Langmuir 1998, 14, 839-844
839
Attenuated Total Reflection Fourier Transform Infrared Spectroscopic Characterization of Fluid Lipid Bilayers Tethered to Solid Supports Yaling Cheng, Neville Boden,* Richard J. Bushby, Steve Clarkson, Stephen D. Evans,* Peter F. Knowles, Andrew Marsh, and Robert E. Miles Centre for Self-Organising Molecular System, University of Leeds, Leeds, LS2 9JT, U.K. Received September 3, 1997. In Final Form: December 2, 1997 Attenuated total reflection Fourier transform infrared spectroscopy has been developed to monitor the tethering of phospholipid bilayers to gold-coated, ZnSe crystals. Bilayer attachment has been accomplished by fusing lipid vesicles onto a self-assembled monolayer comprised of a mixture of 2-mercaptoethanol (EO1) and a hexaethyleneoxythiol derivative of cholesterol (EO6C). The cholesteryl moieties penetrate into the lower leaflet of the bilayer and serve to “anchor” the bilayer to the solid support. For fractional surface area coverage of EO6C < 0.24, no lipid adsorption was detected, while for higher EO6C coverages, bilayers are formed with the outer and inner leaflets comprised, respectively, of pure lipid and the complementary lipid/cholesteryl mixture. From a thermodynamic analysis of this result we conclude that the initial step in bilayer self-assembly onto the surface is adsorption and rupture of a single lipid vesicle. The frequencies of the lipid CH2 stretching vibrations are characteristic of a fluid liquid-crystalline bilayer.
* To whom the correspondence should be addressed. Email:
[email protected] or
[email protected].
assembled monolayers (SAMs), covalently attached using thiol chemistry to a gold-coated surface, have been developed.2, 7-10 For example, Spinke et al.7 have used a SAM prepared from a methacrylic terpolymer whose three structural elements comprise a disulfide unit, a hydrophilic main chain spacer, and lipid-like side chains which can insert into the bilayer to tether it to the surface; Vogel et al.8,9 have used a SAM comprised of “thiol-lipids” with a hydrophilic spacer of oligoethylene glycol chain, variable in length, to couple lipid bilayers to solid surfaces; Naumann et al.10 have used peptides as spacers to create a water-filled space between the bilayer and the solid support allowing incorporation of functional, active ATPase into the bilayer. More recently, Cornell et al.2 have used membrane spanning thiol-lipids to help stabilize tethered bilayers incorporating switchable gramicidinlike ion channels. We have previously reported11,12 a versatile tethering methodology which employs SAMs fabricated from readily synthesized anchor molecules, comprising of a mixture of two ethyleneoxy oligomers: one terminated with hydroxyl and thiol groups (EOm), and the other with cholesteryl and thiol groups (EOnC). The cholesteryl moieties of EOnC penetrate into the hydrophobic region of the inner leaflet of the bilayer and act as “hooks” to anchor the bilayer to the surface. Figure 1 shows a drawn representation of the mixed monolayer and tethered bilayer. The choice of “m” and “n” determines the thickness of the water layer separating the bilayer from the solid substrate: in our previous studies, values of m ) n ) 3 were used. In the present study, we have employed a mixture of “short” (EO1) and “long” (EO6C) thiols (Figure 1a). The distance from
(1) Sackmann E. Science 1996, 271, 43-48. (2) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wleczorek, L.; Pace, R. J. Nature 1997, 387/5, 580-583. (3) Israelachvili J. N. In Intermolecular and Surfaces Forces; Academic Press: San Diego, CA, 1992; pp 307-315. (4) Ulman, A. In An Introduction to Ultrathin Organic Films; Academic Press, Inc.: San Diego, CA, 1991; pp 101-236. (5) Radler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 45394548. (6) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316.
(7) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667-1671. (8) Lang, H.; Duschl, C.; Gra¨tzel, M.; Vogel, H. Thin Solid Films 1992, 210/211, 818-821. (9) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (10) Naumann, R.; Jonczyk, A.; Kopp, R.; van Esch, J.; Ringsdorf, H.; Knoll, W.; Graber, P. Angew. Chem., Int. Ed. 1995, 34, 2056-2058. (11) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751-757. (12) Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles, P. F.; Marsh, A. Tetrahedron 1997, 53, 10939-10952.
