced electron transfer is presently a research frontier in bioelectronics.1,2 Interfacing techniques are especially important for miniaturization of bioelectronic devices. A pressing issue for miniaturization of amperometric biosensors is to obtain efficient or enhanced interfacial electron transfer so that weak signals from small-volume samples3-5 can be detected. An obvious solution is to immobilize electroactive proteins/enzymes on the bare surface of an electrode to achieve efficient transfer of electrons via quantum mechanical tunneling.6 The metalloprotein cytochrome c (Cyt c) is an important electroactive protein. Electrode-immobilized Cyt c has been used in electrical interfacing in bioelectronic applications. Immobilized Cyt c has been used in an amperometric sensor for sulfite as electron acceptor7 and in sensing of superoxide anion radical (O2-).8 For biofuel cells, Cyt c has been immobilized on the cathode as an electrical interface between cytochrome c oxidase and the electrode.9 * To whom correspondence should be addressed. E-mail:
[email protected]. † Hunter College of CUNY. ‡ Marshall Space Flight Center. § Present address: Universities Space Research Association, Marshall Space Flight Center, Huntsville, AL 35875. (1) Gopel, W.; Heiduschka, P. Biosens. Bioelectron. 1995, 10, 853883. (2) Turner, A. P. F Science 2000, 290, 1315-1317. (3) Scheller, F. W.; Schubert, F.; Fedrowitz, J. In Frontiers in biosensorics I; Scheller, F. W., Schubert, F., Fedrowitz, J., Eds.; Birkhauser Verlag: Basel, Boston, Berlin, 1997. (4) Malsch, I. Ind. Phys. 2002, June/July, 15-17. (5) See articles in Nanostructured Materials in Biological and Artificial Systems, Proceedings of the 11th Toyota Conference on Nanostructured Materials in Biological and Artificial Systems. Supramol. Sci. 1998, 5, Nos. 3-4. (6) DeVault, D. Quantum-mechanical tunnelling in biological systems, 2nd ed.; Cambridge University Press: Cambridge, 1984. (7) Abass, A. K.; Hart, J. P.; Cowell, D. Sens. Actuators, B 2000, 62, 148-153. (8) McNeil, C. J.; Athey, D.; Ho, W. O. Biosens. Bioelectron. 1995, 10, 75-83. (9) Willner, I.; Willner, B. Trends Biotechnol. 2001, 19, 222-230.
The electrochemistry of Cyt c has been largely plowed by Hill,10 Taniguchi,11 and Hawkridge.12,13 Eddowes and Hill in 1977 reported quasi-reversible electron transfer of Cyt c dissolved in a solution that also contained 4,4′bipyridyl at a gold electrode.10 Later, Taniguchi and coworkers found that bis(4-pyridyl) disulfide adsorbed on gold resulted in quasi-reversible electron transfer of Cyt c in a promoter-free Cyt c solution.11 At the same time, the electrochemistry of Cyt c dissolved in solution at bare metal oxide electrodes was studied by Hawkridge and coworkers.12,13 Facile electron transfer was found between purified Cyt c and indium and tin oxide electrodes.12 It was demonstrated that an electrode surface that is hydrophilic is necessary for observing facile electron transfer with Cyt c.13 A detailed review of the works in this area is given in ref 14. The electrochemistry of Cyt c immobilized on gold electrodes whose surfaces were derivatized with a self-assembled monolayer (SAM) of alkanethiol has been studied by Bowden as reviewed in ref 15. The Cyt c that is immobilized on a SAM appears to retain its native form as characterized using cyclic voltammetry. Electron-transfer rate constants between the SAM-covered electrode and the immobilized Cyt c were measured.16 Tao and co-workers have performed cyclic voltammetry of Cyt c immobilized on the basal plane of highly oriented pyrolytic graphite (HOPG) using physical adsorption17 and Langmuir-Blodgett deposition.18 The immobilized Cyt c showed quasi-reversible electron trans(10) Eddowes, M. J.; Hill, H. A. O. J. Chem. Soc., Chem. Commun. 1977, 771-772. (11) Taniguchi, I.; Toyosawa, K.; Yamaguchi, H.; Yasukouchi, K. J. Chem. Soc., Chem. Commun. 1982, 1032-1033. (12) Bowden, E. F.; Hawkridge, F. M.; Chlebowski, J. R.; Bacroft, E. E.; Thorpe, C.; Blount, H. N. J. Am. Chem. Soc. 1982, 104, 7641-7644. (13) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. J. Electroanal. Chem. 1984, 161, 355-376. (14) Hawkridge, F. M.; Taniguchi, I. Commments Inorg. Chem. 1995, 17, 163-187. (15) Bowden, E. F. Electrochem. Soc. Interface 1997, 40-44. (16) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 18471849. (17) Boussaad, S.; Tao, N. J.; Arechabaleta, R. Chem. Phys. Lett. 1997, 280, 397-403. (18) Boussaad, S.; Dziri, L.; Arechabaleta, R.; Tao, N. J.; Leblanc, R. M. Langmuir 1998, 14, 6215-6219.
