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Synthesis and Characterization of Amphiphilic Biomimetic Assemblies at Electrochemically Active Surfaces Paweł Krysin´ski*,† and G. J. Blanchard*,‡ Laboratory of Electrochemistry, Department of Chemistry, University of Warsaw, 02-093 Warsaw, Pasteura 1, Poland, and Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1322 Received December 3, 2002. In Final Form: February 17, 2003 We report on the synthesis and characterization of two amphiphilic biomimetic assemblies grown on electrochemically oxidized gold- and indium-doped tin oxide substrates. The molecular assemblies are bound covalently to the substrates by the reaction of the surface oxide with acid chlorides to form ester-like bonds, creating a hydrophilic region adjacent to the substrate. One assembly possesses a moiety capable of forming a second hydrophilic region with amides that can participate in a hydrogen-bonding network within the monolayer (C6-A-C12), and the other is an aliphatic structure (tetradecane, C14). These interfacial structures, presenting hydrophobic aliphatic chains to an adsorbate, constitute a useful system for the subsequent deposition of a lipid monolayer, leading to the formation of a hybrid bilayer membrane. Examination of the capacitance, cyclic voltammetry, and FTIR data for the C6-A-C12 and C14 systems demonstrates their stability and organization, while preserving some fluidlike behavior characteristic of the hydrophilic regions, essential for biomimetic applications.
Introduction The fabrication of functional interfaces between biomaterials such as proteins and cells and metallic and semiconductor substrates is attractive from both the scientific and the technological points of view. The primary limitation to success in this area is that the interaction between the biological entities and the conducting substrates typically alters the biological activity of the material, and to create a biologically amenable interface, the electrochemical communication with the adsorbate is compromised. We have demonstrated the ability to bind layered molecular structures covalently onto metallic (Au) and semiconductor surfaces [indium-doped tin oxide (ITO), silicon-based], a structural motif that can be made to mimic cell membranes, providing a general route to the fabrication of bioelectronic and electrooptical devices.1-10 To this point, the chemistry applicable to the growth of layered assemblies on oxide surfaces has been inaccessible for metallic surfaces and vice versa. If the rich knowledge base extant for oxide surface chemistry could be used in the modification of electrochemically active surfaces, many novel device and sensor structures could be accessed. In this work, we present a new means of self-assembly * Correspondingauthors.E-mailaddresses:
[email protected] and
[email protected]. † University of Warsaw. ‡ Michigan State University. (1) Zasadinski, J. A.; Viswanathan, R.; Madsen, L.; Garnaes, J.; Schwartz, D. Science 1994, 263, 1726. (2) Sackmann, E. Science 1996, 271, 43. (3) Chechik, V.; Crooks, R. M.; Stirling, J. M. Adv. Mater. 2000, 12, 1161. (4) Knoll, W.; Frank, C. W.; Heibel, C.; Naumann, R.; Offenhausser, A.; Ruhe, J.; Schmidt, E. K.; Shen, W. W.; Sinner, A. Rev. Mol. Biotechnol. 2000, 74, 137. (5) Stefan, I. C.; Scherson, D. A. Langmuir 2000, 16, 5945. (6) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1181. (7) Krueger, S.; Meuse, C. W.; Majkrzak, C. F.; Dura, J. A.; Berk, N. F.; Tarek, M.; Plant, A. L. Langmuir 2001, 17, 511. (8) Sinner, E. K.; Knoll, W. Curr. Opin. Chem. Biol. 2001, 5, 705. (9) Stefan, I. C.; Mandler, D.; Scherson, D. A. Langmuir 2002, 18, 6976. (10) Wiegand, G.; Arribas-Layton, N.; Hillebrandt, H.; Sackmann, E.; Wagner, P. J. Phys. Chem. B 2002, 106, 4245.
that offers a unifying approach to the modification of metallic, oxide, and semiconductor surfaces. In addition, the chemistry we report here creates an interfacial structure characterized by a hydrophilic region adjacent to the substrate, a structural feature not readily accessible with alkanethiol or alkylsilane monolayers. This chemistry relies on the reaction of an interfacial oxide with an acid chloride [e.g., adipoyl chloride (C6) or tetradecanoyl chloride (C14)] to form an ester-like bond.11,12 For the ITO substrate, the interfacial oxide is a native ITO deposited on quartz, and for the gold substrate, the interfacial oxide layer is generated electrochemically.11,12 For both substrates, following the initial deposition of C6, the terminal acid chloride or carboxylic acid functionality was reacted with n-dodecylamine to produce an interfacial monolayer of n-dodecyladipamide (C6-A-C12, Figure 1). To gauge the properties of this interfacial species, we compare its behavior to that of a monolayer formed from C14 (Figure 1). The ability to form such matrices on conducting, optically transparent substrates such as ITO renders this chemistry particularly attractive for the fabrication of optoelectronic devices. The focus of this work is not, however, on the demonstration of these devices. We are interested in characterizing the structure and properties of the C6-A-C12 and C14 interfaces and in how these systems respond to the deposition of lipid-layer species. The interfacial structures we report here are characterized by a hydrophilic region adjacent to the electrode surface, with a hydrophobic structure beyond the hydrophilic region.11,12 For C6-A-C12, there is a second hydrophilic region comprised of amide bonds, spaced six aliphatic carbons from the first hydrophilic region. This system is terminated by a 12-carbon aliphatic chain. Both the oxide substrates and the adlayer moieties can be tailored with respect to their structures, their thicknesses, and the spacing of the hydrophilic and hydrophobic regions to produce interfacial films suitable as substrates for (11) Kelepouris, L.; Krysin´ski, P.; Blanchard, G. J. J. Phys. Chem. B 2003, in press. (12) Krysin´ski, P.; Blanchard, G. J. J. Electroanal. Chem. 2003, in press.
