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Influence of Gold Substrate Topography on the Voltammetry of Cytochrome c Adsorbed on Carboxylic Acid Terminated Self-Assembled Monolayers Michael C. Leopold† and Edmond F. Bowden* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204 Received September 20, 2001 Interfacial investigations of a protein monolayer electrochemical system, equine cytochrome c (cyt c) adsorbed to carboxylic acid terminated self-assembled monolayer (COOH SAM) modified gold electrodes, were performed. Electrochemical, spectroscopic, and scanning probe microscopy techniques were utilized to explore the influence of gold topography in the cyt c/COOH SAM/gold system. COOH SAMs were prepared from 14-mercaptotetradecanoic acid and 11-mercaptoundecanoic acid on a variety of substrates including evaporated, bulk, single crystal, and epitaxially grown gold on mica. These substrates encompassed a wide range of surface roughness. As the topography of the gold became smoother, SAMs exhibited an increased ability to block a solution probe molecule, indicative of a lower level of defectiveness. At the same time, after exposure to equine cyt c deposition solutions, the extent of adsorption and the magnitude of the electrochemical response of the adsorbed cyt c decreased significantly with increasingly smooth substrates. The results show cyt c adsorption and electrochemistry to be intimately related to the density of defects in the SAM, which in turn are largely dictated by the topography of the gold substrate. This hypothesis is supported by experiments in which the density of defects in the SAMs was controlled on each type of gold substrate using intentional roughening/smoothing procedures as well as through the use of mixed SAMs. Results are interpreted in terms of the topographically dependent acid/base properties of the COOH SAMs, which can limit the electrostatically driven adsorption of cyt c and the effectiveness of the protein’s electronic coupling at the differently textured SAM surfaces.
Introduction Protein electron transfer (ET) reactions and adsorption processes play a critical role in vital physiological functions such as photosynthesis and cellular respiration. Furthermore, these phenomena are highly relevant to the development of biocompatible materials1 and amperometric biosensors with protein monolayer configurations2 as well as to potential in vivo applications.3 A useful electrochemical model for investigating protein ET is the monolayer arrangement of an electroactive protein. Protein monolayer electrochemistry of cytochrome c (cyt c) at gold electrodes modified with carboxylic acid terminated SAMs (COOH SAMs) (see Figure 1A) has been studied by our group and others.4-6 A COOH SAM, with * To whom correspondence should be addressed. E-mail: Edmond•
[email protected]. Phone: (919) 515-7069. Fax: (919) 515-5079. † Present address: Department of Chemistry, University of North Carolina, CB# 3290 - Kenan Laboratories, Chapel Hill, NC 27599. (1) Rouhi, A. M. Chem. Eng. News, 1999, Jan 18, 51-59. (2) (a) Ryan, M. D.; Bowden, E. F.; Chambers, J. Q. Anal. Chem. 1994, 66, 360R-427R. (b) Anderson, J. L.; Bowden, E. F.; Pickup, P. G. Anal. Chem. 1996, 68, 379R-444R. (3) (a) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55-78. (b) Scouten, W. H.; Luong, J. H. T.; Brown, R. S. Trends Biotechnol. 1995, 13, 178-185. (4) (a) Bowden, E. F. Interface 1997, 6, 40-44. (b) El Kasmi, A.; Wallace, J. M.; Bowden, E. F.; Linderman, R. J. J. Am. Chem. Soc. 1998, 120, 225-226. (c) Song, S.; Clark, R. A.; Tarlov, M. J.; Bowden, E. F. J. Phys. Chem. 1993, 97, 6564-6572. (d) Collinson, M.; Tarlov, M. J.; Bowden, E. F. Langmuir 1992, 8, 1247-1250. (e) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 1847-1849. (5) (a) Zhang, D.; Wilson, G. S.; Niki, K. Anal. Chem. 1994, 66, 38733881. (b) Sagara, T.; Niwa, K.; Sone, A.; Hinnen, C.; Niki, K. Langmuir 1990, 6, 254-262. (c) Hinnen, C.; Niki, K. J. Electroanal. Chem. 1989, 264, 157-165. (d) Niwa, K.; Furukawa, M.; Niki, K. J. Electroanal. Chem. 1988, 254, 275-285. (e) Niki, K.; Kawasaki, Y.; Kimuma, Y.; Higuchi, Y.; Yasuoki, N. Langmuir 1987, 3, 982-986. (f) Hinnen, C.; Parsons, R.; Niki, K. J. Electroanal. Chem. 1983, 147, 329-337.