10.1021/la049942y CCC: $27.50 © 2004 American Chemical Society Published on Web 08/26/2004
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fer with a low degree of facility compared to the other cases described above. In this paper, we report an electrochemical investigation on the redox reaction of Cyt c immobilized on the bare surfaces of micrometer-sized electrodes (microelectrodes) and macroscopic electrodes (macroelectrodes), which are composed of different planes of HOPG. We are interested in knowing if enhanced electron transfer can be achieved with the Cyt c-HOPG system. We show that the Cyt c-immobilized microelectrodes can be fabricated using a simple masking method. For both macroelectrodes and microelectrodes, to activate the redox reaction of the immobilized Cyt c, the potential maximum of cyclic voltammetry needs to be increased to a high positive value (+1 V). We show that, after the redox reaction is “turned on”, the redox currents of the immobilized Cyt c can be enhanced under two conditions as a result of different surface chemical properties of the planes. The redox currents of Cyt c immobilized on the microelectrode that is composed of the edge plane of HOPG show an anomalous enhancement compared to those for the macroelectrode composed of the basal plane. However, the oxidation current of the macroelectrode can be selectively enhanced by decreasing the potential minimum of cyclic voltammetry. Experimental Section HOPG (Advanced Ceramics) was used as the material for the working electrode in an electrochemical cell. Three types of working electrodes were used. Electrode A is the HOPG basal plane having an area of 0.04 cm2. Electrode B is a 0.04 cm2 HOPG basal plane containing three or six holes with a diameter of approximately 300 µm and a depth of about 400 µm. Figure 1 is a schematic description of the fabrication process of the holes. The holes essentially consist of the edge plane of HOPG. Electrode C is the HOPG basal plane having an area of 0.62 cm2. Cyt c was immobilized on all of three kinds of electrodes (see below). Type VI horse heart cytochrome c (Cyt c) was purchased from Sigma. Cyt c was purified to microhomogeneity by ion-exchange high-performance liquid chromatography utilizing a semipreparative VYDAC protein SAX, 2.2 × 100 cm, 300VHP82210 column (The Separations Group, Hesperia, CA). Utilizing 25 mM MOPS buffer, the pH was brought from 6.6 to 6.4 and the protein eluted in multiple peaks from the VYDAC column. Native and SDS electrophoresis indicated protein purified not simply to homogeneity but to microhomogeneity; i.e., multiple peaks of Cyt c were collected, and one was used. The purified Cyt c was dissolved in 20 mM tricine and 20 mM CAPS at pH 7. The protein concentration was 0.2 mg/mL. All solutions were prepared with water (18.2 MΩ cm) from a Direct-Q 5 Millipore system. Cyt c was physically adsorbed on the three kinds of working electrodes. A drop of Cyt c solution was placed on the entire surfaces of electrodes A and C for 30 min with the electrodes sealed inside a container with moisture. After adsorption the electrodes were rinsed with the buffer solution and transferred immediately to the electrochemical cell. Figure 1 describes the construction of electrode B, the Cyt c-immobilized microelectrode. It shows that, for electrode B, Cyt c is immobilized only inside the holes while the flat basal plane is protein-free. Cyclic voltammetry was performed in a three-electrode cell using a bipotentiostat (Veeco, Santa Barbara, CA). Platinum and silver wires were used as the counter electrode and the quasireference electrode, respectively. The latter electrode was calibrated against a commercial (Microelectrode, Inc.) Ag/AgCl (3 M KCl, saturated with AgCl) reference electrode. Cyclic voltammetry of immobilized Cyt c was performed with the cell containing the buffer solution. The potential was held at the initial potential of 0.03 mV for about 10 min before scanning of the potential started.