10.1021/la026946z CCC: $25.00 © 2003 American Chemical Society Published on Web 03/27/2003
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Figure 1. Structures of adlayers bound covalently to oxidized gold or ITO electrode surfaces. Top panel: growth of the C6 adlayer and subsequent reaction to form the C6-A-C12 adlayer. Bottom panel: growth of the C14 adlayer.
hybrid-supported bilayer lipid membranes (HBMs). These structures appear to be amenable to the incorporation of some cell-membrane proteins, and such structures are, in principle, capable of functions such as sensing and energy transduction, rendering them very useful in bioelectronic device development.6,13 The incorporation of hydrophilic and hydrophobic regions in an adlayer at predetermined distances from the substrate can potentially be superior to other methods for hosting biologically relevant molecules at interfaces because of the ability of the systems we report to form hydrogen-bonded networks that can interact strongly with the adsorbed proteins on the solid support and to form an ionic reservoir between the hydrophobic moiety and the surface of a substrate. We report here on the spectroscopic and electrochemical characterization of the C6-A-C12 and C14 self-assembled monolayers (SAMs) on polycrystalline gold and ITO electrode surfaces. In addition to these interfaces, we discuss our growth and characterization of a HBM that consists of a phospholipid monolayer physisorbed to the SAM surface. Experimental Section Chemicals. All chemicals used were the highest purity available commercially: gold, gold-coated silicon, ITO on quartz (Delta Technologies, Ltd), chromic acid (VWR Scientific Products), sulfuric acid (CCI, ACS reagent grade), sodium sulfate (Spectrum Quality Products, Inc.), potassium chloride (Spectrum Chemical Mfg. Corp.), ferrocenemethanol (Aldrich, 97%), C6 (Aldrich), C14 (Aldrich, 97%), 4-methylmorpholine (Aldrich, 99%), dry acetonitrile (Aldrich, 98%), chloroform (Aldrich, 99.8%), ethyl acetate (Spectrum Quality Products, Inc., ACS reagent grade), ndodecylamine (Aldrich, 99+%), 1,2-di(cis-9-octadecenoyl)-snglycero-3-phosphocholine (DOPC; Sigma, 99%), and 1-aminopyrene (Fluka). Deionized water was available in-house. (13) Krysin´ski, P.; Tien, H. T.; Ottova, A. Biotechnol. Prog. 1999, 15, 970.
Krysin´ ski and Blanchard Electrochemistry. Electrochemical measurements were conducted with a personal-computer-controlled model 650A Electrochemical Workstation (CH Instruments, U.S.A.), using a small-volume three-electrode cell with a Pt wire as the counter electrode. All potentials are quoted versus a Ag/AgCl/1 M KCl(aq) reference electrode. For the electrochemical experiments on gold, a gold-ball electrode with a surface area of 0.16 cm2 (measured electrochemically) was used. For the electrochemical measurements on ITO electrodes, the pieces of ITO were 0.12 cm2. All electrochemical experiments were carried out in 0.5 M KCl, and for the alternating current (ac) voltammetry with phase separation experiments, the ac voltage amplitude was 5 mV. IR and Infrared Reflection-Absorption Spectroscopy. For monolayer self-assembly on gold-coated silicon slides, infrared reflection-absorption spectra (IRRAS) were acquired with a 4-cm-1 resolution using a Nicolet Magna 750 Fourier transform infrared (FTIR) spectrometer. The external reflectance sample mount was set to an incidence angle of 80° with respect to the substrate normal. Transmission IR spectra were collected with a 4-cm-1 spectral resolution using a Nicolet Magna 550 FTIR spectrometer. Optical Ellipsometry. Ellipsometric thickness measurements of adlayers formed on the electrochemically oxidized gold surface were conducted with a Rudolph Auto-EL II optical null ellipsometer operating at 632.8 nm. Rudolph DAFIBM software was used for data collection and processing. Time-Resolved Emission Measurements. Time-correlated single-photon counting was used to acquire data on local organization within the interfacial structures. We used 1-aminopyrene as a probe chromophore to bind covalently to the C6adlayer-exposed (terminal) functionalities. These probe molecules were embedded in the outer aliphatic region of the C6-A-C12 structure, serving as a probe of the local structural freedom within the adlayer assembly. We used a mode-locked CW Nd:YAG laser (Quantronix 416) to pump synchronously a cavity-dumped dye laser (Coherent 702) operated at 660 nm using Kiton Red laser dye (Exciton). The output of the dye laser was typically 100 mW of average power at a 4-MHz repetition rate, with about 5-ps pulses. A fraction of the light is used as a reference beam, with the remaining light being frequency-doubled using a type I, angletuned LiIO3 crystal to produce 330-nm light for excitation of the pyrene chromophore. The polarization of the excitation beam is set to be vertical, and the emission polarization is selected as either parallel or perpendicular to the excitation polarization using a Glan-Taylor prism. The instrument response function of this system is typically 35-ps full-width at half maximum. Preparation of Gold Substrates and Oxide-Layer Formation. Gold substrates for the electrochemical measurements (Au, 99.99%; 0.5-mm-diameter wire with tip melted to form a gold-ball electrode14,15 with an electrochemical area of 0.16 cm2, which was determined from the cyclic voltammetry of a K4Fe(CN)6/K3Fe(CN)6 aqueous solution as the reference)16 were cleaned by annealing in a reductive flame and then polarized cyclically (scan rate 0.1 V/s) in the -0.3 to +1.5 V potential range in a 0.5 M H2SO4(aq) solution. The electrodes were then cycled at the same rate