Langmuir 1992,8, 1247-1250
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Voltammetry of Covalently Immobilized Cytochrome c on Self-Assembled Monolayer Electrodes Maryanne Collinson and Edmond F. Bowden' Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204
Michael J. Tarlov Process Measurements Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899 Received February 10, 1992 Cytochromec was covalently immobilized on carboxylicacid terminated self-assembledmonolayer gold electrodes via electrostatically guided carbodiimide coupling. Covalently immobilized cytochrome c was found to be stable, electroactive, and functional. Cyclic voltammetry revealed an electroactive coverage corresponding to ca. one-third monolayer and a surface formal potential slightly negative of the value obtained for electrostatically adsorbed cytochrome c. Electron transfer kinetic results suggest that covalently immobilized and electrostatically adsorbed cytochrome c are similarly oriented at the electrode surface. Introduction functional group of the SAM, specific surface/analyte interactions can be exploited to immobilize molecules at During the past several years, considerable attention the interface. Crooks and co-workers, for example, emhas been focused on self-assembled monolayers (SAM's). ployed a pH-sensitive organothiol monolayer to selectively These well-defined organic films have proven to be bind charged ions.9a Ringsdorf and co-workers' utilized extremely useful for studying protein adsorption,' intera biotin-functionalized SAM to achieve molecular recogfacial electron transfer (ET),2-6 biomolecule7 and cell nition with streptavidin. Prime and Whitesides' modified immobilization,8 ion binding? and coupled chemical the hydrophobic/hydrophilic character of the interface as reactions.lO Systems currently under investigation include SAM's of organothiols on gold and ~ i l v e r , ~organosi~ J ~ - ~ ~ a means for controlling protein adsorption on organic surfaces. In a recent communication from this laboralanes on oxides and mica,1k17 carboxylic acids on metal t0ry,~9 a carboxylic acid terminated alkanethiol monooxides,18and redox-active amphiphiles on gold and other layer was used to provide a favorable surface for electrosurfaces.19~20 statically adsorbing cytochrome c in a functional, elecOrganothiols chemisorbed on gold are particularly welltroactive state. suited for electrochemical and chemical studies due to In this paper, we report the covalent attachment of a their excellent stability, highly characterized structures,21-28 functional, electroactive, electron transfer protein to a and the ease by which interfacial properties can be SAM electrode. A carboxylic acid terminated monolaycontrolled. Through the proper selection of the terminal er12930,31 was utilized to provide a chemically uniform surface for attaching cytochrome c via carbodiimide linkage (1) Prime, K. L.; Whitesides, G. M. Science 1991,252, 1164-1166. (2) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Mujsce, A. M. to side chain lysine groups. In contrast to electrostatically J. Am. Chem. SOC.1990, 112,4301-4306. driven adsorption, which is limited to low ionic strength (3) Chidsey, C. E. D. Science, 1991,251, 919-922. solutions of appropriate pH,29,32333 covalent attachment (4) Miller, C.; Cuendet, P.; Gratzel, M. J . Phys. Chem. 1991,95,877886. allows for the characterization of cytochrome c (sub)mono(5) Lee, K. A. Bunding Langmuir 1990, 6, 709-712. layers over a wide range of solution conditions. Investi(6) Finklea. H. 0.:Lvnch. M. Lanemuir 1987.3. 409-413. gations at higher ionic strength where both solution (7) Haussling, L.; Riigsdorf, H.; Schmitt, F.-J.; Knoll, W.Langmuir resistance effects and desorptive losses are minimized will 1991, 7, 1837-1840. (8) Margel, S.;Sivan, 0.;Dolitzky, Y. Langmuir 1991, 7, 2317-2322. now be possible. (9) Sun, L.; Johnson, B.; Wade, T.;Crooks, R. M. J.Phys. Chem. 1990,
94,8869-8871. (b) Steinbern. S.: Tor. Y.: Sabatani., E.:. Rubinstein. I. J. Am. Chem. SOC.1991.113. 5176-5182. '
(10)Sun, L.; Thomh, R: C.; Crooks, R. M.; Ricco, A. J. J . Am. Chem.
SOC.1991,113, 8550-8552. (11) Bryant, M. A.; Pemberton, J. E. J . Am. Chem. SOC.1991, 113,
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(12) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J . Am. Chem. SOC.1990, 112, 558-569. (13) Evans. S.D.: Ulman. A.: GoDDert-Berarducci. K. E.: Gerenser. L. J. J. Am. Chem. Soc. 1991,113, 5866-5868. (14) De Long, H. C.; Donohue, J. J.; Buttry, D. A. Langmuir 1991,7, 2196-2202.