Introduction The tethering of fluid, biologically functional, lipid bilayers to solid supports is a topic of current interest.1 This is driven by the recognition that success would open up an entirely new dimension of membrane research and applications quite different from those normally associated with the more traditional black lipid membranes or lipid vesicles. For example, at the fundamental level, tethered biomembranes could be used in conjunction with scanning probe microscopy for the study of membrane protein structure and function, while, on the technological side, they could be used to engineer bioactive surfaces and biosensors.2 For such applications, it is essential to be able to vary the thickness of the water layer separating the bilayer from the solid support in order to be able to incorporate the hydrophilic domains of membrane proteins. There are, however, major technological challenges in the fabrication of mechanically stable, tethered, fluid bilayers of this kind. For example, undulation forces in the elastic bilayer sheets need to be overcome.3 Solid supported bilayers can be constructed using Langmuir-Blodgett methodology.4 However, the resulting bilayers are in the gel phase, not the fluid state present in biological membranes and have the additional disadvantage that the water layer, separating the bilayer from substrate, cannot be varied in a controlled manner. The bilayers produced by lipid vesicle fusion onto a glass or mica surface 5,6 have similar drawbacks. Recently, a variety of methods employing lipid vesicle fusion onto self-
S0743-7463(97)00998-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/23/1998
840 Langmuir, Vol. 14, No. 4, 1998
Cheng et al.
Attenuated total reflection Fourier transform infrared (ATR-FTIR) has been widely used for obtaining such information.13-18 In this new study, we use ATR-FTIR spectroscopy to measure the amount of lipid adsorbed, and the conformation and orientation of the lipid chains. To our knowledge, this is the first report on the use of this technique to study lipid bilayers attached to a gold surface in situ. Materials and Methods
Figure 1. (a) Schematic of the experimental setup used in the ATR-FTIR spectroscopic characterization of the tethered bilayers. EP and ES define parallel and perpendicular polarized components of the electric field of the incident light. The chemical structures of EO6C, EO1, and eggPC are given as below:
1(b) Tethered bilayer with the outer leaflet comprised of pure eggPC and the inner leaflet of a mixture of eggPC and cholesteryl anchors.
the hydroxyl groups of EO1 to the carboxylate of EO6C is ∼20 Å, assuming that the ethyleneoxy chains are in their extended conformation. This provides space to accommodate the phospholipid headgroups more effectively. In our previous study, surface plasmon resonance (SPR) spectroscopy was used for monitoring the unrolling of the vesicles and the growth of the adsorbed lipid layers.11 SPR experiments measure the amount of lipid present within a distance of 50 nm from the surface. Unequivocal information about the tethering of bilayers necessitates monitoring the orientation of lipid chains with respect to the surface normal and also their conformational state.
