pubs.acs.org/Langmuir © 2009 American Chemical Society
Influence of an Electric Field on Oriented Films of DMPC/Gramicidin Bilayers: A Circular Dichroism Study J. B. Fiche,†,‡ T. Laredo,†,‡ O. Tanchak,†,‡ J. Lipkowski,*,† J. R. Dutcher,‡ and R. Y. Yada§ †
Department of Chemistry, ‡Department of Physics and §Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada Received June 29, 2009. Revised Manuscript Received August 7, 2009
A film of oriented bilayers containing a mixture of gramicidin and dimyristoylphosphatidylcholine (DMPC) has been deposited on a fused-silica window coated with a 10 nm thick gold layer. The thin layer of gold allows the application of an electric potential across the film and the study of its influence on the structure and integrity of the bilayers. Electrochemical measurements, ellipsometry, and far-UV circular dichroism (CD) were employed to characterize the properties of the film of bilayers as a function of the potential applied to the gold electrode. For potentials across the film that are within the range ∼þ300 to -150 mV the oriented film of bilayers is stable, and no change in the CD spectra of gramicidin molecule is observed. At more negative potentials, an increase in the film thickness and water content measured by ellipsometry indicated that the film swells and incorporates water, which causes a change in the circular dichroism spectrum of gramicidin molecules in the film. This transformation was interpreted as a change in the average orientation of gramicidin molecules within the film due to a decrease in the ordering of the molecules upon swelling.
Introduction A cell membrane is a complex environment in which phospholipid bilayers serve as the base structure for a wide range of other molecules such as cholesterol, proteins, and oligosaccharides. Phospholipids and cholesterol provide a hydrophobic barrier between the intra- and extracellular media while transmembrane proteins allow for selective permeability of ions across the membrane. The selective permeability of ions and the presence of different surface charge densities on the two sides of the membrane result in a transmembrane potential.1,2 This potential imposes an electric field which is of the order of 107-108 V/cm on molecules embedded in the membrane, and this field plays a key role in biological processes such as ATP synthesis.2,3 Solid-supported lipid bilayers have long been used as biomimetic models4 to study the properties of natural membranes and to understand the mechanisms underlying lipid/lipid-lipid/protein interactions.5,6 When a bilayer is supported on a conductive substrate, the potential can be applied across this membrane by charging the substrate. This model provides a convenient means to study the influence of the transmembrane potential on the properties of the bilayer and transmembrane proteins.1,7 On conductive supports, a broad range of surface techniques such as atomic force microscopy (AFM),8-10 scanning tunnelling *To whom correspondence should be addressed. (1) Olivotto, M.; Arcangeli, A.; Carla, M.; Wanke, E. Bioessays 1996, 18, 495. (2) Tsong, T. Y.; Astumian, R. D. Annu. Rev. Physiol. 1988, 50, 273. (3) Dimroth, P.; von Ballmoos, C.; Meier, T. EMBO Rep. 2006, 7, 276. (4) Sackmann, E. Science 1996, 271, 43. (5) Andersen, O. S.; Koeppe, R. E. Annu. Rev. Biophys. Biomol. Struct. 2007, 36, 107. (6) McIntosh, T. J.; Simon, S. A. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 177. (7) Burgess, I.; Li, M.; Horswell, S. L.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Satija, S. Biophys. J. 2004, 86, 1763. (8) Butt, H. J.; Cappella, B.; Kappl, M. Surf. Sci. Rep. 2005, 59, 1. (9) Chen, M.; Li, M.; Brosseau, C. L.; Lipkowski, J. Langmuir. 2008, 25, 1028. (10) Milhiet, P. E.; Gubellini, F.; Berquand, A.; Dosset, P.; Rigaud, J. L.; Le Grimellec, C.; Levy, D. Biophys. J. 2006, 91, 3268. (11) Sek, S.; Laredo, T.; Dutcher, J. R.; Lipkowski, J. J. Am. Chem. Soc. 2009, 131, 119.
