Potential-Driven Structural Changes in Langmuir ... - ACS Publications

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Langmuir 2007, 23, 5180-5194

Potential-Driven Structural Changes in Langmuir-Blodgett DMPC Bilayers Determined by in situ Spectroelectrochemical PM IRRAS Izabella Zawisza,†,‡ Xiaomin Bin,†,§ and Jacek Lipkowski*,† Department of Chemistry and Biochemistry, UniVersity of Guelph, Guelph, N1G2W1, Ontario, Canada, Department of Physical Chemistry, UniVersity of Oldenburg, Carl Von Ossietzky Str. 9/11, 26-129 Oldenburg, Germany, and Steacie Institute for Molecular Sciences, National Research Council, 100 Sussex DriVe, Ottawa, K1A 0R6, Ontario, Canada ReceiVed NoVember 1, 2006. In Final Form: January 23, 2007 Combined Langmuir-Blodgett vertical withdrawing and Langmuir-Schaefer horizontal touch (LB-LS) methods were employed to transfer DMPC bilayers onto a Au(111) electrode surface. Charge density measurements and photon polarization modulation infrared reflection absorption spectroscopy were employed to investigate electric field induced changes in the structure of the bilayer. The results show that the physical state and the molecular arrangement found in the monolayer at the air-water interface is to a large extent preserved in the bilayer formed by the LB-LS method. This approach provides an opportunity to produce supported bilayers with a well-designed architecture. The properties of the bilayer formed by the LB-LS method were compared to the properties of the bilayer produced by spontaneous fusion of unilamellar vesicles investigated in an earlier study (Bin, X.; Zawisza, I.; Lipkowski, J. Langmuir 2005, 21, 330-347). The tilt angles of the acyl chains are much smaller in the bilayer formed by the LB-LS method and are closer to the angles observed for vesicles and stacked hydrated bilayers. The tilt angles of the phosphate and choline groups are also smaller and are characteristic of an orientation in which the area per DMPC molecule is small. The electric field induced changes of these angles are also less pronounced in the bilayer formed by the LB-LS method. We have shown that these differences are a result of the higher packing density of the phospholipid molecules in the bilayer formed by the LB-LS method.

Introduction Supported lipid bilayers are attractive models of biological membranes that have been extensively investigated over the last two decades.1-3 The planar geometry and mechanical stability of these models allows one to study the structure and function of phospholipid bilayers using several techniques, including IR spectroscopy,4-6 NMR,7 X-ray reflectivity,8 neutron reflectivity (NR),9 X-ray and neutron diffraction,10 AFM,11-13 and STM.14 Cellular lipids and proteins are constantly exposed to a static electric field on the order of 105 to 107 V m-1.15 The high electric field may cause proton dissociation, charge separation, cooperative alignment of dipoles, reorientation of dipoles within the molecules, and transition to a state with a higher permanent †

University of Guelph. University of Oldenburg. § Steacie Institute for Molecular Sciences. ‡

(1) Kalb, E.; Rey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307316. (2) Lenz, P.; Ajo-Franklin, C. M.; Boxer, S. G. Langmuir 2004, 20, 1109211099. (3) Sackmann, E. Science 1996, 271, 43-48. (4) Bin, X.; Zawisza, I.; Lipkowski, J. Langmuir 2005, 21, 330-347. (5) Horswell, S. L.; Zamlynny, V.; Li, Q.; Merrill, A. R.; Lipkowski, J. Faraday Discuss. 2002, 121, 405-422. (6) Zawisza, I.; Lachenwitzer, A.; Zamlynny, V.; Horswell, S. L.; Goddard, J. D. J. L. Biophys. J. 2003, 86, 4055-4075. (7) Bayerl, T. M.; Bloom, M. Biophys. J. 1990, 59, 357-362. (8) Salditt, T.; Li, C.; Spaar, A.; Mennicke, U. Eur. Phys. J. E 2002, 7, 105116. (9) Majkrzak, C. F.; Berk, N. F.; Krueger, S.; Dura, J. A.; Terek, D.; Silin, V. C. W. M.; Woodward, J.; Pland, A. L. Biophys. J. 2000, 79, 3330. (10) Wiener, M. C.; White, S. H. Biophys. J. 1992, 61, 434-447. (11) Feng, Z. V.; Spurlin, T. A.; Gewirth, A. A. Biophys. J. 2005, 88, 21542164. (12) Leonenko, Z. V.; Finot, E.; Ma, H.; Dahms, T. E. S.; Cramb, D. T. Biophys. J. 2004, 86, 3783-3793. (13) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806-1815. (14) Xu, S.; Szymanski, G.; Lipkowski, J. J. Am. Chem. Soc. 2004, 126, 1227612277. (15) Tsong, T. Y.; Astumian, R. D. Annu. ReV. Physiol. 1988, 50, 273-290.

dipole moment.15 These changes can lead to structural rearrangements in the membrane, eventually leading to breakdown at high voltages.16 Measurements of macroscopic properties such as capacity,17-19 conductivity,20-22 transmembrane potential,23 electrostriction, and dielectric relaxation24 for membranes exposed to high electric fields has provided valuable information concerning the effect of the field on membrane structure and stability. However, the molecular-level understanding of structural changes occurring in the lipid membrane in the presence of a high electric field remains incomplete. Recently, dry DOPC multibilayers spread between two Ge crystals were exposed to a variable voltage, and the voltage-driven changes in their structure was investigated by ATR IRRAS.25 In another study, dry DMPC multibilayers sandwiched between two Si plates were investigated by the electric field modulated excitation FTIR transmission spectroscopy.26 These studies provided evidence that the tilt angle of phospholipid molecules and the orientation of their polar head groups change under the influence of the electric field applied to the membrane. However, the interface between germanium or silicon and a stack of multibilayers should behave as a semiconductor-solution interface. A potential applied to the semiconductor-solution (16) Winterhalter, M. Colloids Surf., A 1999, 149, 161-169. (17) Bamberg, E.; Benz, R. Biochim. Biophys. Acta 1976, 426, 570-580. (18) Florin, E. L.; Gaub, H. E. Biophys. J. 1993, 64, 375-383. (19) Sargent, D. F. J. Membr. Biol. 1975, 23, 227-247. (20) Corondo, R. Ann. ReV. Biophys. Chem. 1986, 15, 259-277. (21) Krishna, G.; Schulte, J.; Cornell, B. A.; Pace, R.; Wieczorek, L.; Osman, P. D. Langmuir 2001, 17, 4858-4866. (22) Tien, H. Y. Bilayer Lipid Membranes; Marcel Dekker: New York, 1974. (23) Azzone, G. F.; Pieterbon, D.; Zoratti, M. Determination of the proton electrochemical gradient across biological membranes; Academic Press: New York, 1984; Vol. 13, pp 1-77. (24) Hianik, T. ReV. Mol. Biotechnol. 2000, 74, 189-205. (25) LeSaux, A.; Ruysschaert, J. M.; Goormaghtigh, E. Biophys. J. 2001, 80, 324. (26) Schwarzrott, M.; Lasch, P.; Baurecht, D.; Maumann, D.; Fringeli, P. U. Biophys. J. 2004, 86, 285-295.

10.1021/la063190l CCC: $37.00 © 2007 American Chemical Society Published on Web 03/21/2007