in 0.5 M Na2SO4(aq) solution in the potential window of -0.5 to +1.2 V. Once reproducible voltammograms were obtained, the potential cycle was stopped at 1.2 V for 60 s and, under potential control, the electrodes were removed from the cell, washed with distilled water, and dried with a stream of nitrogen. Planar gold substrates were made from silicon wafers, A ) 2 cm2, with evaporated gold (1000 Å) deposited on a chromium adhesion layer. The gold-coated substrates were cleaned by immersion in hot chromic acid (95 °C, 30 s.) and rinsed with distilled water. The clean substrates were transferred to the electrochemical cell and polarized cyclically in 0.5 M Na2SO4(aq), as with the gold-ball electrodes. Polarization in acidic media between -0.3 and +1.5 V was omitted to avoid the dissolution of the chromium layer and consequent delamination of the gold (14) Napper, A. M.; Liu, H.; Waldeck, D. H. J. Phys. Chem. B 2001, 105, 7699. (15) Sumner, J. J.; Weber, K. S.; Hockett, L. A.; Creager, S. E. J. Phys. Chem. B 2000, 104, 7449. (16) Krysin´ski, P.; Brzostowska-Smolska, M. J. Electroanal. Chem. 1997, 424, 61.
Amphiphilic Biomimetic Assemblies layer. This procedure resulted in a stable open-circuit potential of about 0.76 V for both the planar and the ball substrates, characteristic of the formation of a hydrated oxide layer on gold.12 ITO Substrates. ITO on quartz substrates were cleaned first by a brief immersion in hot chromic acid, followed by washing copiously with distilled water and then refluxing for 5 min in acetone/methanol (1:1, v/v). Following removal from the acetone/ methanol reflux, the substrates were immersed in a solution of 30% H2O2/30% NH4OH/H2O (1:1:5, v/v/v) for 5 min under ultrasonication and soaked for another 45 min in the same solution at 60 °C. The cleaned substrates were rinsed with water and dried thoroughly. C6-Adlayer Formation. The cleaned gold and ITO substrates were reacted with C6 in dry acetonitrile under nitrogen,12 using 4-methylmorpholine as a Lewis base (1:50:1, v/v/v) for about 12 h. The reacted substrates were removed and rinsed with ethyl acetate. Gold-plated silicon slides were then dried under a stream of nitrogen and used for IRRAS and ellipsometric measurements, yielding a film thickness of 8.5 ( 1.5 Å, consistent with the addition of a monolayer of C6.12 The surface coverage of 1.5 × 10-10 mol cm2 was evaluated electrochemically on gold-ball electrodes, where ferrocenemethanol was bound covalently to the C6 adlayer.12 Fluorophore Attachment. The acid chloride-terminated substrates were reacted with 1-aminopyrene by exposure to 10 mL of 0.5 mM 1-aminopyrene in dry acetonitrile with 0.1 mL of 4-methylmorpholine under nitrogen. After 12 h of reaction time, the substrates were removed from solution, washed with ethyl acetate, and dried in a nitrogen stream. This procedure yielded a chromophore coverage of ∼3% of the monolayer, evaluated electrochemically.11 The remaining unreacted terminal groups of the C6 adlayer were subsequently reacted with n-dodecylamine, as described below. Such prepared substrates were then studied using time-correlated single-photon-counting experiments. N-n-Dodecyladipoylamide (C6-A-C12)-Monolayer SelfAssembly. The acid chloride terminal functionality on gold (goldball electrodes and gold-plated silicon slides) and ITO was reacted with n-dodecylamine by exposing substrates covered with the C6 adlayer to 10 mL of 0.5 mM n-dodecylamine in dry acetonitrile with 0.1 mL of 4-methylmorpholine under nitrogen for ∼12 h. The substrates were then removed, washed with ethyl acetate, and dried. Gold-plated silicon slides and ITO on quartz slides were used for the spectroscopic studies and ellipsometry, whereas the gold-ball electrodes and ITO on quartz slides were used in the electrochemical experiments. The film thickness evaluated from the ellipsometric measurements was 25 ( 5 Å. A schematic of the C6-A-C12 monolayer is shown in Figure 1. C14-Monolayer Self-Assembly. ITO and oxidized gold substrates were reacted with C14 in dry acetonitrile, as for the case of C6, using 4-methylmorpholine as a Lewis base (1:50:1, v/v/v) under nitrogen for ∼12 h. The reacted substrates were removed and rinsed with ethyl acetate. Gold-plated silicon slides were then dried under a stream of nitrogen and used for IRRAS and ellipsometric measurements, yielding a film thickness of 18 ( 3.5 Å, consistent with the addition of a monolayer of C14. A schematic of the C14 monolayer is given in Figure 1. HBM Formation. A monolayer of DOPC was transferred from the lipid film at the air-water interface onto the C6-A-C12- or C14-modified substrates (gold and ITO). This transfer process has been described in the literature.17,18 Briefly, the DOPC multilayer film was spread on the surface of an aqueous electrolyte, and then the SAM-coated electrode was dipped through the lipid layer into the electrolyte solution. In the resulting bilayer, the alkyl chains of the C6-A-C12 or C14 SAMs are in contact with the aliphatic chains of the phospholipids, while the polar heads of DOPC remain in contact with the aqueous solution. The driving force for this attachment scheme is the dispersion interaction(s) between the adsorbate aliphatic chains and the DOPC aliphatic chains. The HBM-modified interfaces were used immediately in situ for electrochemical measurements. The formation of the C6-A-C12 and C14 SAMs are depicted schematically in Figure 1. (17) Krysin´ski, P.; Zebrowska, A.; Palys, B.; Lotowski, Z. J. Electrochem. Soc. 2002, 149, E189. (18) Peggion, C.; Gormaggio, F.; Toniolo, C.; Becucci, L.; Moncelli, M. R.; Guidelli, R. Langmuir 2001, 17, 6585.