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microscopy (STM),11,12 and photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS)13-15 can be applied to study the effects of the electric field at the molecular level. Lipid bilayers with incorporated ion channels, when supported on a conductive substrate, can also be used for the development of new biosensors acting as ion-to-electron converters.16,17 The objective of the present work is to apply circular dichroism (CD) to investigate the influence of the static electric field on the conformation and orientation of peptide molecules embedded in a film of oriented bilayers. CD is an optical technique which is based on the differential absorption of left- and right-circularly polarized light by optically active molecules.18-20 It has been widely used to study protein secondary structure conformations (β-sheet, R-helix, etc.) in the far-UV range, where the peptide bonds are the main chromophore and absorb at wavelengths below 240 nm.19,20 Additionally, tryptophan and phenylalanine also contribute to the CD spectrum of a protein in the range between 260 and 320 nm. The CD spectrum is dependent on the conformation of the protein, and many algorithms have been developed to determine the secondary structure of proteins from far-UV CD spectra.21,22 (12) Xu, S.; Szymanski, G.; Lipkowski, J. J. Am. Chem. Soc. 2004, 126, 12276. (13) Bin, X.; Horswell, S. L.; Lipkowski, J. Biophys. J. 2005, 89, 592. (14) Garcia-Araez, N.; Brosseau, C. L.; Rodriguez, P.; Lipkowski, J. Langmuir 2006, 22, 10365. (15) Laredo, T. Electric Field Driven Changes in the Orientation and Conformation of Gramicidin in a Lipid Bilayer Supported on a Au(111) Electrode; University of Guelph: Guelph, 2009. (16) Andersson, M.; Keizer, H. M.; Zhu, C.; Fine, D.; Dodabalapur, A.; Duran, R. S. Langmuir 2007, 23, 2924. (17) Bernards, D. A.; Malliaras, G. G.; Toombes, G. E. S.; Gruner, S. M. Appl. Phys. Lett. 2006, 89, 053505. (18) Bulheller, B. M.; Rodger, A.; Hirst, J. D. Phys. Chem. Chem. Phys. 2007, 9, 2020. (19) Condon, E. U. Rev. Mod. Phys. 1937, 9, 432. (20) Fasman, G. D. Circular Dichroism and the Conformational Analysis of Biomolecules; Kluwer Academic Publishers: Dordrecht, 1996. (21) Kelly, S. M.; Jess, T. J.; Price, N. C. Biochim. Biophys. Acta 2005, 1751, 119. (22) Whitmore, L.; Wallace, B. A. Biopolymers 2008, 89, 392.
Published on Web 08/31/2009
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The CD spectra of proteins in oriented samples are different from spectra of randomly oriented proteins in solutions. The effect of sample orientation has been thoroughly described for gramicidin,23,24 alamethicin,25,26 and cytochrome c oxidase.27 In the case of the R-helix conformation, the effect of the protein orientation can be described quantitatively using models based on Moffit’s exciton theory.20,25 Consequently, the orientations of various R-helical peptides embedded in lipid bilayers have been studied as a function of temperature, hydration, and lipid/peptide ratio.26,28,29 These studies led to the development of the oriented circular dichroism method (OCD).26 Despite the lack of proper theoretical background, OCD has also been applied to other secondary structures such as β-sheets.30,31 However, in that case, great care must be taken to differentiate between a change in the protein orientation and conformation. In the present study, a film of oriented bilayers was supported on a thin gold layer sputter-coated onto a fused silica window. Recently, two groups demonstrated the feasibility of using circular dichroism to study the properties of a monolayer of proteins assembled on thin gold layers.32,33 In these papers, gold was used to prepare oriented monolayers of proteins by selfassembly of thiolated molecules. In the present study, the gold film was charged in order to apply an electric field to the bilayers. For the model peptide to be imbedded into the bilayers, gramicidin D, which is a widely studied antibiotic peptide that exerts its activity by forming ion channels across bacterial membranes,34,35 was chosen. Gramicidin D (GD) contains ∼85% gramicidin A and a mixture of gramicidin B and C. Gramicidin A is a short 15-residue hydrophobic peptide with an alternating L-D-amino acid sequence which allows the molecule to fold as a helix: formylL-Val1-Gly2-L-Ala3-D-Leu4-L-Ala5-D-Val6-L-Val7-D-Val8-L-Trp9D-Leu10-L-Trp11-D-Leu12-L-Trp13-D-Leu14-L-Trp15-ethanolamine. In gramicidin B tryptophane-11 is replaced by phenylalanine and in gramicidin C by tyrosine. Gramicidin displays several conformations, depending on its surrounding environment as well as its preparation history.36-40 Its two main conformations are the helical dimer (HD) and the double helix (DH), respectively.34 The first forms conducting channels in lipid bilayers when two single-stranded (SS) β6.3helixes are joined in the center of the bilayer by six hydrogen bonds into a right-handed β6.3-helical dimer.41,42 The second is (23) Harroun, T. A.; Heller, W. T.; Weiss, T. M.; Yang, L.; Huang, H. W. Biophys. J. 1999, 76, 937. (24) Huang, H. W.; Olah, G. A. Biophys. J. 1987, 51, 989. (25) Olah, G. A.; Huang, H. W. J. Chem. Phys. 1988, 89, 2531. (26) Wu, Y.; Huang, H. W.; Olah, G. A. Biophys. J. 1990, 57, 797. (27) Bazzi, M. D.; Woody, R. W. Biophys. J. 1985, 48, 957. (28) B€urck, J.; Roth, S.; Wadhwani, P.; Afonin, S.; Kanithasen, N.; Strandberg, E.; Ulrich, A. S. Biophys. J. 2008, 95, 3872. (29) Clayton, A. H. A.; Sawyer, W. H. Biochim. Biophys. Acta 2000, 1467, 124. (30) Heller, W. T.; Waring, A. J.; Lehrer, R. I.; Huang, H. W. Biochemistry 1998, 37, 17331. (31) Weiss, T. M.; Yang, L.; Ding, L.; Waring, A. J.; Lehrer, R. I.; Huang, H. W. Biochemistry 2002, 41, 10070. (32) Keegan, N.; Wright, N. G.; Lakey, J. H. Angew. Chem. 2005, 44, 4801. (33) Shimizu, M.; Kobayashi, K.; Morii, H.; Mitsui, K.; Knoll, W.; Nagamune, T. Biochem. Biophys. Res. Commun. 2003, 310, 606. (34) Andersen, O. S.; Koeppe, R. E.; Roux, B. IEEE Trans. Nanobiosci. 2005, 4, 10. (35) Wallace, B. A. Bioessays 2000, 22, 227. (36) Bouchard, M.; Auger, M. Biophys. J. 1993, 65, 2484. (37) Chen, Y.; Wallace, B. A. Biopolymers 1997, 42, 771. (38) Kelkar, D. A.; Chattopadhyay, A. Biochim. Biophys. Acta 2007, 1768, 1103. (39) Killian, J. A.; Prasad, K. U.; Hains, D.; Urry, D. W. Biochemistry 1988, 27, 4848. (40) Killian, J. A.; Urry, D. W. Biochemistry 1988, 27, 7295. (41) Davis, R. W.; Patrick, E. L.; Meyer, L. A.; Ortiz, T. P.; Marshall, J. A.; Keller, D. J.; Brozik, S. M.; Brozik, J. A. J. Phys. Chem. B 2004, 108, 15364. (42) Koeppe Ii, R. E.; Providence, L. L.; Greathouse, D. V.; Heitz, F.; Trudelle, Y.; Purdie, N.; Andersen, O. S. Proteins 1992, 12, 49.