Structural Changes in LB DMPC Bilayers

interface predominantly causes band bending inside the semiconductor. The potential difference between the semiconductor and the solution is not changed.27 Therefore, it is uncertain what portion of the voltage applied to the semiconductor electrodes acted at the stack of multibilayers in the above studies. A static electric field can easily be applied to a biomembrane supported at a metal electrode surface.28 The potential drop across this membrane, equivalent to the transmembrane potential, can be measured and easily controlled.29 A phospholipid bilayer can be formed at a gold electrode surface by the spreading of unilamellar vesicles. The field-induced changes in the properties of this bilayer can then be investigated using combined electrochemistry and PM IRRAS,4-6,30,31 neutron reflectivity,32,33 and scanning tunneling microscopy14 techniques. These studies demonstrated that bilayers supported at a metal electrode surface can tolerate transmembrane potentials up to ∼0.4 V. At higher potentials, the membrane is lifted from the metal surface but remains in its close proximity, separated from the metal by a thin (∼1 nm) cushion of electrolyte. Consistent with earlier capacitance measurements,17-19 an increase in tilt angle of the phospholipid molecules was observed for bilayers deposited at the gold surface and exposed to a static electric field. The objective of the present work is to compare the behavior of a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer formed by fusion of unilamellar vesicles on a gold electrode surface (described in a previous study4,30) to that of a membrane formed by a combination of the Langmuir-Blodgett and Langmuir-Schaefer methods (LB-LS), an alternative procedure for producing supported lipid bilayers.34-36 The LB method allows for the transfer of an organic film assembled as a monolayer at the air-water interface onto a solid substrate. The first layer is usually quantitatively transferred onto the solid substrate with a transfer ratio of unity. The DMPC transfer of the second layer gives a transfer ratio equal to -1, indicating removal of the first leaflet from the substrate. To overcome this obstacle, the LB method is used to prepare the first leaflet, and the LS technique (horizontal touch method) is employed to deposit the second layer of the membrane.34,35 An advantage of the LB-LS method is the choice of surface pressure at which the monolayers are transferred, allowing for precise control of the molecular area of the phospholipids in the bilayer and the physical state of the transferred films. In this paper, we present the structural characterization of a DMPC bilayer formed by the transfer of monolayers at a selected surface pressure (42 mN m-1) onto a Au(111) surface. We combined electrochemical and PM IRRAS measurements to provide a molecular-level description of the electric field induced transformation of the DMPC bilayers. Independently, we have performed studies of the bilayer formed by the LB-LS method building a model membrane in which one leaflet consisted of perdeuterated DMPC and the second leaflet of hydrogen(27) Gerischer, H. Electrochim. Acta 1990, 35, 1672-1699. (28) Guidelli, R.; Aloisi, G.; Becucci, L.; Dolfi, A.; Moncelli, M. R.; Tadini Bouninsegni, F. J. Electrochem. Chem. 2001, 504, 1-28. (29) (a) Becucci, L.; Moncelli, M. R.; Herrero, R.; Guidelli, R. Langmuir 2000, 16, 7694-7700. (b) Mohilner, D. In Electroanalytical Chemistry; Bard, A., Ed.; Marcel Dekker: New York, 1966; Vol. 1, pp 241-409. (30) Bin, X.; Lipkowski, J. J. Phys. Chem. B 2006, 110, 26430-26441. (31) Garcia-Araez, N.; Brosseau, C. L.; Rodriguez, P.; Lipkowski, J. Langmuir 2006, 22, 10365-10371. (32) Burgess, I.; Li, M.; Horeswell, S. L.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Satija, S. Biophys. J. 2004, 86, 1763-1776. (33) Burgess, I.; Szymanski, G.; Li, M.; Horswell, S. L.; Lipkowski, J.; Majewski, J.; Satija, S. Colloids Surf., B 2005, 40, 117-122. (34) Crane, J. M.; Tamm, L. K. Biophys. J. 2004, 86, 2965-2979. (35) Tamm, L. K.; McConnel, H. M. Biophys. J. 1985, 47, 105-113. (36) Solttrup, B. J.; Veach, S. L.; Keller, S. L. Biophys. J. 2004, 86, 29422950.

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Figure 1. (A) The compression isotherm for DMPC at the air-0.1 M NaF solution interface recorded at 22 °C. Orientation of DMPC molecules transferred using (B) the Langmuir-Blodgett (LB) method by vertical withdrawing, (C) the Langmuir-Schaefer (LS) horizontal touch method. (D) Schematic structure of a Y-type bilayer.

substituted DMPC. Due to a large volume of experimental material, the results of that study are described in a separate publication.31 Experimental Section Reagents and Thin Film Preparation Methods. DMPC (SigmaAldrich, St. Louis, MO) was used without further purification to make a 1 mg mL-1 stock solution of the phospholipid in chloroform. A few drops of that solution were spread at the surface of a 0.1 M NaF aqueous solution in a Langmuir-Blodgett trough equipped with a movable barrier and a Wilhelmy plate (KSV, Finland) to form a monolayer. The trough was controlled by a computer using KSV LB5000 software; the temperature of the subphase was 22 ( 2°C. The solvent was allowed to evaporate, and the compression isotherm was recorded. The compression isotherm is shown in Figure 1A. At 22 °C, the DMPC monolayer at the air-0.1 M NaF solution interface exists predominantly in the liquid expanded (LE) state. A phase transition from the LE to the liquid crystalline (LC) state is observed at a surface pressure of Π ≈ 45 mN m-1 and the area per molecule 0.43 nm2. The DMPC monolayer collapses at a surface pressure of 53.2 mN m-1, giving a limiting area per molecule Alim of 0.39 nm2. The maximum surface concentration (Γlim) of DMPC in the monolayer is equal to 4.37 × 10-10 mol cm-2. The monolayers were transferred from the air|solution interface onto the Au(111) electrode at a surface pressure of 42 mN m-1, corresponding to a mean molecular area of 0.45 nm2. A combination of the Langmuir-Blodgett (LB) and the Langmuir-Schaefer (LS) techniques were employed to fabricate the phopsholipid bilayer on the Au(111) surface. The first monolayer was transferred using the LB method by vertical withdrawing of the electrode through the interface at a speed of 35 mm min-1. The transfer ratio was 1.0 ( 0.1. The transfer ratio for the monolayers compressed to the LC state (Π > 42 mN m-1) was always higher than unity, and the monolayer could not be transferred quantitatively in that case. For the monolayer transferred onto the gold surface using the LB method, the head groups of DMPC faced the metal surface and the hydrophobic hydrocarbon chains were directed toward the air (Figure 1B). After emersion, the gold electrode covered by the monolayer

5182 Langmuir, Vol. 23, No. 9, 2007 was dried in argon for ∼20 min. The second leaflet was transferred using the LS technique (Figure 1C). The electrode covered by the first DMPC layer was brought into horizontal contact with the monolayer of the DMPC spread at the surface of the Langmuir trough and compressed to a preset surface pressure. In the monolayer transferred using the LS method, the acyl chains of the DMPC molecules are directed toward the metal and the polar heads face the aqueous subphase (Figure 1C). The electrode covered by the bilayer was then detached from the aqueous subphase and dried in argon for ∼1 h. The LB-LS method gives Y-type bilayer (Figure 1D). Electrochemical Measurements. The electrochemical measurements were carried out in an all-glass three-electrode cell using the hanging meniscus configuration. A Au(111) crystal served as the working electrode (WE) and a Au wire as the counter electrode (CE). The reference electrode was a silver-silver chloride electrode (Ag/AgCl/Cl), connected to the cell via a salt bridge (-40 mV vs SCE). All potentials are reported vs the SSCE reference electrode. Cyclic voltammetry and differential capacitance (DC) measurements were used to check the cleanliness of the base electrolyte using experimental procedures described previously.37 A computer-controlled system consisting of a HEKA potentiostat/ galvanostat, a HEKA (Lambrecht/Pfalz, Germany) scan generator, and a lock-in amplifier (EG&G Instruments 7265 DSP) was employed to perform electrochemical experiments. All data were acquired via a plugin acquisition board (National Instruments PCI 6052E) using custom-written software generously provided by Professor Dan Bizzotto of the University of British Columbia. Chronocoulometry was used to determine the charge density at the electrode surface. The gold electrode was held at a base potential Eb ) 0.0 V for 30 s. The potential was then stepped to a variable potential of interest, Ef, where the electrode was held for a time tf ) 30 s. Next, a potential step to the desorption potential Edes ) -0.95 V was applied for 150 ms, and the current transient corresponding to the desorption of the film and recharging of the interface was recorded. Finally, the potential was stepped back to Eb for 30 s before new potential steps to Ef and from Ef to Edes were applied. The integration of the current transients gives the difference between charge densities at potentials Ef and Edes. Similar experiments were performed at the film-free electrode in the pure supporting electrolyte. The absolute charge densities were then calculated using the independently determined potential of zero charge (pzc), Epzc ) 0.31 V vs SSCE. The gold single-crystal electrode was grown, cut, and polished in our laboratory.37 Prior to experiments, the electrode was cleaned by flame annealing. All electrochemical experiments were carried out in 0.1 M NaF supporting electrolyte. Spectra Collection and Processing. A Nicolet Nexus 870 (Madison, WI) spectrometer, equipped with an external tabletop optical mount, High D* MCT-A detector, photoelastic modulator (Hinds Instruments PM-90 with a II/ZS50 ZnSe 50 kHz optical head, Hillsboro, OR), and synchronous sampling demodulator (GWC Instruments, Madison, WI) was used to perform the PM IRRAS experiments. Spectra were acquired using in-house software, an Omnic macro, and a digital-to-analog converter (Omega, Stamford, CT) to control the potentiostat (HEKA PG285, Lambrecht/Pfalz, Germany) and collect spectra. The IR window was a BaF2 1 in equilateral prism (Janos Technology, Townshend, VT). Prior to the experiment, the window was cleaned in water and methanol and then treated for 10 min in an ozone chamber (UVO-cleaner, Jelight, Irvine, CA). The spectroelectrochemical cell was assembled, and the Au(111) bilayer-covered electrode produced by the LB-LS method was inserted into the cell. A starting potential E ) -1.00 V vs SSCE was applied to the gold electrode, and the spectra were collected at a series of potentials which were programmed as a cyclic sequence of steps whose amplitude was progressively increased using 0.05, 0.1, or 0.2 V potential increments. In total, 20 cycles of 400 scans each were performed to give 8000 scans at each applied potential. The instrumental resolution was 2 cm-1. The blocks of (37) Richer, J.; Lipkowski, J. J. Electrochem. Soc. 1985, 133, 121-128.

