Langmuir 2006, 22, 10365-10371
10365
Layer-by-Layer PMIRRAS Characterization of DMPC Bilayers Deposited on a Au(111) Electrode Surface† Nuria Garcia-Araez,‡,§ Christa L. Brosseau,‡ Paramaconi Rodriguez,‡,§ and Jacek Lipkowski*,‡ Department of Chemistry, UniVersity of Guelph, Guelph, Ontario, Canada N1G 2W1, and Departamento de Quı´mica Fı´sica, UniVersidad de Alicante, Apart 99, E-03080 Alicante, Spain ReceiVed May 1, 2006. In Final Form: July 6, 2006 A combination of Langmuir-Blodgett and Langmuir-Schaefer techniques was employed to deposit 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC) bilayers at a gold electrode surface. One leaflet consisted of hydrogen-substituted acyl chains, and the second leaflet was composed of molecules with deuterium-substituted acyl chains. This architecture allowed for layer-by-layer analysis of the structure of the bilayer. Photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to determine the conformation and orientation of the acyl chains of DMPC molecules in the individual leaflets as a function of the potential applied to the gold electrode. The bilayer is adsorbed onto the metal surface when the field applied to the membrane does not exceed ∼108 V/m. When adsorbed, the bottom leaflet is in contact with a hydrophobic metal surface, and the top leaflet is interacting with the aqueous solution. The asymmetry of the environment has an effect on the orientation of the DMPC molecules in each leaflet. The tilt angle of the acyl chains of the DMPC molecules in the bottom leaflet that is in contact with the gold is ∼10° smaller than that observed for the top leaflet that is exposed to the solution. These studies provide direct evidence that the structure of a phospholipid bilayer deposited at an electrode surface is affected by interaction with the metal.
Introduction Planar phospholipid bilayers supported at an electrode surface constitute a natural matrix for the incorporation of proteins.1 Therefore they find application in studies of voltage-gated membrane phenomena such as voltage-gated ion channels and ion pore formation2 and for the development of biosensors.1,3-5 Planar supported bilayers are usually formed either by the fusion of unilamellar vesicles1,6-13 or by a combination of LangmuirBlodgett (LB) and Langmuir-Schaefer (LS) techniques.14-16 We have recently applied photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) to study the structure and potential-driven changes to the structure of phospholipid bilayers formed at the Au(111) electrode surface †
Part of the Electrochemistry special issue. University of Guelph. § Universidad de Alicante. ‡
(1) Sackmann, E. Science 1996, 271, 43. (2) (a) Becucci, L.; Moncelli, M. R.; Naumann, R.; Guidelli, R. J. Am. Chem. Soc. 2005, 127, 13316. (b) Becucci, L.; Moncelli, MR.; Herrero, R.; Guidelli, R. Langmuir 2000, 16, 7694. (3) Sinner, E. K.; Knoll, W. Curr. Opin. Chem. Biol 2001, 5, 705. (4) Knoll, W.; Frank, C. W.; Heibel, C.; Naumann, R.; Offenha¨user, A.; Ru¨he, J.; Schmidt, E. K.; Shen, W. W.; Sinner, A. ReV. Mol. Biotechnol. 2000, 74, 137. (5) Knoll, W.; Yu, F.; Neumann, T.; Schiller, S.; Naumann, R. Phys. Chem. Chem. Phys. 2003, 5, 5169. (6) Horsewell, S. L.; Zamlynny, V.; Li, H. Q.; Merrill, A. R.; Lipkowski, J. Faraday Discuss. 2002, 121, 405. (7) Zawisza, I.; Lachenwitzer, A.; Zamlynny, V.; Horswell, S. L.; Goddard, J. D.; Lipkowski, J. Biophys. J. 2003, 85, 4055. (8) Xu, S.-M.; Szymanski, G.; Lipkowski, J. J. Am. Chem. Soc. 2004, 124, 12276. (9) (a) Bin, X.; Zawisza, I.; Goddard, J. D. and Lipkowski, J. Langmuir 2005, 21, 330. (b) Burgess, I.; Li, M.; Horswell, S. L.; Szymanski, G.; Lipkowski, J.; Majewski, J.; Satija, S. Biophys. J. 2004, 86, 1763. (10) Reviakine, I.; Brisson, A. Langmuir 2000, 16, 1806. (11) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397. (12) Reimhult, E. R.; Hook, F.; Kasemo, B. Langmuir 2003, 19, 1681. (13) Hoffmannova, H.; Hof, M.; Krtil, P. J. Electroabal. Chem. 2006, 588, 296. (14) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105. (15) Cassier, T.; Sinner, A.; Offenhauser, A.; Mohwald, H. Colloids Surf., B 1999, 15, 215. (16) Zawisza, I.; Bin, X.; Lipkowski, J. Langmuir, to be submitted for publication.