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usually found in organic solvents and consists of two gramicidin peptides configured in a double β-helix which is stabilized by 18 intramolecular hydrogen bonds.35 Various DH structures have been identified and characterized, depending on the nature of the solvent used to dissolve the peptide.37,43 Double helices are also observed in lipid bilayers depending on the solvent used for the sample preparation.36,39,40,44 However, as long as the hydrophobic mismatch between the lipid and gramicidin molecules is not too strong,38,45-47 the SS helix remains the thermodynamically stable conformation.39,48,49 Our goal is to apply OCD in combination with electrochemical and ellipsometry measurements to determine the effect on the conformation and orientation of the gramicidin molecules of the electric field applied to the stack of ∼10-15 bilayers. This film was sufficiently thin to ensure a proper control of the potential drop across the film and sufficiently thick to allow measurements of the CD spectra with sufficient S/N ratio.
Materials and Methods Gramicidin D peptide (from Bacillus brevis) and trifluoroethanol (TFE) solvent were purchased from Sigma-Aldrich. 1,2Dimyristoyl-sn-glycero-3-phosphocholine (DMPC, purity higher than 99.9%) was purchased from Avanti Polar Lipids. Sodium fluoride (purity higher than 99.99%) was purchased from EMD Chemicals Inc. Before each experiment, the temperature was brought to 19 C. Consequently, all measurements were performed at 19 ( 1 C. Circular Dichroism Experiments. CD spectra were recorded on a Jasco spectropolarimeter J-810 from 300 to 195 nm with a bandwidth of 1 nm. The time response was set at 1 s, and the scan rate was set between 50 and 10 nm/min. These values were chosen so that the product of the scan rate and the time constant was less than the bandwidth.21 For each spectrum, at least nine scans were averaged and smoothed using the Savitsky-Golay smoothing filter. The temperature in the chamber was maintained at 19 C to ensure than DMPC and DMPC:GD bilayers were in the gel state (the addition of GD shifts the main phase transition of DMPC to ∼21 C).50 This small shift indicates that GD has a small effect on the DMPC membrane structure. Recent STM images of the DMPC:GD monolayer have shown that the hexagonal packing of the acyl chains of DMPC is not perturbed by the presence of GD molecules.11 Figure 1 shows a schematic diagram of the liquid cell used in the OCD experiments. The liquid cell was formed between two fused silica windows that were coated with a thin layer of gold using thermal evaporation, with an intermediate 1 nm thick titanium layer to improve the adhesion of gold onto the fused silica surface. The presence of the metal layers on the fused silica windows allowed for the application of an electric potential across the cell. Two Teflon cups were threaded onto the central part of the cell, and a rubber washer was placed at the bottom of each cup to evenly distribute the pressure on the windows during the assembly process. Viton O-rings coated with gold were also used to ensure a leak-proof seal of the cell without damaging the thin gold layer on the fused silica windows. This design allowed the assembly of the cell with only a small stress applied to the windows. The CD signal measured for the cell strongly depends on the pressure applied to the windows during the cell assembly. This is a well-known (43) Abdul-Manan, N.; Hinton, J. F. Biochemistry 1994, 33, 6773. (44) Sychev, S. V.; Sukhanov, S. V.; Barsukov, L. I.; Ivanov, V. T. J. Peptide Sci. 1996, 2, 141. (45) Galbraith, T. P.; Wallace, B. A. Faraday Discuss. 1999, 111, 159. (46) Greathouse, D. V.; Hinton, J. F.; Kim, K. S.; Koeppe, R. E. Biochemistry 1994, 33, 4291. (47) Mobashery, N.; Nielsen, C.; Andersen, O. S. FEBS Lett. 1997, 412, 15. (48) Ba~no, M. C.; Braco, L.; Abad, C. Biophys. J. 1992, 63, 70. (49) Rawat, S. S.; Kelkar, D. A.; Chattopadhyay, A. Biophys. J. 2004, 87, 831. (50) Wanderlingh, U.; D’Angelo, G.; Conti Nibali, V.; Gonzalez, M.; Crupi, C.; Mondelli, C. J. Phys.: Condens. Matter 2008, 20, 104214.