Zawisza et al. scans were individually checked for anomalies before averaging using in-house software. Measurements of IR spectra were carried out with the PEM set for half-wave retardation at 2900 cm-1 for the CH stretching region. A 2400-3200 cm-1 band-pass filter was employed and the angle of incidence of the infrared beam was set to 52°. The electrolyte thickness between the electrode and the prism was ∼2.2 mm, giving the highest enhancement of the p-polarized light at the electrode surface. In the CH bending and CdO stretching region, the maximum PEM efficiency was set to 1600 cm-1. The angle of incidence was 60° and the electrolyte thickness was 3.2 µm. The symmetric phosphate stretching band was investigated at the maximum of PEM efficiency set to 1000 cm-1. The thickness of the electrolyte was 3.0 µm. In all of these measurements, the electrolyte was 0.1 M NaF in D2O. The maximum of PEM efficiency was set to 1200 cm-1 to investigate the asymmetric stretching of the phosphate group. The thickness of the electrolyte layer was again 3.0 µm, and the electrolyte was a 0.1 M solution of NaF in H2O. The thickness of the thin layer was determined by comparing the experimental reflectivity spectrum of the thin layer cell, attenuated due to the layer of solvent between the electrode and the IR window, to the reflectivity curve calculated from the optical constants of DMPC,4 using a four-phase model (Au, DMPC film, thin layer of the electrolyte, and BaF2 window), as described in ref 38. The demodulation technique developed in Corn’s laboratory39,40 was used in this work. A modified version of a method described by Buffeteau et al.41 was used to correct the intensity average (Is(ω) + Ip(ω))/2 and intensity difference (Is(ω) - Ip(ω)) signals for the PEM response functions and for the difference in the optical throughputs for p- and s-polarized light.42 Finally, the measured spectra were background-corrected due to the absorption of IR photons by the solvent in the thin layer cavity. The spline interpolation technique described by Zamlynny et al.42 was used for this background correction. The background corrected spectrum plots ∆S, which is proportional to the absorbance A of the adsorbed molecules as follows ∆S )

2(Is - Ip) ≈ 2.3Γ ) 2.3A Is + I p

(1)

where Γ is the surface concentration of the adsorbed species and is the decimal molar absorption coefficient of the adsorbed species.

Results Electrochemistry. Figure 2 plots the charge density vs potential curves determined from chronocoulometric experiments for the film-free Au(111) electrode and the bilayer-covered electrode. At the most negative potentials, the charge density curves corresponding to the surface covered by the bilayer merge with the curve for the film-free surface, indicating that the film is desorbed from the metal surface. Independent neutron reflectivity experiments demonstrated that in this state the film is detached from the electrode surface but remains in close proximity, separated from the metal by ∼1 nm thick cushion of the solution.32,33 A characteristic step appears at the charge density curve when the bilayer is adsorbed (in direct contact) with the metal surface. This is due to the decrease of the interfacial capacity. The data in Figure 2 show that the DMPC bilayer is adsorbed on the metal surface when the absolute value of the charge on the metal is less than 10 µC cm-2. The plateau section on the curve (38) Li, N.; Zamlynny, V.; Lipkowski, J.; Henglein, F.; Pettinger, B. J. Electrochem. Chem. 2002, 524-525, 43-53. (39) Barner, B. J.; Green, M. J.; Saez, E. I.; Corn, R. M. Anal. Chem. 1991, 63, 55-60. (40) Green, M. J.; Barner, B. J.; Corn, R. M. ReV. Sci. Instrum. 1991, 62, 1426-1430. (41) Buffeteau, T.; Desbat, B.; Blaudez, D.; Turlet, J. M. Appl. Spectrosc. 2000, 54, 1646-1650. (42) Zamlynny, V.; Zawisza, I.; Lipkowski, J. Langmuir 2003, 19, 132-145.


Figure 2. Charge density vs potential plot for DMPC bilayer transferred (9) on the Au(111) electrode surface at the surface pressure of 42 mN m-1, (0) at the film-free Au(111)/0.1 M NaF in H2O interface. Inset: The shift of the potential of zero charge (EN) vs surface coverage of DMPC in bilayers transferred at various surface pressures onto the Au(111) electrode surface. Potentials measured vs the Ag|AgCl|Cl- reference electrode.

corresponding to the bilayer-covered electrode has a small step at E ≈ -0.1 V. This behavior suggests that the adsorbed bilayer exists in different states in the range of potentials between -0.4 < E < -0.1 V and -0.1 < E < 0.2 V. An increase in the charge at E > 0.2 V indicates a further transformation of the film at these positive potentials. The adsorption of phospholipid molecules onto the Au electrode surface causes a small shift of the potential of zero charge (pzc) in the negative direction, shown in the inset to Figure 2. The shift w 43 of the pzc (EN) is described by EN ) Γ(µorg ⊥ - nµ⊥ )/, where Γ is the surface concentration of organic molecules, is the w permittivity, µorg ⊥ and µ⊥ are the components of the permanent dipoles of the organic and water molecules in the direction normal to the surface, respectively, and n is the number of water molecules displaced from the surface by one organic molecule. Since the transfer ratio was 1 ( 0.1, the surface concentration of DMPC molecules in the bilayer at the gold-solution interface was taken to be twice Γ at the surface of the Langmuir trough. Indeed, the inset to Figure 2 shows that EN depends linearly on the concentration of DMPC molecules in the bilayer. Linear extrapolation to the limiting surface concentration Γlim ) 8.74 × 10-10 mol cm-2 gives EN ) -0.127 V. At the pzc of the Au(111)|solution interface, water molecules are expected to have a small preferential orientation with the oxygen atom directed toward the metal, and hence µw⊥ should be rather small44 and its sign should be negative. Therefore, the absolute value of the dipole potential due to the orientation of permanent dipoles of phospholipid molecules (µorg ⊥ ) should be somewhat higher than 127 mV. An isolated DMPC molecule has a huge dipole moment of ∼20 D.45,46 Therefore, the small value of EN indicates that dipoles are oriented predominantly in the plane of the bilayer and have only a small component in the direction normal to the surface, consistent with the literature.45-48 In addition, the small value of EN indicates that the asymmetry (43) Lipkowski, J.; Stolberg, L. Molecular adsorption at gold and silVer electrodes; Lipkowski, J., Ross, P. N., Eds.; VCH Publishers: New York, 1992; pp 171-238. (44) Lipkowski, J.; Nguyen Van Houng, C.; Hinnen, C.; Parsons, R. J. Electroanal. Chem. 1983, 143, 375-396. (45) Stern, H. A.; Feller, S. E. J. Chem. Phys. 2003, 118, 3401-3412. (46) Wohlert, J.; Edholm, O. Biophys. J. 2004, 87, 2433-2445. (47) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21-51. (48) Raudino, A.; Castelli, F.; Briganti, G.; Cametti, C. J. Chem. Phys. 2001, 115, 8238-8250.

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Figure 3. Left-hand side vertical axis: (9) plot of σM/C representing the potential drop across the DMPC membrane; (0) plot of (E Epzc - φ2). Right-hand side vertical axis gives the electric field corresponding to the σM/C1d or (E - Epzc - φ2)/d for the bilayer transferred at 42 mN m-1. Potentials measured vs the Ag|AgCl|Clreference electrode.

of the surface potentials of the two leaflets of the bilayer, one facing the metal and the other the bulk of the solution, must be small. The charge density data can be used to estimate the potential drop across the membrane equal to the potential difference between the metal and the outer Helmholz plane (∆φM-2), using the following formula29a

∆φM-2 )

σM + χM C1

(2)

where C1 is the capacity of the inner layer and χM is the surface potential of the membrane. The capacity C1 can be calculated from the total capacity of the interface C (determined by numerical differentiation of the charge density curves) and the theory of the diffuse part of the double layer using equations given in ref 29b. Figure 3 plots σM/C1 calculated from the charge density curves, for potentials where the bilayer is directly adsorbed at the gold surface. The open points plot the values of (E - Epzc - φ2) where Epzc is the potential of zero charge for the bilayer-covered electrode and φ2 is the outer Helmholtz plane potential. The potential difference E - Epzc is equal to E - Epzc ) ∆φM-S(E) ∆φM-S(Epzc). Further, since ∆φM-S(Epzc) ) χM, the potential drop across the membrane may also be expressed as equal to ∆φM-2 ) E - Epzc - φ2 + χM. Consequently, the values of σM/C1 should be equal to the values of (E - Epzc - φ2). Figure 3 shows that the agreement between these two sets of data is good. Becucci et al.29a estimated the magnitude of the surface membrane potential χM to be ∼145 mV. With this estimate, the data in Figure 3 show that the bilayer is adsorbed at the gold surface when the potential drop across the membrane is less than 0.4 V. When higher potentials are applied to the electrode, the bilayer is detached from the metal surface. This result is in a good agreement with the magnitude of the transmembrane potential that can cause electric breakdown of a planar bilayer, which is estimated to be 0.3 to 0.4 V by Hianik.24 Considering that the thickness of a membrane bilayer is approximately 5 nm, the electric field across the bilayer changes between ∼-1.0 × 108 V m-1 and ∼+2 × 107 V m-1 in the potential region where the bilayer is adsorbed at the metal surface. Spectroelectrochemistry. Chain Region. Figure 4A shows the PM IRRA spectra in the CH stretching region for DMPC