Results and Discussion “Turn-on” Effect. We found that, to observe the redox peaks of Cyt c immobilized on the edge plane and the
Figure 1. Schematic description of the preparation of electrode B. (a) The top surface of an HOPG sample is masked with Scotch tape. A needle is used to drill holes through the tape on the basal plane of the HOPG sample. (b) A drop of Cyt c solution is placed on the masked sample surface that contains holes of the exposed edge plane of HOPG. (c) After adsorption and removal of the solution, the tape is peeled off, resulting in a fresh basal plane containing holes having walls made of the edge plane of HOPG. Cyt c molecules are immobilized only on the walls of the holes.
basal plane of HOPG, the potential maximum of cyclic voltammetry first has to be increased to about +1 V. Figure 2 shows the cyclic voltammogram (CV) of electrode C with a potential range between 0 and +1 V. This CV is the first scan of potential, and the only feature on the CV is the single peak near +700 mV. The redox peaks of Cyt c start to appear on the second scan (see below) and on subsequent scans. The same effect occurs with electrode B. This phenomenon of “turning-on” of the redox reaction of immobilized Cyt c also has been observed by Tao and coworkers.17 We also decreased the potential minimum of cyclic voltammetry toward -1 V in the first scan and did not observe the turn-on effect. The isolated peak near +700 mV has been identified as due to irreversible electrochemical oxidation of tyrosine and tryptophan residues of proteins, including Cyt c, adsorbed on graphite19-21 and on other solid electrodes.22,23 (19) Brabec, V.; Bianco, P.; Haladjian, J. Biophys. Chem. 1982, 16, 51-59. (20) Brabec, V.; Mornstein, V. Biochim. Biophys. Acta 1980, 625, 43-50. (21) Brabec, V. Bioelectrochem. Bioenerg. 1980, 7, 69-82.
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Figure 2. Cyclic voltammogram of electrode C between 0 and +1 V. The single peak near +700 mV is due to oxidation of tyrosine and tryptophan residues of the immobilized Cyt c.
It is known that tyrosine and tryptophan residues such as Tyr 48, Tyr 67, and Trp 59 are located near the heme of Cyt c.24 Therefore, it is likely that the oxidation of these residues causes repulsive electrostatic interaction between these residues and the heme. Since these residues are located behind the heme with respect to the opening of the pocket, the heme might be pushed outward. Thus, if the immobilized Cyt c assumes an optimum orientation (see below) so that the opening of the pocket is in contact with the electrode, the heme will become closer to the electrode, facilitating electron transfer. Enhanced Redox Currents at the Microelectrode. Figure 3a shows the CVs of electrodes A, B, and C, labeled as a, b, and c, respectively, obtained after the potential maximum was increased to +1 V. For electrode B, on which Cyt c is adsorbed only in the holes, the redox peaks of Cyt c are present on CV b (oxidation peak at +260 mV, reduction peak at +170 mV) with the redox potential located at about 215 mV. This value indicates a positive shift in the redox potential compared to the value of 60 mV for the native form of Cyt c in solution25,26 and for immobilized Cyt c retaining its native form.27,28 The separation between the oxidation and reduction peaks is about 90 mV, which is to be compared with the value of zero for a reversible redox reaction for electroactive molecules adsorbed on an electrode.29 Therefore, this finite value of peak separation indicates a quasi-reversible redox process. The above characteristics are similar to the results of previous cyclic voltammetry studies of Cyt c immobilized (22) Malfoy, B.; Reynaud, J. A. J. Electroanal. Chem. 1980, 114, 213223. (23) Reynaud, J. A.; Malfoy, B.; Bere, A. Bioelectrochem. Bioenerg. 