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Figure 1. Custom circular dichroism liquid cell used for potential control experiments. The Teflon cell holder defined a cavity in the center corresponding to the compartment for CD measurements. The Teflon cell holder contained holes on the outer edges that were sealed by two fused-silica windows, used as the working (WE) and counter (CE) electrodes. The windows are positioned off-center to allow for the electrical connections. A leak-proof seal was ensured by screwing two Teflon cups on each side of the cell. Two radial channels were also drilled in the central part to allow the inner cavity to be filled during the assembly of the cell. The same channels ensure the electrical connections between the CE/WE and an external reference electrode (silver/silver chloride). The inner cavity was 2.5 cm in diameter and 1 cm thick. The area probed by the light was 0.5 cm2. A picture of the counter electrode is given in the bottom inset. consequence of stress-induced birefringence.51 It was necessary to adjust the applied stress to the same value for each assembly of the liquid cell to obtain a reproducible baseline for the CD spectra. The latter was accomplished by placing the working electrode in a specific position within the Teflon cup by aligning small marks made on the back of the window to those on the cup. Finally, the cell was assembled by threading the two cups using the same numbers of turns and aligning their relative position with the aid of special marks designed for that purpose. One of the gold-coated fused silica windows served as the working electrode (WE) with only a 10 nm thick gold layer on the area that was probed by the light beam. The gold layer was sufficiently thick to allow the application of the electric potential across the film and was thin enough to be essentially transparent to the UV light allowing for CD measurements. The second window was used as a counter electrode (CE) and was coated with a thick gold layer (>50 nm), except in the area probed by the light. For the potential control experiments, the cell was assembled and slowly filled with 0.1 M solution of sodium fluoride (NaF). This electrolyte was chosen to allow comparison of the present studies with parallel PMIRRAS experiments performed on a single DMPC:GD bilayer.15 A BaF2 window was used in PMIRRAS experiments, and NaF was needed to suppress the window solubility. The electrolyte and the cell were deaerated with argon for 20 min to remove traces of oxygen. A silver/silver chloride electrode (SSCE) was used as a reference electrode and was connected to the cell through a salt bridge to avoid contamination by chlorides that adsorb strongly on gold. First, several spectra were recorded at the open circuit potential (OCP) to check the stability of the signal. Next, a train of potential steps was applied to the working electrode from þ200 to -600 mV in 200 mV increments. The stability of both the circular dichroism and the absorption spectra was checked at each potential step. Because of (51) Schellman, J.; Jensen, H. P. Chem. Rev. 1987, 87, 1359.
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Article time constraints, the wavelength range for the spectra was limited to the range of 195-260 nm during potential control experiments. Vesicle and Film Preparation. Two concentrated stock solutions of DMPC and gramicidin (GD) were prepared using TFE as the solvent known to promote the β6.3 helical dimer conformation (or channel conformation) in gramicidin.36,39,40 A mixture of DMPC:GD was prepared by mixing solutions of the peptide and the lipid (molar ratio of 1:10) in a test tube that was kept in a water bath at 55 C. The solvent was then evaporated by allowing argon to flow over the solution until the test tube with the film on its internal walls became transparent. The test tubes were kept in a vacuum desiccator overnight to remove all traces of solvent. To prepare vesicles suspensions with gramicidin in the helical dimer conformation, a 0.1 M NaF solution was added to the test tube to give a total gramicidin concentration of 10-4 M. The test tube was sonicated for ∼1 h at 40 C until a clear solution was obtained. A CD spectrum was then measured in a quartz cell with a 0.1 mm path length. The absence of artifacts due to light scattering was checked by measuring the CD spectrum for various cell/detector distances and verifying that no change was observed. To induce gramicidin’s double-helix conformation, the ethanol injection method was used as previously described.49,52 Briefly, ethanol was used to dissolve the dried DMPC:GD film to obtain a 1.1 mM solution of gramicidin. 100 μL of this solution was then injected into 2 mL of 0.1 M NaF solution which had been cooled down to 10 C. The solution was thoroughly vortexed prior to the CD measurements to uniformly disperse the lipids and peptides. The CD spectrum measured using this procedure was in agreement with published CD spectra of the DH conformation of gramicidin.39,40 The substrate for oriented films was the working electrode (WE) that was freshly coated with a 10 nm thick film of gold. Prior to deposition of the lipid and lipid/peptide layers, the WE was rinsed with ethanol and Milli-Q water (18.2 MΩ 3 cm) and was kept for 5 min in a UV-ozone chamber. Oriented films of pure DMPC and the DMPC:GD mixture were prepared by following a procedure used by Heller et al.53 The test tube, with the mixed DMPC:GD film, was filled with the appropriate amount of TFE to achieve a concentration of 1.5 10-4 M of gramicidin (TFE promotes formation of a β6.3 channel39,40). A 150 μL volume of the solution was added dropwise onto the window (area ∼ 20 cm2). To evenly distribute the solution on the surface, the substrate was slowly rotated at ∼30 rpm until the solvent was completely evaporated. The ozone-cleaned gold film surface was uniformly covered by the solution, and the evaporation of the solvent from the gold surface was uniform. We have verified that the ozone-cleaned gold film surface is uniformly wetted by TFE in an independent experiment. The window was then kept overnight under vacuum at 18 C to eliminate traces of solvent. As already discussed by Heller et al.,53 the alignment process relies on selfassembly of the lipid bilayers or lipid/peptide bilayers on the surface. The concentration and amount of solution were chosen to ensure the deposition of ∼15 bilayers onto the window. This was confirmed by measuring the intensity of the 235 nm absorption peak in the UV spectrum of gramicidin (the spectrum is shown in Figure 6) with respect to a reference measurement performed on a single bilayer prepared using the Langmuir-Blodgett/ Langmuir-Schaefer methods54,55 and transferred at the surface pressure of 40 mN/m. Ellipsometry measurements, described below, confirmed that the thickness of the film corresponded to ∼15 bilayers. (52) Kremer, J. M. H.; Van der Esker, M. W.; Pathmamanoharan, C.; Wiersema, P. H. Biochemistry 1977, 16, 3932. (53) Heller, W. T.; He, K.; Ludtke, S. J.; Harroun, T. A.; Huang, H. W. Biophys. J. 1997, 73, 239. (54) Ullman, A. An Introduction to Ultrathin Films; Academic Press: New York, 1991. (55) Zawisza, I.; Bin, X.; Lipkowski, J. Bioelectrochemistry 2004, 63, 137.