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Results and Discussion The purpose of this work is to demonstrate the fabrication of interfacial structures on oxide surfaces that can function as biomimetic systems. Our goal is to design interfaces where selected proteins can be incorporated into (not simply deposited onto) the layered structure in such a manner as to retain their function, and our initial effort has been to construct HBMs where the side of the “membrane” bound to a substrate is expected to be fluidlike but somewhat less fluxional than a true lipid bilayer structure. For systems such as this, the evidence for fluidlike properties will be more or less apparent depending on the manner in which the relevant information is gathered. For example, our IR data indicate a fluidlike environment as gauged by band positions and line widths, whereas our time-resolved fluorescence data sense an environment that does not exhibit significant motional freedom on a ∼20-ns time scale. We discuss these data in more detail below. Additional insight into these systems can be gained from electrochemical measurements, and we will detail these data following a discussion of the spectroscopic information. Time-Resolved Anisotropy Measurements. We have bound tethered pyrene chromophores to C6-A-C12 structures, where the tethered pyrene occupies C12 sites. We have studied the transient optical response of tethered chromophores elsewhere,11 and the experiments we report here are similar. The dynamic information about the chromophore motion, which can be related to the organization of the medium surrounding the probe, is obtained from the experimental data through the induced orientational anisotropy function, R(t):
R(t) )
I|(t) - I⊥(t) I|(t) + 2I⊥(t)
(1)
where I|(t) and I⊥(t) are experimental time-domain signal intensities for emissions parallel and perpendicular to the excitation polarization. The functionality of R(t) is determined by the dynamics of the bound chromophores, and we present the experimental data in Figure 2. For chromophores tethered to the substrate, motional freedom is restricted, leading to nonzero steady-state anisotropies. For the C6-A-C12 adlayer, pyrene yields a time-invariant anisotropy, with R(t) ) R(0) ) R(∞) ) -0.2. We understand these data in the context of the hindered-rotor model,19,20 where the chromophore can sweep out a conic volume within the interfacial layer, and the apex of the cone is the point of the chromophore attachment to the adlayer. In this model, the value of R(t) is related to the average angle, θ, that the molecules make with respect to the surface normal.21 The values of R(0) ) R(∞) ) -0.2 indicate that the chromophore π plane is essentially perpendicular to the substrate surface and the angular distribution of chromophore orientations is small. The time invariance of the experimental R(t) data points to the absence of fast chromophore motion within the C6-A-C12 layer is consistent with the adlayer behaving as a viscous medium.11,21 We attribute this apparent rigidity to the relatively wellorganized aliphatic chains surrounding the chromophores, combined with the inability of these structures to exhibit translational diffusion behavior, owing to the covalent linkages binding the adlayer constituents to the substrate. For pyrene bound to the C6 layer, without the addition of the n-dodecylamine layer diluent, we recover R(0) ) R(∞) (19) Szabo, A. J. Chem. Phys. 1984, 81, 150. (20) Lipari, G.; Szabo, A. Biophys. J. 1980, 30, 489. (21) Karpovich, D. S.; Blanchard, G. J. Langmuir 1996, 12, 5522.
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Figure 3. FTIR spectra of the CH stretching region of the adlayers reported here: (a) C6, (b) C14, and (c) C6-A-C12 adlayers. The spectra have been offset for clarity of presentation.
Figure 2. (a) Instrument response function and time-resolved fluorescence-intensity scans for the polarizations perpendicular (top) and parallel (bottom) to the excitation polarization for pyrene tethered to a C6 adlayer and surrounded by C12 aliphatic chains. (b) R(t) data constructed from I|(t) and I⊥(t) scans. Note that R(0) ) -0.20, indicating perpendicular absorption and emission transition moments. The absence of a time dependence of these data points to a relatively rigid environment for the pyrene chromophore.
