Octadecyl Mercaptan Sub-monolayers on Silver Electrodeposited on

Department of Chemistry, Virginia Commonwealth University, Box 842006, ... reaction of OM on the silver-coated gold QCM electrode surface, a selected ...
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Langmuir 1997, 13, 3781-3786

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Octadecyl Mercaptan Sub-monolayers on Silver Electrodeposited on Gold Quartz Crystal Microbalance Electrodes James D. Burgess and Fred M. Hawkridge* Department of Chemistry, Virginia Commonwealth University, Box 842006, Richmond, Virginia 23284 Received January 23, 1997. In Final Form: May 12, 1997X The objective of this work is to accurately and reproducibly control the amount of alkanethiol selfassembled onto the surface of a gold quartz crystal microbalance (QCM) electrode. This reaction is the first step in preparing bilayer membranes on gold electrodes that contain cytochrome c oxidase. In this work procedures for cleaning the surface of gold QCM electrodes, including use of an oxygen plasma, a UV surface cleaner, and aggressive chemical treatment of the surface, still led to rates of octadecyl mercaptan (OM) self-assembly that were highly irreproducible, varying over several orders of magnitude in time. Electrodeposition of a silver monolayer onto the surface of gold QCM electrodes affords a surface at which the OM self-assembly reaction can be easily controlled. Using the QCM to monitor the self-assembly reaction of OM on the silver-coated gold QCM electrode surface, a selected sub-monolayer coverage can be reliably prepared in about 10 min.

Introduction The assembly of molecular structures at interfaces that exhibit target properties is a frequent goal in attacking diverse problems in chemistry.1 Chemically derivatized electrodes have become practical tools for the study of biological charge transfer processes2-4 in both fundamental studies5 and biosensor applications.6 Lipid bilayers on various solid supports have recently received increased attention because of their ability to host integral membrane biomolecules and mimic the interfacial environment of biological membranes.7 A reproducible method for preparing electrochemically and chemically addressable enzyme/lipid bilayers on gold electrodes could have a significant impact on the field of biosensors as well as in fundamental enzymatic studies. A procedure developed for reconstituting cytochrome c oxidase into vesicles in solution involving cholate dialysis8 has been used in this group to construct lipid membranes hosting cytochrome c oxidase on gold electrodes. This procedure involves initially derivatizing gold with a sub-monolayer of octadecyl mercapton (OM).9 Initial unsuccessful attempts to immobilize cytochrome c oxidase in lipid bilayers on gold using Langmuir-Blodgett techniques led Cullison9 to use sub-monolayer coverages of OM on gold to anchor stable lipid bilayer membranes containing cytochrome c oxidase. * To whom correspondence should be addressed: fax, 804 8288599; e-mail, [email protected]. X Abstract published in Advance ACS Abstracts, June 15, 1997. (1) Swalen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Israelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (2) Murray, R. W. In Electroanalytical Chemistry: A Series of Advances; Bard, A., Ed.; Marcel Dekker, Inc.: New York, 1984; Vol.13, p 191. (3) Hawkridge, F. M.; Taniguchi, I. Comments Inorg. Chem. 1995, 17, 163. (4) Bianco, P.; Haladjian, J. Biochimie 1994, 76, 605. (5) Biomembrane Electrochemistry; Blank, M., Vodyanoy, I., Eds.; Advances in Chemistry Series 235; American Chemical Society: Washington, DC, 1994. (6) Diagnostic Biosensor Polymers; Usmani, A. M., Akmal, N., Eds.; Advances in Chemistry Series 556; American Chemical Society: Washington, DC, 1994. (7) Sackman, E. Science 1996, 271, 43. (8) Hinkle, P. C.; Kim, J. J.; Racker, E. J. Biol. Chem. 1972, 247, 1338. (9) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877.

