Electrochemistry at Ultrathin Organic Films at Planar Gold Electrodes

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Langmuir 1996, 12, 3305-3314

3305

Electrochemistry at Ultrathin Organic Films at Planar Gold Electrodes Britta Lindholm-Sethson Department of Analytical Chemistry, Umeå University, S-901 87 Umeå, Sweden Received November 13, 1995X Electrochemical impedance measurements were performed on two different molecular assemblies that were created in order to mimic living cell membranes. In the first, a bare gold electrode surface was used as a support for Langmuir-Blodgett transfers of mono-, bi-, and multilayers of dipalmitoylphosphatidic acid. In the second, a thin polyelectrolyte film was self-assembled on the gold surface prior to the LangmuirBlodgett transfer. A small membrane resistivity, i.e. 100-300 Ω cm2, was observed across the phospholipid bilayer when deposited on the polyelectrolyte surface provided the outermost layer was polyanionic. The contribution to the total membrane capacitance from one monolayer in these assemblies was 1.16 µF cm-2. Similar results for the membrane capacitance were obtained in multilayer assemblies of more than five monolayers when the support was a bare gold electrode surface, whereas thinner multilayer assemblies displayed significantly higher capacitances. Furthermore, the main contribution to the membrane resistance in the latter case was shown to originate from resistances in defect pores, through which the double-layer capacitances at the ends and inside these defects were charged.

1. Introduction The functional matrix of biological membranes is the ordered bilayer of amphiphiles, which constitutes an effective barrier for water solubles on the two sides of the membrane. The efficiency as a barrier to osmotic forces and/or charge transfer resides in the stability of the lipiphilic core of the bilayer. Artificial bilayer lipid membranes (BLMs) can be formed by painting a lipid solution across an aperture in a hydrophobic septum that separates two aqueous phases. This was accomplished earlier for instance by Mueller et al., who used a voltage clamp method to demonstrate variations in the dielectric properties of the artificial membrane caused by spontaneous adsorption of various water-soluble macromolecules.1 Impedance spectroscopy has long been recognized as a tool to investigate conductive properties of artificial membranes and has been used by all authors mentioned below. Accordingly it has been shown that these unsupported BLMs exhibit very high resistivities, i.e. typically 109 Ω cm2, and specific capacitances in the range 0.4-0.6 µF cm-2,2 but they are probably only useful as model systems, since their lifetimes are often shorter than a few hours.3-10 BLMs may also be formed, with increased stability, on solid supports by various techniques. Most of the early papers report studies conducted in air or vacuum,11,12 but an exception is provided by the work of Buchwald et al., X

Abstract published in Advance ACS Abstracts, June 1, 1996.

(1) Mueller, P.; Rudin, D. O.; Tien, H. T.; Wescott, W. C. Nature 1962, 194, 979-980. (2) Hanai, T.; Haydon, D. A.; Taylor, J. J. Theor. Biol. 1965, 9, 422432. (3) Castillo, J. D.; Rodriguez, A.; Romero, C. A.; Sanchez, V. Science 1966, 153, 185-188. (4) Levie, R. D.; Vukadin, D. J. Electroanal. Chem. 1975, 62, 95109. (5) Levie, R. D.; Thomas, J. W.; Abbey, K. M. J. Electroanal. Chem. 1975, 62, 111-125. (6) Coster, H. G. L.; Smith, J. R. Biochim. Biophys. Acta 1974, 373, 151-164. (7) Ashcroft, R. G.; Coster, H. G. L.; Smith, J. R. Biochim. Biophys. Acta 1981, 643, 191-204. (8) Ashcroft, R. G.; Coster, H. G. L.; Laver, D. R.; Smith, J. R. Biochim. Biophys. Acta 1983, 730, 231-238. (9) Ashcroft, R. G.; Coster, H. G. L.; Smith, J. R. Biochim. Biophys. Acta 1977, 469, 13-22. (10) Zimmerman, U.; Ashcroft, R. G.; Coster, H. G. L.; Smith, J. R. Biochim. Biophys. Acta 1977, 469, 23-32.

S0743-7463(95)01026-2 CCC: $12.00

who studied multilayers of Langmuir-Blodgett films of barium stearate on chromium electrodes in dilute copper sulfate solutions.13 Hongyo and co-workers produced a supported lipid membrane simply by painting a phospholipid/cholesterol solution across an agar filled hole,14 and later Tien et al. demonstrated a simple self-assembly technique for the preparation of phospholipid bilayers on solid substrates.15 Furthermore, Stelzle et al. used the impedance technique to study the tightness and long-term stability of supported LB layers of cadmium arachidate on chromium electrodes in a chloride medium16 and in a later paper examined a supported bilayer deposited by vesicle fusion on a hydrophilic glass/gold electrode covered with a monolayer of carboxy mercaptan.17 In both cases resistivities were reported for the deposited membrane that were several orders of magnitude smaller than those for unsupported BLMs. Fare and co-workers have performed ac-impedance studies on both lipid and fatty acid films transferred with the LB technique at high surface pressures onto platinum electrodes. The resistivities of these films were reported to be within an order of magnitude of those of the BLMs.18 Later the same group deposited multilayers of phospholipid and valinomycin on platinum electrodes, and a decrease in the film impedance was observed as potassium was added to the subphase.19 Furthermore, admittance changes were observed in platinum-supported phospholipid bilayers containing voltage dependent anion channels (VDACs) that were caused by channel gating.20 (11) Mann, B.; Kuhn, H. J. Appl. Phys. 1971, 42, 4398-4405. (12) Procarione, W. L.; Kauffman, J. W. Chem. Phys. Lipids 1974, 12, 251-260. (13) Buchwald, C. E.; Gallagher, D. M.; Haskins, C. P.; Thatcher, E. M.; Zahl, P. A. Proc. Natl. Acad. Sci. USA 1938, 24, 204-208. (14) Hongyo, K.-I.; Joseph, J.; Huber, R. J.; Janata, J. Langmuir 1987, 3, 827-830. (15) Tien, H. T.; Salamon, Z. Bioelectrochem. Bioenerg. 1989, 22, 211-218. (16) Stelzle, M.; Sackmann, E. Biochim. Biophys. Acta 1989, 981, 135-142. (17) Stelzle, M.; Weismu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (18) Fare, T. L. Langmuir 1990, 6, 1172-1179. (19) Fare, T. L.; Rusin, K. M.; Bey, J. Sens. Actuators, B 1991, 3, 51-62.