(15)Wasserman, S. R.; Tao, Y.-T. Tao; Whitesides, G. M. Langmuir 1989,5, 1074-1087. (16) Angst, D. L.; Simmons, G. W. Langmuir 1991, 7, 2236-2242. (17) Kessel, C. R.; Granick, S. Langmuir 1991, 7, 532-538. (18) Chen, S. H.; Frank, C. W. Langmuir 1989,5, 978-987. (19) Gomez, M.; Li, J.; Kaifer, A. E. Langmuir 1991, 7, 1797-1806. (20) Bae, I. T.; Huang, H.; Yeager, E. B.; Scherson, D. A. Langmuir 1991, 7, 1558-1562. (21) Widrig, C. A.; Alves, C. A.; Porter, M. D. J.Am. Chem. SOC. 1991, 113, 2805-2810.
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(22) Widrig, C. A.; Chung, C.; Porter, M. D. J . EZectroanaL Chem. 1991,310, 335-339. (23) Samant, M. G.; Brown, C. A.; Gordon, J. G. Langmuir 1991, 7,
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(24) Sun, L. Crooks, R. M. J. Electrochem. SOC.1991,138, L23-L25. (25) Stole. S. M.: Porter. M. D. Lanemuir 1990. 6. 1199-1202. (26) Port&, M. D.; Bright, T. B.; AllGa, D. L.; Ckidsey, C. ED : : J . Am. Chem. SOC.1987,109,3559-3568. (27) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546-558. (28) Ulman, A.; Eilers, J. E.; Tillman, N. Langmuir 1989, 5, 114711.52. (29) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. SOC. 1991,113,18471849. (30) (a) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J . Am. Chem. SOC.1989,111,321-335. (b) Reference
30a supplementary material. (31) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682-691. (32) Willit, J. L.; Bowden, E. F. J. Phys. Chem. 1990,94,8241-8246. (33) Collinson, M.; Willit, J. L.; Bowden, E. F. In Charge and Field Effects in Biosystems II; Allen, M. J., Cleary, S.F., Hawkridge, F. M., Eds.; Plenum: New York, 1989; pp 63-76.
0 1992 American Chemical Society
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1248 Langmuir, Vol. 8, No. 5, 1992
Experimental Section Reagents/Equipment. Horse heart cytochrome c (type VI) and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) were purchased from Sigma Chemical Co. Cytochrome c was chromatographically purified as previously described.16-Mercaptohexadecanoic acid (HS(CHz)&OOH), abbreviated 16-MHDA, and 11-mercaptoundecanoic acid (HS(CHz)l&OOH), abbreviated 11-MUDA, were synthesized and purified according to published procedures.30b Reagent grade chemicalswere used to prepare all solutions. Water was purified using a Milli-Q system with an Organex-Q final stage (Millipore, Bedford, MA). The gold electrodes were prepared as previously described." A Model 273 PAR potentiostat was used to acquire the cyclic voltammetric (CV) data. A one-chamber electrochemical cella2 containing a horizontally mounted electrode was used. All potentials refer to Ag/AgCl (1 M KCl), which is 0.23 V vs NHE. Procedures. Carboxylic acid terminated alkanethiol electrodes were prepared by soaking a bare gold electrode in a 1 mM solution of the alkanethiol in absolute ethanol for 3-5 days. After equilibration the electrode was removed from the solution, rinsed with absolute ethanol, dried under argon, and assembled in the electrochemical cell, and background Cv's were obtained in 4.4 mM potassium phosphate buffer (pH 7.0, 10 mM ionic strength). Ferricytochrome c was then electrostatically adsorbed to the SAM electrode as previously described," and Cv's were acquired. Ferricytochrome c was then covalently immobilized to the same SAM electrode by a procedure similar to that previously described for protein/protein ~ r o s s - l i n k i n g .A~ cy~~~ tochrome c solution was first reintroduced to the cell cavity and allowedto equilibrate for 10-15 min. An aliquot of concentrated EDC was then added to this solution, and the immobilization reaction was allowed to proceed at room temperature for 30 min. The fiial solution conditions were -30 pM cytochrome c and -5 mM carbodiimide in pH 7.0,2.2 mM phosphate buffer. The cytochrome c/EDC solution was then removed from the sample cavity, the cell thoroughly rinsed several times and refilled with 4.4 mM, pH 7.0 phosphate buffer, and Cv's of the covalently immobilized cytochrome c were acquired. The electroactive surface coverage of cytochrome c was estimated by integrating the anodic peak of background-subtracted Cv's. A geometric electrode area of 0.32 cm2was used in this calculation. Laviron's model37 was used to calculate quasi-reversible E T rate constants from CV peak separations obtained from the backgroundsubtracted Cv's.