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The specific aim attacked here is preparing electrodes modified with the same degree of thiol surface coverage to serve as the anchor for supported lipid bilayer membranes. The use of silver electrodeposited on gold to enhance the reproducibility of OM self-assembly kinetics is described. An electrochemical quartz crystal microbalance (EQCM) developed by Bruckenstein et al.10 has been constructed and used to monitor both silver deposition and thiol self-assembly onto gold QCM electrodes. Initial experiments using a QCM to monitor the rate of OM self-assembly on gold QCM electrodes revealed wide variability in the kinetics of these processes (i.e., full monolayers were formed in times ranging from 10 s to over 10 h). This variability persisted even when a host of surface pretreatments and cleaning procedures were applied to the QCM electrodes. Procedures employing potential control of the gold QCM electrode immersed in OM-containing solutions were also tried to control the degree of OM coverage. Neither the procedures reported by Porter’s group11 nor those of Buttry12 proved acceptable based on the frequency shifts that were monitored at the gold QCM electrode and the charge passed. Although the explanation for the failure of these methods as applied to the application described here remains uncertain, some speculation is offered later (vide infra). This problem has been solved in practical terms by electrodepositing a silver monolayer on the gold QCM electrode suface and then forming the OM self-assembled sub-monolayer on this interface. The silver monolayer surface exhibits controllable self-assembly kinetics when exposed to dilute ethanolic OM solutions. Monitoring selfassembly at silver monolayers on gold QCM electrodes in real time allows the surface coverage of OM to be reproduced for each electrode regardless of small differences in the reaction rates. OM sub-monolayers on bulk silver QCM electrodes were investigated as substrates for lipid bilayer membranes containing cytochrome c oxidase. Dialysis experiments (10) Bruckenstein, S.; Michaliski, M.; Fensore, A.; Li, Z.; Hillman, A. R. Anal. Chem. 1994, 66, 1847. (11) Weisshaar, D. E.; Lamp, B. D.; Porter, M. D. J. Am. Chem. Soc. 1992, 114, 5860. (12) Schneider, T. W.; Buttry, D. A. J. Am. Chem. Soc. 1993, 115, 12391.

© 1997 American Chemical Society

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conducted both in the presence and in the absence of cytochrome c oxidase showed that the oxidase enzyme solution facilitates corrosion of OM-modified bulk silver QCM electrodes. Bulk silver surfaces readily oxidize, a problem which complicates thiol self-assembly on bulk silver.13,14 However, for silver monolayers on gold QCM electrodes no corrosion is apparent after several days of exposure to cytochrome c oxidase solutions due to their increased stability compared with bulk silver. Although gold/thiol self-assembly chemistry has been used to modify electrodes for a host of reasons, starting with Taniguchi’s report on bis(4-pyridyl) disulfide15 and closely followed by Nuzzo and Allara’s report,16 comparatively little has been done with this chemistry using silver as the electrode material. Alkanethiols react at bulk silver and gold to form monomolecular films with the sulfur atoms chemisorbed to the metal and the hydrocarbon tails extending from the surface plane.17-20 Although the mechanism of this reaction remains uncertain, evidence points to the S-H bond cleaving with the resultant formation of a metal bound thiolate.17,18 One prominent difference between alkanethiol monolayers formed on silver and gold is the tilt angle of the hydrocarbon tail with respect to the surface. The reported tilt angles of OM monolayers on silver range from normal20 to 15°18 with respect to the surface normal. On gold, there is agreement that OM monolayers have a chain tilt angle of about 30°.20 Another difference between alkanethiol monolayers formed on silver and gold is the larger thiol packing density on silver.13,17,21,22 On gold, alkanethiol spacing is apparently governed by sulfur-substrate spacing, while on silver the packing density is consistent with control by the dimensions of the hydrocarbon tail.22 Differences between the sulfur-metal bonds at gold and silver accommodate these structural dissimilarities.13,20 Recently,23 the stability and self-exchange properties of OM monolayers on gold and silver were investigated. OM dissociates from gold faster than from silver in THF.23 This finding is consistent with a Raman scattering study which suggests that the sulfur-silver bond may have more ionic character and thus may be stronger than the sulfurgold bond.18 However, ab initio calculations by Sellers et al.20 do not support this possibility. These differences that exist between the bonding of alkanethiols at gold and silver may, in part, be due to the large difference between the potentials of zero charge of the two metals. The potentials of zero charge have been measured in 0.02 N Na2SO4 yielding values of +0.23 V vs NHE for gold and -0.70 V vs NHE for silver.24 Silver oxidation may also be involved. Porter et al.17 have shown that alkanethiols and alkanethiolates form similar monolayer structures on silver (13) Walczak, M. M.; Chung, C.; Stole, S. M.; Widrig, C. A.; Porter, M. D. J. Am. Chem. Soc. 1991, 113, 2370. (14) Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y.; Parikh, A. N.; Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152. (15) Taniguchi, I.; Toyosawa, K.; Yamaguchi, H.; Yasukouchi, K. J. Electroanal. Chem. 1982, 140, 187. (16) Nuzzo, R. C.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. (17) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335. (18) Bryant, M. A.; Pemberton, J. E. J. Am. Chem. Soc. 1991, 113, 8284. (19) Laibinis, P. E.; Fox, M. A.; Folkers, J. P.; Whitesides, G. M. Langmuir 1991, 7, 3167. (20) Sellers, H.; Ulman, A.; Shnidman, Y.; Eilers, J. E. J. Am. Chem. Soc. 1991, 115, 9389. (21) Heinz, R.; Rabe, J. P. Langmuir 1995, 11, 506. (22) Fenter, P.; Eisenberger, P.; Li, J.; Camillone, N., III; Bernasek, S.; Scoles, G.; Ramanarayanan, T. A.; Liang, K. S. Langmuir 1991, 7, 2013. (23) Schlenoff, J. B.; Li, M.; Ly, H. J. Am. Chem. Soc. 1995, 117, 12528. (24) Bockris, J. O’. M.; Reddy, A. K. N. Modern Electrochemistry; Plenum Press: New York, 1970; p 708.