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Figure 4. (A) The PM IRRA spectra in the CH stretching region of the DMPC bilayer transferred on the Au(111) electrode at 42 mN m-1 in 0.1 M NaF/D2O solution at potentials indicated on the figure. Thick line: the calculated spectrum for randomly oriented molecules in a 5.4 nm thick DMPC bilayer. (B) Deconvoluted spectrum at E ) -0.2 V with band assignment.

bilayers transferred onto the Au(111) electrode at a surface pressure of 42 mN m-1, for different potentials as indicated in the figure. The thick black line plots the spectrum for randomly oriented DMPC molecules. Figure 4B shows the deconvoluted spectrum in the CH stretching region for the DMPC bilayers transferred at 42 mN m-1 at E ) -0.2 V. Four bands corresponding to the methyl asymmetric (νas(CH3) at ∼2960 cm-1), methylene asymmetric (νas(CH2) at ∼2924 cm-1), methyl symmetric (νs(CH3) at ∼2972 cm-1), and methylene symmetric (νs(CH2) at ∼2854 cm-1) stretches can be found for the deconvolution of these spectra. The shoulder on the lowerfrequency side of the asymmetric methylene stretching band at ∼2898 cm-1 corresponds to a Fermi resonance between the symmetric methylene stretching and methylene bending modes. The deconvoluted peak at ∼2934 cm-1, seen as the shoulder on the higher-frequency side of the νas(CH2) band, arises from Fermi resonance between the symmetric methylene stretching and the methylene bending modes.49 Figure 5A shows complementary PM IRRA spectra in the CH bending region for DMPC bilayers transferred onto the Au(111) electrode at the potentials indicated on the figure. Again, the spectrum of randomly distributed molecules in the bilayer is represented by the thick black line. The deconvoluted spectra in the CH bending region are shown in Figure 5B. This band is composed of four overlapping peaks located at 1457.0 ( 0.2 cm-1, 1468.5 ( 0.2 cm-1, 1477.8 ( 0.3 cm-1, and ∼1490 cm-1, originating from the C-terminal methyl asymmetric bending, methylene bending, and two peaks originating from the asymmetric methyl bending in the choline group, respectively.50 (49) MacPhail, R. A.; Strauss, H. L.; Snyder, R. G.; Elliger, C. A. J. Phys. Chem. 1984, 88, 334-341. (50) Fringeli, P. U. Z. Naturforsch. 1977, 32c, 20-45.

Zawisza et al.

Figure 5. (A) The PM IRRA spectra in the CH bending region in DMPC bilayers transferred at 42 mN m-1 onto the Au(111) electrode surface at potentials indicated on the figure. Thick line: the spectrum calculated for randomly distributed molecules in a 5.4 nm thick bilayer. (B) Deconvoluted spectrum at E ) -0.2 V with band assignment.

Table 1 lists the positions and full widths at half-maximum (fwhm) for the asymmetric and symmetric methylene stretching and bending bands in bilayers transferred at 42 mN m-1. For comparison, positions and fwhm of the bands in the spectrum calculated from optical constants determined for the suspension of the DMPC vesicles in D2O at 23 °C and in the bilayer formed by fusion of vesicles4 are also included in this table. In DMPC bilayers supported at the gold electrode, the positions of the νas(CH2) and the νs(CH2) and their full widths at half-maximum are dependent on potential. Thus, two sets of values characteristic for the adsorbed film (at E > -0.45 V) and for the detached film (at E > -0.45 V) are given in Table 1. The error bars for the reported values represent the uncertainty of the band deconvolution procedure. Two deconvolution methods were used: one assuming a Lorentzian band shape and one assuming a mixed Lorentzian-Gaussian band shape. The error bars represent differences between the data calculated using these procedures. The gel-liquid crystalline phase transition in a DMPC bilayer is observed at 24°; hence, these data refer to the gel phase in a pretransition state. The frequencies of the band maximum lower than 2920 cm-1 for νas(CH2) and lower than 2850 cm-1 for νs(CH2) are characteristic for the gel state of the bilayer in which the acyl chains are fully stretched and assume an all-trans conformation.51-53 Higher frequencies for the methylene bands indicate the presence of gauche conformations and melting of the chains. (51) Casal, H. L.; Mantsch, H. H. Biochim. Biophys. Acta 1984, 779, 381401. (52) Mantsch, H. H.; McEchaney, R. N. Chem. Phys. Lipids 1991, 57, 213226. (53) Mitchell, M. L.; Dluhy, R. A. J. Am. Chem. Soc. 1988, 110, 712-718.


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Table 1. Frequencies and fwhm of the Stretching and Bending Modes of the CH2 Group DMPC bilayer at Au(111) transferred at 42 mN m-1/cm-1

DMPC vesicles in D2O/cm-1

mode

2922 ( 0.5

νas(CH2)/cm-1 for E > -0.45 V for E < -0.5 V fwhm of νas(CH2)/cm-1 for E > -0.45 V for E < -0.5 V νs(CH2)/cm-1 for E > -0.45 V for E < -0.5 V fwhm of νs(CH2)/cm-1 for E > -0.45 V for E < -0.5 V δ(CH2)/cm-1 fwhm of δ(CH2)/cm-1

2922 ( 0.3 2923 ( 0.5

18.5 ( 0.5

16.5 ( 1 14 ( 1

2853 ( 0.3 12.2 1468.5 ( 0.2 9.8 ( 0.2

The band frequencies compiled in Table 1 are close to the transition between the gel and the liquid crystalline state and indicate that a significant fraction of the chains may remain in the trans conformation; however, some of them have melted. The frequencies and bandwidths for the CH2 stretching vibrations in the bilayer formed by the LB-LS method and for the bilayer formed by fusion of vesicles are quite comparable, indicating the same physical state of the acyl chains for the two bilayers. However, bands in the bilayer formed by vesicle fusion have somewhat lower frequencies. In bilayers supported at the Au(111) surface, the frequencies of νas(CH2) and νs(CH2) are slightly red-shifted with respect to the corresponding band positions in the spectrum for vesicles, indicating that the adsorbed film has less gauche conformations. In addition, the bands in the adsorbed films are narrower than for the vesicles, indicating that DMPC molecules are slightly less mobile in the adsorbed bilayer. In the desorbed state (at E < -0.5 V) for the bilayer formed by the LB-LS method, the band positions and fwhm’s are close to the frequencies and widths of the corresponding bands for vesicles, indicating that this bilayer is less ordered in the adsorbed than in the detached state. For all DMPC bilayers discussed in this paper, the methylene bending mode appears as a single absorption band located at ∼1468 cm-1. This frequency value for the δ(CH2) band and the fact that no splitting of this band is observed indicate that the hydrocarbon chains are arranged in a tilted hexagonal or triclinic subcell.54,55 From the spectra in Figures 4 and 5, it is clear that the intensities of the methylene stretching and bending bands are dependent on the electrode potential. Changes of the integrated intensities of the IR absorption bands are related to a change in the orientation of the vector of the transition dipole moment with respect to the vector of the electric field of the photon, which is perpendicular to the metal surface. Therefore, the integrated intensities may be used to calculate the angle (θ) between the direction of the transition dipole and the surface normal using the following equation56,57

cos2 θ )

1 A(E) 3 A(random)

(3)

DMPC bilayer by fusion of vesicles at Au(111) from (Bin et al. 2005)/cm-1 2920.5 ( 0.3 2919.7 ( 0.3 16.5 ( 0.5 16 ( 0.5