its negatively charged surface, has proven to be an excellent platform for the immobilization and electrochemical characterization of basic proteins such as cyt c. A number of electrochemical and spectroelectrochemical studies have been performed from which key thermodynamic and kinetic information about the ET reaction of this redox protein has been obtained.4-6 More recently, attention has become directed toward better defining critical interfacial phenomena such as the electronic coupling between cyt c and the SAM.4b Although the voltammetric response of adsorbed cyt c at COOH SAMs on gold can be highly stable and reproducible, deviations from the expected response for an ideal adsorbed redox system7 have been observed (Figure 1B).8 Most notably, the voltammetric peaks often display anomalous broadening with full width at halfmaximum (fwhm) values exceeding the theoretical value of 90/n mV at 25 °C for a simple reversible redox couple. Previous research in our group established that broadening was a consequence of dispersions of electrochemical properties such as formal potential (E°′) and ET rate constant (kET°). The dispersion is believed to arise from the existence of heterogeneous adsorption sites on the surface.6a,8 When considering interfacial aspects of the cyt c/SAM/ Au system, the surface of the gold substrate itself is a primary interest. Gold surface features are often characteristic of the type of substrate being employed and can (6) (a) Nahir, T. M.; Bowden, E. F. J. Electroanal. Chem. 1996, 410, 9-13. (b) Nahir, T. M.; Clark, R. A.; Bowden, E. F. Anal. Chem. 1994, 66, 2595-2598. (c) Nahir, T. M.; Bowden, E. F. Electrochim. Acta 1994, 39, 2347-2352. (7) Bard, A. J.; Faulkner, L. R. Electrochemical Methods - Fundamentals and Applications, 1st ed.; John Wiley and Sons: New York, 1980. (8) Clark, R. A.; Bowden, E. F. Langmuir 1996, 13, 559-565.
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morphology in SAM formation and structure seems apparent. In this report, we describe results of experiments aimed at achieving a deeper understanding of how gold substrate topography influences both the formation and structure of COOH SAMs and, ultimately, the adsorption and electrochemical response of cyt c. To address this issue, experiments were performed on equine cyt c/SAM systems prepared on a variety of gold substrates that exhibited a range of topographical characteristics. We have found that substrate topography can exert profound effects on the extent of adsorption and the electrochemistry of cyt c on COOH SAMs. Thus, it is clear that substrate topography and pretreatment must be carefully considered as they can substantially affect both the structure and performance of the SAM. Experimental Details
Figure 1. (a) Highly simplified conjectural model of the cyt c/SAM/Au model system. Although the heme group, shown as a black line, is depicted perpendicular to the surface, its actual orientation is uncertain. (b) Cyclic voltammogram illustrating typical electrochemical behavior of equine cyt c adsorbed onto an evaporated gold film modified with a 14-mercaptotetradecanoic acid SAM (HS(CH2)13COOH). Solution conditions are 4.4 mM potassium phosphate buffer (µ ) 10 mM, pH ) 7), and the sweep rate is 100 mV/s.