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but not on gold. This finding suggests that the sulfur bound hydrogen of the alkanethiol is involved in the selfassembly mechanism at gold but is not involved in the self-assembly mechanism at silver. This difference in selfassembly was explained by a silver oxide binding site for the case of thiols reacting at silver.17 Because silver monolayers on gold exhibit surface properties unlike that of bulk silver or gold, it is not reasonable to infer any specific features about thiol bonding at silver monolayers on gold from the comparison given above. Furthermore, the characterizations described above are for full monolayer structures of alkanethiol and they may not be relevant to sub-monolayer coverages. Bain et al.25 first studied the kinetics of alkanethiol self-assembly from the solution phase onto gold by ex-situ optical ellipsometry and contact angle measurements. The kinetics of OM self-assembly onto clean gold depend on the thiol concentration, temperature, and solvent. At a 1 mM OM concentration initial self-assembly is fast such that the ellipsometric thickness and contact angles reach about 80% of their final value in a few seconds. This initial fast phase is followed by a slower phase in which a full monolayer is formed over several hours. These findings are supported by similar results from in-situ second harmonic generation spectroscopy studies26 and by insitu QCM studies.27 Also, X-ray photoelectron spectroscopy,26 near-edge X-ray absorption fine structure spectroscopy,28 scanning tunneling microscopy (STM),29,30 and QCM measurements29 have been used to investigate thiol self-assembly on gold ex-situ and have yielded essentially the same results. Both Langmuir27,28 and diffusioncontrolled23 kinetics have been assumed to model selfassembly of alkanethiols onto gold. The self-assembly kinetics of alkanethiols at silver have not been as widely studied. Hexanethiol self-assembly onto silver (111) has been investigated in-situ by STM21 and sub-monolayer coverages of decanethiol on silver (111) have been imaged ex-situ by STM.31 Both of these studies report fast initial self-assembly. Silver monolayers deposited on gold by underpotential deposition have been studied by atomic force microscopy (AFM),32 extended X-ray absorption fine structure (EXAFS),33 rotating ring-disk electrode techniques,34,35 and by X-ray diffraction.36 Unlike the case of gas phase deposition, uniform monolayer coverages of silver can be deposited onto gold electrochemically. This fact is believed to be due to the repulsive forces between aqueous silver cations.32 The structure of the silver adlayer has been found to depend on the potential of deposition32,36 and on the supporting electrolyte.32 Gewirth’s group used AFM to show the differences in structure of silver monolayers underpotentially deposited onto gold (111) in the presence of several electrolytes.32 In most cases a correlation was (25) Bain, C. D.; Troughton, E. B.; Tao, Y.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (26) Buck, M.; Grunze, M.; Eisert, F.; Fischer, J.; Trager,F. J. Vac. Sci. Technol., A 1992, 10, 926. (27) Karpovich, D. S.; Blanchard, G. J. Langmuir 1994, 10, 3315. (28) Ha¨hner, G.; Wo¨ll, C.; Buck, M.; Grunze, M. Langmuir 1993, 9, 1955. (29) Kim, Y.; McClarley, R. L.; Bard, A. J. Langmuir 1993, 9, 1941. (30) Poirier, G. E.; Pylant, E. D. Science 1996, 272, 1145. (31) Dhirani, A.; Hines, M. A.; Fisher, A. J.; Ismail, O.; GuyotSionnest, P. Langmuir 1995, 11, 2609. (32) Chen, C.; Vesecky, S. M.; Gewirth, A. A. J. Am. Chem. Soc. 1992, 114, 451. (33) Samant, M. G.; Borges, G.; Melroy, O. R. J. Electrochem. Soc. 1993, 140, 421. (34) Swathirajan, S.; Mizota, H.; Bruckenstein, S. J. Phys. Chem. 1982, 86, 2480. (35) Swathirajan, S.; Bruckenstein, S. J. Electrochem. Soc. 1982, 129, 1202. (36) Chabala, E. D.; Ramadan, A. R.; Brunt, T.; Rayment, T. J. Electroanal. Chem. 1996, 412, 67.