1980, 7, 595-606. (24) Brayer, G. D.; Murphy, M. E. P. In Cytochrome c: A Multidisciplinary Approach; Scott, R. A., Mauk, A. G., Eds.; University Science Books: Sausalito, CA, 1996. (25) Armstrong, F. A.; Cox, P. A.; Hill, H. A. O.; Lowe, V. J.; Oliver, B. N. J. Electroanal. Chem. 1987, 217, 331-366. (26) Bowden, E. F.; Hawkridge, F. M.; Blount, H. N. In Comprehensive treatise of electrochemistry; Srinivasan, S., Chiamadzhev, Y. A., Bockris, J. O., Conway, B. E., Yeager, E., Eds.; Plenum Press: New York and London, 1985; Vol. 10. (27) Sagara, T.; Niwa, K.; Sone, A.; Hinnen, C.; Niki, K. Langmuir 1990, 6, 254-262. (28) Willit, J. L.; Bowden, E. F. J. Electroanal. Chem. 1987, 221, 265-274. (29) South Hampton Electrochemistry Group. Instrumental methods in electrochemistry; Ellis Horwood Limited: Chichester, England, 1985; Chapter 6.
Figure 3. (a) Cyclic voltammograms of electrodes A, B, and C, on which Cyt c was immobilized. Voltammogram a is for electrode A, which is the HOPG basal plane having an area of 0.04 cm2. The absence of the redox peaks of Cyt c persists even if the current scale is lowered. Voltammogram b is for electrode B, which is a 0.04 cm2 HOPG basal plane containing six holes with a diameter of approximately 300 µm and a depth of about 400 µm. The holes essentially consist of the edge plane of HOPG. Voltammogram c is for electrode C, which is the HOPG basal plane having an area of 0.62 cm2. Purified Cyt c is immobilized on the entire electrode surface with the exception that for electrode B Cyt c is immobilized only in the holes. The voltammograms were recorded at 100 mV/s in a buffer solution of 20 mM CAPS and 20 mM Tricine at pH 7. (b) Current of the oxidation peak versus scan rate for electrode B. The slope of the linear regression of the data points is 0.23 µF.
on macroelectrodes of the basal plane of HOPG.17,18 For electrode A, on which Cyt c is adsorbed on the entire electrode surface, CV a in Figure 3a shows an absence of the redox peaks of Cyt c. For electrode C, whose area is 16 times larger than that of electrode A, the redox peaks of Cyt c appear on CV c. The characteristics of the redox peaks are similar to those of electrode B. Cyclic voltammetry of the three kinds of electrodes without Cyt c adsorption was also performed and shows no redox peaks in the potential range used here. Figure 3b is a plot of the oxidation peak current versus scan rate for electrode B. The linear dependence indicates that the redox process is due to the immobilized protein.29 The same kind of plot for electrode C also shows a linear dependence. From the areas under the peaks, the amount of electroactive Cyt c is estimated to be about 2-3 pmol for electrode B, while that for electrode C is about 2 pmol. Therefore, the numbers of Cyt c molecules that contribute to the redox currents for electrodes C and B appear to be similar. However, the total amount of immobilized Cyt c on electrode C should be much greater than that on electrode B. This is because the basal plane is hydrophobic,
Electron Transfer at Cyt c-Immobilized Electrodes
Cyt c contains hydrophobic residues on its surface, and the hydrophobic interaction gives rise to immobilization on the entire surface of any basal plane as confirmed using atomic force microscopy.17 Electrode C, which is the basal plane having an area of 0.62 cm2, should contain Cyt c on its entire surface, while Cyt c is immobilized only within the 300 µm wide holes for electrode B. Therefore, CVs b and c of Figure 3a reveal an interesting phenomenon that a microelectrode generates anomalously larger redox currents compared to a macroelectrode, both electrodes