Materials. 2-Mercaptoethanol (EO1) was purchased from Aldrich, U.K. The synthesis of hexaethyleneoxythiol derivative of cholesterol (EO6C) is described in ref 12. Egg yolk phosphatidylcholine (egg PC) was supplied as a stock solution in chloroform/methanol (1:1) by Lipid Products (Surrey, U.K.). Preparation of the Gold-Coated ATR Crystal. Au (99.99%, purchased from ADVENT Research Materials Ltd., U.K.) films 200 Å thick were deposited on one surface of a ZnSe ATR crystal in a Edwards Auto 306 TMP vacuum evaporator operating at pressures less than 2.6 × 10-6 mbar. The evaporation rate was held constant at 0.1-0.2 nm/s. Atomic force microscopy has been used to study 200 Å thick gold film evaporated on glass or Si with a 15 Å chromium underlayer and shows a continuous gold layer with Rq (root mean square) value, averaged over 1 µm scale, of ∼5 Å. Preparation of Self-Assembled Monolayers. Immediately after gold deposition, the Au-coated ZnSe crystal was placed in a 1 mM solution of varying proportions of EO1/EO6C in dichloromethane (HPLC grade) for 1 h. Samples were then rinsed repeatedly with dichloromethane to remove excess thiol. The effective surface coverage of cholesteryl units in the selfassembled monolayers (SAMs) was estimated using advancing water contact angle measurements. This involved increasing the volume of a drop of Milli-Q water in contact with the surface until the edge of the drop just began to advance. The image was viewed through a Hamamatsu C3077 CCD camera and recorded and analyzed using Accuware software. The angles at both edges of the drop were measured at several sites on each surface, and the average values were used in subsequent calculations. Preparation of Egg-Phosphatidyl Choline (eggPC) Vesicles. EggPC solution (20 mL) in 1:1 chloroform/methanol (1 mg/mL) was evaporated under a stream of pure nitrogen gas and then dried under vacuum for several hours to remove all traces of the solvents. Aqueous NaCl solution (0.1 M) was added to the dry lipid to give a solution with a lipid concentration of 10 mg/mL. The solution was subjected to probe sonication under a stream of nitrogen gas for 45 min using a Branson 250 Sonifier (Danbury, CT) and diluted with NaCl solution (0.1 M) to 0.4 mg/mL. Unilamellar vesicles prepared in this way typically have a diameter of 250 Å.19 ATR-FTIR Experiments. Nonpolarized and polarized ATRFTIR spectra were recorded, respectively, on a 1760X PerkinElmer spectrometer and a Bruker IFS-48 spectrometer. The latter was equipped with a gold-grid polarizer on a KRs-5 substrate. The sample compartment of the Bruker IFS-48 was continuously purged with nitrogen gas. A Specac Variable-Angle ATR attachment and a 45°-cut ZnSe crystal were used to give 14 reflections in total. An airtight liquid cell with a closed volume of 0.5 mL, created by interposing a 0.8 mm Viton spacer (Goodfellow, U.K.) between one face of the ATR crystal and the cell wall, was used. Each nonpolarized spectrum represents the average of 512 scans with a resolution of 4 cm-1. The polarized ATR spectra are the average of 2000 scans with a resolution of 4 cm-1. All the spectra presented are corrected for background (13) Frey, S.; Tamm, L. Biophys. J. 1991, 60, 922-930. (14) Wenzl, P.; Fringeli, M.; Goette, J.; Fringeli, U. P. Langmuir 1994, 10, 4253-4264. (15) Mu¨ller, E.; Giehl, A.; Schwarzmann, G.; Sandhoff, K.; Blume, A. Biophys. J. 1996, 71, 1400-1421. (16) Stephens, S. M.; Dulhy, R. A. Thin Solid Films 1996, 285, 381386. (17) Reinl, H. M.; Bayerl, T. M., Biochemistry 1994, 33, 14091-14099. (18) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213-226. (19) Huang, C. Biochemistry 1969, 8, 344-352.
Fluid Lipid Bilayers Tethered to Solid Supports
Langmuir, Vol. 14, No. 4, 1998 841
Figure 2. Effective fractional surface coverage of EO6C as a function of the mole fraction of EO6C in the EO6C/EO1 mixture (1 h of exposure to a 1 mM solution in dichloromethane). signal by subtracting the respective spectrum of the SAM/NaCl solution recorded under the same conditions. Band fitting procedures were applied to calculate the integrated intensities of the stretching vibrations of lipid CH2 bands.