© 1996 American Chemical Society

3306 Langmuir, Vol. 12, No. 13, 1996

In some of the articles cited above the authors have ignored the possibility of interfering faradaic reactions, in spite of the fact that neither chromium nor platinum electrodes are inert in a chloride medium. The charging current of a membrane capacitance is very small, and consequently the contribution from charge transfer resistances and redox capacitances linked to faradaic processes at the electrode surface might be a severe interference. Plant et al. report resistivities close to the values of unsupported lipid membranes for supported bilayers created with vesicle fusion on alkanethiol coated planar gold electrode surfaces. However, it cannot be excluded that the observed resistance is highly affected by the charge transfer resistance for the reduction of water that probably occurs at the imposed potential, -0.55 V vs SCE, where the impedance measurements were performed. Surprisingly, the authors claim that they observe the charge transfer resistance of the ferro/ferricyanide couple that is present in the electrolyte, although the potential was almost 800 mV negative of the reversible electrode potential of the couple. At this potential only ferrocyanide is stable and the heterogeneous charge transfer cannot be studied with impedance techniques. Further, the charge transfer resistance of any electrochemical reaction at a membrane-coated electrode is not equivalent to the resistance of the membrane itself, as is indicated in the article. The conclusions drawn concerning the size of the membrane resistance are therefore probably incorrect.21 The importance of avoiding faradaic reactions in the construction of admittance sensors was pointed out early by Miller et al.22 They report a K+ sensor based on the admittance change in synthetic bilayer membranes containing tetradecyl-18-crown-6 supported on Si/SiO2 electrodes. The electrodes were polarized negatively to the inversion region of the n-doped semiconductor where they behave as inert metal electrodes. A Ca2+ sensor was constructed in a similar way, and the specific response of the membrane was based on changes in the imaginary admittance: i.e. a quantity related to the membrane capacitance.23 In a later report the same group describes an impedance spectroscopy study on lipid monolayers containing different amounts of GM1 on alkanethiol-coated gold surfaces where small impedance changes were induced by the adsorption of cholera toxin.24 Thus, the introduction of supported BLMs leads to more stable membranes but with lower impedances than those of unsupported systems. Excessive “fixation”, however, may interfere with the “self-mending” capacity of membranes, which is a natural consequence of the hydrophobic-hydrophilic interplay at the boundary between two water phases. Accordingly, it has been suggested that, in order to function correctly, an artificial membrane should be surrounded on both sides with an aqueous phase. On the basis of this discussion, Vogel and co-workers developed a new class of lipid molecules, the so-called thiolipids. Thus, a lipid bilayer can be coupled to a gold surface with an aqueous layer between the first monolayer and the electrode. The impedance data for these molecular aggregates show an almost pure capacitive behavior comparable to the capacitance values of unsupported, socalled black-lipid membranes. However, the reported (20) Stenger, D. A.; Fare, T. L.; Cribbs, D. H.; Rusin, K. M. Biosens. Bioelectron. 1992, 7, 11-20. (21) Plant, A. L.; Gueguetcherkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126-1133. (22) Miller, C.; Cuendet, P.; Gra¨tzel, M. J. Electroanal. Chem. 1990, 278, 175-192. (23) Terrettaz, S.; Vogel, H.; Gra¨tzel, M. J. Electroanal. Chem. 1992, 326, 161-176. (24) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361-1369.

Lindholm-Sethson

Figure 1. Schematic picture of the gold electrode surface modified with a thin polyelectrolyte film and a phospholipid bilayer deposited on the polyelectrolyte surface. The picture is not to scale.

membrane resistance across the bilayer was 10 -100 kΩ, which is far from the value obtained for an unsupported BLM.25,26 The somewhat contradictory results in the literature have motivated us to perform impedance measurements on Langmuir-Blodgett films based on mono-, bi-, and multilayers of dipalmitoylphoshatidic acid. In this paper we demonstrate how impedance data can be evaluated so that different charging processes at a modified electrode surface can be distinguished from one another. In order to create a supported BLM with aqueous phases on both sides, a gold electrode surface was modified with a thin polyelectrolyte film by alternate adsorption of polycations and polyanions (Figure 1). A small membrane resistivity, i.e. 100-300 Ω cm2, was observed across the phospholipid bilayer and multilayers deposited on the polyelectrolyte surface, provided the outermost layer was polyanionic. The contribution to the total membrane capacitance from one monolayer in these assemblies was 1.16 µF cm-2. When the support was a bare gold surface, however, multilayer assemblies with less than five layers displayed significantly higher capacitance values, whereas good agreement with the former case was obtained for thicker multilayers. Furthermore, the main contribution to the membrane resistance in the latter case was shown to originate from resistances in defect pores, through which the double-layer capacitances at the ends and inside these defects were charged. 2. Principles and Basic Concepts in Impedance Spectroscopy When a material is subjected to a small-amplitude alternating voltage, v(t) ) ∆E sin ωt, the total electrical response, i(t) ) ∆i sin(ωt + θ) is characterized by the different time constants of the relaxation processes that are prevalent. ∆E is the amplitude of the perturbing alternating voltage, ω is the angular frequency, ∆i is the amplitude of the resulting alternating current, and θ is the phase shift. The impedance is defined as Z* ) v(t)/ i(t), which is a vector quantity having both magnitude and direction. It is therefore convenient to use complex notation to illustrate the features of the impedance, i.e. Z * ) Z′ + jZ′′, where j2 ) -1, Z′ is resistance, and Z′′ is a component related to the capacitance, i.e. the reactance. Moreover, it is often helpful to derive other quantities (25) Lang, H.; Duschl, C.; Gra¨tzel, M.; Vogel, H. Thin Solid Films 1992, 210/211, 818-821. (26) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210.