being made of the same material. We believe the fact that, between electrode A and electrode B, only electrode B shows the redox currents of immobilized Cyt c is due to the chemical nature of the electrode surface. As shown by Armstrong et al.,30 electron transfer of Cyt c at the graphite electrode should occur only at functionalized sites which contain carbon-oxygen functional groups. The basal plane of HOPG is chemically inert and ideally should contain no such sites. The small amount of functionalized sites that in reality exist on the basal plane is caused by damage due to a manufacturing or cleavage process. When an HOPG sample is cleaved using Scotch tape to expose a fresh basal plane, steps are generated on the basal plane. The step surface, whose orientation is perpendicular to that of the basal plane, is the edge plane of HOPG containing functionalized sites (see below). Therefore, the redox current of Cyt c is less pronounced at the basal plane.30 In our case, the area of the basal plane of electrode A may not be large enough to contain enough functionalized sites to produce a detectable signal. However, when electrode C, whose area of the basal plane is 16 times larger than that of electrode A, was used, the redox reaction was detected as shown in Figure 3a. This effect can be caused by the larger amount of damage-related functionalized sites or by the fact that some of the larger numbers of immobilized Cyt c molecules happen to assume the optimum oritentation (see below). For electrode B, Cyt c is immobilized exclusively in the holes, whose walls are composed of the edge plane of HOPG. When the holes are made by drilling the basal plane, the edge plane is exposed to oxygen. The ruptured carbon-carbon bonds on the aromatic rings on the edge plane are oxidized, thereby creating carbon-oxygen functional groups such as the carboxylic, phenolic, ketonic, ether-like, and quinone-like groups.30,31 These groups render hydrophilicity and ionic character to the edge plane, providing an optimum orientation for Cyt c via electrostatic interaction.25 In the optimum orientation, the positively charged lysine residues surrounding the heme crevice are attracted to the negatively charged functional groups, bringing the heme close to the electrode surface. Therefore, the large number of functionalized sites on the edge plane promotes electron transfer. The higher charging current of electrode B compared to that of electrode C, as shown in Figure 3a, is due to the fact that the nonfaradic differential capacitance of the edge plane is significantly higher than that of the basal plane due to the presence of the polar carbon-oxygen functional groups on the surface of the edge plane, which are exposed to the electrolyte.32,33 Positive Shift of the Redox Potential. As mentioned above, the redox potential of Cyt c immobilized on the (30) Armstrong, F. A.; Bond, A. M.; Hill, H. A. O.; Psalti, I. S. M.; Zoski, C. G. J. Phys. Chem. 1989, 93, 6485-6493. (31) Kamau, G. N.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1985, 57, 545-551. (32) Randin, J.-P.; Yeager, E. J. Electroanal. Chem. 1975, 58, 313322. (33) McGreery, R. L. In Interfacial Electrochemistry, Wieckowski, A., Ed.; Marcel Dekker: New York, 1999.
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Figure 4. Effect of varying the potential minimum of cyclic voltammetry on the faradic and charging currents of Cyt c-immobilized electrodes: (a) electrode C and (b) electrode B with three holes. The potential minimum was decreased from -100 mV to different values in the range between -200 mV and -1 V. The potential minimum values are (a) -100 mV, (b) -200 mV, (c) -400 mV, (c) -600 mV, (e) -800 mV, and (f) -1 V. The scan rate was 100 mV/s.
