J . Phys. Chem. 1993,97, 6564-6572
6564
Characterization of Cytochrome c/Alkanethiolate Structures Prepared by Self-Assembly on Gold Shihua Song, Rose A. Clark, 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: March 23, 1993
Composite monolayer structures comprised of cytochrome c strongly adsorbed to alkanethiolate self-assembled (SA) monolayers on sputter-deposited gold film electrodes, Le., cyt c/HOOC(CHz),S/Au (n = 5,10,15), were examined using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and X-ray photoelectron spectroscopy (XPS). Monolayer coverage of cytochrome c in a stable, functional, electroactive state was obtained in neutral phosphate buffer of low ionic strength for the thickest film (n = 15). Somewhat lower electroactive coverages were generally observed with the thinner SA monolayers (n = 5, 10). XPS revealed similar total coverage of adsorbed cytochrome c on all of the monolayers, however, suggesting the presence of some cytochrome c in a nonelectroactive conformation on the two thinner monolayers. The voltammetric responses were well-behaved and reasonably ideal although evidence for some dispersion of the formal potential value was apparent that is attributed in part to defect sites in the SA monolayer. The surface formal potential of cytochrome c, +215 mV vs NHE, is nearly identical to values previously reported for cytochrome c bound to physiological membranes. Electron transfer within the cyt C / H O O C ( C H ~ ) ~ ~ S /structure AU appears to be strongly nonadiabatic, with a rate constant (keto)of 0.4 s-l, a postulated electron-transfer distance of ca. 28 A, and a tunneling decay factor (8) of 1.0 A-l. The reorganization energy for electron transfer was estimated to be 0.35 (f0.15)eV. The potential usefulness of these composite monolayers for investigating biological electron transfer is highlighted.
Introduction The deliberate chemical modification of gold surfaces using organosulfur compounds has developedinto an extremely popular and important method for the preparation of molecular monolayers on electrodes and other surfaces. The first report of this approach apparently arose from the protein electrochemistry field' and is due to Taniguchi and co-workers.2 They reported that the modification of gold electrodes by the irreversible chemisorption of bis(4-pyridyl) disulfide resulted in well-behaved direct cytochrome c (cyt c) voltammetry. They subsequently concluded from SERS measurements on silver and gold electrodes that monolayers form through chemisorption via sulfur attachment, leaving the pyridine moiety facing the ~olution.~ Their finding improved upon the first generation promoters of cytochrome c electrochemistry, e.g., 4,4'-bipyridine, discovered by Eddowes and Hill,4which desorbed in the absence of promoter in solution. A large number of organosulfur compounds have since been evaluated in the design of electrode surfaces for catalyzing the interfacial voltammetryof electron-transfer protein^.^,^ Themode of adsorption and the mechanism of action of these various compounds have been studied by SERS and electroreflectance technique^.^'^^ Nuzzo and Allara initiated another vein of work on organosulfur modification of gold with their seminal publication on disulfide adsorbates? After characterizing a number of different adsorbate layers by ellipsometry, contact angle measurements, and vibrational spectroscopy,they concluded that close-packedmonolayers were formed through chemisorptive gold-sulfur bonding. Some of the monolayer systems they investigated, e.g., bis(hexadecy1) disulfide/gold, provided the first examples of alkanethiolate selfassembled monolayers (SA monolayer, or SAM), a field of research that has grown exponentially in recent years?-lZ To whom correspondence should be addressed.
0022-3654/93/2097-6564$04.00/0
Weaver and co-workers also provided early examples of organosulfur modification of gold surfaces in their work on diffusionless v01tammetry.I~They created pentaamminecobalt(111) electroactivemonolayers through ligand bonding to a variety of chemisorbed species. As in the previously cited studies, they took advantage of the bifunctional nature of organosulfur compounds to generate stable monolayers that exhibited desired properties. Our interest is in SAM/Au systems that will be useful for the investigation of biological electron-transfer (ET) processes. As noted above, the use of small organosulfurcompounds for electrode modificationhas been widespread in the protein electrochemistry field for some time.2*3,5-7 Recently, alkanethiol SA monolayers have been successfully applied in ele~trochemical'~ and nonelectrochemi~all~ studies of proteins. We provided initial reports of the electrochemistry of strongly adsorbed14" and covalently attached14bcytochrome c monolayers on carboxylic acid terminated SAM/Au electrodesusing sputter-depositedgold substrates. These systems display diffusionless ET, a concept that has been crucial to the rapid advancement of the biological ET field over the past decade.I6 Although the vast majority of research in this field has addressed reactions of donor-acceptor species,16a small but growing number of voltammetric studies involving diffusionless interfacial ET reactions of functional protein (sub)monolayers have been r e p ~ r t e d . ~ ~ J ~The J ' - ~advantages ~ of diffusionless voltammetry for elucidating fundamental aspects of ET kinetics have been described24and are applicable to electroactive protein monolayer^.^^^^^^^^ Two key advantages are the ready control of reaction driving force and the absence of reorganization energy for the electrode. Although our prior ~ ~ r k ~has~ shown J ~ ~ , ~ v ~ this approach to be feasible, many challenging problems remain with regard to interfacial characterization, surface heterogeneity, and relatively low signal-to-background levels. In the present paper, we describe a detailed characterization of the electrochemistry and photoelectron spectroscopy of cy0 1993 American Chemical Society
Cytochrome clAlkanethiolate Structures
A -COO- or -COOH
Figure 1. Cartoon depictinga hypothetical structure for an ideally ordered and oriented cytochromec/ 16-MHDA/Au composite. For Au( 1 1 1) the alkane chains are tilted 30° to the normal? Cytochromec structure was taken from ref 26 and is oriented with its molecular dipole axis2' normal to electrodesurface. Spacing between cytochromec molecules is arbitrary.