Electrochemistry at Ultrathin Organic Films

that will highlight other properties more relevant for the particular system under study. The admittance is the inverse of the impedance; Y* ) Z*-1 ) Y′ + jY′′, and by means of this transformation of data, relaxation processes in the high-frequency range are accentuated. The real part of the admittance reflects the frequency independent conductivity of the material while the imaginary part is frequency dependent and due to dielectric relaxation, i.e. the susceptance. Further, the complex relative permittivity is defined as * ) Y*/jωCc ) ′ + j′′, where Cc is the capacitance of the empty cell. This transformation emphasizes relaxation processes in the low-frequency range, and the real part of * is equivalent to the dielectric “constant” of the material although ′ is only frequency independent in the low-frequency range. The imaginary part, ′′, is often referred to as the dielectric loss and is the frequency dependent part of the conductivity. Instead of the complex permittivity, the related concept complex capacitance, i.e. C* ) *Cc, is frequently preferred and is used throughout this paper. A fourth quantity is called the modulus function, M* ) *-1.27 In order to elucidate the advantages which can be obtained from these different representations, we will discuss some simple equivalent circuits that are relevant to the present work. Since we assume no faradaic reactions, all the suggested equivalent circuits must reflect a blocking electrode system; i.e., there should be no conductive path. An inert electrode/electrolyte interface is frequently modeled by an equivalent circuit consisting of a series RC circuit, where R ) Rs is the solution resistance and C ) Cdl is the double-layer capacitance. The impedance of such an interface is given by

Langmuir, Vol. 12, No. 13, 1996 3307

a

b

Figure 2. Simulated response for the equivalent circuit in the inset of part (a) CPE ) A(jω)-n. In all cases Rs ) 300 Ω. (o) CPE ) 1/((10 × 10-6)jω); (0) CPE ) 1/((11.5 × 10-6)(jω)0.97); (4) CPE ) 1/((13.0 × 10-6)(jω)0.94). (b) Complex impedance plot. (c) Complex capacitance plot.

a

Z ) Rs + 1/jωCdl which yields a vertical line in the complex impedance plane. In real systems the capacitive line always exhibits less than the theoretical slope, even in the absence of faradaic or adsorption background reactions at the electrode surface. An improved fit to experimental data is obtained when the ideal double-layer capacitance is replaced by a so-called constant phase element, CPE: Z ) A(jω)-n, which is originally an empirical formula. The choice of the electrode material and the properties of the electrolyte affect the value of A whereas the frequency exponent is mainly sensitive to the surface structure and can be related to the fractal dimensions of the electrode surface.28,29 When n ) 1, the CPE transforms into an ideal double-layer capacitance, whereas an increasing deviation from 1 indicates an increasing surface roughness. The simulated response is shown in Figure 2. Note that as n is decreasing the slope of the capacitive line in the complex impedance plane is deviating more and more from the ideal vertical line, and the depression of the semicircles in the capacitance planes is increasing. Furthermore, at low frequencies the imaginary capacitance does not approach zero which, in a real experiment, would indicate a slow prevailing faradaic process. In this case, it originates from an anomaly in the constant phase element at extreme frequencies.30 In Figure 3a an equivalent circuit is proposed for a supported lipid membrane located on an electrode surface which is covered with a thin polyelectrolyte film. The parallel RC circuit represents the membrane which is in (27) Impedance Spectroscopy; Macdonald, J. R., Ed.; John Wiley & Sons: New York, 1987. (28) Bates, J. B.; Chu, Y. T. Ann. Biomed. Eng. 1992, 20, 349-362. (29) Levie, R. D. J. Electroanal. Chem. 1989, 261, 1-9. (30) Impedance Spectroscopy; Macdonald, J. R., Ed.; John Wiley & Sons: New York, 1987; pp 40 and 91.

b

c

Figure 3. Simulated response for the equivalent circuit in part a. In all cases Rs ) 300 Ω; CM ) 0.6 × 10-6 µF; CPE ) 1/((13.0 × 10-6)(jω)0.94). (O) RM ) 0 Ω; (0) RM ) 100 Ω; (]) RM ) 500 Ω; (4) RM ) 2000 Ω. (b) Susceptance vs log f. (c) Complex impedance plane.

series with a constant phase element, i.e. a nonideal double-layer capacitance, and the solution resistance, Rs.

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We therefore expect two charging processes, one of which constitutes charging of the double-layer capacitance and the other the charging of the membrane. The two different relaxation processes are clearly seen when the imaginary admittance is plotted as a function of the frequency, even when the membrane resistance is small (Figure 3b). Charging of the membrane occurs at a significantly smaller time constant than charging of the large doublelayer capacitance, and consequently the latter dominates in the low-frequency part of the impedance spectrum. In the high-frequency regime a semicircle is obtained with a diameter that equals the membrane resistance (Figure 3c). For the case of phospholipid multilayers transferred directly onto gold electrode surfaces, we suggest a similar equivalent circuit to that proposed for polymer protective coatings on metal surfaces by Armstrong (Figure 4a).31 A spreading of the electrolyte beneath the coating is assumed, and two charging processes are also expected. One of them constitutes charging of the phospholipid membrane and the other charging of double-layer capacitances. As the pore resistance, R1, increases, a difference in the time constants of these charging processes develops, which is also clearly demonstrated in the simulated response in Figure 4b and c. When R1 is small, the charging process of the double-layer capacitances in the electrolyte film at the electrode surface dominates the electrical response at all frequencies. In this case a depressed semicircle is obtained in the complex capacitance plane with a diameter that is equal to the sum of the parallel capacitances, i.e. Cm + 6C1. However, as R1 increases, the major charging process at high and medium frequencies becomes the charging of the membrane capacitance, which is observed as a semicircle in the complex capacitance plane, whereas charging of 6C1 is shifted to lower frequencies.