help define the level of defectiveness, uniformity, and stability of SAM adlayers.9-12 Indeed, gold topography such as rough terrain, step edges, crystal facet grain boundaries, and pitting can affect the defect density found in a SAM.11,12 Descriptions of the assembly and structure of SAMs on different types of gold or on gold subjected to different types of pretreatment procedures have been published.9-12 Creager and co-workers showed that SAMs with excellent blocking characteristics can be formed on polycrystalline bulk gold by examining the electrochemistry of a defectsensitive solution probe molecule.9 Likewise, researchers in Golan’s laboratory,10e as well as those with Crooks,10d created apparent pinhole-free SAMs with superior blocking behavior on sputtered and evaporated films on glass and mica. Porter’s group described thermally annealed gold-on-mica films that give rise to ordered, low-defect SAMs and has suggested that surface roughness may be less important for dictating SAM permeability than other topographical features such as crystallinity.10f No obvious consensus that delineates the role of gold topography and (9) Creager, S. E.; Hockett, L. A.; Rowe, G. K. Langmuir 1992, 8, 854-861. (10) (a) Hoogvliet, J. C.; Dijksma, M.; Kamp, B.; van Bennekom, W. P. Anal. Chem. 2000, 72 (9), 2016-2021. (b) Subramanian, R.; Lakshminarayanan, V. Curr. Sci. 1999, 76 (5), 665-669. (c) Guo, L. H.; Facci, J. S.; McLendon, G.; Mosher, R. Langmuir 1994, 10, 4588-4593. (d) Chailapakul, O.; Crooks, R. M. Langmuir 1993, 9, 884. (e) Golan, Y.; Margulis, L.; Rubinstein, I. Surf. Sci. 1992, 264, 312. (f) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335. (11) Finklea, H. O. Electroanalytical Chemistry - A Series of Advances; Marcel Dekker: New York, 1996; Vol. 19, pp 108-335. (12) Leopold, M. C.; Black, J. A.; Bowden, E. F. Langmuir 2002, 18, 978.
Materials. Evaporated gold film substrates from Evaporated Metal Films Inc. (Ithaca, NY) were comprised of 1000-5000 Å of Au evaporated on glass slides with a 50 Å thick Ti adhesion layer. They were used following an electrochemical pretreatment that employed potential cycling in a solution of 0.1 M H2SO4 and 0.01 M KCl.6a Bulk polycrystalline gold foil from Alfa Aesar/Johnson Matthey, Premion 99.9985% (Ward Hill, MA), was chemically etched for 10 min in dilute aqua regia (HCl/HNO3/H2O ) 3:1:6) and subsequently polished via a Minimet polisher/grinder by Buehler (Lake Bluff, IL) using successively finer aqueous alumina suspensions (Buehler) of 5, 1, 0.3, and 0.05 µm particle size. Bulk gold samples were then rinsed thoroughly with Milli-Q (M/Q) purified water (Millipore) and sonicated in Alconox (Fisher Scientific) and M/Q water before further pretreatment. At this point, the samples were either electrochemically pretreated (cleaned) as previously described6a to obtain rougher topography or hydrogen flame annealed for a significantly lower degree of roughness. Single-crystal gold (111) samples were purchased from Cornell University (Ithaca, NY) and were used after annealing in a hydrogen flame according to a procedure recommended by Molecular Imaging Corp. (see below). Gold epitaxially grown on mica was purchased from Molecular Imaging Corp. (Phoenix, AZ). These samples lacked an adhesion layer of Ti or Cr and had an approximate gold film thickness of 1500 Å. The substrates were pretreated by hydrogen flame annealing (HFA) procedures. Equine (horse heart) cyt c (Sigma, type VI) was purified via a cation exchange column (Whatman, CM-52) using procedures previously described.4 Self-assembled monolayers were prepared using 11-mercaptoundecanoic acid and 14-mercaptotetradecanoic acids obtained commercially (Aldrich Chemical Co., St. Louis, MO) or via inhouse synthesis, respectively.13 The 10-mercaptodecanol and 7-mercaptodecanol compounds used in preparing mixed SAMs were synthesized or purchased commercially. Equipment. Cyclic voltammetry was performed using an EG&G PAR potentiostat, model 273 or 263, operating in ramp mode, generating a staircase waveform with a 0.25 mV step height and, in most cases, a 1 mV scan increment. Unless otherwise stated, all voltammetry data presented in this paper were acquired at a sweep rate of 100 mV/s versus a Ag/AgCl (1 M KCl) reference electrode and a platinum wire auxiliary electrode. The geometric area of the gold working electrodes was 0.32 cm2. Procedures. Hydrogen flame annealing was performed according to a procedure recommended by Molecular Imaging Inc. Ignited hydrogen gas flowing through a finely drawn quartz tube was passed repeatedly over a gold substrate for 30-60 s in a darkened room until the substrate glowed a healthy orange color. Changes in topography due to annealing were verified using (13) Nahir, T. M.; Tiani, D.; Miller, D.; Linderman, R. J.; Bowden, E. F. Manuscript in preparation.