) -0.14.11 Even though there are no observable dynamics, the smaller (in magnitude) R(∞) value indicates a larger orientational distribution of the chromophore in the absence of the aliphatic moiety. IRRAS. IRRAS can provide steady-state orientational and conformational information on the hydrocarbon chains in the C6-A-C12 and C14 SAMs when they are grown on reflective substrates. The relevant information is contained in the frequencies and relative intensities of the aliphatic-chain methylene stretching vibrations.22-25 The selection rules for IRRAS allow only the modes with dipolar components perpendicular to the surface to contribute to the spectrum, providing a means to evaluate bound aliphatic-chain molecular orientation. Well-ordered, densely packed alkyl chains in n-alkanethiol SAMs on gold have -CH2-(as) and -CH2-(sym) bands at 2918 and 2850 cm-1, respectively, being slightly but significantly (5-9 cm-1) shifted red relative to the spectral features seen for disordered monolayers.17,24,25 The relative intensities of the methylene stretches have been used to obtain quantitative information about alkyl-chain orientation for n-alkanethiols on several coinage metals.9,24-27 For our system, however, such a quantitative analysis is not justified as a result of uncertainty about the spectral contributions from the C6 adlayer that forms ester-like (22) Persson, H. H. J.; Caseri, W. R.; Suter, U. W. Langmuir 2001, 17, 3643. (23) Parikh, A. N.; Gillmor, S. D.; Beers, J. D.; Beardmore, K. M.; Cutts, R. W.; Swanson, B. I. J. Phys. Chem. B 1999, 103, 2850. (24) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (25) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 45. (26) Clegg, R. S.; Hutchison, J. E. J. Am. Chem. Soc. 1999, 121, 5319. (27) Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y. T.; Parikh, A. N.; Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152.
bonds to the substrate and the amide linkage to the n-dodecylamine moiety. In addition, the surface morphology of the oxides we use in this work is not well-understood and, in any event, is less well-organized than that of crystalline coinage-metal surfaces. Despite these limitations, the qualitative evaluation of our IRRAS spectra in comparison to the spectra of alkanethiol SAMs is instructive in evaluating the organization of our monolayer. Parts a-c of Figure 3 show typical IRRAS spectra for the C6 adlayer (Figure 3a), C14 adlayer (Figure 3b), and C6-AC12 adlayer (Figure 3c), all on oxidized gold substrates in the region 2700-3100 cm-1. As is evident from these data, the CH stretching vibrations of the C6 and C6-A-C12 adlayers are most similar to those of liquidlike films (Table 1). The relative intensities of the -CH2-(as) and -CH3(as) stretches in Figure 3b,c and, in particular, the intensity increase of -CH2-(as) suggest at least some ordering in the C14 and C6-A-C12 adlayers. These findings are consistent with our earlier work on DPPE-mercaptopropionamide monolayers on gold, a system that also contains a short side chain that tethers the DPPE monolayer to the metallic substrate.28 We show in Figure 4 the IRRAS data for the C6, C6A-C12, and C14 adlayers in the 800-2000 cm-1 spectral region. These data demonstrate that the C6 underlayer in the C6-A-C12 SAMs are hydrogen-bonded and oriented through their amide linkages, with the CdO and NsH bonds almost parallel to the substrate plane. The diagnostic amide I band (CdO stretch at ∼1668 cm-1) is weak and broad, and the ester resonance (CdO stretch at ∼1741 cm-1) is pronounced for the C6 adlayer and weak for both the C6-A-C12 and the C14 SAMs. Consistent with the orientational dependence of these band intensities, the weak intensities suggest the CdO and NsH bonds lie almost parallel to the substrate plane. We assign the band at ∼1540 cm-1 to the amide II vibration,26 and its relatively high frequency and width are indicative of hydrogen bonding within the C6-A-C12 SAM.26,29 Additionally, the C6 adlayer and C14 SAM are both characterized by a small contribution of unreacted acid chloride, as detected by the CdO resonance at ∼1810 cm-1. The reason for the (28) Krysin´ski, P.; Zebrowska, A.; Michota, A.; Bukowska, J.; Becucci, L.; Monelli, M. R. Langmuir 2001, 17, 3852. (29) Clegg, R. S.; Hutchison, J. E. Langmuir 1996, 12, 5239.
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Table 1. IRRAS Band Positions for the Adlayers Reported Here and Crystalline- and Liquid-Phase Alkanethiols crystalline alkanethiola (cm-1)
liquid alkanethiola (cm-1)
C6 adlayer (cm-1)
C6-A-C12 adlayer (cm-1)
C14 adlayer (cm-1)
-CH2-(as)
2918
2924
2924
2924
-CH2-(sym)
2851
2855
2854
2854
-CH3(as)
2956
2957
2924 (broad) 2855 (broad) n/ab
2961
2959
band
From ref 24. The -CH3(as) vibration, noticeable for the adipoyl adlayer at about 2962 for the adlayer assembly. a
b
Figure 4. FTIR spectra of the 1000-2000-cm-1 spectral region of the adlayers reported here: (a) C6, (b) C14, and (c) C6-A-C12 adlayers. The spectra have been offset for clarity of presentation.