Control of SAM on a QCM Electrode

Figure 1. QCM cell used for silver deposition and octadecyl mercaptan modification. The QCM electrode is mechanically clamped to the cell.

found between the size of the electrolyte anions and the silver packing density. More open silver structures (i.e., lower coverages) resulted in the presence of the larger supporting electrolyte anions. Also reported by Gewirth is an anion discharge effect that complicates surface coverage calculations based on the total charge passed.32 In the work presented here, silver electrodeposition experiments are carried out in the absence of supporting electrolyte to avoid these problems (vide infra). The thiol-derivatized silver monolayer electrodes described here support stable lipid bilayer membranes containing cytochrome c oxidase and that work will be reported elsewhere. Reported here is the formation of electrodeposited silver monolayer/gold QCM electrodes and the subsequent derivatization of these surfaces with OM sub-monolayers, as followed by EQCM and QCM measurements. Experimental Section Instrumentation. The potential step experiments were performed using a potentiostat of conventional design built inhouse. The potentiostat was used with a QCM built in-house based on a circuit published by Bruckenstein et al.10 A 12-bit National Instruments MIO-16DE-10 analog-to-digital and digitalto-analog data acquisition board was used in conjunction with National Instrument’s LabView graphical programming software to generate applied potential waveforms and acquire data. The difference frequency of the QCM is passed through a 7414 NAND gate Schmitt Trigger before being counted by the 9513 chip aboard the MIO-16DE-10 DAQ board. The QCM frequency digitization shown in the figures given in this paper arises from a combination of thermally induced noise and integration of the QCM difference frequency. This digitization is not bit noise. QCM Cell. The cell was machined in-house from Lucite. The QCM electrode was clamped to the bottom of the cell, and a silicon rubber gasket (RTV, adhesive sealant, self-leveling) cast on an O-ring sealed the contact. The cell chamber above the clamped crystal has a volume of about 4 cm3. This cell design is depicted in Figure 1. QCM Electrodes. Gold and silver 10 MHz QCM electrodes were purchased from International Crystal Manufacturers Co., P.O. Box 26330, 729 West Sheridan, Oklahoma City, OK 731260330. The electrodes consist of 1000 Å of vapor-deposited gold (99.99% pure) or silver on polished quartz (i.e., having a texture of e1 µm) with a 50 Å chromium adhesion layer between the electrode and quartz. Atomic force microscopy (AFM, Seiko 3700) was used to characterize the surface roughness of these electrodes. The images of the bare gold QCM surfaces show rolling hills with a valley to peak height of about 5 nm and a valley to valley distance of about 300 nm. Also, some narrow deep defects were apparent covering no more than ca. 5% of the surface. Reagents. The water used in all the experiments was deionized and then further purified with a Milli RO-4/Milli-Q system (Millipore Corp.) to exhibit a resistivity of 17 MΩ‚cm upon delivery. Silver nitrate (Puratronic; ALFA, 99.9995%) was used to make 1 mM aqueous silver nitrate stock solutions. Octadecyl mercaptan (Aldrich, 98%) was used as received. Purification of the OM by recrystallizating it twice from ethanol