by both the vesicle fusion6-9 and LB plus LS methods.16 These studies revealed that bilayers formed by the LB plus LS method are more ordered and the chains of phospholipid molecules are less tilted than in bilayers formed by the fusion of vesicles. However, hydrogenated phospholipipids were used in the previous experiments, and the measured tilt angles are therefore values averaged over both leaflets of the bilayer. A bilayer deposited at the gold electrode surface is exposed to an asymmetric environment. One leaflet is in contact with the metal, which at small charge densities is a hydrophobic phase, and the second leaflet is exposed to the aqueous solution. To assess the impact of the metal substrate on the structure of the supported bilayer, it is important to determine the tilt angles of the phospholipids in the two leaflets separately. The object of this work was the layer-by-layer characterization of a bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) supported at the Au(111) electrode surface. We have employed all-hydrogenated DMPC (h-DMPC) and the phospholipid with deuterated acyl chains (d-DMPC). Using the approach described earlier by Zawisza et al.,16 we employed the LB plus LS technique to build bilayers with the bottom leaflet (in contact with metal) composed of d-DMPC and the top leaflet (in contact with aqueous solution) composed of h-DMPC and vice versa. The PM-IRRAS spectra were then recorded in the C-H stretching region, and the IR bands were determined for the leaflet containing h-DMPC molecules only. This strategy allowed us to determine the tilt angles of the chains in the two leaflets of the bilayer independently. Experimental Section Reagents and Thin Film Preparation Methods. Hydrogenated (h-DMPC) (Sigma) and deuterated (d-DMPC) (Avanti Polar Lipids) DMPC were both used without further purification. These compounds were dissolved in chloroform to give a 2 mg mL-1 stock solution. The monolayers of h-DMPC and d-DMPC were prepared at the air/water interface using a Langmuir-Blodgett trough equipped with a movable barrier and a Wilhelmy surface balance (KSV5000,
10.1021/la061217v CCC: $33.50 © 2006 American Chemical Society Published on Web 08/15/2006
10366 Langmuir, Vol. 22, No. 25, 2006
Garcia-Araez et al.
Figure 1. (A) Compression isotherms for monolayers of h-DMPC and d-DMPC spread at the air-water interface of a Langmuir trough. (B) Scheme of the deposition of Langmuir-Blodgett films onto a gold surface by withdrawing. (C) Scheme of the deposition of the second leaflet by the horizontal touch (Langmuir-Schaefer) method. (D) Schematic model of a Y-type bilayer. Finland). The trough was controlled by a computer using KSV 5000 software. The temperature of the subphase was 20 ( 1 °C. A few microliters of the stock solution of DMPC or d-DMPC were spread at the interface, the solvent was allowed to evaporate, and the compression isotherm was recorded. The compression isotherms are shown in Figure 1A. The results for h-DMPC and d-DMPC are almost superimposable, taking into consideration experimental error. The compression isotherms show that h-DMPC and d-DMPC monolayers exist predominantly in the liquid expanded (LE) state at the air/water interface. At the maximum surface pressure of Π ) 47 mN m-1 (just prior to monolayer collapse), the limiting area per molecule Alim is equal to 0.42 nm2. The maximum surface concentration of DMPC in the compressed monolayer (Γlim) therefore corresponds to 3.9 × 10-10 mol cm-2. The monolayers were transferred from the air/water interface onto the Au(111) electrode at a surface pressure of 40 mN m-1, corresponding to a mean molecular area of 0.48 nm2. A combination of the Langmuir-Blodgett (LB) and the Langmuir-Schaefer (LS) techniques was employed to fabricate the phopsholipid bilayers on the Au(111) surface. The first monolayer was transferred using the LB method by vertically withdrawing the electrode at a speed of 25 mm min-1. The transfer ratio was 1.0 ( 0.1. In the monolayer transferred onto the gold surface by the LB method, the headgroups of DMPC face the metal surface, and the hydrophobic hydrocarbon chains were directed toward the air (Figure 1B). After the emersion, the monolayer-covered gold electrode was allowed to dry for 1.5 h. The second leaflet was then transferred using the LS technique (Figure 1C). The electrode covered by the first layer was horizontally brought into contact with the monolayer of DMPC, which was spread at the surface of the Langmuir trough and compressed to a surface pressure of 40 mN m-1. In the monolayer transferred using the LS method, the acyl chains of the phospholipid were directed to the metal, and polar heads were directed to the aqueous subphase (Figure 1C). The electrode covered by the bilayer was then lifted from the aqueous subphase, allowed to dry for ∼1.5 h, and then used for further experiments. The LB-LS method gives a Y-type bilayer (Figure 1D). This layer-by-layer method of deposition allows for