basal and edge planes is shifted positively by an amount of about 155 mV. This shift is possibly due to a changed heme exposure to the solvent with the adsorbed Cyt c in the folded form. It is known that changes in the redox potential of Cyt c can be caused by variations in heme exposure to solvent.34-36 A significant negative shift indicates total exposure of heme to solvent due to denaturation of Cyt c,35 while a positive shift is caused by a certain degree of solvent exclusion from the heme environment with the protein in the folded form.35,36 Previous studies show that the redox potential of Cyt c can be tuned by 500 mV and that, with a positive shift of 240 mV, Cyt c is still in the folded form.36 However, the positive shift can also be caused by a structural reorganization due to the breaking of bonds involving tyrosine residues, accompanied by a more marked burying of the heme. A modified electron pathway within the protein may also cause the shift of the redox potential. Selectively Enhanced Oxidation Current. We have studied the effect of varying the potential extrema of cyclic voltammetry on the redox peaks. For electrode C, as the potential minimum is decreased from -100 mV to a value between -200 mV and -1 V, the oxidation peak current increases while the reduction peak current is not affected as shown in Figure 4a. This selective or exclusive enhancement of the oxidation peak current persists upon repetitive scanning of the potential with the potential minimum assuming a given value in the range shown (34) Kassner, R. J. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 22632267. (35) Stellwagen, E. Nature 1978, 275, 73-74. (36) Tezcan, F. A.; Winkler, J. R.; Gray, H. B. J. Am. Chem. Soc. 1998, 120, 13383-13388.
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above. Increasing the potential maximum from 400 mV to 1 V does not have any effect on the currents at the redox peaks or on the charging current. For electrode B, when the potential minimum is decreased from -100 mV toward -1 V or when the potential maximum is increased from 400 mV toward +1 V, the size (height and width) of the redox peaks is not affected as shown in Figure 4b. To show that the enhanced oxidation current is not caused by subjecting the basal plane to high potential, cyclic voltammetry of the bare basal plane has been performed in buffer solution in the range of (1 V. No features were observed between -200 and +450 mV. Presently, we are not certain about the origin of the selective enhancement of the oxidation peak current observed with electrode C. However, a feature of the potential-dependent enhancement should be noted. Figure 4a shows a sudden pronounced increase in the oxidation current when the potential minimum is decreased to -600 mV. It is known that hydroxide anions are generated when water is reduced near -620 mV Ag/AgCl.37 Therefore, it is possible that the hydroxide anions produced at the electrode may play a role in orienting the Cyt c molecules that are adsorbed on the basal plane, giving rise to enhanced electron transfer. The unaffected reduction peak may be due to a field-related reorientation of Cyt c as proposed by Bowden.28 The reason for the redox peaks of electrode B to remain unchanged when the potential extrema is varied might be that the Cyt c molecules have already assumed the optimum orientation due to the large number of negatively charged carbon-oxygen functional groups on the edge plane. Future experiments will be carried out to study the dependence of peak height/shape (37) Handbook of Chemistry and Physics, 55th ed.; Weast, R. C., Ed.; CRC Press: Cleveland, OH, 1974-1975.
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and peak potential on potential scan rate and to study the role of the nature of the supporting electrolyte to understand the origin of this effect. Conclusion We found that the redox reaction of Cyt c immobilized on the planes of HOPG needs to be activated by applying a high potential. This effect is likely to be related to the oxidation of tyrosine and tryptophan residues near the heme. We show that the redox currents of Cyt c immobilized on HOPG can be enhanced using two approaches. Cyt c immobilized on microelectrodes composed of the edge plane gives rise to enhanced redox currents compared to that on macroelectrodes that are made of the basal plane. The enhancement is a result of coupling the positive charges located near the heme with negative charges of the large number of functional groups on the edge plane. Our attempt of manipulating the redox process by varying the potential minimum shows that the oxidation peak can be selectively enhanced for the Cyt cimmobilized macroelectrode made of the basal plane. We are not certain about the origin of this effect, and further detailed studies on this effect are planned. Acknowledgment. We thank N. J. Tao for critical reading of the manuscript and discussion. This work was supported by the ONR (Grant N000140310517), an NIHSCORE Grant to Hunter College (S06 GM60654), Hunter College Presidential Initiatives in Research, and a Research Centers in Minority Institutions award (Grant RR03037), from the National Center for Research Resources of the NIH. LA049942Y