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Table 1. Gold Substrate Topographical Characteristicsa (Listed in Order of Decreasing Overall Roughness)b Rfb,c
topographical descriptiona
surface crystallinityd
evaporated gold films
2.1
“sand dune” topography (ref 16)
bulk gold foil
1.5
single-crystal gold (111)
1.1
macroscopically rough; microscopically smooth (ref 9) “plateau” topography
polycrystalline Au(111) > Au(110) polycrystalline Au(111) = Au(110) predominantly Au(111)
gold epitaxially grown on mica
1.0
“plateau” topography
predominantly Au(111)
substrate
potential SAM defect surface featuresa rough terrain grain boundaries rough terrain grain boundaries high density of step edges separating small plateaus low density of step edges separating large plateaus
a Determined experimentally using scanning probe microscopy and electrochemical techniques and/or from literature accounts (refs 11 and 16). b Determined experimentally through electrochemical roughness factor (Rf) measurements based on procedures by Oesch, Janata, and Arvia (ref 14). Detailed procedures are provided in ref 12. c These roughness factors correspond to electrochemical pretreatment of the evaporated gold films and hydrogen flame annealing for the other three types of gold (see experimental section). d Determined experimentally through Pb underpotential deposition (UPD) stripping analysis (ref 15) and Au-oxide formation analysis (ref 14b).
scanning probe microscopy, electrochemical roughness factor determinations,14 and underpotential deposition stripping analysis.15 For SAM deposition and protein adsorption, following electrochemical and/or hydrogen flame annealing pretreatment the gold substrates were rinsed copiously through two cycles of M/Q H2O followed by 95% ethanol. Immediately after the second ethanol rinse, the sample was immersed in an ethanolic thiol solution (5 mM) and allowed to sit for at least a 72-h period. After modification, the electrodes were rinsed with excess ethanol and M/Q H2O. Mixed SAM modification was accomplished by immersing pretreated gold substrates in ethanolic solutions containing equal concentrations of hydroxy- and carboxy-terminated alkanethiols for 24 h. Specific combinations of thiols included 11-mercaptoundecanoic acid with 7-mercaptoheptanol and 14-mercaptotetradecanoic acid with 10-mercaptodecanol. These combinations were chosen because of their relatively small differences in chain length, which should encourage more uniform and complete mixing. In all cases, the total concentration of thiol in the deposition solution was 5 mM. SAM/gold assemblies were then immersed in potassium phosphate buffer (KPB; 4.4 mM, µ ) 10 mM, pH ) 7) for initial background scans before exposure to cyt c solution (10-25 µM in KPB) for 1 h at room temperature. The cell was then rinsed five times with KPB, filled with KPB, and sparged with humidified argon. Scanning probe microscopy experiments were conducted with a Digital Instruments Nanoscope III Multimode scanning probe microscope. Scanning tunneling microscopy images were collected in constant current mode (low current settings), using mechanically cut Pt/Ir tips (Digital Instruments). Silicon nitride cantilevers (Digital Instruments) were used for atomic force microscopy imaging. X-ray photoelectron spectroscopy testing was performed by John Phillips, the residential microscopist at the Engineering Analytical Instrumentation Facility at North Carolina State University, Centennial Campus.