the construction of asymmetric bilayers, for example, bilayers with one leaflet made entirely of h-DMPC and the other leaflet made of d-DMPC. Previous studies with bilayers of n-octadecanol demonstrated that in the adsorbed state the molecules present in separate leaflets do not undergo transverse diffusion (diffusion from the bottom to the top leaflet).17 We performed all measurements at 20 °C, which is below the gel-to-liquid crystalline state transition. Therefore, the mobility of the DMPC molecules in the two leaflets was restricted, and the probability of transverse diffusion was low. We also emphasize that drying of the electrode covered by the LB monolayer has no effect on the state of hydration of the final bilayer. The PM-IRRAS experiments on the headgroups of the DMPC bilayer, described in ref 17, indicate that the headgroups in a bilayer deposited at the Au(111) electrode surface using the LB-LS approach are more hydrated than in the aqueous suspension of DMPC vesicles. Clearly, when the bilayer deposited onto a Au(111) electrode surface is brought into contact with an aqueous solution the state of hydration equilibrium is reestablished. Electrochemical Measurements. The electrochemical measurements were carried out in an all-glass three-electrode cell using the hanging meniscus configuration. The Au(111) crystal served as the working electrode (WE), and a Au wire served as the counter electrode (CE). The reference electrode was a saturated calomel electrode (SCE) connected to the cell via a salt bridge. All potentials are reported versus the SCE reference electrode, unless otherwise stated. The cleanliness of the base electrolyte was checked by cyclic voltammetry and differential capacitance measurements using experimental procedures described previously.18 The results of these measurements are not shown in this article. A computer-controlled system consisting of a HEKA PG 590 potentiostat/galvanostat and a lock-in amplifier (EG&G Instruments 7265 DSP) was used to perform electrochemical experiments. All data were acquired via a plug-in acquisition board (National Instruments NI-DAQ BNC-2090) (17) Zawisza, I.; Lipkowski, J. Langmuir 2004, 20, 4579. (18) Richer, J.; Lipkowski, J. J. Electrochem. Soc. 1985, 133, 121.
DMPC Bilayers Deposited on a Au(111) Surface 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 of Eb ) -0.1 V for 120 s. The potential was then stepped to a variable potential of interest Ei, where the electrode was held for a time ti ) 120 s. Next a potential step to the desorption potential Edes ) -1.2 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 120 s before a new potential step to a different Ei and from Ei to Edes was applied. The integration of the current transients gives the difference between charge densities at potentials Ei 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) value of Epzc ) 0.31 V versus SCE. The pzc was determined from differential capacity measurements in dilute solutions of NaF. Because of a weak OH- adsorption in 0.1 M NaF solution, the pzc is shifted to 247 mV. The Au(111) single-crystal electrode was grown, cut, and polished in our laboratory.18 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. The Nicolet Nexus 870 (Madison, WI) spectrometer equipped with an external tabletop optical mount, an MCT-A detector, a photoelastic modulator (Hinds Instruments PM-90 with a II/ZS50 ZnSe 50 kHz optical head, Hillsboro, OR), and a demodulator (GWC Instruments Synchronous Sampling Demodulator, Madison, WI) was used to perform PMIRRAS experiments. The spectra were acquired using in-house software, an Omnic macro, and a digital-to-analog converter (Omega, Stamford, CT) to control the potentiostat (EG&G PAR 362, Princeton, NJ) and collect spectra. The reference electrode was a Ag/AgCl electrode (SSCE). The IR window was a CaF2 1 in. equilateral prism (Janos Technology, Townshend, VT). Prior to the experiment, the window was cleaned with water and methanol and then ozonated for 10 min in an ozone chamber (UVO cleaner, Jelight, Irvine, CA.). Prior to the assembly of the