survival of this reactive moiety is not clear at present, but it is possible that the adlayers provide a moderately efficient barrier to the penetration of water through to the substrate. The spectroscopic data, both time-resolved and steadystate, show that the growth of the C6-A-C12 and C14 adlayers is characterized by significant order, but not on a par with that seen for the alkanethiol monolayers on gold. For the C6-A-C12 adlayer, it appears that H bonding between amide moieties within the adlayer may account for some of the organization we observe. For a C14 adlayer, we recover a thickness of 18 ( 3.5 Å; for the C6-A-C12 adlayer, the thickness is 25 ( 5 Å; and for the C6 adlayer, we measure 8.5 ( 1.5 Å.12 These values are fully consistent with the literature values for the C12 moiety (16.5 Å) and C14 moiety (19.5 Å), as measured for the corresponding alkanethiol monolayers using capacitance measurements.18,24,30,31 With the spectroscopic characterization of these adlayers as a basis, we turn to the consideration of their electrochemical behavior. In addition, for the electrochemical measurements, we are able to evaluate the properties of a hybrid lipid bilayer formed on the covalently bound adlayers discussed above. Mott-Schottky Analysis. For the systems we report here, it is possible to gain additional insight into the nature of the adlayer bonding to the surface by taking advantage of the fact that ITO is a semiconductor. We apply a MottSchottky analysis to our data to detect changes in the flatband potential, Ufb, of the ITO semiconductor electrode. (30) Krysin´ski, P.; Moncelli, M. R.; Tadini-Buoninsegni, F. Electrochim. Acta 2000, 45, 1885. (31) Plant, A. L. Langmuir 1999, 15, 5128.
cm-1,
is probably due to impurities in C6 used
The band potential near the surface of a semiconductor is mediated by surface states, resulting in “band bending”. The covalent attachment of a C6 adlayer should change the nature and density of ITO surface states as a result of the formation of new covalent, ester-like bonds. This type of effect has been reported for ITO and silanized ITO surfaces.32-34 We measured the dependence of the spacecharge capacitance, Csc, on the applied potential for bare ITO electrodes and ITO with C14 and C6-A-C12 adlayers bound covalently. Using the treatment reported before,32-34 we plot Csc-2 versus the applied potential. For our experimental conditions, Csc is the primary capacitive component for the bare ITO electrode (the electrical doublelayer capacitance is at least 1 order of magnitude larger than Csc). For the measurements we report here, the Csc values were taken directly from the out-of-phase component of the ac voltammetry data. The best way to acquire Csc information for C14- and C6-A-C12-modified ITO surfaces is from impedance spectroscopy measurements. The analysis of impedance data in terms of appropriate equivalent circuits usually leads to capacitance and resistance values of the circuit elements used, allowing the separation of the space-charge capacitance and monolayer capacitance. In the low-frequency regime, the change in the overall impedance of the ITO-monolayer interface is not correlated with the adlayer impedance but is attributed to the decrease of the space-charge capacitance.32-34 This approach holds particularly true when the adlayers grown on the ITO surface do not form compact monomolecular films but leave uncovered areas on the surface of the semiconductor, limiting the effect of adlayer capacitance. This situation is obtained in our experiments, where we observe a capacitance drop from about 41 µF/cm2 for the bare ITO surface to about 10 µF/ cm2 for the monomolecular-adlayer-covered ITO. The expected value for a highly organized alkanethiol monolayer of comparable thickness is about 1.14 µF/cm2.30 Taking into account the above considerations, we performed our ac voltammetry with phase separation experiments in the low-frequency regime (ω ) 1 Hz). Assuming that the monolayer capacitance is constant over the potential range we investigated (vide infra), the dependence of the interfacial capacitance on the applied potential will be dominated by changes in the space-charge capacitance. Figure 5 shows the Mott-Schottky plots of a bare ITO electrode (Figure 5a), an ITO electrode with a C14 adlayer (Figure 5b), and an ITO electrode with a C6A-C12 adlayer (Figure 5c). Extrapolation of the linear region of the plot of C-2 versus the potential to the C-2 ) 0 intercept yields the approximate value of Ufb. The flatband-potential value of -0.58 V estimated for the bare ITO surface (Figure 5a) is in good agreement with the (32) Hillebrandt, H.; Tanaka, M. J. Phys. Chem. B 2001, 105, 4270. (33) Hillebrandt, H.; Wiegand, G.; Tanaka, M.; Sackmann, E. Langmuir 1999, 15, 8451. (34) Gritsch, S.; Nollert, P.; Jahnig, F.; Sackmann, E. Langmuir 1998, 14, 3118.
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Figure 5. Mott-Schottky plots of the relationship between the surface-charge capacitance (Csc-2) and the potential: (a) bare ITO electrode, (b) ITO electrode with a C14 adlayer, and (c) ITO electrode with a C6-A-C12 adlayer.
literature data.32 The self-assembly of either the C14 (Ufb ) -0.51 V) or the C6-A-C12 (Ufb ) -0.50 V) adlayers shifts the Ufb value toward more positive potentials by about 70 mV, suggesting that the surface-state distribution has been altered by the covalent attachment of the adlayers to the surface hydroxyl groups. The similarity of the Ufb potential shifts is expected because the same chemistry is used for the binding of both adlayers to the electrode surfaces. A positive shift of similar magnitude was reported for alkylsiloxane monolayers bound to ITO.32 Because the formal charge of the adlayer constituents is 0, the electrostatic effects on the flatband potential can be excluded. Capacitance Characteristics and Blocking Properties of C6-A-C12 SAMs and HBMs (C6-A-C12/ DPPE HBMs. The use of gold and ITO substrates covered either with C6-A-C12 or C14 SAMs as working electrodes in electrochemical experiments can provide insight into the structure and organization of the adlayers. The capacitance of the interfacial region at the metal electrode is a useful measure of the adlayer. The extent to which the measured capacitance fits the predictions of the Helmholtz parallel-plate-capacitor model provides a measure of the alkyl-chain organization within the adlayer. This interpretation cannot be applied to the ITO electrode as a result of the significant contribution of the spacecharge capacitance, Csc. If the adlayer is well-organized and impermeable to the electrolyte, the measured monomolecular-adlayer capacitance, Cm, is small and can be related directly to the monolayer thickness, d, by the parallel-plate-capacitance equation
Cm ) 0S/d
(2)
where S is the electrode area, 0 is the dielectric constant of the vacuum, and is the apparent dielectric constant of the adlayer. If the adlayer contains defects, such as disordered chains or pinholes, the electrolyte will penetrate the film, yielding a capacitance value larger than that predicted by the model.26,31,35,36 The capacitance charac(35) Peng, Z.; Tang, J.; Han, X.; Wang, E.; Dong, S. Langmuir 2002, 18, 4834.