Lindholm-Sethson

a

b

3. Experimental Section 3.1. Chemicals and Instrumentation. Dipalmitoylphosphatidic acid (DPPA), HPLC grade chloroform, and polyallylamine hydrochloride (PAH; Mw ) 50 000-65 000) were obtained from Aldrich. Ca(N03)2, p.a., was purchased from Merck, polystyrenesulfonate, sodium salt (Na-PSS) was obtained from Scientific Polymer Products, Inc., and aminoethanethiol (>98%) was obtained from Fluka Chemie. All chemicals were used without further purification. A monolayer spreading solution (ca 4 mM) was prepared fresh every day by weighing and dissolving DPPA in chloroform. Ultrapure water (F > 18.4 MΩ/ cm) was obtained by running house deionized water through a Milli-Q Plus water purification system (Millipore) and was used for the preparation of the subphase and in all cleaning procedures. All LB work was carried out in a double-trough commercially available from KSV instruments, KSV 5000ALT. The surface pressure was monitored with a 20 mm wide Wilhelmy plate made of platinum. Impedance spectroscopy was performed with a Solartron 1250 frequency response analyzer together with a Solartron 1286 electrochemical interface. The experiments were carried out in the trough at room temperature, 20-22 °C, with the calomel reference electrode placed inside a specially designed compartment, where the 1 M Ca(N03)2 inner solution was separated from the subphase solution with a Nafion membrane. The impedance measurements were performed with the working electrode facing the Nafion membrane at a separation of a few millimeters. An auxiliary electrode consisting of a platinum wire was arranged in order to obtain a homogeneous current density at the working electrode. The Nafion membrane serves two purposes, both of which depend on its cation-exchange properties. Firstly, mixing of the two Ca(N03)2 solutions of different concentrations is prevented, and secondly, any leakage of chloride ions from the calomel electrode into the subphase is effectively hindered by Donnan (31) Armstrong, R. D.; Wright, D. Electrochim. Acta 1993, 38, 17991801.

c

Figure 4. Simulated response for the equivalent circuit in part a. In all cases Rs ) 300 Ω; CM ) 0.6 µF; C1 ) 1.0 µF. (O) R1 ) 0 Ω; (0) R1 ) 100 Ω; (]) R1 ) 2000 Ω; (4) R1 ) 10 000 Ω; l (b) R1 ) 100 000 Ω. (b) Phase shift, Φ, vs log f. (c) Complex capacitance plot. exclusion. The presence of chloride ions in the subphase might cause increasing faradaic currents which would severely disturb the impedance measurements. This is also the reason for using a chloride-free supporting electrolyte in the trough. Gold electrodes were chosen as substrates because of their excellent electrochemical properties as compared with, e.g., platinum. Gold is, however, easily contaminated. Thin gold film electrodes (thickness ∼ 1000 Å and electrode area either 0.64 or 0.50 cm2 connected with a 1 mm wide strip to a contact pad) were

Electrochemistry at Ultrathin Organic Films deposited by electron gun evaporation in ultrahigh vacuum onto glass slides using (3-mercaptopropyl)trimethoxysilane as a molecular glue between glass and gold.32 3.2. Buildup of Polyelectrolyte Film. The procedure employed for preparing an ultrathin polyelectrolyte film essentially follows the original work of Decher et al.33 and requires initial functionalization of the bare gold electrode surface to give it a positive charge. The gold electrode was cleaned in a boiling mixture of ethanol and chloroform 1:1 for one minute, rinsed thoroughly with water, dried in an argon stream, and then immersed for about 30 min into a 5 mM aminoethanethiol/ethanol solution. A self-assembled monolayer was obtained on the gold electrode surface, which carries a positive charge in neutral and acid solutions. The electrode was then immersed for 20 min at 0 °C into a 9.8 mM Na-PSS aqueous solution (18 mM H2SO4 + 0.5 M Na2SO4), whereupon the anionic polyelectrolyte was adsorbed onto the positively charged surface. Subsequently multilayers of polyelectrolyte could be formed on the electrode surface by alternate dipping of the electrode into aqueous solutions of anionic and cationic polyelectrolytes. The anionic solution in these studies was 9.8 mM NaPSS in 0.5 M Na2SO4, and the cationic solution was 21.4 mM PAH in 18 mM H2SO4. In order to avoid an uneven film assembly, the PAH-solution was prepared by dissolution of the polymer into 9 mL of pure water and thereafter 1 mL of 0.18 M H2SO4 was added drop by drop into the polymer solution under vigorous stirring. The immersion time was always 20 min, and the adsorption was carried out at room temperature. It has been shown that small adjustments in the acid concentration of the PAH adsorption solution have a dramatic effect on the average thickness of each pair of layers. Thus, for a sulfuric acid concentration of 9 mM, the average thickness of each pair of layers was determined with ellipsometry to be 18 Å which is in good agreement with the findings of Decher et al. for a similar system.34 When the concentration was raised to 18 mM (as in this work), the solution became milky and the average thickness of each pair of layers increased to 110 Å.35 3.3. LB Technique and Deposition of DPPA Multilayer. The Langmuir trough was thoroughly cleaned on an everyday basis. This comprised soaking both the trough and barrier in 96% ethanol, wiping with Techni-Cloth texwipes, and rinsing for at least 20 min in ultrapure water. Hereafter the trough was placed in a laminar flow hood and filled with 10 mM Ca(N03)2. Then the electrode was mounted in the dipper arm and the cleanliness of the surface of the subphase was established by repeatedly sweeping the barriers back and forth. When the surface area was at a minimum, surface active contaminants were aspirated away with a capillary. The cleaning procedure was not terminated until the change in surface pressure observed was less than 0.1 mN/m upon compression from maximum to minimum area. At this point the electrode was lowered into the subphase ensuring that the whole electrode area and approximately 3 mm of the thin strip connecting the electrode area and the contact pad were wetted. The first impedance spectrum was then recorded. Two different types of multilayer LB films of DPPA were created on the gold electrodes for subsequent impedance measurements. In one of them, the hydrophobic carbon tails were facing the electrode surface, whereas, in the other type, the first monolayer was transferred head down. In all the LB transfers described below the transfer rate was always 5 mm/min. In the first case, only bare gold electrodes were used. Firstly, the electrode was withdrawn from the subphase and the cleanliness of the air/water interface was checked again as described above. The monolayer was spread at the air/water interface by micropipetting a precise amount of the DPPA spreading solution to a mean molecular area of ∼0.7 nm2. After a resting period of 20-30 min, in order to allow for evaporation of the chloroform, the monolayer was compressed to the target surface pressure of 45 mN/m at a rate of ∼0.01 nm2/(molecule min). To ensure equilibration of the monolayer, a relaxation period of 30-45 min was allowed at this surface pressure. (32) Goss, C. A.; Charych, D. H.; Majda, M. Anal. Chem. 1991, 63, 85-88. (33) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210/ 211, 831-835. (34) Decher, G.; Schmitt, J. Progr. Colloid Polym. Sci. 1992, 89, 160164. (35) Han, S.; Lindholm-Sethson, B. In preparation.