Results and Their Interpretation Characterization of Self-Assembled Monolayers. Mixed EO6C/EO1 monolayers were deposited on the goldcoated ZnSe ATR crystal from dichloromethane solution. The fractions of EO1 and EO6C adsorbed on surfaces are expected to be different to their relative concentration in solution. We have therefore employed water contact angle measurements to estimate the effective surface coverage (fractional areas) of the two components using Cassie’s law (eq 1)20,21
cos(θ) )
∑i Xi cos(θi)
(1)
Xi represents the fractional surface coverage of the component group (e.g., cholesteryl or hydroxyl). θ is the observed contact angle and θi is the contact angle of the separate pure components. The advancing water contact angles measured for 100% EO6C and 100% EO1 SAMs are 105° and 25°, respectively. Figure 2 shows a typical variation of the fractional surface coverage of cholesteryl groups as a function of solution composition. This curve does not, however, correspond to a true equilibrium between surface and solution due to the fact that the immersion time was limited to 1 h. Note that the fractional surface coverage of EO6C does not correspond to its mole fraction in the monolayer as the cross sectional area of a cholesteryl unit is much larger than that of a hydroxyl group. Tethering of Lipid Bilayer. Typically, an eggPC vesicle dispersion was injected into an airtight ATR cell (20) Cassie, A. B. D. Discuss. Faraday Soc. 1948, 3, 11-16. (21) Adamson, A. W. In Physical Chemistry of Surface, 5th ed.; John Wiley & Sons: New York, 1990; pp 385-389.
Figure 3. IR spectra of eggPC adsorbed onto EO6C/EO1 coated surfaces. The spectra were recorded at 20 °C and corrected by subtracting the spectrum of the SAM recorded under the same conditions. The spectra were baseline corrected and fitted with a mixed Lorentzian/Gaussian function in the region between 3000 and 2800 cm-1. The dash lines represent the resultant fits to the CH2 peaks.
and left in contact with the SAM-coated substrate for ∼20 h. The cell was then gently washed 5 to 6 times with 0.1 M aqueous NaCl solution in order to remove loosely adsorbed vesicles. Detectable lipid adsorption was observed only for cholesteryl surface coverages in excess of 24%. Figure 3 shows how the IR spectrum of adsorbed eggPC changes with cholesteryl coverage. The poor signalto-noise ratio is due to the presence of the gold layer on the ATR crystal. The bands at 2922 and 2853 cm-1 correspond, respectively, to asymmetric and symmetric stretching vibrations of the chain CH2 bond. The frequencies are characteristic of conformationally disordered hydrocarbon chains,18 indicative of a fluid or liquidlike, liquid-crystalline bilayer. The eggPC coverage was quantitatively estimated as follows: The depth of penetration (dP) in the frequency range 3000-2800 cm-1 was calculated to be ∼2000 Å using a 4 × 4 scattering matrix.22 In the calculation, the real and imaginary parts of the refractive indices were taken to be, respectively, 2.4 and 0 for ZnSe, 0.7 and -21.8 for gold,23 (22) Azzam, R. M. A.; Bashara, N. M. In Ellipsometry and Polarised Light; Elsevier Science B.V.: Amsterdam, 1987; Chapter 4. (23) Ordal, M. A.; Long, L. L.; Bell, R. J.; Bell, S. E.; Bell, R. R.; Alexander, R. W., Jr.; Ward, A. Appl. Opt. 1983, 22, 1099-1119.
842 Langmuir, Vol. 14, No. 4, 1998
Cheng et al.
Table 1 Infrared Characteristics of EggPC Bilayers Adsorbed on EO6C/EO1 SAMs fractional area coverage of cholesteryl moiety
νas(CH2)/ cm-1
intensitya
νs(CH2)/ cm-1
intensitya
1 0.35 0.24
2922 2922 2922
0.07 0.11 0.13
2853 2853 2852
0.025 0.035 0.041
Infrared Parameters Measured for ODT Monolayer and Calculated for EggPC Monolayer monolayers ODT eggPC (expected) a
νas(CH2)/ cm-1 2917.5
intensitya 0.125 0.078
νs(CH2)/ cm-1 2850.7
intensitya 0.045 0.027
The error in the integrated intensity is ∼10%.