2852.5 ( 0.5 2854 ( 0.3

2852 ( 0.2 2851.6 ( 0.2

9.5 ( 0.5 11.5 ( 0.5 1468.5 ( 0.2 10.5 ( 0.6

9.8 ( 0.3 9.0 ( 0.3 1468 ( 0.3 7.2 ( 0.4

in the bilayer at the gold surface and in a hypothetical bilayer of randomly distributed molecules, respectively. In order to find θ, the spectra for films with randomly oriented molecules has to be obtained, so that A(random) can be determined. These spectra were calculated from optical constants for DMPC,4 assuming a 5.4 nm thick film with a surface coverage of 0.9 for the bilayer formed at a surface pressure of 42 mN m-1. Figure 6 plots the change of the angle θ with potential for the νas(CH2) (top panel A), νs(CH2) (middle panel B), and δ(CH2) (bottom panel C). The angles between the directions of the transition dipole moments of the CH2 vibrations are large and change from ∼79° at negative potentials where the bilayer is detached from the metal surface to ∼72° at potentials where the bilayer is adsorbed at the metal surface. The estimated error in the determination of θ is (3o. It is chiefly due to the uncertainty of the background correction and band deconvolution procedures. Since the same background correction and band deconvolution procedures were used in the treatment of all spectra, all θ values are affected by the same error. This error shifts all points in Figure 6a-c up or down by the same increment of (3o without affecting the potential dependence of θ displayed in this figure. The spread of experimental points in a given series of measurements is much smaller than the estimated error. The error analysis is described in detail in ref 30. The cartoons depicted in Figure 6 show that the vector of the transition dipole of the asymmetric stretch lies along the line joining two hydrogen atoms of the methylene group. The vectors of the symmetric methylene stretch and the bending mode have the same direction along the bisector of the CH2 plane.50 The angles in the middle and the bottom panels of Figure 6 differ by only 2-3°. These differences are within the estimated error bars. The satisfactory agreement between these two data sets indicates that similar structural information can be extracted from the analysis of the methylene symmetric stretching and bending modes. It also indicates that the background correction and the band deconvolution procedures are free of major errors. The vectors of the transition dipoles of the symmetric and asymmetric methylene stretches are perpendicular to each other and are perpendicular to the line of a fully extended all-trans hydrocarbon chain. The angles between these directions and the surface normal are related by the following equation58

where A(E) and A(random) are the integrated intensities of the bands (54) Cameron, D. G.; Casal, H. L.; Gudgin, E. F.; Mantsch, H. H. Biochim. Biophys. Acta 1980, 596, 463-467. (55) Small, D. M. J. Lipid Res. 1984, 25, 1490-1500. (56) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 52-66. (57) Allara, D. L.; Swalen, J. D. J. Phys. Chem. 1982, 86, 2700-2704.

cos2 θνas + cos2 θνs + cos2 θtilt ) 1

(4)

(58) Umemura, J.; Kamata, T.; Kawai, T.; Takenaka, T. J. Phys. Chem. 1990, 94, 62-67.

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Figure 6. Dependence of the angle (θ) between the direction of the transition dipole moment and the surface normal on the electrode potential for (A) νas(CH2), (B) νs(CH2), and (C) δ(CH2) vibrations in the DMPC bilayer transferred onto Au(111) at 42 mN m-1; potential steps moving in the negative (9) and positive (0) directions. Cartoon shows the direction of the νas(CH2), νs(CH2), and δ(CH2) dipole moments relative to the direction of the hydrocarbon chain. Each point has an error bar of (3°. To show the trend, the scale of the angle axis is expanded and the error bars are omitted. Potentials measured vs the Ag|AgCl|Cl- reference electrode.

where θtilt is the angle between the line of fully extended alltrans hydrocarbon chain and the surface normal. For the DMPC bilayer transferred at the surface pressure of 42 mN m-1, the hydrocarbon chains exist predominantly in the all-trans, fully extended conformation; however, a certain portion of the chains are melted and gauche conformations are formed.53,59,60 Changes of the tilt angle of the acyl chains in the DMPC bilayer are plotted versus potential in Figure 7. The LB-LS bilayer was transferred from the air-solution interface of the Langmuir trough at the open circuit potential (∼0.35 V). At this potential, the tilt angle of the chains is 25 ( 3°. In our experiments, (59) Sengupta, K.; Raghunathan, V. A.; Katsaras, J. Phys. ReV. E 2003, 68, 031710-1-12. (60) Snyder, R. G.; Tu, K.; Klein, M. L.; Mendelssohn, R.; Strauss, H. L.; Sun, W. J. Phys. Chem. B 2002, 106, 6273-6288.

θνas ≈ θνs and ∆θνas ≈ ∆θνs. Error propagation analysis gives ∆(cos2 θtilt) ) ∆(cos2 θνas) x2. Taking into account that ∆(cos2 θ) ) 2 cot θ∆θ, the error in the tilt angle is equal to ∆θtilt ) x2 cot θνas/cot θtilt ∆θνas. Since θνas > θtilt, the error ∆θtilt should be less than ∆θνas. The above estimate of the error in the tilt angle is therefore rather conservative. The tilt angle of DMPC molecules in the film compressed to 42 mN m-1 at the surface of the Langmuir trough can be determined from the formula47

ADMPC cos θtilt ) 2Σ

(5)

where ADMPC is the area per DMPC molecule in the monolayer and ∑ is the cross-sectional area of a single hydrocarbon chain.


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Figure 8. Dependence of the tilt angle of the hydrocarbon chains vs (E - Epzc - φ2) in the LB-LS DMPC bilayer adsorbed directly on the Au(111) surface. Each point has an error bar of (3°. To show the trend, the scale of the angle axis is expanded and the error bars are omitted. Potentials measured vs the Ag|AgCl|Cl- reference electrode.

Figure 7. (A) Dependence of the tilt angle of the chains vs potential for the bilayer produced by the LB-LS transfer method at a surface pressure of 42 mN m-1(9,0) and spread from vesicles (b,O); (9,b) negative, (0,O) positive potential steps. Potentials measured vs the Ag|AgCl|Cl- reference electrode. (B) Packing of the DMPC molecules at a small tilt angle and a zigzag arrangement of the polar heads for a bilayer with water on both sides of the membrane. (C) Packing of the DMPC molecules at the metal-solution interface with polar heads located in one plane.

Using ADMPC ) 0.45 nm2 and ∑ ) 0.195 nm2, the tilt angle of DMPC molecules at the air-solution interface amounts to 30°. The tilt angle for the bilayer at the gold-solution interface is about 5° smaller. The area available per one phospholipid molecule affects the packing of the polar head groups in the bilayer. When the area per molecule is ∼0.45 nm2, the polar head groups may be located in one plane,47 as shown schematically in Figure 7C. The bilayer is desorbed from the metal surface when E < -0.5 V. As mentioned previously, neutron reflectivity studies32 have demonstrated that, after desorption, the bilayer remains in the proximity of the gold surface, separated from the metal by a thin ∼1 nm thick layer of solvent. Figure 7 shows that in the desorbed state the tilt angle of the acyl chains decreases to 17 ( 3°. A tilt angle of ∼17° corresponds to the area per molecule of ∼0.41 nm2, which is expected for a DMPC bilayer in the gel state in which the polar heads are packed in a zigzag fashion, as illustrated schematically by the cartoon in Figure 7B.47 The data in Figure 7 indicate that the structure of the bilayer is different for the adsorbed and desorbed states. In the adsorbed state, all polar heads are in contact with the metal and are located in one plane. Upon desorption, the two leaflets of the bilayer are exposed to water (solution bulk and the thin layer of the solvent between the bilayer and the metal). With water on both sides, the head groups can be packed in a zigzag fashion, allowing for a smaller tilt of the chains and a stronger chain-chain interaction. Therefore, the large change between the tilt angle in the adsorbed and the desorbed states is driven by the difference in the packing of the polar heads of DMPC molecules for the two states. Figure 7 shows that there are small but measurable changes of the tilt angle with potential (or surface pressure) for the bilayer

in the adsorbed state. These changes are analyzed in Figure 8, where the tilt angles for the adsorbed state are plotted against the rational potential corrected for the potential drop across the diffuse layer (E - Epzc - φ2). We mentioned earlier that the rational potential may be used as equivalent of the transmembrane potential applied to a membrane in a patch clamp or BLM experiment. When a potential difference (E - Epzc - φ2) is applied to the membrane, it compresses the bilayer with a pressure p equal to24

p ) C(E - Epzc - φ2)2/2d

(6)

where C is the capacity and d is the thickness of the bilayer. This pressure causes a change in the thickness ∆d (thinning of the bilayer) which depends on the magnitude of the membrane’s Young’s modulus of elasticity, K⊥ 24

∆d ) -

pd K⊥

(7)

The thickness of the bilayer can be expressed in terms of the length (l) of the two chains and their tilt angle, θtilt

d ) l cos θtilt

(8)

Equations 6-8 can be combined to give the following expression for the change of the cos θt due to the electrostriction phenomenon

∆ cos θtilt ) -

C(E - Epzc - φ2)2 2lK⊥

(9)

Knowing that at Epzc the tilt angle is equal to ∼24° and taking C ) 0.5 µF cm-2, l ≈ 5 nm, and K⊥ ≈ 2 × 107 Pa 24 for a supported bilayer, one can estimate that the expected change in tilt angle, due to electrostriction at (Epzc - E - φ2) ) 0.5 V should be less than 5°. Figure 8 shows that the change observed in this study is ∼2.5° and is smaller by a factor of 2. This may be due to the uncertainty of the estimate of K⊥. Recently, Schwarzott et al.26 observed electric field driven changes in the tilt angle of DMPC multibilayers formed between two silicon electrodes that was less than 1°. However, the field applied to the multibilayers was a few times smaller than in the present study. By comparing the tilt angle data in Figure 8 with the charge density data in Figure 1, it can reasonably be concluded that the