Langmuir, Vol. 12, No. 13, 1996 3309 The first monolayer was then transferred onto the electrode at a downstroke. The transfer ratio was 0.3-0.4 for this first transfer, which indicates that only the gold electrode surface was covered with the monolayer. After a resting period of 5 min to allow an impedance spectrum to be recorded, a second transfer was accomplished on an upstroke. The transfer ratio was now close to unity, indicating that the monolayer was transferred intact onto the whole surface of the solid support with the head down. A drying period of 30-45 min was found to be essential in order to avoid the return of the second monolayer to the air/ water interface on the subsequent downstroke. For all successive transfers, a new monolayer was added to the electrode surface with a transfer ratio of one. The resting period was now only 10 min in the upper position and 5 min in the lower position. Thus a multilayer system could be produced with the first layer tail down on the electrode surface. In the second case, we employed either bare gold electrodes or electrodes covered with seven or eight layers of polyelectrolyte obtained by successive adsorption of PSS and PAH as described above. The substrate was not withdrawn from the subphase after the first impedance spectrum was recorded. Instead the monolayer was spread and compressed as already described with the substrate still immersed in the subphase. The first transfer was therefore performed on an upstroke. The transfer ratio was always close to unity, which indicates that the complete monolayer was transferred to the substrate head down. A prolonged drying period of at least 30 min was employed after the first transfer of the monolayers, and a shorter period was adopted in the subsequent transfers. In this way a multilayer system could be obtained, in which the head groups in the innermost monolayer were facing the gold electrode surface or the polyelectrolyte film. 3.4. Impedance Measurements. Impedance measurements were carried out in the frequency range 65 kHz to 100 mHz with an amplitude of 10 mV. The measurements were done at two static potentials, i.e. 200 and 400 mV vs the reference electrode compartment. Since a membrane potential of 65 mV evolves across the Nafion membrane, the potential at the working electrode is referred to as 135 and 335 mV vs SCE, respectively, in the following discussion. No significant difference was obtained in the response at the two different potentials, which leads us to conclude that no faradaic process dominates the electrode response. Measurements were performed in situ in the trough, firstly at bare or polyelectrolyte-covered electrodes and then as successively more phospholipid layers were transferred to the electrode surface. Thus, in the first type of multilayers, impedance measurements could be accomplished with an increasing number of odd layers whereas, in the second type, measurements were made at electrodes with an increasing number of even layers. The data were analyzed either graphically from a suitable complex plane plot or by executing complex nonlinear least squares (CNLS) fitting (LEVM636 ) to an appropriate equivalent circuit. This allows detailed analyses of the dielectric properties of the electrode/electrolyte interfacial region to be obtained as more and more layers are added to the multilayer systems.

4. Results and Discussion 4.1. Bare Gold Electrodes and Gold Electrodes Covered with Several Layers of Polyelectrolyte Film. In Figure 5a and b results from impedance measurements at a bare gold electrode are shown both in the complex impedance plane and in the complex capacitance plane. An almost straight capacitive line is displayed in the impedance plane and in the complex capacitance plane a somewhat depressed semicircle is observed. Note that the imaginary capacitance does not approach zero at low frequencies, which indicates an ongoing slow faradaic process. This leads us to the equivalent circuit in the inset in Figure 5a, which is identical with the inset in Figure 2a except for the addition of the geometrical capacitance Cg. In this case the anomaly in the constant phase element at extreme frequencies (36) Impedance Spectroscopy; Macdonald, J. R., Ed.; John Wiley & Sons: New York, 1987; pp 180.

3310 Langmuir, Vol. 12, No. 13, 1996

Lindholm-Sethson

Figure 5. Results from impedance measurements on a bare gold electrode (A ) 0.5 cm2) in 10 mM Ca(NO3)2 at 135 mV vs SCE. The solid line in part b indicates the results from a CNLSfit with LEVM6. Rs was fixed to 145 Ω cm2, and the fit yielded A-1 ) (14.5 ( 0.3) × 10-6 (µF/cm2)0.94 s0.6, n ) 0.94 ( 0.003, and Cg ) 2.6 ( 0.3 nF/cm2. The parameters used in the fit were (IRE, MAXFEV, IRCH) ) (-9, 91, 2).