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Article The film was hydrated following a two-step procedure. First, as described by Clayton et al.,29 the bottom of the cell was wetted with a few drops of the NaF electrolyte, and the WE was assembled and sealed into the cell. After the film was hydrated, the cell was slowly filled with a deaerated 0.1 M NaF solution, and the potential control experiments were performed. Differential light scattering and birefringence artifacts have been reported for oriented films of bilayers but are generally observed when using very thick films.56 As for vesicle suspensions, the absence of light scattering artifacts in the CD spectra was assessed by shifting the position of the film with respect to the detector. To check for birefringence artifacts, the cell was rotated by 90 and CD spectra measured at the two positions were compared. Since the fused silica windows display stress-induced birefringence, the signal must be corrected by removing the contribution due to the birefringence of the windows. The film was considered to be free of birefringence artifacts if the CD spectra recorded at the two angles of rotations were essentially identical. Films prepared in this manner were very stable in the NaF electrolyte, and CD spectra recorded for these films were similar to those published for oriented lipid/gramicidin films.23,24 Electrochemistry Measurements. Electrochemistry experiments were performed using a solid gold polycrystalline electrode in the hanging meniscus configuration (surface area of 0.8 cm2). The reference electrode was either a saturated calomel electrode (SCE) or a silver/silver chloride electrode (SSCE), depending on the experiment. The SSCE potential versus the SCE was -40 mV, and this value was used to convert potentials measured versus the SCE onto the SSCE scale. In the present study, all of the electrode potentials are reported versus the SSCE. The supporting electrolyte was a 0.1 M solution of NaF and oxygen was removed from the solution by bubbling argon for 20 min before the experiment. The potential of zero charge Epzc of the gold electrode measured in this electrolyte was equal to Epzc = -100 mV versus the SSCE. Before each experiment, a cyclic voltammetry (CV) scan was performed to check the cleanliness of the cell. All measurements were performed using a HEKA PG590 potentiostat and an EG&G 7265 lock-in amplifier for the differential capacitance experiments. Data were recorded using a National Instruments data acquisition board in conjunction with LabView programs generously provided by Prof. Dan Bizotto of the University of British Columbia. The DMPC:GD films were prepared using the same protocol as described above, with the gold electrode flame-annealed before the deposition procedure. The method used to measure the surface charge has been described previously.57,58 Briefly, the electrode is held at a base potential Eb of -50 mV for 300 s. This value was chosen since it coincides with the smallest value of the capacitance of the film as measured using differential capacitance (DC). The potential was then stepped to a variable potential Ec for a time that was sufficiently long that equilibrium was reached (>300 s for the measurements involving a film, 30 s for the bare gold electrode). After this period of time, a potential Edes = -1100 mV was applied to promote film desorption from the surface. The current transient was measured for 250 ms, and the potential was stepped back to Eb. Measurements were performed for decreasing values of Ec, starting at 200 mV to -1100 mV in steps of -50 mV. Integration of the current transients gives the difference between charge densities at potentials Ec and Edes. The charge density curves, measured with and without the film, merged at the most negative potentials. The absolute charge densities for the electrode without the thiolipid can be calculated knowing the potential of zero charge, Epzc = -100 mV vs SSCE, determined independently. Ellipsometry Measurements. The thickness and refractive index of gold films and DMPC:GD samples were measured using (56) Gibson, N. J.; Cassim, J. Y. Biophys. J. 1989, 56, 769. (57) Lipkowski, J.; Ross, P. N. Adsorption of Molecules at Metal Surfaces; VCH: New York, 1992. (58) Richer, J.; Lipkowski, J. J. Electrochem. Soc. 1986, 133, 121.