5188 Langmuir, Vol. 23, No. 9, 2007

small step in the charge density curves at E ≈ -0.1 V versus Ag/AgCl electrode corresponds to a transition between compressible and noncompressible states of the film. At (E - Epzc - φ2) > -0.2 V, the changes in the tilt angle due to electrocompression are very small and the bilayer behaves as a noncompressible film. In contrast, at higher values of the rational potential the changes in tilt angle due to the electrostriction become significant and the bilayer becomes electrocompressible. Finally, Figure 7 also compares changes of the tilt angle with potential for the bilayer formed at the Au(111) electrode surface by the LB-LS technique to the changes of the tilt angle measured for a DMPC bilayer formed by fusion of unilamellar vesicles.4 The differences are striking. In the bilayer formed by fusion of vesicles, the tilt angles are essentially double that measured for the bilayer formed using the LB-LS technique. Further, the tilt angle in the adsorbed state of the bilayer formed from vesicles is almost independent of electrode potential. No change in the tilt angle due to the electrostriction can be seen for this bilayer. However, in the bilayer formed by fusion of vesicles, the tilt angles are close to the magic angle of 54.5° and the bilayer may be disordered. The lack of order may prevent detection of a rather small electrostriction effect. Our results show that a bilayer formed at the gold electrode surface by a combination of the LB and LS techniques is superior to a bilayer formed by vesicle fusion. Polar Head Region. The IR spectra provide useful information concerning the hydration, conformation, and orientation of the polar head group region. When eq 3 is used to calculate the orientation of transition dipoles in the polar head region, one should take into account that the hydration of the DMPC molecules at the electrode surface may be different than in the spectrum calculated from optical constants. Consequently, changes in the band intensity may be due not only to a change in orientation, but also to a change in hydration. To estimate the effect of hydration, calculations of tilt angles were performed using optical constants determined for a solution of DMPC in CCl4 and for a suspension of DMPC vesicles in D2O or H2O. When the difference between the tilt angles calculated using the two sets of the optical constants were small, the calculated tilt angles are reported. When the differences were significant, the hydration influenced the band intensity, and as a result, the tilt angles were not determined. Choline Moiety. The choline group is poorly hydrated, and hence the differences between IR band intensities in the bilayer adsorbed at the electrode surface and in vesicles are affected chiefly by a change in orientation. The IR bands of the choline group involve bending and stretching vibrations. Figure 5B shows the two asymmetric methyl bending modes of the choline moiety, (δas(N+(CH3)3)) are observed at ∼1490 cm-1 and ∼1480 cm-1, where they overlap with δ(CH2) and δ(CH3) modes. In an aqueous dispersion of DMPC vesicles, the choline bending modes are located at 1478.5 cm-1 and 1490.9 cm-1. In the bilayer supported at the gold electrode surface, the band positions depend on the electrode potential. In the bilayer desorbed from the electrode surface (E < -0.5 V), the δas(N+(CH3)3) modes are found at 1480.0 ( 0.2 cm-1 and 1492.0 ( 0.3 cm-1. In the bilayer adsorbed at the metal surface (E > -0.4 V), methyl bending modes of the choline group are red-shifted to 1477.5 ( 0.3 cm-1 and 1490.5 ( 0.2 cm-1. Similarly, small changes of the band position with potential were also observed for the bilayer formed by fusion of vesicles.30 These results indicate that the overall volume of the choline group decreases at positive potentials, where the bilayer is adsorbed at the electrode surface.61 (61) Wong, P. T. T.; Mantsch, H. H. Chem. Phys. Lipids 1988, 46, 213-224.

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Figure 9. The PM IRRA spectra in the C-N stretching region in the DMPC bilayers: (A) calculated from optical constants for randomly distributed molecules in 5.4 nm thick film; (B) in the LB-LS bilayer at electrode potentials marked on the figure.

Figure 9 shows the PM IRRA spectra of DMPC bilayers in the 1000-800 cm-1 spectral region, where the C-N-C stretching bands are located. The position of the symmetric C-N-(CH3)3 stretches provides information on the conformation of the O-CC-N frame of the choline moiety.50 In the trans O-C-C-N conformation, the νs(CN+(CH3)3) peaks are observed at 925 cm-1 and 875 cm-1.50,62 In the gauche O-C-C-N conformation, these two bands appear at ∼900 cm-1 and ∼860 cm-1.50 The gauche conformation of the O-C-C-N frame is observed in hydrated films and the trans conformation in dry films.50 Figure 9A plots a transmission spectrum for an aqueous dispersion of vesicles. The spectrum displays peaks at ∼972 cm-1 and ∼953 cm-1 which correspond to the asymmetric νas(CN+(CH3)3) stretches and a weaker band at ∼925 cm-1 which corresponds to the symmetric νs(CN+(CH3)3) stretch. The frequency of the νs(CN+(CH3)3) indicates that the choline group in the bilayer of vesicles assumes a trans conformation. The six curves in Figure 9B show PM IRRAS spectra of the DMPC bilayer formed at the gold electrode surface using the LB-LS technique. The νas(CN+(CH3)3)bands are located at ∼970 cm-1 and ∼948 cm-1, and are slightly red-shifted with respect to the position of these bands in the spectrum for the suspension of vesicles. However, the νs(CN+(CH3)3) band is significantly red-shifted to ∼905 cm-1. This large red shift of the νs(CN+(CH3)3) to ∼905 cm-1 indicates that, in contrast to the bilayer made from vesicle fusion, the O-C-C-N frame of the choline moiety in the bilayer formed by the LB-LS technique assumes the gauche conformation. (62) Fringeli, P. U. Biophys. J. 1981, 34, 173-187.


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Figure 10. Dependence of the angles (θ) between the direction of the transition dipole moment and the surface normal on the electrode potential for the following vibrations: (A) δas(N+(CH3)3) at 1490 cm-1, (B) δas(N+(CH3)3) at 1480 cm-1, and (C) νas(CN+(CH3)3) at 970 cm-1, for a DMPC bilayer transferred onto Au(111) at 42 mN m-1; (9) negative and (0) positive potential steps. Potentials measured vs the Ag|AgCl|Cl- reference electrode. Cartoon shows the arrangement of the choline moiety and directions of the δas(N+(CH3)3) and νas(CN+(CH3)3) transition dipole moments. Each point has an error bar of (3°. To show the trend, the scale of the angle axis is expanded and the error bars are omitted. In panel A symbols O and b represent bilayer formed by fusion of unilamellar vesicles.

The intensities of the δas(N+(CH3)3) and the νas(CN+(CH3)3) bands provide information concerning the orientation of the choline moiety with respect to the surface normal. Figure 10 plots the angle between the directions of the transition dipoles of the δas(N+(CH3)3) bands at ∼1490 cm-1 and at ∼1480 cm-1 and the νas(CN+(CH3)3) band at ∼970 cm-1 and the surface normal, calculated from the integrated band intensities and eq 3. The cartoons in Figure 10 show the directions of these transition dipoles.50 The angle θ of the δas(N+(CH3)3) at 1490 cm-1 provides information concerning the tilt of the CN bond with respect to the surface normal. In the desorbed state of the bilayer, at E < -0.5 V and σM < -10 µC cm-2, the C-N bond of the positively charged choline group has a ∼63° angle with respect to the surface normal. The angle decreases by ∼10° when the bilayer adsorbs on the electrode surface, at E > -0.4 V and σM > -10 µC cm-2.

The transition dipoles of the δas(N+(CH3)3) at 1480 cm-1 and the νas(CN+(CH3)3) at 970 cm-1 have the same direction. The experimental values of their tilt angles, shown in Figure 10B,C agree very well. This agreement indicates that the errors caused by the uncertainty of the band deconvolution and background correction procedures are small. Overall, the changes of these angles with potential are small. In the bilayer formed by fusion of vesicles, the tilt angles of the δas(N+(CH3)3) band at 1480 cm-1 and the νas(CN+(CH3)3) are equal to 66 ( 3° and are potential-independent.30 Clearly, these tilt angles are comparable in magnitude for the two types of bilayer. In contrast, Figure 10A shows that the angle between the C-N bond and the surface normal is significantly smaller in the bilayer formed by the LBLS method. The differences in the orientation of the choline group in the two films are due chiefly to the change in the tilt of the C-N bond.

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Figure 11. The PM IRRA spectra in the phosphate stretching region (A,B) of the νas(PO2-) and (C,D) of the νs(PO2-) of a DMPC bilayer transferred on the Au(111) electrode at a surface pressure of 42.0 mN m-1 at electrode potentials indicated on the figure. Thick line: the spectra of randomly distributed molecules in 5.4 nm thick DMPC film. (B,D) deconvoluted spectra at E ) 0.2 V with band assignment. Potentials measured vs the Ag|AgCl|Cl- reference electrode.