allows for compensation of a slow faradaic process. In Figure 5b, the results from a CNLS fit with Rs fixed to 145 Ω cm2 are displayed. In order to translate the impedance data into more straightforward physical parameters, an ideal RC circuit is assumed for the highfrequency part of the spectrum. The double-layer capacitance is now calculated from Cdl ) 2 Re[C]max and was estimated to be 12.2 µF cm-2, which is reasonable for a gold electrode that has been immersed in a supporting electrolyte for a prolonged time.37 Values at lower frequencies that are subject to frequency dispersion because of the rough electrode surface are hereby excluded from the analysis. The results from impedance measurements on electrodes covered with seven or eight layers of polyelectrolyte are identical with measurements on bare electrodes, which indicates that the ionic conductivity is high in the polyelectrolyte film. 4.2. Multilayer Films of DPPA on Bare Gold Electrodes. Results from impedance measurements on planar gold electrodes covered with an increasing number of dipalmitoylphosphatidic acid layers are shown in Figures 6 and 7 for odd and even number of layers, respectively. Straight capacitive lines are observed in both cases in the complex impedance plane, where the absolute value of the imaginary impedance is increasing faster the more layers are transferred to the electrode surface (Figures 6a and 7a). A simple approximation of the dielectric properties of the interface is suggested by several authors as a series RC circuit. Now, C ) CM is the membrane capacitance and R ) RS the solution resistance, which is easily obtained graphically from the extrapolated intercept with the real axis.22,25,26 This choice of equivalent circuit implies that the membrane resistance is assumed to approach infinity and that the double-layer capacitance between the membrane and the electrode is large compared with the membrane capacitance. Hence, (37) Swietlow, A. Doctoral Dissertation Thesis, University of Lund, 1994, p 51.

Figure 6. Results from impedance measurements at 135 mV vs SCE in 10 mM Ca(NO3)2 on a planar gold electrode (A ) 0.5 cm2) covered with an increasing odd number of DPPA layers. (a) The frequency 10 kHz is indicated in the graph with a filled symbol. (b and c) The numbers refer to the total number of DPPA layers deposited on the gold electrode surface.

a transformation of the impedance data into the complex capacitance plane is expected to yield a semicircle with a diameter that is equivalent with CM. Indeed, single semicircles are observed in all complex capacitance plots from measurements on electrodes with an odd number of layers of phospholipids in the multilayer on the electrode surface, as can be seen in Figure 6b and c. The diameter of the semicircle is observed to decrease as the number of phospholipid layers increases. A somewhat different picture is seen, when the impedance data stems from measurements on electrodes with an even number of phospholipid layers on the electrode surface. The complex capacitance plot for an electrode with two layers of DPPA transferred to its surface shows two superimposed semicircles, where only the high-frequency semicircle is complete, as can be seen in Figure 7b and c. In the former case the hydrophobic carbon tails are facing the electrode surface and therefore a hydrophobic milieu

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Figure 8. Total membrane capacitance, CM, as a function of 1/n. The multilayer assembly consists of n layers of DPPA at a bare gold electrode surface. The slope of the line through the origin is 1.20 µF cm-2/layer.

Figure 7. Results from impedance measurements at 135 mV vs SCE in 10 mM Ca(NO3)2 on a planar gold electrode (A ) 0.5 cm2) covered with an increasing even number of DPPA layers. (a) The frequency 10 kHz is indicated in the graph with a filled symbol. (b and c) The numbers refer to the total number of DPPA layers deposited on the gold electrode surface.

is created that restricts the development of a water film at the electrode surface beneath the phospholipid multilayer. In the latter case, however, the hydrophilic head groups of the phospholipid are facing the electrode surface and the existence of a thin water film at the electrode surface cannot be excluded. In both cases the equivalent circuit in Figure 4a can be used as a basis for a qualitative discussion of the results. The occurrence of a single semicircle in the complex capacitance plane for all assemblies with an odd number of DPPA layers on the electrode surface indicates that R1 is always rather high. Thus, the high-frequency semicircle represents charging of the membrane capacitance through the solution resistance. In the case of an even number of DPPA layers on the electrode surface the observation of overlapping semicircles in the complex capacitance plane for two layers, and to some extent also for four layers, indicates that the pore resistances are smaller than in the previous case. We propose that the semicircle at high

frequencies corresponds with charging of the membrane capacitance, CM, of the bilayer through the solution resistance whereas the other, that is not complete, is attributed to charging of the double-layer capacitance in the water film beneath the deposited layer through the defects in the film. As more layers are transferred to the electrode surface, the contribution from the defects becomes smaller and single semicircles are again obtained in the complex capacitance plot for six and eight layers of DPPA. Furthermore, in many cases it was not possible to obtain multilayer films with the head groups facing the gold electrode surface where the resistance in the defect pores was large enough to allow for investigation of the dielectric properties of the artificial membrane. In those cases the impedance response at all frequencies was dominated by charging of the double-layer capacitance in the thin water film at the electrode surface. Only when several layers of DPPA were transferred to the electrode surface could the charging of the membrane capacitance be clearly discriminated from other charging processes. The double-layer capacitance is never an ideal capacitance because of surface roughness, and the impedance of the defect pores within the membrane is probably also subject to frequency dispersion. Therefore we conclude that an equivalent circuit that would envisage the structure of the interface in detail is very complicated, and a CNLS fit would probably give an ambiguous result and is therefore not pursued. Instead, the equivalent circuit in Figure 3a is reduced to a simple RC circuit by taking advantage of the observation that the time constants of the two prevalent charging processes in most cases are substantially different. CM is now estimated from the high-frequency semicircle, with the same graphical method as is suggested in the preceding paragraph. A linear relationship between CM and 1/n is expected if a series connection of n equivalent monolayer capacitances is assumed. This is also observed in the case of multilayers exceeding five phospholipid monolayers (Figure 8). When the capacitance for a single monolayer of phosphatidic acid is estimated from the slope of that line, we obtain CMONO ) 1.20 µF cm-2, from several independent measurements, which value is in good agreement with literature values on the specific capacitance of black-lipid membranes. The deviation from the linear slope in the CM vs 1/n plot for multilayers containing just a few monolayers is not surprising, since the phospholipid film is deposited on a polycrystalline gold electrode surface. The first transferred layers of DPPA are therefore not organized according to the ideal structures that are always found in textbooks on Langmuir-Blodgett films. The disorder and loose packing of the phospholipid is reflected in a higher specific capacitance in these first layers than for the well-organized DPPA monolayers later in the transfer sequence. When the first layer is transferred head down, this disorder is greater than when the carbon