Results and Discussion The gold substrates used in this study were selected to provide a range of roughness and topographical features, including gold with extremely flat, plateaulike regions. These topographical features were verified experimentally and are summarized for each type of substrate in Table 1. A commonly held view is that atomically smooth gold surfaces are the most effective templates for creating very ordered, low-defect SAMs. Their effectiveness in this role is credited to their lack of grain boundaries, step edges, and roughness, all of which are known to cause defects in SAM adlayers.11 In this respect, the experiments discussed (14) (a) Oesch, U.; Janata, J. Electrochim. Acta 1983, 28 (9), 12371246. (b) Vela, M. E.; Salvarezza, R. C.; Arvia, R. J. Electrochim. Acta 1990, 35 (1), 117-125. (15) Schultze, J. W.; Dickertmann, D. Surf. Sci. 1976, 54, 489.
Table 2. HMFc Redox Probe Voltammetry at Homogeneous and Mixed SAMsa,b system
Ep,a (mV)
E°′ (mV)
evaporated gold (ref) C13COOH/evaporated gold C13COOH/bulk gold C13COOH/single-crystal gold (111) C13COOH/gold/mica mixed SAMd/evaporated gold
230 348 388 384 419 322
193 c c c c 161
a C COOH denotes the HS(CH ) COOH SAM. b Relative stan13 2 13 dard deviation in Ep,a and E°′ was B > C > D; C14OOH denotes HOOC(CH2)13SH; HFA ≡ hydrogen flame annealing; E/C ≡ electrochemically pretreated. Solution conditions: 4.4 mM potassium phosphate buffer (µ ) 10 mM, pH ) 7); scan rate, 100 mV/s.
of an improvement in cyt c voltammetry as a result of decreasing substrate roughness, the faradaic response due to cyt c disappeared altogether (Figure 3c,d). Identical behavior, although less pronounced, was observed when using C10COOH SAMs on the same group of gold substrates (results not shown). Two possibilities were considered to account for the loss of electroactivity on the smoother substrates: (1) cyt c adsorbs but not in an electroactive state, possibly from improper orientation or denaturation, or (2) cyt c does not adsorb to these surfaces. To evaluate these possibilities, X-ray photoelectron spectroscopy (XPS) was employed. XPS has been suc-
cessfully used in prior work to determine relative amounts of cyt c on carboxylic acid terminated SAM modified gold substrates.4c The N(1s) signal arising from the amino acids of the protein shell is assumed to be proportional to the amount of adsorbed cyt c, regardless of its level of electroactivity or degree of denaturation. For the XPS experiments, a series of SAMs (HS(CH2)13COOH) were formed on two types of bulk gold surfaces, one “smooth” and the other “rough.” Hydrogen flame annealing was used to create the smoother topography, whereas an electrochemical cycling pretreatment6a was used to produce the roughened surface (see Experimental
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Figure 4. XPS and corresponding voltammetry results for (a) C13COOH/evaporated Au; (b) cyt c/C13COOH/bulk Au (HFA); (c) cyt c/C13COOH/bulk Au (electrochemically cleaned); (d) cyt c/C13COOH & C10OH mixed SAM/bulk Au (HFA). The unitless numbers in the figure are proportional to the N(1s) peak area (at 405 eV).