A
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Results Figure l a shows a typical background CV of a 16-MHDA monolayer electrode in buffer. The low capacitance of this electrode is consistent with the presence of a compact monolayer of 16-MHDASz9The CV of cytochrome c electrostatically adsorbed on this monolayer, Figure lb, is similarto that previouslydescribed.29 When cytochrome c was immobilized to this 16-MHDA monolayer via carbodiimide treatment, the CV shown in Figure ICwas obtained. The cathodic and anodic peaks are diminished in size and shifted to more negative potentials relative to those for electrostatically adsorbed cytochrome c. After exposure to a solution of saturated potassium nitrate, the faradaic response of electrostatically adsorbedcytochrome c nearly disappeared due to desorption, Figure 2A. In contrast, negligible change in the CV's of covalently immobilized cytochrome c was observed, Figure 2B. The voltammetric response of covalently immobilized cytochrome c was stable over 3 weeks of storage in buffer at 4 "C. Qualitatively similar voltammetry was obtained for (34) Brautigan, D. L.; Fergueon-Miller, S.; Margoliash, E. Methods Enzymol. 1978,630, 128-164. (36) Waldmeyer, B.; Boeehard, H. R. J. Biol. Chem. 1986,260,51845190.
(38)Mauk, M. R.; Mauk, A. G. Eur. J. Biochem. 1989,186,473-476. (37) Laviron, E. J . Electroanal. Chem. 1979, 101, 19-28.
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Figure 2. Cyclic voltammograms of cytochrome c electrostatically adsorbed, panel A, and covalently immobilized, panel B, on a 16-MHDA monolayer electrode before (a) and after (b) exposure to a solution of saturated potassium nitrate for ca. 5 min: scan rate, 50 mV/s; solution conditions, pH 7.0, 4.4 mM phosphate buffer.
cytochrome c electrostatically adsorbed, Figure 3b, and covalently attached, Figure 3c, to an 11-MUDA monolayer electrode. Background subtracted CV's of cytochrome c covalently
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Figure 3. Cyclic voltammograms of (a) an 11-MUDA monolayer covered Au electrode, (b) cytochrome c electrostatically adsorbed on this monolayer, and (c) cytochrome c covalently immobilizedvia carbodiimideto this monolayer. Conditionswere the same as given in as Figure 1.
immobilized to 16-MHDA are shown in Figure 4 as a function of scan rate. The electroactive surface coverage of ca. 6 pmol/cm2 is 30-40% of the near-monolayerm obtained for electrostatically adsorbed cytochrome c, typically 16 pmol/cm2. The estimated surface formal potential of -60 mV is some 20 mV more negative than the value for electrostatically adsorbed cytochrome c and nearly 90 mV more negative than the value for solution cytochrome c, ca. 30 mV.% The apparent ET rate constant ( k 0 3 for covalently immobilized cytochrome c on 16MUDA/Au is ca. 1 s-l. Some scan rate dependence was evident in these data, however, with keet varying monotonically from ca. 0.7s-l at 20 mV/s to ca. 1.4 s-l at 150 mV/s. For cytochrome c electrostatically adsorbed to the 16-MHDA monolayer electrode, e.g., Figure lb, ascan rate independent value of 0.9 s-l39 was obtained. Background-subtractedCV's of cytochrome c covalently immobilized to an 11-MUDAmonolayer electrode are also shown in Figure 4. The electroactive coverage of this covalent complex is ca. 6 pmol/cm2,which can be compared to 9-13 pmol/cm2 for the electrostatic complex. The surface formal potential of -50 mV is ca. 15 mV more negative than the formal potential determined for the electrostatic complex. On this electrode, both the electrostatically adsorbed and the covalently immobilized cytochrome c molecules exhibit more reversible ET rates as evident from the smaller peak separations. This faster ET rate is attributed largely to the decreased ET distance for the thinner 11-MUDA monolayer.
Discussion EDC, a "zero-length" cross-linking reagent, has been routinely used to form stable, covalent protein/protein complexes through the formation of amide bonds between (38)Dickerson, R. E.; Timkovich, R.; Boyer, P. D. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1975; Vol: XI-A, pp 397547.