spectroelectrochemical cell, the DMPC bilayer was deposited onto the electrode surface in an identical manner to that outlined above for electrochemical measurements, the electrode was transferred to the cell, the cell was filled with 0.1 M NaF solution in D2O, and the solution was purged with argon for 1 h to remove O2. A starting potential of E ) 0.36 V versus SCE was applied to the gold electrode, and the spectra were collected at potentials of 0.36, 0.26, 0.16, 0.06, -0.04, -0.14, -0.24, -0.34, -0.44, -0.54, -0.64, and -1.04 V vs SCE. At each potential, 8000 scans were measured and averaged. The measurements started at the most positive potential, and the potential was stepped progressively in the negative direction. The instrumental resolution was 2 cm-1. Measurements of IR spectra were carried out with the PEM set for half-wave retardation at 2900 cm-1 for the CH stretching region, and the angle of incidence of the infrared beam was set to 57°. The electrolyte thickness between the electrode and prism was ∼4.4 µm, giving a large enhancement of the p-polarized light at the electrode surface and a good cancellation of the background. The thickness of the thin layer was determined by comparing the experimental reflectivity spectrum of the thin layer cell, attenuated by the layer of solvent between the electrode and the IR window, to the reflectivity curve calculated from the optical constants of the cell constituents, as described in ref 19. The demodulation technique developed in Corn’s laboratory20,21 was used in this work. A modified version of a method described by Buffeteau et al.22 was used to correct the intensity average (Is(ω) + Ip(ω))/2 and the intensity difference (Is(ω) - Ip(ω)) for the PEM (19) Li, N.; Zamlnny, V.; Lipkowski, J.; Henglein, F.; Pettinger, B. J. Electroanal. Chem. 2002, 524/525, 43. (20) Barner, B.J.; Green, M. J.; Saez, E. I.; Corn, R.M. Anal. Chem. 1991, 63, 55. (21) Green, M. J.; Barner, B. J.; Corn, R. M. ReV. Sci. Instrum. 1991, 62, 1426. (22) Buffeteau, T.; Desbat, B.; Blaudez, D.; Turlet, J. Appl. Spectrosc. 2000, 54, 1646.
Langmuir, Vol. 22, No. 25, 2006 10367
Figure 2. Charge density σM vs potential E curves at the Au(111) electrode covered by a bilayer with isotopically substituted DMPC in different leaflets: (9) h-DMPC in the bottom leaflet and d-DMPC in the top leaflet; (0) d-DMPC in the bottom leaflet and h-DMPC in the top leaflet; (-) film-free Au(111) electrode, supporting electrolyte 0.1 M NaF. response functions and for the difference in the optical throughputs for p- and s-polarized light.23 Finally, the measured spectra had to be background corrected because of the absorption of IR photons in the thin layer cavity. The spline interpolation technique described by Zamlynny et al.23 was used for this background correction. The background-corrected spectrum plots ∆S, which is proportional to the absorbance A of the adsorbed molecules ∆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 shows three charge density versus potential curves determined from chronocoulometric experiments for the Au(111) electrode that was film-free and covered by mixed DMPC bilayers with one of the leaflets (either top or bottom) deuterated. To determine these curves, the bilayercovered electrode was transferred to the electrochemical cell, and potential step measurements were performed. The charge density was measured by stepping the potential from the initial value Ei, plotted along the ordinate in Figure 2, to a desorption potential Edes ) -1.2 V for 0.15 s. The whole set of potential step measurements was performed on the same bilayer. The differences between the charge density curves of the two bilayers are within experimental error. Hence, the position of the leaflet with deuterium-substituted DMPC does not significantly affect the behavior of the DMPC bilayer. The experimental points correspond to an average of three independent experiments performed using freshly prepared bilayers. The error bars represent the standard deviation of the three independent measurements. 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 experiments9b demonstrated that in this state the film is detached from the electrode surface but remains in close proximity, separated from the metal by an ∼1-nm-thick cushion of solution. (23) Zamlynny, V.; Zawisza, I.; Lipkowski, J. Langmuir. 2003, 19, 132.24.
10368 Langmuir, Vol. 22, No. 25, 2006
Garcia-Araez et al.
Figure 3. Plot of the potential drop and the field across the bilayer as a function of the electrode potential.