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Figure 2. QCM frequency shifts during two single potential step experiments resulting in silver deposition. Curve a shows sub-bulk deposition and curve b shows bulk deposition. Electrodeposition is from a 1 mM aqueous silver nitrate solution. The electrode area is 0.2 cm2. The frequency integration time is 350 ms. had no apparent bearing on the self-assembly rates observed. Ethanol (USI Chemical Co., 100%) was the solvent for the 500 µM OM stock solutions. Procedure. The gold QCM electrodes were cleaned for 30 min in a ultraviolet light surface cleaner (Boekel Industries, Inc., UV-Clean 135500). The electrodes were then rinsed with ethanol and blown dry with Dust-off (containing difluoroethane, Falcon Safety Products Inc., Branchburg, NJ 08876) prior to being clamped to the cell. Aqueous silver nitrate was then added to the cell. Silver monolayer depositions were achieved during asymmetric double potential step experiments. Following silver deposition, the cell and electrode were rinsed extensively with water and then with ethanol without removing the electrode from the cell. The electrode was again dried with Dust-off and reconnected to the QCM oscillator circuit. Ethanol was added to the cell and, as discussed later, nitrogen was bubbled into the ethanol through a blunt tip 22 gauge stainless steel needle placed about 2 mm below the ethanol surface. The electrodes were allowed to equilibrate with the ethanol for ca. 10 min until a stable baseline frequency was observed. During these equilibration times a decrease in frequency (i.e., increase in mass) was observed. Baseline data were collected for 1 min prior to injection of 10 µL of 500 µM OM near the site of nitrogen agitation. This procedure is shown pictorially in Figure 1. The final thiol concentration in the cell was 1.66 nM for all thiol experiments shown. Each electrode was allowed to react with OM until a frequency shift of 20 Hz was reached. The Ag/AgCl, 1 M KCl reference electrode, used inside an isolation chamber filled with 1 mM silver nitrate, was calibrated with a platinum wire immersed in a saturated solution of quinhydrone (Eastman) of known pH. All potentials are reported vs NHE.

Results Uniform coverages of silver are deposited onto polycrystalline gold QCM electrodes electrochemically in the absence of added supporting electrolyte to avoid anion problems described above. Under such conditions the uncompensated solution resistance is large (vide infra), and therefore, the deposition is not under potential control. Initial experiments were required to optimize the electrodeposition of silver monolayers. Figure 2 shows the QCM frequency shifts obtained during two separate single potential step experiments resulting in silver deposition. These two experiments demonstrate the electrodeposition rate dependence on applied potential. Figure 2a shows silver deposition during a potential step experiment from 725 to 605 mV. About one monolayer of silver is deposited in 2 min following the potential step. The amount of silver deposited at this potential varies from experiment to experiment as a result of chromium (vide infra) and/or changes in the iR potential drop (e.g., e14 mV for the data shown in this paper). This problem is evident for all silver deposition experiments and will not be discussed

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Figure 3. Asymmetric double potential step EQCM experiment for the deposition of a silver monolayer. The left curve is the current response and the right curve is the QCM response. Electrodeposition is from a 1 mM aqueous silver nitrate solution. The electrode area is 0.2 cm2. The frequency integration time is 350 ms.