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Fiche et al. a custom single wavelength (633 nm) self-nulling ellipsometer at a fixed angle of incidence of 70 with respect to the sample normal and a quarter-wave plate angle of -45. For each measurement, two different pairs of polarizer and analyzer angles corresponding to a null in the light intensity at the detector were measured, corresponding to null zones one and three. In situ experiments were performed using a custom-built fluid cell for ellipsometry experiments that had two BK7 windows that were perpendicular to the incident and reflected beams. The accuracy of the cell was tested using a hydrophobic polystyrene film spincoated onto a Si wafer,59 which did not swell in the presence of water. To ensure that the instrument was correctly calibrated for the liquid cell, the measurements were performed first in air and then in water. The uncertainty for the film thickness and its index of refraction were estimated to be 2% and 0.3%, respectively. The oriented DPMC:GD films were prepared using the protocol described above. The substrate was a 1.5 1.5 cm2 fused silica slide freshly coated with 200 nm of gold with an intermediate 1 nm thick titanium adhesive layer. The complex refractive index of gold (n þ ik) was measured on different samples, and the average value was equal to 0.17 - i3.4. The estimated uncertainties for n and k were 6% and 1.5%, respectively. A thick layer of gold was used for these experiments to avoid multiple reflections at the gold/fused silica interface. For potential controlled experiments, the electrolyte was a 0.1 M NaF solution. The gold slide was used as the working electrode, and the counter electrode was a flame-annealed gold wire, immersed in the NaF electrolyte. The reference electrode was a silver-silver chloride electrode connected to the cell through a salt bridge. The average thickness of the DMPC:GD film was equal to 70 ( 15 nm, corresponding to 14 ( 3 bilayers if one assumes that the thickness of a single DMPC bilayer is ∼5 nm.9,60,61 The best fit refractive index values ranged from 1.37 to 1.41, which are consistent with values determined in previous studies.61 The error estimates for both the thickness and the refractive index are due to the inherent nonhomogeneity of the samples and irreproducibility in the sample preparation procedure. The open circuit potential (OCP) was usually 150 ( 50 mV. The starting potential was set at 100 mV, and the system was allowed to stabilize for 30 min before measurement of the ellipsometric angles {Δ, Ψ}.62 Both parameters were measured for at least five different spots on the surface and averaged. The potential was then changed in the negative direction in -100 mV increments until a final value of -600 mV was reached. To avoid exposing the thin film of gold to a significant surface stress that could cause its delamination, the measurements were limited to potentials larger than -600 mV versus SSCE.
Results Electrochemical Experiments. The electrochemical experiments were performed to determine how the electric field influences the properties of the film deposited onto the electrode. To allow comparison with CD experiments, a polycrystalline Au electrode was used in these studies. Figure 2 shows the differential capacitance curves measured for the Au(pol) electrode in the absence of the film and the electrode covered by a film consisting of ∼15 bilayers. For comparison with other studies performed in our laboratory, the capacity curve recorded at the Au(pol) with a single bilayer is also included in this figure. Interestingly, the capacitance curve measured for the film consisting of ∼15 bilayers is quite similar to the curve measured for a single bilayer. At potentials higher than -500 mV, the (59) Tanchak, O. M.; Barrett, C. J. Macromolecules 2002, 35, 3164. (60) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21. (61) Howland, M. C.; Szmodis, A. W.; Sanii, B.; Parikh, A. N. Biophys. J. 2007, 92, 1306. (62) Tompkins, H. G. A User’s Guide to Ellipsometry; Academic Press: Boston, 1993.
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Figure 2. Differential capacitance (DC) curves measured using a gold polycrystalline electrode in 0.1 M NaF supporting electrolyte, with a sweep rate of 5 mV s-1, and applying an AC perturbation of frequency 25 Hz and amplitude 5 mV. The dotted line plots the curve measured in the supporting electrolyte without the film. The triangles and circles mark curves measured for the DMPC:GD bilayer (2) and the film of oriented bilayers (O). The measurements were performed at 19 ( 1 C.