Figure 6. Capacitance as a function of applied potential for the adlayer structures indicated by each curve.
teristics of the C6-A-C12 and C14 adlayers on gold are derived from the ac voltammetric measurements in Figure 6. These data are the capacitance-versus-voltage curves for the adlayers deposited on gold electrodes in 0.5 M KCl. We have also deposited DOPC layers onto the C6-A-C12and C14-adlayer structures, and we show these data for comparison in Figure 6. We conclude from these latter data that all of the films formed are stable within the experimental potential window. We note the nascent parabolic shape of the capacitance-versus-potential curves for the C6-A-C12 and C14 adlayers, suggesting either potential-dependent changes in the aliphatic chain organization or electrostriction operating within these structures.35,37-41 In light of this interpretation, the application of the Mott-Schottky model to the analysis of our data should be considered qualitative in nature. In comparing both the C6-A-C12 and C14 adlayers, one should note that the capacitance value for the C6-A-C12 adlayer, with Cm ) 1.22 µF/cm2, corresponds more closely to the value expected for a defect-free monolayer of densely packed alkanethiols of comparable length (for dodecanethiol on gold, Cm ) 1.14 µF/cm2)30 than that for the C14 adlayer (Cm ) 2.66 µF/cm2). This finding implies that the C6-A-C12 adlayer is characterized by less permeability than the C14 adlayer, owing to the presence of the H-bonding region within the adlayer, which serves to “seal” the surface. These data show also the quality of the C6A-C12 monolayer compared to the similar-length, amidecontaining alkanethiol SAMs,26 where a monolayer capacitance as high as 5.9 µF/cm2 was obtained. The quality of the adlayer bound directly to the substrate is also reflected in the behavior of the HBMs formed on their surfaces. The value of CHBM ) 0.65 µF/cm2, obtained from the flat capacitance-versus-voltage curve of the C6A-C12/DOPC HBM (Figure 6), lies within the range of (36) Finklea, H. O. In Electroanalytical Chemistry: A Series of Advances; Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1996; Vol. 19, p 109. (37) Mirsky, V. M. TrAC, Trends Anal. Chem. 2002, 21, 439. (38) Horswell, S. L.; Zamlynny, V.; Li, H.-Q.; Merrill, A. R.; Lipkowski, J. Faraday Discuss. 2002, 121, 405. (39) Schoenfisch, M. H.; Pemberton, J. E. Langmuir 1999, 15, 509. (40) Byloos, M.; Al-Maznai, H.; Morin, M. J. Phys. Chem. B 2001, 105, 5900. (41) Anderson, M. R.; Gatin, M. Langmuir 1994, 10, 1638.
Amphiphilic Biomimetic Assemblies
Figure 7. (a) CVs, in order of decreasing maximum current density, of the bare gold electrode, gold electrode coated with a C14 adlayer, and gold electrode coated with a C14/DOPC hybrid bilayer structure. The redox probe is 1 mM K4Fe(CN)6, and the scan rate is 0.1 V/s. (b) CVs of the bare ITO electrode and ITO electrode coated with a C14/DOPC hybrid bilayer structure. The redox probe is 1 mM K4Fe(CN)6, and the scan rate is 0.1 V/s. The CVs for ITO coated with a C14 adlayer were qualitatively similar to those obtained for gold coated with a C14 adlayer and are not shown.
0.5-1 µF/cm2 reported for bilayers tethered to gold through hydrophilic spacers42-44 and compares favorably with the capacitance value of a solvent-free lipid bilayer membrane (CBLM ) 0.8 µF/cm2).45 The use of the Helmholtz model with two capacitors in series to represent the HBM structure allows for the evaluation of the capacitance of the outer DOPC monolayer. The value we recover (CDOPC ) 1.6 µF/cm2) is in good agreement with the value of a DOPC monolayer densely packed on mercury and with that of other HBM systems.28,46,47 Cyclic Voltammetry. As a result of the molecular length scale over which electron-transfer and chargeseparation processes operate, the presence or absence of defects and pinholes can be examined using dynamic techniques, such as cyclic voltammetry or ac voltammetry. Well-ordered layers, free of structural defects, block electron transfer efficiently, even for kinetically facile redox probes present in solution. The systematic analysis of cyclic voltammograms (CVs) often allows for differentiation among the possible mechanisms of electron transfer at monolayer-modified electrodes.29,36,48 For wellordered, defect-free films on the electrode surface, the CV (42) Raguse, B.; Braach-Maksvytis, V.; Cornell, B. A.; King, L. G.; Osman, P. D. J.; Pace, R. J.; Wieczorek, L. Langmuir 1998, 14, 648. (43) Steinem, C.; Janshoff, A.; von dem Bruch, K.; Reihs, K.; Goosens, J.; Galla, H. J. Bioelectrochem. Bioenerg. 1998, 45, 17. (44) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197. (45) Montal, M.; Meueller, P. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 3651. (46) Moncelli, M. R.; Becucci, L.; Nelson, A.; Guidelli, R. Biophys. J. 1996, 70, 2716. (47) Nelson, A.; Benton, A. J. Electroanal. Chem. 1986, 202, 253. (48) Zhang, L.; Vidu, R.; Waring, A. J.; Lehrer, R. I.; Longo, M. L.; Stroeve, P. Langmuir 2002, 18, 1318.