further. Figure 2b shows silver deposition following a potential step from 725 to 595 mV (i.e., the deposition potential is 10 mV more negative than for the experiment shown in Figure 2a). At this potential bulk deposition occurs and, after ca. 1 min, the deposition rate approaches a constant value as mass transfer to the electrode is dominated by migration. Stepping to more negative potentials results in faster deposition rates. Lingane and Kolthoff37 showed that the initial limiting current of lead ion reduction in the absence of supporting electrolyte is ca. twice as large as the diffusion current measured when excess supporting electrolyte is added. As stated above, our silver surfaces are electrodeposited from a 1 mM aqueous silver nitrate solution and the uncompensated solution resistance is ca. 3 kΩ in the cell used for each experiment. In the presence of 0.1 M supporting electrolyte bulk deposition occurs at ca. 225 mV (i.e., 370 mV more negative than the bulk deposition potential under low ionic strength conditions). The silver monolayer surfaces used in this work were electrodeposited at an applied voltage of 525 mV. This applied potential results in a silver deposition rate that is convenient for monolayer formation. Figure 3 shows the QCM frequency shift and the current passed during the deposition of ca. one monolayer of silver onto a gold QCM electrode. The potential is initially stepped from 725 to 525 mV, until a monolayer of silver has been deposited. Then the potential is stepped to 625 mV, stopping the electrodeposition. Stepping to potentials more positive than about 625 mV, for the case of the second potential step, results in oxidative stripping of the silver adlayer. Stopping electrodeposition at monolayer coverages can also be achieved by going to open circuit (i.e., disconnecting the working electrode). However, this causes about a 120 Hz increase in QCM frequency which is due to a change in impedance. About 57 µC was passed during this particular experiment and the charge calculated for a silver monolayer based on the geometric area of the electrode (0.2 cm2) and the area occupied by a silver atom (6.61 Å2) is 48 µC. Since the electrode is not atomically smooth, more than the calculated mass of a silver monolayer was deposited to cover the gold surface. The EQCM sensitivity constant calculated from silver deposition charge/frequency shift data (1.11 Hz/ng) is in good agreement with that predicted by the Sauerbrey equation38,39 (1.13 Hz/ng) and it is also independent of the silver deposition potential. Bruckenstein et al.39 have shown that when silver is electrodeposited from a solution (37) Lingane, J. J.; Kolthoff, I. M. J. Am. Chem. Soc. 1939, 61, 1045. (38) Sauerbrey, G. Phys. Z. 1959, 155, 206. (39) Bruckenstein, S.; Shay, M. Electrochim. Acta 1985, 30, 1295.

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Figure 4. (a) Typical QCM response upon octadecyl mercaptan sub-monolayer formation on an electrodeposited silver monolayer electrode. (b) QCM response upon slow octadecyl mercaptan sub-monolayer formation on an electrodeposited silver monolayer electrode. (c) Control experiment showing the QCM baseline frequency upon injection of pure ethanol. The electrode area is 0.2 cm2. The frequency integration time is 850 ms.

containing 0.2 M perchloric acid the EQCM sensitivity constant agrees with the theoretical sensitivity constant when 10 µg or more of silver is electrodeposited. In this work, when 53 ng (i.e., one monolayer) of silver was electrodeposited in the presence of 0.2 M perchloric acid or 0.1 M nitric acid, the EQCM sensitivity constant calculated from the charge/frequency shift data was often larger than that predicted by the Sauerbrey equation. A dependence of the sensitivity constant on the deposition potential was also observed when perchloric acid or nitric acid was used as the supporting electrolyte. This finding is in agreement with charge/AFM data reported in the above mentioned study of underpotentially deposited silver monolayers on gold. This work suggests that these electrolyte anions provide some of the charge required for silver reduction (i.e., anion discharge).32 An EXAFS study conducted by Samant et al.33 confirmed that silver is fully reduced when electrodeposited onto gold in the presence of 0.2 M perchloric acid. Anion discharge is the main reason that supporting electrolyte is not used when electrodepositing silver and adventitious surface contamination is further minimized. The rate of OM self-assembly onto the silver monolayer electrodes depends on the thiol concentration and on mass transfer (i.e., agitation). As explained in the Experimental Section, convection is achieved by slow nitrogen bubbling near the surface of the ethanol. Placing the needle tip from which nitrogen is delivered more than a few millimeters below the surface of the ethanol results in a faster QCM frequency shift upon thiol injection due to an increased flux of thiol to the electrode. Parts a and b of Figure 4 show the measured variation of the QCM integrated frequency counts upon OM sub-monolayer formation under fixed conditions. These two experiments were conducted using two different QCM crystals. Figure 4c is a control experiment showing the baseline QCM frequency upon injection of pure ethanol. The noise in the two OM experiments shown (Figure 4a,b) is ca. 2 Hz. The majority of the electrodes require 6-8 min for derivatization (e.g., see Figure 3a) while other electrodes require additional time for equal modification (e.g., see Figure 3b). The average reaction time of 21 experiments is 10.64 ( 4.42 min. Reproducing mass transfer of OM to the electrode using gentle nitrogen bubbling probably causes these variations. Attempts were made to correlate a QCM frequency shift with the formation of a complete monolayer coverage. For all concentrations studied (e.g., concentrations ranging