The data in Figure 2 show that DMPC bilayers are adsorbed on the metal surface when the absolute value of the charge on the metal is smaller than 10 µC cm-2 and the bilayer becomes detached at higher charge densities. The charge density curves can be used to estimate the potential drop between the metal and the bulk electrolyte solution (∆φM-S), which is approximately equal to the potential drop across the membrane, using the equation9b
∆φM-S )
σM + χM C
(2)
where χM is the surface potential of the membrane. The capacity C can be calculated by differentiation of the charge density curve. Figure 3 plots σM/C calculated from the charge density curves versus the electrode potential for potentials where the bilayers are directly adsorbed at the gold surface. These potentials are equivalent to the transmembrane potentials applied to a membrane in the patch clamp or bilayer lipid membrane (BLM) experiment. Independently, the solid line in Figure 3 plots the rational potential E - Epzc as a function of the electrode potential. As expected, the values of σM/C are comparable to the values of the rational potential. The values of σM/C are quite scattered around the line plotting the rational potential. The scatter reflects errors in the calculation of the capacity C by numerical differentiation of the charge potential plots. The results show that the bilayers are adsorbed at the gold surface when the absolute value of σM/C is less than ∼0.6 V. (Figure 2 shows that detachment of the bilayer begins at ∼ -0.4 V(SCE), and Figure 3 shows that σM/C is ∼0.6 V at E ≈ -0.4 V(SCE).) However, neutron reflectivity experiments indicate that the adsorbed bilayer swells and absorbs water at E < -0.2 V versus Ag/AgCl. This suggests that a compact bilayer exists when the transmembrane potential is less than 0.4 V, in good agreement with the magnitude of the transmembrane potential applied to bilayer lipid membranes in membrane conductivity measurements.24a Considering that the thickness of a membrane bilayer is approximately 5 nm, one can estimate the electric field across the bilayer. The estimated values are plotted on the righthand side of Figure 3. The field changes between ∼ -2 × 108 and ∼ +6 × 106 V m-1. The magnitude of this electric field is comparable to that of the field acting on a natural membrane.24b Finally, we would like to mention that the minimum capacity of the Au(111) electrode covered by the DMPC bilayer is (24) (a) Sandblom, J.; Galvanovskis, J.; Jilderos, B. Biophys. J. 2001, 81, 827. (b) Tsong, T. Y.; Astumian, R. D. Annu. ReV. Physiol. 1998, 50, 62.
∼5.5 µF cm-2. This value is much larger than the capacity of a defect-free bilayer, which is expected to be ∼0.8 µF cm-2. This large value of the capacity indicates that the film has defects or that it is much thinner than the thickness expected for the bilayer. To verify this point, we have performed additional atomic force measurements on the bilayer deposited using the combination of the LB and LS techniques. We have measured forcedistance curves that allowed us to determine the thickness of the bilayer. These experiments will be described in a separate publication. The average of 40 measurements gave an average thickness of 5.5 ( 1 nm. This result indicates that the high value of the capacity is due to the presence of defects in the bilayer. Spectroelectrochemistry of the CH Stretching Region. The PM IRRA spectra in the CH stretching region were recorded using electrodes with bilayers formed by consecutive layer-bylayer deposition of h-DMPC and d-DMPC monolayers. These spectra provide information about the tilt angles and conformation of acyl chains in the leaflet containing h-DMPC molecules. Parts A and B of Figure 4 show the spectra for bilayers in which h-DMPC constitute the bottom and top leaflets, respectively. The thick top line plots the spectrum of randomly distributed molecules in a 2.7 nm thick film, calculated from optical constants determined for DMPC vesicles in D2O at 20 ( 2 °C.9 The thickness of a DMPC bilayer measured by the neutron diffraction method25 is equal to 5.5 nm. Therefore, a value of 2.7 nm represents the thickness of one leaflet. The four remaining curves in Figure 4A and B plot PM-IRRAS spectra for the mixed bilayers, recorded at selected electrode potentials as indicated. Figure 5 shows the deconvolution of the spectrum for randomly oriented molecules. Four fundamental bands corresponding to methyl asymmetric, methylene asymmetric, methyl symmetric, and methylene symmetric stretches are clearly resolved and are located at ∼2958.5, ∼2922.1, ∼2872.6, and ∼2853.2 cm-1, respectively.26a Their fwhm values are ∼15.5, ∼18.8, ∼8.0, and ∼12.0 cm-1, respectively. In addition, two bands corresponding to the Fermi resonances between the overtones of the symmetric bending mode and the symmetric methyl and methylene stretching modes appear at 2900.1 and 2931.4 cm-1, respectively.26a The spectra corresponding to the bottom and top leaflets of the DMPC bilayers transferred onto the gold electrode are similar. The positions of νas(CH2) and νs(CH2) are ∼2922 ( 1 and ∼2854 ( 1 cm-1. The fwhm values are 19 ( 2 and 11 ( 2 cm-1. Because of the low intensity, measurements of the width and position of the bands in the PM-IRRAS spectra were sensitive to the background correction procedure. The error bars are quite large; consequently, changes in the band positions and widths with the electrode potential are within experimental error. The positions and widths of the CH stretching bands in the h-DMPC leaflet of the bilayer deposited onto the Au electrode are similar to positions and widths of these bands in the spectrum of h-DMPC vesicles. These frequencies and widths are characteristic of a pretransition state (or ripple phase) of the bilayer in which a majority of the acyl chains assume an all-trans conformation. However, some chains are already melted and have gauche conformation defects.26-30 (25) Le´onard, A.; Escrive, C.; Laguerre, M.; Pebay-Peyroula, E.; Ne´ri, W.; Pott, T.; Katsaras, J.; Dufourc, E. J. Langmuir 2001, 17, 2019. (26) (a) MacPhail, R. A.; Strauss, H. L.; Snyder, R. G.; Elliger, C. A. J. Phys. Chem. 1984, 88, 334. (b) Snyder, R. G.; Hsu, S. L.; Krimm, S. Spectrochim. Acta 1977, 34, 395. (27) Cameron, D. G.; Casal, H. L.; Mantsch, H. H. Biochemistry 1980, 19, 3665. (28) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J. Phys. Chem. 1982, 86, 5145. (29) Brandenburg, K.; Snyder, R. G. Z. Naturforsch. 1986, 41C, 453. (30) Heimburg, T. Biophys. J. 2000, 78, 1154.