Details). Figure 4a-c illustrates the XPS results along with the corresponding cyclic voltammetry. For these homogeneous SAMs, a 3-4-fold larger N(1s) signal was observed for the rough substrate, indicative of a proportionately higher concentration of adsorbed cyt c. Thus, the XPS results indicate that the absence of cyt c voltammetry in Figure 3c,d is largely a result of cyt c not adsorbing to those surfaces, although it is clear that some electroinactive cyt c is present. The type of SAM surface that results from self-assembly of longer chain COOH thiols on smooth gold appears to be resistant to cyt c adsorption. To further define the role of surface roughness and topography, experiments were designed to assess whether intentional roughening of smooth substrates would give
rise to an improved cyt c response on the C13COOH SAM overlayers. In the example described below, cyt c was first tested with modified bulk gold substrates that had been smoothed using hydrogen flame annealing. The same substrates were subsequently roughened electrochemically followed by modification and retesting of cyt c. Roughness factors were determined electrochemically12,14 to document the changes in topography (roughness). Figure 5 is a typical result, illustrating that intentional roughening of a smoothed bulk gold surface markedly improves both the adsorption and voltammetry of cyt c. Similar experiments performed using the other types of gold substrates produced similar results. The effect of intentional roughening of smooth bulk gold on the electrochemistry of cyt c adsorbed to C13COOH SAMs is
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Table 3. Effects of Roughening and Mixed SAMs on Cyt c Voltammetric Propertiesa substrate
SAM
Rf
E°′ (mV)
Γb (pmol/cm2)
fwhm (mV)
∆Ep (mV)
ket° (CV/EISc) (s-1)
Cdld (CV/EISc) (µF/cm2)
bulk gold (annealed) C13COOH 1.5 (0.1) X X X X X 3.5/2.6 (0.3/0.3) bulk gold (EC cycled) C13COOH 2.1 (0.02) -14 (1) 14.2/15.4 (0.4/0.2) 127 (5) 104 (3) 2.0/0.4 (0.2/0.2) 1.5/1.2 (0.1/0.1) bulk gold (annealed) mixed SAM 1.5 (0.1) -20 (1) 16.0/15.5 (0.2/0.4) 122 (4) 40 (3) 13.0/16.2 (0.1/ 0.2) 2.5/1.9 (0.2/0.2) C13COOH/C10OH a EC ≡ electrochemical; R ≡ roughness factor (unitless); E°′ ≡ formal potential; Γ ≡ surface coverage; fwhm ≡ full width at halff maximum; ∆Ep ≡ cyclic voltammetry peak splitting; ket° ≡ standard electron-transfer rate constant; CV ≡ cyclic voltammetry; EIS ≡ electrochemical impedance spectroscopy; Cdl ≡ double layer capacitance. X ) Poor voltammetry rendered these properties impractical to measure. Standard deviations are shown in parentheses. b Values determined for both cathodic (C) and anodic (A) peaks and listed above as C/A. c Properties measured using electrochemical impedance spectroscopy (EIS). d Capacitance trends are related to the structure of COOH SAMs on various gold topographies and are the subject of a separate report (ref 12).
Figure 5. Cyclic voltammetry of equine cyt c/C13COOH SAM/ bulk Au assemblies with the gold surface smoothed by HFA (thin trace) or roughened by electrochemical cycling (thick trace). The electrochemically determined roughness factors (Rf) for these two substrates were 1.5 and 2.1, respectively. Similar results were obtained on other types of gold in Table 1 (results not shown). Solution conditions: 4.4 mM KPB (µ ) 10 mM, pH ) 7); scan rate, 100 mV/s.
documented in the first two rows of Table 3. Similar behavior was observed in experiments utilizing C10COOH SAMs though the difference in voltammetry quality between rough and smooth substrates was not as great (results not shown) ostensibly because of the greater intrinsic disorder already present in shorter carboxylic acid terminated SAMs.11 A final set of results were obtained from experiments designed using two-component mixed SAMs. Certain types of mixed SAMs are known to exhibit randomly segregated components that impact the molecular defectiveness of the SAM surface.17 We previously demonstrated that such surfaces can be highly conducive to cyt c adsorption and electron transfer.4b The goal of this experiment, therefore, was to evaluate the ability of a mixed acid SAM on a smooth gold surface to mimic a single-component acid SAM on a rougher surface with respect to adsorbed cyt c voltammetry. A shorter hydroxyl-terminated alkanethiol was used to create an acidic two-component mixed SAM [HS(CH2)13COOH & HS(CH2)10OH]. This choice of components retains the electrostatic character of the pure COOH films while maintaining a distinctly hydrophilic interface. Just (16) Clemmer, C. R.; Beebe, T. P. Scanning Microsc. 1992, 6 (2), 319-333. (17) Segregation of greater than nanometer sized domains is not expected to occur in these mixed SAMs due to the modest difference in chain length between the two components and the hydrophilic nature of both types of endgroups. On the basis of related literature reports (ref 18), the mixed SAMs used here should have a rather randomly segregated structure although their actual structures have not yet been determined experimentally.