Control of SAM on a QCM Electrode

from 2 nM to 0.1 mM) the QCM frequency shifts observed at different QCM silver monolayer surfaces during OM reactions did not show a reproducible plateau as would be expected for the formation of a monolayer. On the basis of the QCM sensitivity constant calculated from the silver deposition experiments, a 20 Hz shift in QCM frequency (i.e., the frequency shift allowed before stopping the OM reactions) corresponds to formation of half of a monolayer of OM. Kim et al.29 suggest that the mass of OM self-assembled onto gold QCM electrodes from ethanol can be measured by applying the Sauerbrey equation.38 In their work STM was used to confirm when a monolayer of OM had formed on gold QCM surfaces from ethanol solutions. However, QCM frequency shifts may not correlate with true mass changes when conducting experiments that impart changes in surface hydrophobicity or roughness.40 Although the silver surfaces are prepared and then modified with OM immediately, displacement of differing amounts of contamination from the electrodes would have the result of inconsistent submonolayer coverages being formed. Significant changes in the viscoelastic property of the quartz crystal are not predicted for ultrathin films41 and impedance measurements show that complete alkanethiol coverages do not affect the viscoelastic property of the quartz in air.42 In this work, the QCM frequency shift is used to reproducibly control the apparent mass of OM deposited on silver even though an absolute mass measurement is not available. Another factor considered was the presence of chromium. Chromium is used as an adhesion layer between the quartz crystals and the gold electrodes of the QCM elements used in this work. Electron probe microanalysis43 data showed a variance of chromium from electrode to electrode. Depositing a silver monolayer on the gold QCM electrode may mask this problem. Discussion Thiol self-assembly onto metal surfaces has experienced explosive growth over the past decade due to the use of this chemistry in preparing highly ordered monolayer films through covalent sulfur-metal bond formation.1 In general, excess reaction time is allowed to ensure complete monolayer formation and, until recently, little insight has been gained concerning the mechanism of formation. Thiol sub-monolayers on gold are thought to form highenergy surfaces.12 The EQCM study by Buttry’s group mentioned above reports physisorbed multilayer formation on chemisorbed sub-monolayer coverages formed under potential control from acetonitrile. The multilayer structures were not stable and they desorbed upon complete formation of a chemisorbed monolayer (i.e., the multilayers desorbed at more positive potentials). It was suggested that multilayer growth occurred at the interface because the sub-monolayer surface was of high energy.12 However, multilayer formation of OM at gold QCM electrodes from ethanol has been reported following long exposure times (i.e., days).29 A detailed investigation of the alkanethiol self-assembly mechanism at gold (111) has been conducted using STM to probe the orientation of the thiol molecules under various surface exposures.30 At low coverages the STM images were not resolved and this was attributed to lateral diffusion of surface-confined thiol molecules.30 The high-energy sub-monolayer model is also consistent with (40) Buttry, D. A.; Ward, M. D. Chem. Rev. 1992, 92, 1355. (41) Hingsberg, W. D.; Wilson, C. G.; Kanazawa, K. K. J. Electrochem. Soc. 1986, 133, 1448. (42) Yang, M.; Thompson, M.; Duncan-Hewitt, W. C. Langmuir 1993, 9, 802. (43) The electron probe microanalysis experiments were conducted using a JXA-8900R/RL from JEOL Co. Ltd (Tokyo, Japan).