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Figure 8. CVs, in order of decreasing maximum current density, of the bare gold electrode, gold electrode coated with a C6-A-C12 adlayer (dashed line), and gold electrode coated with a C6-A-C12/DOPC hybrid bilayer structure. The redox probe is 1mM K4Fe(CN)6, and the scan rate is 0.1 V/s.
curve exhibits a nearly exponential current-potential dependence, reflecting a through-chain mechanism of electron transfer across the adlayer. However, spherical diffusion-limited electron transfer, indicative of pinhole defects acting as a microelectrode array, yields a sigmoidal CV curve. Figure 7 shows the CVs of C14/DOPC hybrid bilayers on gold (Figure 7a) and ITO (Figure 7b). Also shown in Figure 7 are the CV curves for the bare electrodes under the same conditions. As is evident from the data, both HBM-covered electrodes show that, even though the Faradaic current is almost 2 orders of magnitude smaller than that for bare electrodes, a significant contribution from radial-diffusion-limited electron transfer from the kinetically facile, hydrophilic K4[Fe(CN)6] redox probe is apparent. Similar behavior is also seen in the CV curves of C6-A-C12/DOPC hybrid bilayers on gold (Figure 8). The radial-diffusion-limited electron-transfer behavior is more pronounced for the C6-A-C12 and C14 adlayers without the addition of the DOPC layer (data not shown), suggesting the presence of widely spaced pinhole defects large enough [>5 Å radius estimated for Fe(CN)64-] to allow the close approach of the redox probe to the electrode surface for direct electron transfer.16,29,36 We believe that these defects originate from our use of relatively short adlayer constituents and possibly because of nonuniformity in the substrate oxide layer upon which the adlayers are covalently bound. It is known that the gold oxide layer can be discontinuous,49 which can introduce point defects and grain boundaries that are not readily accessible to covalent bonding with reactive adlayer constituents. The availability of such sites to the redox-active probe is also (49) Ron, H.; Matlis, S.; Rubinstein, I. Langmuir 1998, 14, 1116.
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an issue, but given the size of the Fe(CN)64- molecule, we expect some interactions to occur, even at sterically restricted defect sites where the adsorbate molecules were unable to follow the rough topology of the surface during the self-assembly. Regardless of the details of these defects, the addition of a DOPC layer to the adlayer seals these defects to a significant extent, indicating the formation of a relatively well-ordered HBM, with the underlayer being covalently bound to either metal oxide or semiconductor substrates using the same chemistry. Through careful control of the hydrated oxide layer and better selection of the acid chloride chain length, we anticipate even better adlayer organization and more favorable electrochemical characteristics for this family of HBM films. Conclusions We report a new alternative to metal-supported alkanethiol or oxide-supported alkylsilane structures for the construction of layered assemblies on electrochemically active supports, allowing for the facile modification of a variety of substrates. The adlayer structures we report here can be utilized to form HBM assemblies, with structures that can be chosen to accommodate various biofunctional molecules in their active forms. We anticipate that the actual incorporation of transmembrane proteins will require the establishment of a hydrophilic region next to the conductive substrate somewhat larger than that which we present here, and we intend to explore the structural aspects of this issue in future work. The permeability of the bilayer films bound to polycrystalline, electrochemically oxidized gold and ITO electrodes were evaluated experimentally to assess their potential use as biomimetic matrixes. The covalently bound adlayers were found to exhibit IR spectral features characteristic of a
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fluidlike environment but were seen to be devoid of motion on the nanosecond time scale, as measured by timeresolved anisotropy data for tethered pyrene. These pieces of information can be reconciled in the context of the adlayers behaving as a viscous fluid medium. These same systems were characterized by electrochemical behavior consistent with the presence of pinhole defects, and the addition of a DOPC adlayer enhanced the blocking effect for Fe(CN)64- electrooxidation, pointing to substantial organization within the hybrid bilayer structure, with some defects remaining. We anticipate that the use of longer aliphatic chains in the adlayer(s) bound covalently to the electrode surfaces will increase the quality of these films and improve the electrochemical response accordingly. We have also shown that films such as those we report here can be tailored to provide hydrophobic and hydrophilic regions stacked at predetermined intervals along the surface normal; this is a structural prerequisite for hosting biomolecules such as membrane integral proteins and peptides in their active forms. The covalent binding of adlayers to either gold or ITO substrates provides these assemblies with sufficient mechanical stability to allow them to be both electronically and spectroscopically addressable and compatible with current sensor transduction technology. Acknowledgment. We are grateful to NATO and the National Science Foundation for support of this work through Grant 0090864. Partial support of P.K. by Grant 3T09A 117 19 from the Polish Committee for Scientific Research is also acknowledged. LA026946Z