DMPC Bilayers Deposited on a Au(111) Surface
Langmuir, Vol. 22, No. 25, 2006 10369
Figure 5. Example of a deconvolution of the overlapping νas (CH3), νas (CH2), νs (CH3), and νs (CH2) bands and two Fermi resonances between the overtones of the symmetric bending mode and symmetric methyl FRVs(CH2) and methylene stretching FRVs(CH3) modes for the spectrum of a 2.7-nm-thick film of randomly oriented h-DMPC molecules.
Figure 4. PM-IRRAS spectra for the CH stretching region of a DMPC bilayer on a Au(111) electrode in a 0.1 M NaF/D2O solution at the indicated potentials. The top trace plots spectra calculated for a 2.7-nm-thick film of randomly oriented DMPC molecules calculated using optical constants of DMPC for vesicles dispersed in D2O.9a (A) Bilayer with h-DMPC in the bottom and d-DMPC in the top leaflet; (B) bilayer with d-DMPC in the bottom and h-DMPC in the top leaflet.
Figure 4A and B shows that the intensities of methylene stretching bands are dependent on the potential. This behavior indicates a potential-dependent change in the orientation of the acyl chains. The integrated intensity of an IR band depends on the angle between the directions of the transition dipole moment of a given vibration and the electric field of the photon. The electric field vector of the p-polarized photon is perpendicular to the metal surface. Therefore, the angle (θ) between the direction of the transition dipole and the surface normal can be determined using the following equation31,32
cos2 θ )
1 A(E) 3 A(random)
(3)
where A(E) is the integrated intensity of the band at a given potential and A(random) is the integrated intensity of a band in a monolayer of randomly oriented molecules. To find the angle θ, the spectrum for randomly oriented molecules in the film has to be known. (31) Allara, D. L.; Swalen, J. D. J. Phys. Chem. 1982, 86, 2700. (32) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 52.
This is the top spectrum in Figure 4A and B and the spectrum whose deconvolution is shown in Figure 5. As mentioned earlier, it was calculated from optical constants for DMPC9 using the optical matrix method.23,31 Deconvolution of the bands was done using a mixed Gaussian-Lorentzian function. Figure 6A plots the angle between the direction of the transition dipole moment of νs(CH2) and the surface normal. Filled points represent the data for the case where h-DMPC was in the bottom leaflet, and open points represent the data for the case where h-DMPC was in the top leaflet of the bilayer. Figure 6B plots the corresponding angles for the νas(CH2) stretch. In each experiment, the bilayers with h-DMPC and d-DMPC leaflets were freshly transferred onto the gold electrode, and the PM IRRAS spectra were recorded for two independently made films. The points in Figure 6A and B plot the average values of these two experiments. The error bars, which on average correspond to (3°, denote the upper and lower limits of the measured angles θ. Inset cartoons in Figure 6A and B show that the direction of the transition dipole of the symmetric methylene stretch lies along the bisector of the CH2 plane and the direction of the transition dipole of the asymmetric stretch lies along the line joining the two hydrogen atoms of the CH2 group.33 Both vectors lie in the plane of the methylene group that is perpendicular to the line of the fully extended all-trans hydrocarbon chain. Because these three directions are perpendicular, knowledge of θνas and θνs allows one to calculate the tilt of the hydrocarbon chains (θchain) of the h-DMPC molecules using the equation34
cos2 θνas + cos2 θνs + cos2 θchain ) 1
(4)
Equation 4 is applicable only when the hydrocarbon chains are fully extended as in the all trans-conformation. In the present case, a fraction of the chains have a gauche conformation, and hence eq 4 may be used only as an approximation. Figure 7 plots the chain tilt angles as a function of the electrode potential. Filled points mark angles for the h-DMPC monolayer constituting the bottom leaflet, and open points mark angles in the h-DMPC monolayer composing the top leaflet of the bilayer. (33) Fringeli, U. P. Z. Naturforsch. 1977, 32C, 20.