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the above mentioned stability and self-exchange study of OM monolayers on gold and silver. This work suggests that only thiols bound at defect sites are immobile.23 On the basis of this background a high-energy sub-monolayer surface may be involved in the adhesion of stable lipid bilayers to the OM sub-monolayer substrates prepared in this work. The STM study30 of alkanethiol molecules deposited onto gold (111) showed that the arrangement of the thiol molecules changed at threshold surface coverages. Similar sub-monolayer structures were observed between gold derivatized with decanethiol from the gas phase and from ethanol. Despite differences in the thermal stability of thiol films formed on gold from solution (stable above room temperature) and under ultrahigh vacuum (UHV) conditions (desorb below room temperature),25 this finding has led to a generalized model proposed for alkanethiol selfassembly onto gold.30 Exposures of the gold (111) surface to thiol that were greater than that necessary to create the above mentioned high-energy coverage led to island formation. The carbon chains of the thiols that formed these islands were oriented parallel to the surface plane. Islands with the hydrocarbon tails extended toward the surface normal were only observed after exposures that led to monolayer coverages with the hydrocarbon tails oriented parallel to the surface.30 Small increases in surface coverage may induce reorganization of solvated sub-monolayers on silver as well. An STM study31 of alkanethiols on silver conducted under ambient conditions reported dynamic sub-monolayer structures and island formation. However, another STM study21 of alkanethiol monolayers on silver, also conducted under ambient conditions, did not report island formation. Subtle differences in the substrate, the conditions of the selfassembly process, or the method of characterization may have momentous effects on the structure of the observed thiol film. Clearly it is necessary to strictly control the extent of thiol modification when surfaces exhibiting identical properties are sought. Under the reaction conditions reported here, electrodeposition of silver monolayers onto the gold QCM electrodes increases the reproducibility of OM selfassembly kinetics compared with the reaction rates observed at the bare gold QCM electrodes. The present work does not contribute to the fundamental understanding of the sub-monolayer structure or the reaction mechanism. However, the silver monolayer does appear to provide a system that is more easily controlled than bulk gold in reacting with OM, and studies of the reaction mechanism of both sub-monolayer and monolayer formation may be useful. Conclusions Electrodeposited silver monolayer electrodes derivatized with OM sub-monolayers have been prepared for subsequent modification with lipid bilayer membranes containing cytochrome c oxidase. When EQCM data were used to characterize the electrodeposition of silver onto gold under low ionic strength conditions, the sensitivity of the EQCM calculated from charge/frequency shift data was found to agree with the sensitivity predicted by the Sauerbrey equation.38,39 The QCM was also used to control OM self-assembly at these silver monolayer electrodes allowing routine production of a sub-monolayer coverage. OM sub-monolayers on silver monolayer/gold electrodes support stable oxidase/lipid bilayers and control of this step has led to an increase in the reproducibility of the membrane formation process. Studies are needed to investigate the dependence of the oxidase voltammetry on the thiol surface coverage. Other than the advantages

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concerning adhesion of lipid bilayer membranes and direct electroanalysis of integral membrane biomolecules, thiol sub-monolayers on silver monolayer electrodes may also be applicable surfaces for use in areas such as electrocatalysis, corrosion repression, and lubrication.1 Regulating the thiol surface coverage on solid electrodes is an important variable for controlling the properties of the interface and may be useful for characterization studies as well as for technological advances. Note Added in Proof. We have become aware of a study describing the stability of alkanethiol monolayers (44) Jennings, G. K.; Laibinis, P. E. Langmuir 1996, 12, 6173.

Burgess and Hawkridge

on underpotentially deposited silver.44 This work suggest that the sulfur-silver bond is stronger than the sulfurgold bond. Acknowledgment. We appreciate the assistance of Professor Stanley Bruckenstein in constructing the EQCM circuit and Professor Isao Taniguchi who provided the AFM and electron probe microanalysis characterizations of the QCM electrodes. Support of this work by the National Science Foundation (Grant NSF CHE-9508640) is gratefully acknowledged. LA9700735