Figure 6. Cyclic voltammetry of adsorbed equine cyt c at singlecomponent, C13COOH (thin trace), and mixed, C13COOH & C10OH (thick trace), SAMs prepared on annealed bulk gold. Similar results were obtained on other types of gold in Table 1 (results not shown). Solution conditions: 4.4 mM KPB (µ ) 10 mM, pH ) 7); scan rate, 100 mV/s.
as importantly, this mixed SAM should present an irregularly textured molecular surface when formed on a smooth gold substrate. As shown in Figure 6, the cyt c/mixed SAM/smooth bulk gold system displayed excellent cyt c electrochemistry compared to the pure COOH SAM on the same surface. Similar results were achieved for all substrate types in Table 1. XPS data (see Figure 4d) confirm that excellent adsorption is attainable on smooth gold surfaces when modified with a mixed SAM. A comparison of the first and last rows of Table 3 further illustrates the advantage of using mixed SAMs. Replacement of the homogeneous (single-component) SAM with the mixed SAM on smoothed bulk gold greatly enhances the observed voltammetry and allows for the evaluation of the electrochemical properties of cyt c (see Table 3). A similar enhancement of voltammetry was also noted when a thinner C11COOH SAM was replaced by a C11COOH/ C7OH mixed SAM (results not shown). We believe the improved voltammetry that occurs with mixed SAMs is due to enhanced electronic coupling at the cyt c/SAM interface4b and more extensive coverage as evident from the XPS results (Figure 4, compare traces b and d). In prior experiments utilizing rough evaporated gold films,4b we observed that the ET rates for yeast and horse cyt c could be increased by substituting a COOH/ OH mixed SAM for a pure COOH SAM. Rate increases of 103-104 and 5-10 for yeast and horse cyt c, respectively, were attributed to increased electronic coupling at the cyt/SAM interface. Analogous behavior can be observed in Figure 4 by comparing traces c and d. The substantial difference in peak splitting supports the hypothesis that
Voltammetry of Cytochrome c Adsorbed on SAMs
mixed SAMs lead to better electronic coupling between protein and SAM. Conclusions The major findings of the research can be summarized as follows: the topography of the gold electrode plays a highly significant role in determining the structural properties of longer chain COOH SAMs, which in turn substantially impact the adsorption and electrochemistry of cyt c. For smoother substrates, the density of defects is expected to be lower due to fewer grain boundaries, step edges, and other surface features that are known to cause defects in SAMs. However, it was shown here that more uniform, lower defect C13COOH SAMs atop smooth gold actually behave quite poorly as platforms for cyt c adsorption and electrochemistry. On the contrary, rougher polycrystalline gold surfaces, supporting SAMs with a higher degree of defectiveness and disorder, have been found to be most suitable for this application. The observed topographic effects were highly repeatable for both C13COOH and C10COOH SAMs. As stated above, the topographic effects observed for the thinner SAMs, while qualitatively identical to those we have presented in detail here for the C13COOH SAMs, were not as pronounced. We presume that since shorter chain length SAMs, especially those possessing carboxylic acid endgroups, are known to be more disordered (less crystalline), their properties are intrinsically less sensitive to variations of substrate topography.11,19 With both chain lengths, however, higher defect density arising either from rough gold topography or from the presence of a mixed SAM exhibited surface properties beneficial toward cyt c adsorption and electron transfer. Although the molecular details behind the cyt c adsorption and electrochemical behavior shown in Figures 3-6 remain to be established, we hypothesize that two major effects are involved, one primarily affecting binding extent and the other affecting electronic coupling. In a separate report, we have proposed that the surface pKa of COOH SAMs is highly dependent upon gold topography12 with smooth substrates appearing to give rise to basic pKa values of 8-10.20 Thus, if SAMs on smoother gold can indeed exhibit pKa values of approximately 9 or more, a very low density of negative charge would result at neutral pH, on the order of 1% or less, leading to weak electrostatic binding of cyt c. More acidic pKa values, on the other hand, (18) (a) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7175. (b) Stanick, S. J.; Parikh, A. N.; Tao, Y.-T.; Allara, D. L.; Weiss, P. S. J. Phys. Chem. 1994, 98, 7636-7646. (c) Folkers, J. P.; Laibinis, P. E.; Deutch, J.; Whitesides, G. M. J. Phys. Chem. 1994, 98, 563-571. (d) Atre, S.; Liedberg, B.; Allara, D. L. Langmuir 1995, 11, 3883-3893. (19) (a) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (b) Delamarche, E.; Biebuyck, H. A. Adv. Mater. 1996, 8 (9), 719-729. (20) (a) Hu, K.; Bard, A. J. Langmuir 1997, 13, 5114-5119. (b) Kakiuchi, T.; Iida, M.; Imabayashi, S.; Niki, K. Langmuir 2000, 16, 5397-5401. (c) Godinez, L. A.; Castro, R.; Kaifer, A. E. Langmuir 1996, 12, 5087-5092.