capacitance of the film-covered electrode significantly decreased below the value observed for the film-free electrode. The minimum capacitance measured at positive potentials was 5 ( 1 μF/cm2 for both the stack of oriented bilayers and single bilayers. This value is about 5 times larger than the value expected for real biological samples,7 suggesting that the films deposited at the gold surface contain water-filled defects. Similar behavior was observed previously for a single bilayer deposited onto a Au (111) electrode.7,55 In the potential range between -100 and -500 mV, the capacitance increases gradually when the potential is scanned in the negative direction. Such behavior has been observed previously for a DMPC bilayer63 and was associated with the electroporation of the film induced by the negative potential. Below -700 mV, the capacitance rises above that for the supporting electrolyte as the film detaches from the gold surface. The detachment is gradual with the maximum change of the electrode coverage by the film taking place at ∼-950 mV for which a peak is seen in the capacitance curve. At potentials lower than -1100 mV, the curves measured in the presence of the DMPC:GD film merge with the curve obtained for the bare gold electrode. This behavior indicates that at these very negative potentials, the film is no longer in contact with the gold electrode. However, neutron reflectivity experiments performed for a single bilayer demonstrated that the film remains close to the metal surface, lifted on a ∼1 nm thick cushion of the electrolyte.7,64 When the potential is stepped back to positive values, the film is readsorbed onto the electrode. The charge density curves shown in Figure 3 are complementary to the differential capacitance data. These results confirm that the potential has a similar effect on the stack of ∼15 bilayers and on the single bilayer. The two films are adsorbed on the metal surface at charge densities between -10 and þ10 μC/cm2. The steps observed on the charge density curves at potentials down to -400 mV, and charge densities down to -10 μC/cm2 indicate a progressive desorption (detachment) of the film from the (63) Bizzotto, D.; Zamlynny, V.; Burgess, I.; Jeffrey, C. A.; Li, H. Q.; Rubinstein, J.; Galus, Z.; Nelson, A.; Pettinger, B.; Merill, A. R. Amphiphilic and Ionic Surfactants at Electrode Surfaces; Wieckowski, A., Ed.; Marcel Dekker: New York, 1999. (64) Burgess, I.; Li, M.; Horswell, S. L.; Szymanski, G.; Lipkowski, J.; Satija, S.; Majewski, J. Colloids Surf., B 2005, 40, 117.
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Figure 3. Surface charge density curves measured using a polycrystalline gold electrode surface plotted versus the electrode potential for the supporting electrolyte of 0.1 M NaF (0), a single 10:1 DMPC:GD bilayer (2), and an oriented film of 10:1 DMPC: GD multiple bilayers (O). The inset plots the potential drop across the film calculated from the charge density data as explained in the text. The measurements were performed at 19 ( 1 C.
electrode surface. The ratio of charge density to capacitance gives the value of the potential drop across the capacitor. The values of the potential drop are plotted in the inset to Figure 3. When the film is directly adsorbed at the metal surface, it separates the charge on the metal from the charge on the solution side of the interface. The ratio of charge density to capacitance can then be taken as the value of the potential across the bilayer (equivalent to the transmembrane potential). The charge density curves indicate that the films are adsorbed at the metal surface if the potential drop across the film is less than 400 mV (see the inset in Figure 3) and is detached from the metal surface when the potential drop exceeds this value. These values of the potential drop across the stack of bilayers will be used below to discuss the results of the ellipsometry and CD experiments below. Additionally, a distinction between the electrode potential applied to the electrode versus a reference electrode and the potential drop across the membrane determined from the charge density curves will be made. The former is the operational quantity, and the latter is the physical quantity that affects the structure of the bilayer. Ellipsometry Measurements. Ellipsometry measurements were performed on a stack of oriented DMPC:GD bilayers in contact with the electrolyte solution using the spectro-electrochemical cell designed for ellipsometric measurements. In Figure 4 the parameters {Δ, Ψ} determined from the ellipsometry experiments, as a function of the electrode potential, are plotted. Within experimental uncertainty, Ψ is independent of potential. In contrast, Δ changes stepwise with potential. In the potential range from þ100 to -300 mV versus SSCE, the values of Δ are approximately equal to 89; for potentials more negative than -500 mV versus SSCE, the values of Δ increase steeply to reach a quasi-plateau at ∼91. This behavior is very reproducible from one sample to another. However, the magnitude of the step on the plot of Δ seen at ∼-400 mV versus SSCE may range from 1 to 3. It is due to the presence of the film deposited on the gold surface. The data reported in the Supporting Information illustrate that when the experiment was performed using a film-free electrode, Δ was essentially independent of potential: its values varied by less than 0.2 as shown in Figure S1. To extract further information from the ellipsometric data, calculations employing a three-layer model for the gold/film/ water interface were performed, and both the thickness (h) and the refractive index (n) of the film were determined. Figure 5 shows DOI: 10.1021/la902325n
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(circle) as a function of the applied potential for a film of DMPC: GD deposited on the gold electrode. For potentials greater than -300 mV, both parameters were essentially constant. However, for potentials less than -300 mV, a strong increase of Δ was measured while Ψ remained unchanged, since the variations measured were within experimental error. At the end of the experiment, the sample was set again at OCP and stabilized for 10 h. No change of {Δ, Ψ} was measured as compared with the values obtained at -600 mV. The error bars were calculated by averaging ten sets of {Δ, Ψ} values over the surface. The top scale shows the potential drop across the film, calculated using the charge density data (see Figure 3). The measurements were performed at 19 ( 1 C.