1.5 and -0.3 for lipid,24 1.33,25 and -0.02 for 0.1 M NaCl solution. The imaginary part of the refractive index of the NaCl solution was calculated, using the method described by Allara and Nuzzo,26 from the transmission spectrum of the NaCl solution (0.1 M). For a depth of penetration of 2000 Å, the E-field amplitude at a distance of 50 Å from the gold surface falls off by only 2.5%. As the position of the hydrocarbon chain of the attached lipid is only ∼20-50 Å away from the gold surface (Figure 1b), the absorbance by the lipids may be treated as being a linear function of the amount on the surface. Since the average number of methylene groups in the hydrocarbon chain of eggPC is ∼18 27 and octadecyl mercaptan (ODT) forms highly ordered monolayers on gold, as demonstrated from contact angle measurement,28 the absorbance of an ODT monolayer AODT (integrated intensity) was used as a reference to quantify the absorbance of eggPC. The frequencies of νas(CH2) and νs(CH2) of ODT, given in Table 1, correspond to those for crystal-like acyl chains.18 As the hydrocarbon chains of eggPC are in the liquid crystalline phase, the cross-sectional areas of ODT and the acyl chains of eggPC are different. The cross-sectional area for a crystal-like hydrocarbon chain is 19 Å2.29 Taking the tilt angle of ODT on a gold lattice to be 30°,30 the projected cross-sectional area of ODT is ∼22 Å2, while the cross-sectional area of a eggPC molecules at full hydration is ∼70 Å2.31 To cover a given surface area, the ratio of ODT/eggPC needed is therefore 70/22. The absorbance of an equivalent eggPC monolayer will be 2AODT (22/70) (as the number of methylene units of the hydrocarbon chains of eggPC is about twice that of ODT). The integrated intensities of νas(CH2) and νs(CH2) of eggPC on various SAMs are summarized in Table 1. The intensities of νas(CH2) and νs(CH2) of eggPC on 100% EO6C SAM is close to that expected for a “perfect” eggPC monolayer (in term of number of eggPC molecules), which indicates that an eggPC monolayer is formed on the top of a hydrophobic surface of cholesteryl moieties (Figure 1b with the inner leaflet comprised solely of cholesteryl groups). The intensities of νas(CH2) and νs(CH2) of eggPC (24) Evans, S. D. Unpublished results. (25) Handbook of Physical Chemistry, E224, 5th ed.; CRC Press: Boca Ratan, FL. (26) Allara, D. L.; Nuzzo, R. G. Langmuir 1985, 1, 52-66. (27) Gunstone, F. D. In Fatty Acid and Lipid Chemistry; Blackie Academic and Professional: Glasgow, 1996. (28) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 11, 321-335. (29) Pearson, R. H.; Pascher, I. Nature 1979, 281, 499-501. (30) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546-558. (31) Small, D. M. In The Physical Chemistry of Lipids; Plenum Press: New York and London, 1986; p 512.
Figure 4. Polarized spectra of eggPC absorbed on a 30% cholesteryl SAM. The spectra were recorded at 20 °C and corrected by subtracting the spectrum of the SAM recorded at the same conditions. The spectra were baseline corrected and fitted with a mixed Lorentzian/Gaussian function in the region between 3000 and 2800 cm-1. The dash lines represent the resultant fits to the CH2 peaks.
on mixed EO6C/EO1 surfaces are larger than predicted for a monolayer, but less than for a complete bilayer. This indicates that a fraction of the inner leaflet of the bilayer is now comprised of eggPC in addition to anchoring cholesteryl groups. This tethering of bilayers to the surfaces is also supported by our polarized ATR-IR measurements described below. The arrangement used in the polarized ATR-IR experiments is shown in Figure 1a. s- and p-polarization spectra of eggPC on a EO6C/EO1 surface (30% cholesteryl coverage) are shown in Figure 4. The symmetric and asymmetric stretching vibrations of lipid CH2 are well resolved in the p-polarized spectrum, but not in the s-polarized spectrum. This is not because the lipids adopt certain orientations which prevent the observation of these bands in the s-polarized spectrum but rather because the E-field amplitude in the y-direction is attenuated severely by the metal surface. The E-field amplitudes, Ex2, Ey2, and Ez2, at the interface of sample and gold film, are calculated to be, respectively, 0.029, 0.011, and 0.071, using the threephase theory of Hansen.32 Thus, ES is about 9 times smaller than EP at this interface. In principle, information about the orientation of lipid hydrocarbon chains can be obtained from the dichroic ratio of the CH2 stretching vibration band.15,33 The relationship between the dichroic ratio RATR and the IR order parameter function s is given by eq 2, where s is related to the chain (32) Hansen, W. N.; Kuwana, T.; Osteryoung, R. A. Anal. Chem. 1966, 38, 1810-1821. (33) Fringeli, U. P. Z. Naturforsch. 1977, 32c, 20-45.