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would result in a higher level of negative charge and a greater extent of electrostatic binding. More acidic pKa values can arise when mixed COOH SAMs contain a second nonacidic component.21 We propose elsewhere that the same effect can result when homogeneous COOH SAMs are prepared on suitably rough substrates.12 Although the roughening effect on pKa remains to be experimentally proven, the fact that mixed SAMs and substrate roughening impact cyt c adsorption and electrochemistry in a similar fashion suggests that a similar underlying phenomenon is at work. Spectroscopic studies are currently under way to delineate the relationship between substrate topography and surface pKa values of homogeneous COOH SAMs. Rougher substrates or mixed SAMs also give rise to a more disordered and irregularly textured surface, which, we argued previously,4b promotes more optimal docking and superior electronic coupling for adsorbed cyt c. The idea of disorder or a higher degree of defectiveness instigating beneficial effects on adsorption processes is not unprecedented.22,23 One of the more pertinent examples comes from Ringsdorf and co-workers,22 who introduced an element of disorder into an interfacial biological system, namely, the binding of streptavidin to biotinylated SAMs (HS(CH2)11[biotin]). Greater amounts of adsorbate were achieved by introducing a mixed SAM. Additionally, in a separate report Klein and co-workers used computer simulations to propose that cyt c most likely penetrates, possibly at defect sites, the surface of COOH SAMs upon adsorption.24 Their study also lends support to the conclusion reached here, namely, that cyt c adsorption is dependent upon the density of defects in the SAM. In retrospect, it is really not surprising that rougher, defect-riddled, acidic surfaces are more effective at immobilizing cyt c, considering that the natural redox partners of cyt c in biological systems are also irregularly textured both chemically and topographically. It follows that physiological protein/protein binding interactions can be mimicked by protein binding at a uniform, singlecomponent SAM only to a limited degree.4b Acknowledgment. We acknowledge the U.S. National Science Foundation for generously supporting this research (CHE-9816268). We thank Professor Christopher B. Gorman and Jill C. Mikulecky for their help and support. LA011456C (21) Creager, S. E.; Clarke, J. Langmuir 1994, 10, 3675-3683. (22) (a) Haussling, L.; Michel, B.; Rohrer, H.; Ringsdorf, H. Angew. Chem., Int. Ed. Engl. 1991, 30 (5), 569-572. (b) Haussling, L.; Ringsdorf, H.; Schmitt, F.-J.; Knoll, W. 1991, 7 (9), 1837-1840. (23) (a) Niwa, M.; Mori, T.; Nishio, E.; Nishimura, H.; Higashi, N. J. Chem. Soc., Chem. Commun. 1992, 547-549. (b) Mandler, D.; Turyan, I. Electroanalysis 1996, 8 (3), 207-213. (c) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7175. (24) Tobias, D. J.; Mar, W.; Blasie, J. K.; Klein, M. L. Biophys. J. 1996, 71, 2933-2941.