10370 Langmuir, Vol. 22, No. 25, 2006
Garcia-Araez et al.
Figure 6. Plots of the angle between the direction of the transition dipole moment and the surface normal as a function of the electrode potential: (9) for the bilayer with h-DMPC in the bottom layer; (0) for the bilayer with h-DMPC in the top layer; (A) for the νs(CH2) stretch; and (B) for the νas(CH2) stretch. Cartoons show the directions of the transition dipole moments.
At E > -0.4 V, the bilayer is directly adsorbed at the gold surface. At these potentials, the average values of the tilt angles amount to ∼25° in the bottom layer and ∼35° in the top layer. The tilt angles of DMPC molecules in the bottom leaflet, which is in direct contact with the metal, are about 10° smaller than that of the top leaflet. This is a very important result. It shows that the asymmetric environment of the two leaflets has a significant impact on the orientation of the phospholipid molecules. In the top leaflet, the polar heads of the DMPC molecules interact with the aqueous solution whereas in the bottom leaflet they interact with the gold surface. It is interesting to compare these tilt angles with the tilt of chains in a monolayer spread at the air/water interface that was transferred onto the gold electrode. This angle can be calculated from the equation35
SDMPC cos θtilt ) 2Σ
(5)
where SDMPC is the area per DMPC molecule in the monolayer (34) Umemura, J.; Kamata, T.; Kawai, T.; Takenaka, T. J. Chem. Phys. 1990, 94, 62. (35) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21.
Figure 7. Plots of the angle between the acyl chain of the h-DMPC molecule and the surface normal as a function of the electrode potential: (9) for the bilayer with h-DMPC in the bottom layer and (0) for the bilayer with h-DMPC in the top layer. The cartoon gives a pictorial definition of θchain.
and Σ ) 0.195 nm2 is the cross-sectional area of a single hydrocarbon chain.35 In the film of DMPC compressed to 40 mN m-1, the area per molecule is equal to SDMPC ) 0.48 nm2, and the tilt angle calculated from eq 5 is equal to 35°. This number is in good agreement with the tilt of h-DMPC in the top layer of the bilayer adsorbed at the gold electrode. Apparently, the LS transfer of the second monolayer takes place without a change in the tilt angle of the DMPC molecules. In contrast, the LB transfer of the first leaflet onto the gold support involves a change in the tilt angle. This behavior suggests that polar groups of DMPC molecules in contact with gold assume a somewhat different conformation/orientation than the headgroups that are in contact with the aqueous phase. Alternatively, one may argue that the differences between the tilt angles in the two leaflets are due to the different film-transfer procedures used to deposit the two layers. However, in previous studies of the octadecanol bilayer17 we observed an opposite trend. The tilt angle in the bottom layer was larger than in the top layer. This behavior suggests that the changes in the chain orientation are indeed induced by the differences between interactions of the polar head of an amphiphilic molecule with two different subphases. For DMPC, these differences can be determined by measuring IR bands for the polar head region. Such measurements are planned for the near future. Figure 7 shows that the tilt angle depends on the electrode potential. The changes are small in the bottom leaflet but are much more pronounced in the top leaflet. At E < -0.6 V, the
DMPC Bilayers Deposited on a Au(111) Surface
bilayer is detached from the electrode surface but remains in close proximity to the metal suspended on an ∼1-nm-thick cushion of water.9b The data show that the tilt angle is smaller in the detached state than in the adsorbed state of the bilayer. This result is consistent with the previous PM-IRRAS studies of bilayers formed by spreading DMPC vesicles onto the gold electrode surface.9a However, the difference between the tilt angles in the detached and adsorbed states are much smaller in bilayers made by the LB plus LS method than in bilayers formed by the fusion of unilamellar vesicles. Indeed, in the top leaflet, the maximum difference between the tilt angle in the detached and adsorbed states of the bilayer is equal to ∼10°. In contrast, in the bilayer formed by the fusion of unilamellar vesicles the tilt angle increases by ∼20° by moving from the detached to the adsorbed state of the bilayer.9a The data for the top leaflet also show that in the adsorbed state of the bilayer (E > -0.4V) the tilt angle decreases slowly with the electrode potential. Consequently, the tilt angle-potential curve displays a maximum at E ≈ -0.3V. A similar small change in the