tochrome c adsorbed on 16-mercaptohexadecanoic acid SA monolayers on sputter-deposited gold (cyt c/ 16-MHDA/Au). Figure 1, a cartoon of a hypothetical idealized structure for a Au( 111)substrate,is intended mainly to provide an idea of relative dimensions in this system. Good agreement was found between transient and steady-state electrochemical measurements with regard to the physical properties of the electroactive cytochrome c monolayer. It was also found that the interfacial electrontransfer rate can be satisfactorily understood at this early stage in terms of simple nonadiabatic ET theory. Furthermore, we report the first determination of an electron-transfer reorganization energy for a protein confined to an electrode surface. Finally, some initial results for adsorbed cytochrome c monolayers on thinner carboxylic acid terminated SA monolayers are also presented. Experimental Section
Reagents. Cytochrome c (Sigma Type VI, horse heart)55was purified2*on a cation exchange column (Whatman, CM-52, carboxymethylcellulose)and used within 2-3 weeks. Purification and storage temperature was 4 "C. The following alkanethiols were synthesized and purified according to established proced u r e ~HS(CH2) :~~ I~COOH ( 16-mercaptohexadecanoicacid, 16MHDA), HS(CH2)loCOOH (1 1-mercaptoundecanoicacid, 11MUDA), and HS(CH2)sCOOH (dmercaptohexanoic acid, 6-MHA). Structures were verified using NMR and IR. Water
The Journal of Physical Chemistry, Vol. 97, No. 24, 1993 6565 for all experiments was purified on a Milli-Q/Organex-Q system (Millipore). Other chemicals were reagent grade. Instrumentation. Cyclic voltammetry (CV) was performed using EG&G PAR 362 and 273 potentiostats. The PAR 273 was operated in RAMP mode which generated a staircaseapplied potential waveform with 0.4-mV step height for our experiments. The PAR 362 is an analog instrument that uses a linear ramp for sweep experiments. Electrochemicalimpedance spectroscopy (EIS) was performed using an EG&G PAR impedance system (270/273/5208). Impedancedatawereanalyzedusing a complex nonlinear least-squares (CNLS) computer program written by J. R. Macdonald, LEVM version 3.30-31 Procedures. Gold film electrodes (200 nm) were prepared by sputter deposition on single-crystal silicon wafers as previously described.32 Most electrodes were then immediately immersed in absolute ethanol containing 1 mM alkanethiol for 2-5 days. Some gold electrodes were stored unmodified in clean vials for modification at a later time. After rinsing and drying,cytochrome c was adsorbed at 4 "C by exposure to a 30pM solution containing 4.4 mM potassium phosphate (pH 7,lO mM ionic strength). The low adsorption temperature gave significantly improved results relative to room temperature adsorption. Most electrochemical experiments were performed at room temperature, 22 f 2 OC; a Fisher 5000 circulator and a nonisothermalcell were used in certain variable temperatureexperiments (vide infra). Apparent electron-transferrate constants, keto,were determined from cyclic voltammetry peak separations using Laviron's simplest model.33 Doublelayer capacitances( c d l ) for SAM/Au electrodes were determined from CVs acquired using the PAR 362 analog potentiostat at sweep rates of 50-200 mV/s for potentials in the range 0 to +0.2 V. For cyt c/SAM/Au structures, the electroactive surface concentration (I?) of cyt c was determined either by manual CV peak integration (PAR 362) or by numerical integration of background subtracted CVs (PAR 273, RAMP mode). Agreement of f l O % was typically obtained with these two methods. Geometric electrode area was 0.32 cm2. The uncompensated resistance of the electrochemical cell was approximately 0.8-2 kQ for pH 7,4.4 mM phosphate buffer (ionic strength 10 mM), depending on positioning of the reference electrode. Uncompensated iR for CV experiments was