Fluid Lipid Bilayers Tethered to Solid Supports
Langmuir, Vol. 14, No. 4, 1998 843
order parameter S by eq 3.15,33
RATR )
AP Ex2 Ez2 (2 cos2 γ + s) ) + AS E 2 E 2 (sin2 γ + s)
(2)
1.5s 1 + 1.5s
(3)
y
S)1-
y
Strictly speaking, eq 2 is only applicable to “rigid chains” in their all trans configuration. In this case, γ is the angle between the CH2 dipole moment and the lipid chain axis. For perfect order, S ) 1 (s ) 0), while for an isotropic orientational distribution of chain axes, S ) 0 (s ) ∞). Since the lipids are in the liquid-crystalline phase, and the hydrocarbon chains adopt a mixture of trans and gauche configurations,18 the angle γ is no longer well defined. However, we have made the assumption that, for an “average chain”, γ has the value 90°.13,14 The measured dichroic ratios of the asymmetric and symmetric CH2 stretching vibrations were estimated, using band fitting procedures, to be 3.3 ( 0.3 and 3.3 ( 0.3, respectively, which gives values for s and S of 0.051 and 0.93, respectively. This value of S can be compared with the average CD2 segmental order parameter, measured by NMR for fluid phase dimyristoylphosphatidylcholine in the presence of 30 mol % cholesterol, which is 0.72 (unpublished result). A direct comparison is not necessarily valid as a bilayer tethered to a solid support could be more “rigid” than a free bilayer. Nevertheless, the value gives an indication that the lipids are oriented predominantly perpendicular to the ATR surface, consistent with the formation of planar tethered lipid bilayers. Discussions We have demonstrated that ATR-FTIR spectroscopy can be used to study the tethering of lipid bilayers to gold surfaces in-situ. Compared with other techniques, such as surface plasmon resonance spectroscopy and impedance measurements,5,7,34,35 for characterization of solid supported bilayers in-situ, ATR-FTIR measurements give complementary information such as lipid chain conformation and orientation. In tethered bilayers incorporating membrane proteins, the secondary structure of protein could also be characterized in the same way. The advantages of using the cholesteryl moiety as the bilayer anchoring group compared with using a lipid moiety is that the cholesterol-thiol derivative is simple to synthesize and more chemically and biologically robust. The extent of the water-filled space between bilayer and solid support can be varied by adjusting the length of the ethyleneoxy chain (n in EOnC).12 This is a prerequisite for incorporating bulky membrane proteins which protrude into the aqueous phase as is the fluidity of the bilayers. The attached bilayers are of necessity asymmetric since the cholesteryl groups can only penetrate into the lower leaflet of the bilayer. This could, in some cases, be a disadvantage. However, cholesterol could be incorporated into the outer monolayer to preserve the symmetry of the bilayer if required. The ATR-FTIR measurements show the feasibility of using mixed EO6C/EO1 monolayers to attach fluid, lipid bilayers to solid supports. EggPC bilayers can be reliably attached only for cholesteryl coverages in excess of 24%, (34) Plant, A. L. Langmuir 1993, 9, 2764-2767. (35) Stelzle, M.; Weissmu¨ller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981.
suggesting a minimum degree of surface hydrophobicity is needed in order to induce lipid vesicle fusion onto the surface. These results can be used to gain insight into the initial vesicle adsorption and rupture events leading to bilayer self-assembly. We shall assume, for the argument, that this proceeds via a distinct nucleation step which involves the simultaneous adsorption, rupture, and fusion of n vesicles. The bilayer then grows by subsequent addition of individual vesicles. We proceed by calculation of the balance between the free energy gained in inserting cholesterol into the lipid bilayer and the free energy cost in exposing to water the rim of a circular bilayer disk formed by fusion of n vesicles. The free energy gained is ADFchol∆µ0 and the free energy cost is LDdhcνo/w. Therefore, the balance is described by eq 4, which leads to an expression for n described by eq 5.
