Alkanethiolate

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Voltammetric Peak Broadening for Cytochrome c/ Alkanethiolate Monolayer Structures: Dispersion of Formal Potentials Rose A. Clark† and Edmond F. Bowden* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695 Received July 1, 1996. In Final Form: November 22, 1996X Anomalous peak broadening is a persistent feature observed in the cyclic voltammetry of cytochrome c (cyt c) adsorbed on COOH-alkylthiolate/Au self-assembled monolayer electrodes. The origins of this broadening have been explored through variation of the electroactive cytochrome c surface concentration (Γ). Cytochrome submonolayers were prepared using two different methods. In the first method, submonolayers were created by partial desorption of an electroactive cyt c monolayer by exposure to higher ionic strength buffers. The apparent voltammetric properties of these submonolayers were dependent on Γ, exhibiting decreasing formal potentials, increasing electron-transfer rates, and decreasing peak widths as Γ f 0. On the other hand, submonolayers prepared according to the second method, in which partial desorption of a preadsorbed blocking monolayer of electroinactive porphyrin cyt c was followed by adsorption of active cyt c to the unblocked sites, exhibited opposite trends, i.e., increasing formal potentials and decreasing electron-transfer rate as Γ f 1. These results are indicative of a heterogeneous population of cyt c adsorbates on the SAM/Au surface due to a distribution of interfacial environments and adsorption energies. Peak broadening under reversible electrochemical conditions has been specifically attributed to a thermodynamic distribution of cyt c differential adsorption energies (i.e., ferri- vs ferro-cyt c). Fitting based on a Gaussian distribution of formal potentials resulted in a standard deviation, σ(E°′), of 40 mV for the cyt c/HOOC(CH2)11S/Au system. Physical models based on intrinsic surface heterogeneity and adsorption-induced heterogeneity arising from steric exclusion are proposed to explain the dispersion of formal potentials.

Introduction The preparation and characterization of electrode-bound redox protein monolayers has important implications for bioelectrocatalytic applications such as amperometric biosensors as well as for the field of biological electrontransfer (ET). The immobilization of redox enzyme monolayers for bioelectrocatalytic purposes dates back more than 2 decades,1 with the major goal being to realize selective catalysis for biosynthesis or biosensing applications. Recent studies have described a number of new amperometric biosensor configurations based on the concept of monolayer electrochemistry.2,3 With regard to biological ET, binding of a protein to an electron donor/acceptor, whether a discrete molecular site or an electrode, gives rise to a major simplification in kinetic analysis as a result of the absence of diffusional limitations on the rate.4 With the use of electrode donor/ acceptors, one can potentially realize other attractive features including direct control over the reaction free energy and also the absence of reorganization energy contributions from the donor/acceptor. This diffusionless electrochemical approach to fundamental ET kinetic * Author to whom correspondence should be addressed. E-mail: [email protected]. Phone: (919) 515-7069. Fax: (919) 515-5079. † Present address: Department of Chemistry, The Pennsylvania State University, 152 Davey Laboratory, University Park, PA 16802. X Abstract published in Advance ACS Abstracts, January 15, 1997. (1) Varfolomeev, S. D.; Berezin, I. V. In Advances in Physical Chemistry: Current Developments in Electrochemistry and Corrosion; Kolotyrkin, Ya. M., Ed.; MIR: Moscow, 1982; pp 60-95. (2) Ryan, M. D.; Bowden, E. F.; Chambers, J. Q. Anal. Chem. 1994, 66, 360R-427R. (3) Anderson, J. L.; Bowden, E. F.; Pickup, P. G. Anal. Chem. 1996, 68, 379R-444R. (4) Bolton, J. R.; Archer, M. D. In Electron Transfer in Inorganic, Organic, and Biological Systems; 1991, Bolton, J. R., Mataga, N., McLendon, G., Ed.; Advances in Chemistry Series 228; American Chemical Society: Washington, DC, 1991; pp 7-23.

analysis was advanced in the 1980s by Weaver and coworkers5,6 and has subsequently been applied to proteins.7-9 Cytochrome c, especially the equine species, has been the focal point for a considerable portion of the protein electrochemistry field. Several useful biocompatible surfaces have been described for preparing stable cytochrome c monolayers including, most recently, alkanethiolate/gold self-assembled monolayer (SAM) substrates. The initial SAM reported for this purpose featured a COOH terminus on the alkanethiol that gave rise to an acidic SAM surface for binding the basic cytochrome c molecule.8 A range of ET kinetic behavior has been documented for the cyt c/SAM/gold system, ranging from reversible to completely irreversible, as dictated by the time frame of the experiment and the time constant of the ET process.8-10 Recently, the feasibility of extracting ET reorganization energies directly from voltammograms has been demonstrated for cytochrome11 and ferrocene12,13 monolayers. A complicating factor in such analyses, however, is the nonideal voltammetric behavior typically exhibited by electroactive monolayers.14-17 (5) Hupp, J. T.; Weaver, M. J. J. Electroanal. Chem. 1983, 145, 4351. (6) Li, T. T.; Weaver, M. J. J. Am. Chem. Soc. 1984, 106, 6107-6108. (7) Willit, J. L.; Bowden, E. F. J. Electroanal. Chem. 1987, 221, 265274. (8) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 18471849. (9) Song, S.; Clark, R. A.; Bowden, E. F.; Tarlov, M. J. J. Phys. Chem. 1993, 97, 6564-6572. (10) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Electroanal. Chem. 1995, 394, 149-154. (11) Nahir, T. M.; Clark, R. A.; Bowden, E. F. Anal. Chem. 1994, 66, 2595-2598. (12) Weber, K.; Creager, S. E. Anal. Chem. 1994, 66, 3164-3172. (13) Tender, L.; Carter, M. T.; Murray, R. W. Anal. Chem. 1994, 66, 3173-3181. (14) Angerstein-Kozlowska, H.; Klinger, J.; Conway, B. E. J. Electroanal. Chem. 1977, 75, 45-60. (15) Laviron, E. J. Electroanal. Chem. 1979, 100, 263-270. (16) Brown, A. P.; Anson, F. C. Anal. Chem. 1977, 49, 1589-1595.