Phosphate Group. We will restrict analysis of the phosphate group region to the discussion of the non-esterified P-O stretching bands. The asymmetric stretching vibration(νasPO2-) appears at ∼1230 cm-1 and the symmetric stretching vibration(νsPO2-) at ∼1090 cm-1. 50,63,64 The PM IRRAS spectra in the phosphate stretching region are shown in Figure 11A. The asymmetric phosphate stretching band at ∼1230 cm-1 overlaps with the band at ∼1170 cm-1, which originates from the C-O asymmetric stretching mode in the ester group of the DMPC molecule as shown in Figure 11B. For the aqueous DMPC vesicle dispersion, the νas(PO2-) band is found at 1237.0 ( 0.2 cm-1 and its fwhm is 47 ( 1 cm-1. In DMPC bilayers assembled on a Au(111) electrode surface, the position of the νas(PO2-) band shifts with potential. For the desorbed DMPC bilayer (E < -0.5 V), the νas(PO2-) peak appears at 1237.5 ( 0.5 cm-1 and its fwhm is 38 ( 1 cm-1. In the adsorbed state (E > -0.5 V), the position of this band is redshifted to 1231.0 ( 0.5 cm-1 and its fwhm slightly decreases to 36 ( 1 cm-1. The position and the width of the νas(PO2-) band are known to depend on the state of hydration of the phosphate group. The νas(PO2-) peak position changes from ∼1280 to ∼1200 cm-1 and the band becomes broader on transition from a dry to a hydrated phosphate.63,64 The positions and the widths of the νas(PO2-) band indicate that the phosphate group is somewhat more hydrated in vesicles than in the bilayer supported at the electrode surface. (63) Shimanouchi, T.; Tsuboi, M.; Kyogoko, Y. Infrared spectra of nucleic acids and related compounds; Interscience: New York, 1964; Vol. 7, pp 467499. (64) Binder, H. Appl. Spectrosc. ReV. 2003, 38, 15-69.

Panel 11C shows the PM IRRA spectra in the symmetric phosphate stretching region. The band assignment and deconvolution are shown in Figure 11D. The νs(PO2-) band overlaps with the C-O[P] asymmetric stretches of the phosphate ester group. In DMPC molecules, the phosphate esterified C-O bonds are not equivalent: one is bonded to the rigid glycerol moiety and the second to the nonrigid choline group.50 Therefore, the C-O [P] asymmetric stretching band is composed of two peaks, one centered at 1068.5 cm-1 originating from the (νasC-O[P]) stretch of the ester group attached to glycerol and the second (νasC-O[C]) at 1055.3 cm-1 due to the esterified phosphate group bonded to the choline group.6,50,62,61,64 The deconvolution of the C-O[P] bands is difficult, and hence further discussion will be restricted to the analysis of the νs(PO2-) band. In DMPC vesicles, the νs(PO2-) peak appears at 1091 cm-1, and its fwhm is equal to 33 ( 1 cm-1. In the desorbed state of the bilayer, the band is located at 1094.5 cm-1, with a fwhm equal to 15.0 ( 1 cm-1. For the bilayer in the adsorbed state, the νs(PO2-) position shifts to 1089.5 cm-1 and its width increases to 17.0 ( 1 cm-1. As in the case of the asymmetric phosphate stretch discussed earlier, the position and the shape of the νs(PO2-) band are also dependent on the hydration of the phosphate group. A red frequency shift on the order of 8 cm-1 and a band broadening were observed in the transition from dry to hydrated phosphate groups.61,63 Therefore, the analysis of the νs(PO2-) band confirms that the phosphate group of DMPC molecules in the bilayer supported at the Au(111) surface is somewhat less hydrated than in the aqueous suspension of vesicles. Similar values of the νs(PO2-) band position were reported for the DMPC bilayer


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Figure 12. Dependence of the angle (θ) between the direction of the transition dipole moment and the surface normal with potential for (A) νas(PO2-), (B) νs(PO2-), and (C) the tilt of the O-P-O line in the esterified region of the phosphate group for the DMPC bilayer transferred onto the Au(111) surface at 42 mN m-1 (9,0) and spread from vesicles (b,O); (9,b) negative, and (0,O) positive potential steps. Potentials measured vs the Ag|AgCl|Cl- reference electrode. Cartoon shows the direction of the νas(PO2-) and νs(PO2-) dipole moments relative to the arrangement of the phosphate group.

deposited at the Au(111) surface by fusion of vesicles. Apparently, hydration of the phosphate group is comparable for these two films. Since hydration of the phosphate group in the bilayer and in the aqueous suspension of vesicles is somewhat different, the calculation of the angle between the direction of the transition dipoles of νs(PO2-) and νas(PO2-) based on eq 3 may be affected by a systematic error. To estimate the magnitude of this error, calculations of the angle θ using optical constants determined for a solution of DMPC in CCl4 and for the aqueous suspension of DMPC vesicles were preformed.30 The differences between the values of the angles calculated using the two sets of optical constants were on the order of 5°. The differences between the hydration of the phosphate group of DMPC molecules in vesicles and in the bilayer supported at the Au(111) surface are much

smaller than that observed between DMPC vesicles and the solution of DMPC in CCl4. Consequently, by calculating the angle θ using eq 3 and optical constants determined for a suspension of vesicles, we may expect that the error due to the hydration effects will be smaller than 5°, and such an error is acceptable. Figure 12A,B plots the angles between the directions of the transition dipoles of the νas(PO2-) and νs(PO2-) bands and the surface normal, respectively. Cartoons show the directions of the two transition dipoles in coordinates of the phosphate group.63 These two vectors are located in the plane of the non-esterified phosphate group; they are perpendicular to each other and are perpendicular to the line of the O-P-O esterified phosphate group. Therefore, eq 4 can be used to calculate the angle between direction of the -O-P-O- line and the surface normal. This

5192 Langmuir, Vol. 23, No. 9, 2007

Figure 13. Cartoon showing the scheme of the glycerol moiety in phosphatidylcholine with marked directions of the transition dipole moment vectors of the ν(CdO) and the νas(COC) modes. Plot of the position of the νas(COC) stretching vs potential in the DMPC bilayer transferred by the LB-LS method at surface pressure of 42 mN m-1 (9) and spread from vesicles (b). Potentials measured vs the Ag|AgCl|Cl- reference electrode.

angle is plotted in Figure 12C. For comparison, the corresponding angles determined for the bilayer produced by vesicle fusion30 are also included in these figures. The data show that angles for the νas(PO2-) and the νs(PO2-) bands are larger, and as a consequence, the angles between the -O-P-O- line and the surface normal are smaller for DMPC molecules present in the bilayer produced by the LB-LS than by fusion of unilamellar vesicles. Interestingly, the tilt of the phosphate esterified group follows the tilt of the hydrocarbon chains in both bilayers (LBLS method and vesicle fusion). This behavior is consistent with crystallographic data concerning the structure of the DMPC bilayer.47 The Ester Group. Two bands which emanate from the glycerol ester group, the ν(CdO) stretch at ∼1740 cm-1 and the asymmetric νas(COC) stretch at ∼1180 cm-1, provide useful information concerning the hydration of the carbonyl group and the planarity of the glycerol frame. The νas(COC) band overlaps with the asymmetric phosphate νas(PO2-) stretching mode.50 The deconvolution of this spectral region into individual bands is shown in Figure 11B. The νas(COC) band is complex, consisting of vibrations of the -C(O)-O-C- groups in the β and γ chains. The ester group in the γ chain is coplanar with the chain, while the ester group of the β chain is out of the plane of the chain (see cartoon in Figure 13). The vibrations of the two ester -C(O)O-C- groups are poorly coupled with each other; however, they are strongly coupled with the wagging modes of the chains.

Zawisza et al.