(39) Some batch-to-batch variability in the value of hoe, for electrostatically adsorbed cytochrome c, i.e., 0.14.9 8-1, has been observed since theinitialvalueof0.1 e-1wasreported.a Thereasonforthia isnot presently known but may be due to factors associated with the thin film deposition or the self-assembly processes. (40) Smaller coverages are typically obtained for cytochrome c adsorbed on 11-MUDA compared to 16-MHDA. The reason for this is not presently known.
complementary amino and carboxyl groups.353694143 Cytochrome c has been cross-linked in this manner to cytochrome c p e r o ~ i d a s e , ~cytochrome . ~ ~ . ~ ~ c oxidase,"31u cytochrome b6,s and plastocyaninto form covalent ET complexes that are structurally similar to their electrostatic counterparta.a*a In addition to crose-linking proteins, carbodiimides have also been used to covalently attach proteins to solid supports such as activated graphite p a r t i ~ l e s , 4 ~siliconized *~ glass beads,S1vS2and graphite electrodes.63-s6 In this paper, we report the use of carboxylic acid terminated alkanethiol electrodes as a support for the covalent immobilization of cytochrome c in a functional state. We fiid, as previously observed with protein/protein ~omplexes,3~*~~ that the formation of a stable electrostatic precursor complex facilitates the subsequent covalent attachment of cytochrome c in orientations optimal for electron transfer. Minimal electroactiue coverage was obtained when the monolayer electrode was first treated with carbodiimide followed by addition of cytochrome c. In low ionic strength buffer, cytochrome c forms a stable electrostatic complex with the carboxylated surface, presumably through the specific attraction of the lysine side chains near the exposed heme edge of cytochrome c and the carboxylate groups on the surface of the electrode.B Assuming the coverage attained in the electrostatic complex represents a monolayer,29the coverage obtained after carbodiimide treatment corresponds to a yield of 30-40 % This yield is comparableto values obtained when cytochrome c is cross-linked to other proteins under similar condition^.^^^^^ The lower electroactive coverage of cytochrome c covalently immobilized to S A M electrodes may reflect the formation of an incomplete monolayer or the presence of nonelectroactive cytochromec. Denaturation or nonoptimal electron transfer orientations via covalent attachment to backside lysine groups on cytochromec are possible.67 When cytochrome c electrostatically adsorbs to the 16MHDAmand 11-MUDA monolayer electrodes, its formal potential shifts 60-80 mV negative of the solution value. This shift, which arises from the stronger binding of the oxidized form, is commonly observed when cytochrome c
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(41) Moench, S. J.; Satterlee, J. D.; Erman, J. E. Biochemistry 1987, 26, 3821-3826. (42) Erman, J. E.; Kim, K. L.; Vitello, L. B.; Moench, S. J.; Sattarlee, J. D. Biochim. BioDhvs. Acta 1987.911. 1-10. (43) Seiter, C. H.A;: Margalit, R.'; Perreault, R. A. Biochem. Biophys. Res. Commun. 1979,86,473-477. (44)Millet, F.; Darley-Usmar, V.; Capaldi, R. A. Biochemistry 1982, 21,3857-3862. (45) Geren, L. M.; Stonehuemer, J.; Davis, D. J.; Millett, F. Biochim. Biophys. Acta 1983, 724,62-68. (46) Peerey, L. M.; Kwtib, N. M. Biochemistry 1989,28,1861-1868. (47) Peerey, L. M.; Brothers, H. M., XI; Hazzard, J. T.; Tollin, G.; KostiE, N. M. Biochemistry 1991,30,9297-9304. (48) Zhou, J. S.; Brothers, H. M., 11; Peerey, L. M.; KostiC, N. M. Submitted for publication in Biochemistry. (49) Cho, Y. K.; Bailey, J. E. Biotech. Bioeng. 1979,21, 461-476. (50) Osborn, J. A.; Ianniello, R. M.; Wieck, H. J.; Decker, T. F.; Gordon, S. L.; Yacynych, A. M. Biotech. Bioeng. 1982,24, 1653-1699. (51) Weetall, H. H. Methods Enzymol. 1976,44, 134-148. (52) Janolino, V. G.; S w a i s g d , H. E. Biotech. Bioeng. 1982,24,10691080. (53) Koshy,A.;Bennetto,H.P.;Delaney,G. M.;MacLeod,A.J.;Mason, J. R.; Stirling, J. L.; Thurston, C. F. Anal. Lett. 1988, 21, 2177-2194. (54) DCosta, E. J.; Higgins, I. J.; Turner, A. P. F. Biosenaors 1986,2, 71-87. (55) Laval, J.-M.; Bourdillon, C.; Moiroux, J. J. Am. Chem. Soc. 1984, 106,4701-4706. (56) Kamin, R. A,; Wilson, G. S. Anal. Chem. 1980, 52, 1198-1205. (57) Other possible complications include dimerization or polymerization of cytochrome c and the formation of multiple layers. However, this is considered less likely in light of solution studies which show very little dimerization and polymerization of cytochrome c upon addition of EDC.SB,41-42
1250 Langmuir, Vol. 8, No. 5, 1992
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Figure 4. Background-subtractedcyclic voltammograms of cytochrome c covalently immobilizedto a 16-MHDAand to an 11-MUDA monolayer Au electrode. For the 16-MHDA electrode, the scan rates are (a) 100, (b) 70, (c) 50, (d) 40, (e) 30, and (f) 20 mV/s. For the 11-MUDA electrode the scan rates are (a) 130, (b) 100, (c) 70, (d) 50, and (e) 30 mV/s. Solution conditions were pH 7.0,4.4 mM phosphate buffer.