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Langmuir, Vol. 13, No. 3, 1997

Clark and Bowden

In this report, we describe the results of experiments aimed at gaining further insight into voltammetric peak broadening for cytochrome c monolayers on SAM/gold surfaces. Clearly, a number of factors must be considered when interpreting peak-broadening phenomena. The focus of this article is directed primarily at thermodynamic peak broadening observed under conditions of reversible voltammetry. From our results, we propose that a distribution of differential adsorption energies, i.e., ferri- vs ferrocytochrome c, gives rise to a dispersion in the formal potential. Physical models based on surface heterogeneity and steric constraints on adsorption are discussed in this context. Experimental Section Reagents and Materials. Water for all experiments was purified on a Milli-Q/Organex-Q system (Millipore). The following alkanethiols were synthesized and purified according to established procedures:18 HS(CH2)15COOH (16-mercaptohexadecanoic acid, 16-MHDA), HS(CH2)10COOH (11-mercaptoundecanoic acid, 11-MUDA), HS(CH2)5COOH (6-mercaptohexanoic acid, 6-MHA). Structures were verified using NMR and IR. All other chemicals were reagent grade. Cytochrome c (Sigma Type VI, horse heart) was purified on a cation exchange column (Whatman, CM-52, (carboxymethyl)cellulose) and used within 4 weeks. The purification and storage temperature was 4 °C. Porphyrin cytochrome c was prepared according to Vanderkooi and Erecinska’s method,19 a modification of the methods of Flatmark and Robinson20 and Fisher et al.21 Safety Caution: HF used in the preparation of porphyrin cyt c is extremely corrosive! The iron-extraction reaction was performed in a sealed Teflon apparatus generously made available to us in the Vanderkooi laboratory. After metal extraction and purification, porphyrin cytochrome c (pcyt c) was stored in pelletized form at 77 K. Thawed samples of pcyt c were exchanged into 4.4 mM phosphate buffer, pH 7.0, and used within 2-3 weeks. Equipment. Cyclic voltammetry (CV) was performed using an EG&G PARC Model 273 potentiostat controlled by PARC Model 270 software in the ramp mode. A staircase wave form is generated with a 0.4-mV step height for the potential window employed. Electrode Preparation. Gold film electrodes (1000-Å Au/ 50-Å Ti on glass) were purchased from Evaporated Metal Films (Ithaca, NY). The modified electrodes were prepared using a two-stage self-assembly procedure. Following pretreatment (i.e., heating in concentrated nitric acid until boiling, rinsing in roomtemperature water, and then rinsing with ethanol), electrodes were immersed in ethanolic alkanethiol solution (ca. 1 mM) for 2 days of self-assembly (Stage 1). The electrode was then mounted to the electrochemical cell and rinsed with buffer, and the initial background voltammograms were obtained in the selected buffer. The cell was then rinsed and refilled with the alkanethiol solution, and an additional day’s immersion was allowed for further selfassembly (stage 2). As a result of the second immersion in alkanethiol solution, background currents typically decreased an additional 10-20%. Cyt c was adsorbed after stage 2 by 30min exposure to a 30 µM solution containing 4.4 mM phosphate (pH 7.0, 10 mM ionic strength) at 4 °C. All electrochemical experiments were conducted at room temperature in deoxygenated phosphate buffers. The geometric electrode area was 0.32 cm2. The uncompensated solution resistance (R) of the electrochemical cell fell within the range 0.8-2 kΩ for pH 7, 4.4 mM phosphate buffer (10 mM ionic strength), depending on the positioning of the reference electrode. Uncompensated iR (where (17) Smith, D. F.; Willman, K.; Kuo, K.; Murray, R. W. J. Electroanal. Chem. 1979, 95, 217-227. (18) 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. (19) Vanderkooi, J. M.; Erecinska, M. Eur. J. Biochem. 1975, 60, 199-207. (20) Flatmark, T.; Robinson, A. B. Stucture and Function of Cytochromes Okunuki, K., Kamen, M. D., Sekuzu, J., Eds.; University Park: Baltimore, MD, 1967; pp 318. (21) Fisher, W. R.; Taniuchi, H.; Anfinson, C. B. J. Biol. Chem. 1973, 248, 3188-3195.

Figure 1. Cyclic voltammograms for cyt c/SAM/Au (s) and background SAM/Au (- - -): (a) cyt c/6-MHA/Au; (b) cyt c/11MUDA/Au; (c) cyt c/16-MHDA/Au. Scan rates: 100 (a, b) and 5 mV/s (c). Solution conditions: pH 7.0, 4.4 mM phosphate buffer, µ ) 10 mM. i is the current) for CV experiments was