tilt angle was observed for bilayers formed by the spreading of unilamellar vesicles.9a Within the potential region of +0.4 to -0.4 V, the adsorbed bilayer is exposed to an electric field that changes from ∼ +106 to ∼ -108 V/m. The tilt angle increases only by ∼3° in response to such a large change in the electric field. Apparently, the field has a rather small effect on the structure of the adsorbed bilayer. The effect of the static electric field on the structure of the lipid membrane has been investigated by electrochemical methods.36-39 Very precise measurements of the voltage-induced changes in membrane capacity suggest that the polar head region of the phospholipid molecules reorient under the influence of the electrode potential. However, such changes are small. Sargent40 estimated that a 100 mV change in potential across the membrane should lead to a change in the orientation of the dipole by ∼1°. This estimate is consistent with ATR-FTIR spectroscopy studies of a stack of dry DMPC bilayers deposited at the surface of an ATR element41 and transmission experiments performed on a film of oriented multibilayers of DMPC formed between two Si windows.42 These studies demonstrated that an electric field on the order of 107 V/m has a small effect on the orientation and conformation of the phospholipid molecules. Our results are consistent with the literature. (36) Sargent, D. F. J. Membr. Biol. 1975, 23, 227. (37) Sargent, D. F. In Molecular Aspects of Membrane Phenomena; Kaback, H. R., Neurath, H., Radda, G. K., Schwyzer, R., Wiley, W. R., Eds.; SpringerVerlag: Berlin, 1975; pp 104-120. (38) Bamberg, E.; Benz, R. Biochim. Biophys. Acta 1976, 426, 570. (39) Hianik, T. J. Biotechnol. 2000, 74, 189. (40) Sargent, D. F. Biophys. J. 2001, 81, 1823. (41) Le Saux, A.; Ruysschaert, J. M.; Goormaghtigh, E. Biophys. J. 2001, 80, 324. (42) Schwarzott, M.; Lasch, P.; Baurecht, D.; Naumann, D.; Fringeli, U. P. Biophys. J. 2004, 86, 285.
Langmuir, Vol. 22, No. 25, 2006 10371
Summary and Conclusions We have deposited DMPC bilayers at a gold electrode surface with one leaflet consisting of hydrogen-substituted molecules and another leaflet consisting of molecules with deuteriumsubstituted acyl chains. PM-IRRAS spectroscopy experiments performed on these isotopically labeled films provided information concerning the orientation of the acyl chains in the individual leaflets of the bilayer. At the gold electrode surface, the bilayer is exposed to an asymmetric environment. The bottom leaflet is in contact with a hydrophobic metal surface while the top leaflet is interacting with the aqueous solution. The results show that the asymmetry of the environment has an effect on the orientation of the DMPC molecules in each leaflet. The tilt angle of the acyl chains of the DMPC molecules in the leaflet in contact with gold is ∼10° smaller than in the top leaflet that is exposed to the solution. The bilayer deposited at the electrode surface is stable when the field applied to the membrane does not exceed ∼108 V/m. In the adsorbed state, the tilt angle of the acyl chains changes by only 3° in response to the change in electric field from ∼ +106 to ∼ -108 V/m. At higher fields, the bilayer is detached from the electrode surface but remains in close proximity to the surface, separated from the metal by a thin cushion of electrolyte. Upon detachment, the tilt angle of the chains decreases by up to 10°. In the detached state, the two leaflets are in contact with water, and the bilayer is exposed to a much more symmetric environment. In response to this symmetric environment, the difference in the tilt angles for the two leaflets becomes smaller (∼5°). In conclusion, the results of these studies provide direct evidence that the structure of a phospholipid bilayer supported at an electrode surface is affected by the interaction with the metal. The results of this work may also contribute to the advancement of the understanding of the stability of phospholipid bilayers in the presence of strong electric fields.43 Acknowledgment. This work was supported by a grant from the Natural Sciences and Engineering Council of Canada. J.L. acknowledges the Canada Foundation for Innovation for the Canada Research Chair Award. The results of these studies were presented as part of the Sackler Lecture at the Mortimer and Raymond Sackler Institute of Advanced Studies at Tel Aviv University, Israel, March 2006. J.L. expresses his gratitude for the Sackler Lecture Award. N.G.-A. is grateful to the MEC (Spain) for the award of an FPU grant. We express our gratitude for the help from T. Laredo and J. Leitch. LA061217V (43) Bowen, P. J.; Lewis, T. J. Thin Solid Films 1983, 99, 157.