ADFchol∆µ0 ) LDdhcγo/w n)
(
dhcγo/w 0
)
rv∆µ Fchol
(4)
2
(5)
Here AD and LD are, respectively, the area and circumference of a circular bilayer disk, which are functions of the average radius of sonicated vesicles, rV, and is taken to be 125 Å;19 Fchol is the number of cholesteryl molecules per unit area of surface: for 24% cholesteryl coverage, Fchol is 6.3 × 1017 m-2; ∆µ0, the change in the standard chemical potential of cholesterol on transferring from an amphiphilic, membrane-like solvent to water, is calculated from the partition coefficient of cholesterol between 2-propanol and water36 to be 9.985 kJ M-1; dhc is the thickness of the hydrocarbon region of an eggPC bilayer (25 Å); γo/w, the interfacial tension of tetradecane in water, is taken to be 52 mJ m-2.3 On substitution of these values in eq 5, we then have n ) 0.99 (∼1); i.e., the initial step in bilayer self-assembly is the adsorption and rupture of a single vesicle. This is presumably because, once adsorbed, the bilayer “disk” formed by rupture of the vesicle is not able to diffuse and fuse with a neighbor to reduce the excess energy associated with its edge. Otherwise, we would have expected bilayer self-assembly to have been nucleated by fusion of n vesicle disks at lower surface coverages of cholesteryl. This is why a “critical” fractional surface coverage of anchoring cholesteryl moieties is required. We have not included, in the calculation, the free energy cost of damping bilayer fluctuations, which will be of the order kBT per mode. However, we do not expect this to significantly affect the result. Finally, we compare the results of these new ATR-FTIR and our previous SPR studies. The ATR-IR results show that for fractional surface area coverages less than 24% cholesteryl, there is no detectable lipid adsorption. This appears to contradict the observation by surface plasmon resonance measurements that lipid bilayers are formed on hydrophilic surfaces.11 The differences between the SPR and ATR experiments are that the former employed a 500 Å gold layer deposited on high refractive index glass (LiTHS) and a SAM comprised of EO3/EO3C, whereas the ATR experiments employed a 200 Å gold layer on ZnSe and EO1/EO6C. It seems unlikely that the thickness of the gold layer in itself is the reason for the different behavior. (36) Takino, T.; Konishi, K.; Takakura, Y.; Hashida, M. Biol. Pharm. Bull. 1994, 17, 121-125.
844 Langmuir, Vol. 14, No. 4, 1998
Conclusion ATR-FTIR spectroscopy has been used to study in-situ the self-assembly of lipid bilayers on gold-coated substrates employing a new cholesteryl tethering methodology. The data obtained provides information on lipid chain conformation, the amount of lipid adsorbed, and the orientation of lipids within the tethering bilayers. In particular, our results show that eggPC vesicles spontaneously adsorb and fuse into bilayers on SAMs with fractional surface coverage of EO6C g 0.24. The outer leaflet of this tethered bilayer is comprised of pure eggPC and the inner one is comprised of a mixture of cholesteryl and eggPC. Because there are hydrophilic spacers (oligoethylene glycols) between the gold surfaces and the lipid bilayer, the
Cheng et al.
tethered bilayer is believed to have a distinct water containing layer between itself and the substrate, providing that the concentration of EO6C is sufficiently low. We estimate a maximum water layer thickness of ∼10 Å when the ethyleneoxy chains are fully extended. By use of analogues of EO6C with longer ethyleneoxy chains,12 a more extensive water layer should be achievable so that membrane proteins could be incorporated. Acknowledgment. We thank the EPSRC and the BBSRC for financial support and Dr. L. M. Williams for helpful advice. LA970998K