binds to membranes and other s u r f a ~ e s . ~Upon ~?~ covalent immobilization to 16-MHDA, we find that the redox potential undergoes a further negative shift of ca. 20 mV. This shift is probably due, in part, to the neutralization of cationic lysine side chains as previously proposed by Kosti6 and co-workersfor the cytochrome c/plastocyanin covalent complex. The ET rate constants appear to be essentially identical for cytochrome c electrostatically adsorbed and covalently immobilized to the 16-MHDA electrode. This result suggests that electroactive cytochrome c is similarly oriented in both the electrostatic and covalent protein/ electrode complexes as would be suggested by analogy to protein-protein complexing studies.Some dependence of keet on scan rate was, however, obtained with the simple model ~ s e d . 3This ~ apparent dependence may arise from a distribution of orientations, which has been reported for cross-linked covalent protein/protein complexes.41*Other explanations, however, cannot be ruled o ~ t . ~ ~ More reversible E T kinetics for both electrostatically adsorbed and covalently immobilized cytochrome c are observed for the thinner 11-MUDA monolayer electrode. (58) Vanderkooi, J.;Erecinska, M. Arch. Biochem. Biophys. 1974,162, 383-391. (59)Dutton, P. L.; Wilson, D. F.; Lee, C. P. Biochemistry 1970, 9, 5077-5082. (60) Vanderkooi, J.; Erecinska, M.; Chance, B. Arch. Biochem. Biop h p . 1973,154, 531-540. (61)An apparently similar dependence of hoe, on scan rate has previously been reported for adsorbed cytochrome c on tin oxide.33 However, the relevance to the present work is unclear. Although orientation heterogeneity may also be a factor for the cytochrome chin oxide system, recent chronoamperometry experiments have indicated that charge transfer limitations arising from semiconductor effects play a significant role in the voltammetry (Song, S.; Willit, J. L.; Bowden, E. F. Unpublished results).
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Electrostatically adsorbed cytochrome c was essentially reversible with bE, 0 for scan rates below 50 mV/s. Some kinetic limitations were introduced upon covalent attachment, however, as evidenced by the small but definite peak splitting8 in Figure 4b.
Conclusions Carboxylated self-assembled monolayer electrodes provide a chemically uniform surface to covalently immobilize cytochrome c in a stable, functional state. This work demonstrates that covalent attachment and electrostatic adsorption will provide complementary immobilization routes for further investigations of protein ET kinetics using diffusionless voltammetric strategies. The prospects for extending this work to other cationic proteins or to anionic protein immobilizationon amine-terminated SAM electrodes appear to be excellent. Immobilization of enzymes on SAM electrodes has promising implications 3for ~ the ~ design of amperometric biosensors. Future work will involve optimizing immobilization procedures to achieve maximum surface coverage and characterizing the cytochrome c/SAM/Au structures.
Acknowledgment. We thank Shihua Song for her assistancewith the SAM electrode,Nenad Kostie for useful discussions regarding cross-linking procedures and a preprint of ref 48, and William Kwochka and Tarakeshwar Anklekar for synthesizing 16-MHDA and 11-MUDA. We gratefully acknowledge support of this work by the National Science Foundation through CHE-8820832. Certain commercial products and instruments are identified to adequately specify the experimental procedure. In no case does such identification imply endorsement by the National Institute of Standards and Technology.