According to refs 50 and 65, the νas(COC) band is observed at 1180 cm-1 when the C-C(O)-O-C frame of the ester group has a planar conformation. A deviation from the planar conformation leads to a red shift of this band down to ∼1160 cm-1. Figure 13 plots the position of the νas(COC) band for the bilayer formed by the LB-LS method (squares) and for the bilayer formed by vesicle spreading (circles). In a dispersion of DMPC vesicles, the νas(COC) band is located at 1175.5 cm-1. In the bilayer formed by fusion of unilamellar vesicles, the band position is ∼1195 cm-1 for the entire range of potentials studied, indicating that the C-C(O)-O-C frame has a planar conformation. In contrast, in the bilayer formed by LB-LS method the position of the νas(COC) band changes from 1180.5 cm-1 in the desorbed state to ∼1167 cm-1 in the adsorbed state (see Figure 13). This behavior suggests that a significant deviation from planarity takes place upon moving from the desorbed to the adsorbed state. In addition, this also indicates that the conformations of the C-C(O)-O-C frame in the bilayer formed by fusion of vesicles and by the LB-LS method are significantly different. Moreover, differences between the integrated intensities of this band for a solution of DMPC in CCl4 and for the aqueous suspension of vesicles was observed. These differences indicate that the intensity of this band is very sensitive to changes in the hydration of the glycerol moiety. Therefore, the tilt angles of the transition dipole of this band are not reported. The ester carbonyl CdO stretching band located at ∼1740 cm-1 is also quite complex. DMPC is a mixture of two conformers, DMPC-A (80%) and DMPC-B (20%).66 The normal coordinate calculations by Bin et al.4 show that vibrations of the CdO bands in the β- and γ-acyl chains are coupled, and as a result, the band splits into two bands corresponding to the in-phase and out-of-phase motion of the atoms. In the case of the predominant DMPC-A conformation, the coupling is weak, and the in-phase and out-of-phase components are separated by 5 cm-1. The transition dipoles of these bands are almost normal to the acyl chains; however, their direction rotates in the plane normal to the chains. The position of the CdO stretch depends on the hydration of the ester group. In hydrated phospholipids, the band splits into two components with maxima at 1743.3 cm-1 and 1728.8 cm-1, corresponding to the dry and hydrated ester groups, respectively.67,68 Figure 14A plots the CdO stretch band for the supported bilayer formed by the LB-LS method as a function of the electrode potential. Figure 14B plots the position of the band maximum as a function of the electrode potential (squares for the bilayer formed by the LB-LS method and circles for the bilayer formed by fusion of vesicles). To facilitate discussion, the differential capacity curve is also plotted in this figure. The changes of the peak position correlate well with the shape of the differential capacity curve. At negative potentials, where the capacity is high and the bilayer is in the desorbed state, the band maximum corresponds to ∼1742 cm-2. This value indicates that the ester group is poorly hydrated. However, upon moving to positive potentials, where the capacity is low and the bilayer is adsorbed at the gold surface, the peak position undergoes a significant red shift to ∼1731 cm-1. Such a shift indicates that (65) Bradbury, E. M.; Elliott, R. D.; Fraser, R. D. B. J. Chem. Soc. 1960, 1117-1124. (66) Aussenac, F.; Laguerre, M.; Schmitter, J. M.; Dufourc, E. J. Langmuir 2003, 19, 10468-10479. (67) Lewis, R. N. A. H.; McElhaney, R. N.; Pohle, W.; Mantsch, H. H. Biophys. J. 1994, 67, 2367-2375. (68) Blume, A.; Hubner, W.; Messer, G. Biochemistry 1988, 27, 8239-8249.


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Figure 14. (A) PM IRRA spectra in the CdO stretching region of DMPC bilayers transferred onto Au(111) surface at 42 mN m-1 at potentials indicated in the figure. Thick line: spectra of randomly distributed DMPC molecules in 5.4 nm thick film. (B) The position of the ν(CdO) stretch vs potential (symbols) and the capacity vs potential (line) plots of DMPC bilayer transferred onto Au(111) surface at surface pressure 42 mN m-1 (9,0) and spread from vesicles (b,O). (C) The angle between the transition dipole moment of the ν(CdO) and the electric field vectors vs potential plots of DMPC bilayer transferred onto Au(111) surface at 42 mN m-1 (9,0) and spread from vesicles (b,O); (9,b) negative, and (0,O) positive potential steps. Potentials measured vs the Ag|AgCl|Cl- reference electrode.

in the adsorbed state the ester group is well-hydrated. For comparison, Figure 14B also plots the peak position of the CdO stretch for the bilayer formed by fusion of unilamellar vesicles (circles). Although a red shift is also observed upon moving from the desorbed to the adsorbed state of the bilayer, numerically this shift is much smaller. This behavior shows that in the adsorbed state the ester group is more hydrated in the bilayer formed by the LB-LS method than it is for the bilayer formed by the fusion of vesicles. Figure 14C plots the angle between the average direction of the transition dipoles of the CdO stretch modes and the surface normal calculated from the integrated band intensity with the help of eq 3. The angles were calculated using both the optical constants for the aqueous suspension of vesicles and for the solution of DMPC in CCl4. The differences between the angles determined using these two sets of optical constants were on the

order of 3°, an acceptable level of uncertainty. The angles plotted in Figure 14C were calculated from optical constants determined for the aqueous suspension of vesicles. The angles are large and decrease when potential is changed in the positive direction. Figure 14C also plots the angle θν(CdO) determined for the bilayer formed by fusion of unilamellar vesicles.30 The angles in this set of data are much larger, and their changes with the transition from the desorbed to the adsorbed state of the bilayer are much more profound. A comparison of Figures 7 and 14C shows that the chain tilt angle and the angle of the transition dipole moment change with potential in opposing directions. This behavior confirms that the transition dipole of the ν(CdO) band is approximately 90° with respect to the acyl chains and that the DMPC-A conformation is the dominant structure of the DMPC molecule.

5194 Langmuir, Vol. 23, No. 9, 2007

Figure 15. Orientation of DMPC molecules in the bilayer formed by the LB-LS method: (a) E ) -0.8 V (detached film) and (b) E ) 0.3 V (adsorbed film); and in the bilayer formed by fusion of unilamellar vesicles: (c) E ) -0.8 V (detached film) and (d) E ) 0.3 V (adsorbed film).

Summary and Conclusions Using a combination of LB and LS methods, two monolayers of DMPC were transferred from the air-solution interface onto the Au(111) electrode surface. This approach allowed us to form the bilayer at a controlled surface pressure, providing a welldefined physical state. The effect of the potential applied to the electrode on the properties of the bilayer was then investigated by recording charge density-potential curves. We have demonstrated that the rational potential (E - Epzc - φ2) is equivalent to the transmembrane potential applied to a membrane in patch clamp or BLM experiments. The DMPC bilayer was found to be adsorbed (in direct contact with the metal surface) at (E Epzc - φ2) > -0.5 V. In the adsorbed state of the bilayer, the charge density curve displayed a small step at (E - Epzc - φ2) ≈ -0.2 V, indicating a transition from a compressible state at -0.5 V < (E - Epzc - φ2) < -0.2 V to a noncompressible state at (E - Epzc - φ2) > -0.2 V. The bilayer is desorbed (detached) from the gold surface at (E - Epzc - φ2) < -0.5 V. However, independent neutron reflectivity experiments revealed that in the desorbed (detached) state the bilayer remains supported at the gold electrode surface, separated from the metal by a ∼1.0 nm thick cushion of the electrolyte.32,33

Zawisza et al.

The PM IRRAS results provided significant information concerning the effect of the electric field on the structure of the bilayer supported at the metal electrode surface. We have demonstrated that the main change in the structure of the bilayer is observed when the bilayer is detached from the metal surface. In the adsorbed state, changes of the electric field acting on the bilayer amounting to several orders of magnitude have only a small effect on the orientation of the DMPC molecules. However, the adsorbed bilayer has an asymmetric environment with one leaflet being in contact with the metal and the second exposed to the electrolyte. Parallel experiments performed with one leaflet of the bilayer consisting of deuterated DMPC molecules demonstrated that the tilt angle of the acyl chains in the leaflet in contact with the metal is somewhat smaller that of the leaflet exposed to electrolyte.31 Tilt angles determined in the present study are values averaged over both leaflets. The PM IRRAS data for the bilayer formed by the LB-LS method were compared to the results of earlier work concerning a bilayer formed by fusion of unilamellar vesicles.4 The molecular models in Figure 15 summarize the main results of these studies. There are significant differences between the tilt angles of the acyl chains, the O-P-O line of the phosphate group, and the C-N bond of the choline moiety for the two bilayers. All these angles are significantly smaller in the LB-LS bilayer. These changes reflect significant differences between packing densities of phospholipid molecules in these two films. The packing densities are lower in the bilayer formed by fusion of vesicles, where the area per molecule changes from 0.47 to 0.66 nm2 on going from the desorbed to the adsorbed state. In contrast, these numbers vary from 0.41 to 0.45 nm2 for the bilayer formed by the LB-LS method. Thus, the LB-LS method allows one to form bilayers with a higher packing density, characterized by chain tilt angles, and consequently the thickness similar to that observed in bilayers of vesicles69 or in a stack of hydrated multibilayers.70 In conclusion, the structure and physical state of DMPC molecules in the monolayer at the air-water interface are to a large degree preserved in the bilayer deposited at the Au(111) electrode surface. The bilayer formed by the LB-LS method allows one to form bilayers in which the structure is much better controlled than for bilayers formed by the spontaneous fusion of vesicles. The LB-LS method is therefore recommended for further studies of model membranes supported at metal electrode surfaces. Acknowledgment. This work was funded by a NSERC Discovery Grant. J.L. acknowledges the Canada Foundation for Innovation (CFI) for a Canada Research Chair Award. LA063190L (69) Balgavy, P.; Dubnickova, M.; Kucerka, N.; Kiselev, M. A.; Yaradaikin, S. P.; Uhrikova, D. Biochim. Biophys. Acta 2001, 1512, 40-52. (70) Tristram-Nagle, S.; Liu, Y.; Legleiter, J.; Nagle, J. F. Biophys. J. 2002, 83, 3324-3335.

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