3312 Langmuir, Vol. 12, No. 13, 1996

tails are facing the electrode surface, which is reflected in small or nonexistent pore resistances. 4.3. Multilayer Films of DPPA on PolyelectrolyteCovered Gold Electrodes. A thin polyelectrolyte film with a positively charged outermost layer was obtained on the electrode surface by alternate self-assembly of eight layers of polyelectrolyte, since in this case PAH is facing the electrolyte. However, although multilayers of DPPA could be successfully transferred to this surface with a transfer ratio of one, there was no detectable change in the impedance data even for 10 transferred monolayers of DPPA. We believe that the Ca2+ ions that bind the phospholipid monolayers together are exchanged for the cations at the surface of the polyelectrolyte layer. We suggest that this causes a distortion in the first transferred monolayer that induces defects in all consecutive layers which prevent the development of a membrane resistance. Since the membrane is leaky, no charging of the membrane capacitance is possible. With seven layers of the polyelectrolyte self-assembled on the electrode surface, a negative outermost layer is obtained, since in this case PSS is adjacent to the electrolyte. Hence, when a bilayer of DPPA was transferred to the surface, a small distorted semicircle was always observed in the high-frequency range in the complex impedance plane. We suggest that in this case the first monolayer is connected to the negatively charged polyelectrolyte with a calcium link according to the discussion by Stelzle et al.17 The impedance plane plot from measurements on an electrode modified with seven layers of polyelectrolyte and with an increasing number of layers of DPPA is shown in Figure 9a. In Figure 9b the imaginary admittance, i.e. the electric susceptance, is plotted as a function of the applied frequency and the two charging processes discussed in section 2 are clearly seen. We therefore propose an equivalent circuit that is almost identical with Figure 3a (see inset in Figure 9a), where a CPE represents the nonideal double-layer capacitance connected in series with a parallel RC circuit which corresponds to an idealized model of the phospholipid membrane. Furthermore, Rs is the solution resistance and Cg the geometrical capacitance of the cell. Firstly, CPE and Cg were determined with CNLS fitting of impedance data obtained from measurements on the polyelectrolyte film-covered gold electrode prior to the deposition of DPPA. Then, CPE and Cg were fixed at these values for subsequent fits of the data from all measurements with DPPA transferred to the electrode surface. Likewise, Rs was estimated from the intercept with the real axis in the complex impedance plane and kept constant in the fitting procedure. The numerical results from two different experiments are summarized in Table 1. Note that the membrane resistance does not always increase as more layers of DPPA are transferred to the polyelectrolyte surface. The highest resistivity obtained across a bilayer of DPPA resting on the negatively charged polyelectrolyte layer was 300 Ω cm2, which is much smaller than the values reported for black-lipid membranes. It is also much smaller than the literature values on membrane resistivities from supported membranes. However, in the present work it is obvious that the reported membrane resistance is linked to the membrane itself and not to charge transfer resistances or charging processes of double-layer capacitances at the electrode surface at the bottom of defect channels. The solid line in Figure 9 represents results from the CNLS fit of measurements on a polyelectrolyte-covered gold electrode with 10 layers of DPPA transferred to its surface. There is good qualitative agreement between the experimental data and the fitted curve. Thus in the simulated curve a small semicircle is observed in the high-

Lindholm-Sethson

Figure 9. Results from impedance measurements at 135 mV vs SCE in 10 mM Ca(NO3)2 on a planar gold electrode (A ) 0.64 cm2). The electrode was covered first with seven layers of selfassembled polyelectrolyte and then with an increasing even number of DPPA layers. (a) Complex impedance plane plot; insert shows the proposed equivalent circuit. (b) Susceptance vs log f. The numbers refer to the total number of DPPA layers, and the symbols in both figures are corresponding. The solid line shows the result from CNLS fitting of the data from measurements on the 10-layer DPPA assembly. See Table 1 for details.

frequency region in the complex impedance plane, and in the complex admittance plane two charging processes are observed. The size of the small semicircle and the time constants of the two charging processes are similar to those of the real experiment. However, no account has been taken for relaxation processes with different time constants. This is obviously a gross simplification and may be the reason for the discrepancy between the fitted curve and the experimental data. As has already been pointed out, it is always possible to suggest an equivalent circuit that allows perfect fitting to experimental data. Thus, an exchange of the membrane capacitance for a constant phase element, for instance, will allow for different time constants and also improves the fitting over the whole frequency range. However, the refined fitting prohibits a straightforward physical interpretation of the data, and the fitting procedure is therefore not extended in this direction. In Figure 10, the membrane capacitance from table 1 is plotted against 1/n. It is found that, for all multilayer assemblies, CM is inversely proportional to the number of layers of DPPA at the polyelectrolyte surface. This indicates that the first bilayer is also well organized. The contribution from a single monolayer was estimated to be 1.16 µF cm-2, which is in excellent agreement with the results from measurements on multilayers of DPPA

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Table 1. Results from CNLS Fitting to Data Obtained from Impedance Measurements at 135 mV vs SCE in 10 mM Ca(NO3)2 on a Planar Gold Electrode (A ) 0.64 cm2)a number E/mV of vs layers SCE 2 4 6 8 10

135 335 135 335 135 335 135 335 135 335

CM1/µF cm-2

RM1/Ω cm2

0.543 ( 0.009 0.570 ( 0.013 0.280 ( 0.004 0.283 ( 0.006 0.191 ( 0.003 0.195 ( 0.005 0.147 ( 0.003 0.146 ( 0.004 0.117 ( 0.002 0.118 ( 0.003

78.1 ( 0.5 54.9 ( 0.5 116 ( 1 110 ( 1 143 ( 1 136 ( 2 172 ( 2 171 ( 2 206 ( 2 200 ( 2

CM2/µF cm-2

RM2/Ω cm2

0.580 ( 0.007 0.586 ( 0.008 0.297 ( 0.013 0.301 ( 0.005 0.202 ( 0.01 0.206 ( 0.013