how these parameters change as a function of the applied potential. In addition, the values of the potential drop across the membrane are plotted at the top of the figure. The result shows that the thickness and the refractive index of the film are constant for transmembrane potentials between ∼þ200 mV and ∼-150 mV, indicating that the membrane is stable in this region. At transmembrane potentials more negative than -150 mV, the thickness increases and the refractive index decreases stepwise with the step centered at the transmembrane potential of ∼-200 mV. Since the refractive index of water (1.33) is smaller than that of the film (∼1.38), this trend indicates a potentialinduced swelling of the film, with the membrane incorporating more solvent molecules at transmembrane potentials lower than -150 mV. These measurements were repeated four times using freshly prepared samples. In each case a change of the film thickness at a transmembrane potential value of ∼-200 mV was observed. However, the magnitude of the change varied from sample to sample, ranging from 30% to 80% of the original value measured at the transmembrane potential of þ200 mV. It should be noted that, due to the onset of gold oxidation, measurements could not be conducted at more positive potentials. The present results are in good agreement with previous measurements of the electrical properties of black lipid membranes (BLMs) of various lipid compositions.65-67 Numerous studies demonstrated that transmembrane potential values in excess of 200-300 mV induce electroporation of the film.65-67 Therefore, the ellipsometry measurements indicate that the film of oriented bilayers remains stable in the range of transmembrane potentials between (65) Baba, T.; Toshima, Y.; Minamikawa, H.; Hato, M.; Suzuki, K.; Kamo, N. Biochim. Biophys. Acta 1999, 1421, 91. (66) Liu, A. L. Advances in Planar Lipid Bilayers and Liposomes; Academic Press: New York, 2007; Vol. 6. (67) Robello, M.; Gliozzi, A. Biochim. Biophys. Acta 1989, 982, 173.
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Figure 5. Variation of ellipsometry angles {Δ, Ψ} as a function of the potential was interpreted using a three-layer model gold/ DMPC:GD film/water. The refractive index of water was set to 1.33. For the supporting gold electrode, the measured refractive index was 0.175 þ i3.401. The refractive index (circles) and thickness (squares) of the second layer were allowed to vary as a function of the Δ and Ψ values. The parameter values remained fairly stable as long as the potential applied across the film was maintained above -400 mV. However, below this threshold, the film started to swell, as indicated by the 50 nm increase of the thickness between -400 and -600 mV. Simultaneously, due to water penetration into the system, the refractive index of the film decreased by about 0.02. The top scale shows the potential drop across the film, calculated using the charge density data (see Figure 3). The measurements were performed at 19 ( 1 C.
þ200 and -150 mV. At more negative potentials, electroporation is observed, resulting in an increase in the water content and the thickness of the film. Similar behavior has already been observed for a single phospholipid bilayer supported on a gold electrode surface by neutron reflectivity and AFM studies.7,9 Lastly, it is important to note that the potential-induced changes of the film structure are irreversible. Once the film is exposed to negative potentials, the parameters {h, n} never returned completely to their initial values when the potential was stepped back to the open circuit potential (OCP) equal to þ200 mV versus SSCE. This property suggests that the structure of the film is irreversibly changed during the swelling process. This is in contrast to the behavior of a single phospholipid bilayer for which the potential induced changes were reversible.7,9 Potential Control Experiments: Light Absorption. The CD spectropolarimeter usually gives the opportunity to monitor both the far-UV CD and absorption spectra of the sample. For a suspension of DMPC:GD vesicles, the absorption spectrum of gramicidin is shown in Figure 6A. It displays two strong bands around 190 and 223 nm. As already discussed by Chen et al.,37 two main contributions can be assigned to the gramicidin absorption spectrum: absorption by the peptide backbone and by the tryptophan residues. The band at ∼190 nm corresponds to the π-π* (^) transition of the peptide backbone perpendicularly polarized with respect to the helix axis. The band at 223 nm is a mixed band with the π-π* ( ) transition parallel polarized, n-π transitions of the peptide backbone, and transitions of the tryptophan residues. The contribution from the tryptophan residues is the most intense and dominates this part of the spectrum.37 In Figure 6B, the far-UV absorption spectra for a stack of oriented bilayers at the electrode surface for selected electrode )
Figure 4. Variation of the ellipsometry angles Δ (square) and Ψ
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(68) Henglein, F.; Lipkowski, J.; Kolb, D. M. J. Electroanal. Chem. 1991, 303, 245.
(69) Grahame, D. C. Chem. Rev. 1947, 41, 441. (70) Ibach, H.; Bach, C. E.; Giesen, M.; Grossmann, A. Surf. Sci. 1997, 375, 107.
Figure 6. (A) Typical UV absorption spectrum of gramicidin measured for a suspension of DMPC:GD vesicles. The spectrum was baseline corrected by removing the signal measured under the same conditions for pure DMPC vesicles. (B) Absorption spectra of an oriented film of DMPC:GD bilayers as a function of the potential. A strong contribution due to the fused silica window and the 10 nm thick gold layer is observed. In the inset the absorptions by the film of gold and the quartz were removed by subtracting spectra measured for a DMPC film without gramicidin for each potential. These spectra are due only to the absorption by gramicidin. The measurements were performed at 19 ( 1 C.
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potentials is plotted. The shape of these spectra is different than that for the suspension of DMPC:GD vesicles and suggests that absorption by the silica window and the gold film strongly contribute to the measured spectra. Therefore, the absorption spectrum for a stack of DMPC bilayers without GD was measured independently at each potential (data shown in Figure S2), and this spectrum was subtracted from the spectrum of the film that contained GD molecules. The result is plotted as the inset to Figure 6B. After removal of the background due to the silica window and the film of gold, the CD spectra of the stack of supported bilayers resemble the spectrum of the suspension of vesicles. Furthermore, the amplitude of the band at ∼223 nm does not change with the applied potential, indicating that no loss of the membrane portion took place in this experiment. The spectra for the stack display a small potential dependence at wavelengths