302 ( 2 289 ( 2 316 ( 8 268 ( 2 277 ( 7 257 ( 8

a The electrode was covered first with seven layers of selfassembled polyelectrolyte and then with an increasing even number of DPPA layers. The equivalent circuit employed in the fit is shown in the inset of Figure 9a. CMi and RMi are the membrane capacitances and resistances for two different experiments, respectively. CPEi, Cg,i, and Rs,i were all determined separately and kept constant during the CNLS fit. Measurements at 135 mV vs SCE: CPE1 ) 1/[((17.56 ( 0.04) × 10-6)(jω)(0.966(0.006)]; Cg,1 ) 1.33 ( 0.06 nF cm-2; Rs,1 ) 200 Ω cm2; CPE2 ) 1/[((15.87 ( 0.05) × 10-6)(jω)(0.961 ( 0.007)]; Cg,2 ) 3.12 ( 0.10 nF cm-2; Rs,2 ) 156 Ω cm2. Measurements at 335 mV vs SCE: CPE1 ) 1/[((19.33 ( 0.05) × 10-6)(jω)(0.961(0.006)]; Cg,1 ) 1.35 ( 0.06 nF cm-2; Rs,1 ) 200 Ω cm2; CPE2 ) 1/[((17.12 ( 0.05) × 10-6)(jω)(0.961(0.007)]; Cg,2 ) 3.18 ( 0.09 nF cm-2; Rs,2 ) 156 Ω cm2. The parameters used in the CNLS fit with LEVM6 were (IRE, MAXFEV, IRCH, ROE) ) (-9, 91, 3, 0.3).

Figure 11. Time dependence of a DPPA bilayer on a gold electrode surface covered with seven layers of polyelectrolyte. Results from impedance measurements every 15 min at 135 mV vs SCE in 10 mM Ca(NO3)2; electrode surface, A ) 0.64 cm2. (a) Complex impedance plot. (b) Susceptance vs log f. Figure 10. Total membrane capacitance, CM, as a function 1/n. The multilayer assembly consists of n layers of DPPA on a gold electrode surface modified with seven layers of polyelectrolyte. The slope of the line through the origin is 1.16 µF cm-2/layer.

transferred to bare gold electrode surfaces. These results underline the adequacy of the suggested simple equivalent circuit and suggest that a refined fit is probably not worthwhile. 4.4. Stability of a Bilayer of DPPA Deposited on a Negatively Charged Polyelectrolyte Surface. Impedance spectra were generally recorded directly after transfer. In order to check the stability of a bilayer transferred to a negatively-charged polyelectrolyte surface, impedance spectra at 135 mV vs SCE were initiated every 15 min, starting as soon as possible after the transfer was completed and pursued for 90 min. The results from these measurements are summarized in Figure 11, and it is clear that the stability of this particular type of artificial cell membrane is poor. During the experiment the capacitance of the bilayer increases from 0.63 to 0.76 µF cm-2 and the membrane resistivity decreases from 85 to 20 Ω cm2. Both these observations are consistent with a slow degradation of the membrane by a gradual opening of aqueous channels. 5. Conclusions and Future Aspects There were three main objectives with the present paper. Per primo, we wanted to investigate the meaning of the resistances and capacitances of artificial membranes

on solid supports. Per secundo, we wanted to emphasize the advantages of using various transformations of impedance data when interpreting different relaxation processes at the modified electrode surface. Per tertio, we wanted to apply these procedures to two different molecular assemblies on gold electrode surfaces. To do so, multilayers of dipalmitoylphosphatidic acid on gold electrode surfaces were created with LangmuirBlodgett techniques. In the first case the phospholipid monolayers were transferred onto the bare electrode. It was found that defects in the transferred film gave a dominating contribution to the membrane resistance and thus the resistance in a defect-free membrane could not be determined. Moreover, the specific capacitances of the first layers are higher than expected from measurements on black-lipid membranes and are also quite irreproducible. This implies that the first transferred monolayers do not comprise an ordered structure. For multilayer assemblies thicker than five layers a linear relation exists between the inverse of the number of transferred phospholipid monolayers and the total capacitance. The contribution from one monolayer was determined to be 1.20 µF cm-2 in several independent measurements. In another experiment, a thin polyelectrolyte was formed on the electrode surface by alternate adsorption of polycations and polyanions prior to the LB transfer of DPPA monolayers. In this case the charge on the outermost layer in the polyelectrolyte film appeared to be of critical importance. When a cationic polyelectrolyte is facing the head groups of the first transferred phospholipid monolayer, no detectable change in the impedance spectra

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was obtained even though multilayer assemblies of up to 10 layers were investigated. With an anionic polyelectrolyte in the outermost layer a small membrane resistivity of 100-300 Ω cm2 develops which allows determination of the membrane capacitance. The contribution to the membrane capacitance from a single monolayer of DPPA was found to be 1.16 µF cm-2 for all multilayer assemblies containing two or more transferred phospholipid monolayers. This indicates that a well-organized structure is formed on the surface of the polyelectrolyte. In Conclusion. The present paper demonstrates how the response from impedance measurements on artificial membranes on solid supports can be translated into physically meaningful parameters that describe their dielectric properties. The suggested method facilitates recognition of resistances and capacitances in defect pores and charge transfer resistances. These interferences in impedance analyses of supported membranes have not been discussed in detail in the literature before.

Lindholm-Sethson

At present, our membrane preparations on the polyelectrolyte film rapidly deteriorate. The combined effect of the phase transition temperature of DPPA and the stabilizing effect of Ca2+ on the self-mending properties of the bilayer will be the subject of future studies. We want to investigate the possibility to create a fluid lipid bilayer on a solid support with a Langmuir-Blodgett technique that would be suited for protein incorporation. Acknowledgment. The author wishes to thank Dr Åke Sellstro¨m at the National Defence Establishment in Umeå for valuable suggestions and for providing space in his laboratory, Mrs. Sue Han for preparation of the polyelectrolyte-coated electrodes, and Prof. Michael Sharp for linguistic checking and fruitful discussions. The Swedish Natural Science Foundation is also acknowledged for financial support. LA951026K