Alkylthiol Layer

Lipid bilayer assembly at a gold nanocavity array. Bincy Jose , Colm T. Mallon , Robert J. Forster , Chuck Blackledge , Tia E. Keyes. Chemical Communi...
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Langmuir 2000, 16, 5093-5099

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Mobile Phospholipid Bilayers Supported on a Polyion/ Alkylthiol Layer Pair Liqin Zhang, Marjorie L. Longo,* and Pieter Stroeve* Center on Polymer Interfaces and Macromolecular Assemblies (CPIMA), Department of Chemical Engineering and Materials Science, University of California at Davis, Davis, California 95616 Received October 13, 1999. In Final Form: March 7, 2000 The formation of phospholipid bilayers on a polymer/alkylthiol layer pair was investigated by surface plasmon resonance (SPR). The organic layer pair between the lipid membrane and the solid gold surface consisted of a self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) on a gold surface followed by a thin layer of hydrated cationic poly(diallyldimethylammonium chloride) (PDDA). The lipid layers were formed by vesicular fusion of small unilamellar vesicles of an anionic lipid 1-stearoyl-2-oleoylphosphatidylserine (SOPS), a zwitterionic lipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and mixtures of these two lipids with different compositions. In the case of pure SOPS and lipid mixtures with a POPC composition below 25%, the lipid layer thickness was approximately that of a single bilayer, while multilayers tended to build up with higher concentrations of POPC. The electrostatic interaction of the cationic PDDA with the negatively charged lipid membrane is most probably the driving force for the adsorption of a single bilayer. Fluorescence recovery after photobleaching (FRAP) experiments showed that single bilayers supported on the PDDA/alkylthiol layer pair were mobile at room temperature with lateral diffusivities of approximately (1-2) × 10-9 cm2/s. A membrane fusion peptide wt-20 of the influenza virus was bound to the supported bilayers as detected by SPR. This suggests the potential of this model membrane system for use as a biosensor.

Introduction Phospholipid bilayers supported on flat solid substrates are of both practical and scientific interests as model systems to study the structure and function of natural membranes, as well as those of membrane-bound biomolecules.1-4 Most biomolecules require a fluid membrane environment to retain their bioactivities, which makes it important for the supported biomembrane to be laterally mobile. A fluid membrane environment requires the minimization of the influence of the solid support on the membrane since the solid substrate has been found to restrict the motion of supported organic layers.3,5 Hydrated polymer layers have been used as “cushions” to lift biomembranes from solid surfaces.3 The hydrated space created by the addition of a polymer layer is not only advantageous for decreasing the substrate effect on the membrane itself but also desirable for maintaining the activities of incorporated biomolecules. For example, transmembrane proteins can protrude from the membrane and become immobilized by direct interaction with the solid surface.6,7 Moreover, a polymer cushion is similar to the cytoskeleton structure supporting mammalian plasma cell membranes.8 * To whom correspondence should be addressed. E-mail [email protected]; telephone (530) 752-8778; Fax (530) 7528778. (1) Majewski, J.; Wong, J. Y.; Park, C. K.; Seitz, M.; Israelachvili, J. N.; Smith, G. S. Biophys. J. 1998, 75, 2363-2367. (2) Sohling, U.; Schouten, A. J. Langmuir 1996, 12, 3912-3919. (3) Sackmann, E. Science 1996, 271, 43-48. (4) Stelzle, M.; Weissmuller, G.; Sackmann, E. J. Phys. Chem. 1993, 97, 2974-2981. (5) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667-1671. (6) Salafsky, J.; Groves, J. T.; Boxer, S. G. Biochemistry 1996, 35, 14773-14781. (7) Kalb, E.; Tamm, L. K. Thin Solid Films 1992, 210/211, 763-765. (8) Jacobson, K.; Sheets, E. D.; Simson, R. Science 1995, 268, 14411442.

In recent years, vesicle fusion has been used to form bilayers on solid substrates because it allows the incorporation of transmembrane proteins and is relatively easy to perform.1,2,9 Certain polyelectrolytes have been reported to induce fusion of vesicles.2,10,11 Negatively charged dioleoyl-L-R-phosphatidic acid sodium salt (DOPA) vesicles have been fused on a polyethylenimine (PEI) layer adsorbed on quartz.2 An X-ray scattering study gave evidence of single bilayer formation. Neutron scattering data reported by Majewski et al.1 show that the zwitterionic lipid dimyristoylphosphatidylcholine (DMPC) forms inhomogeneous multilayer structures when added on a PEI-covered quartz substrate. However, a single bilayer of DMPC can be formed on bare quartz, and subsequent addition of PEI appears to penetrate through the DMPC bilayer and lift the membrane away from the quartz substrate.1 It has also been reported that the adsorption of zwitterionic vesicles forms a single bilayer on a copolymer containing side chains resembling phospholipid tails.5 Some other methods of decoupling the membrane from the solid surface by a soft polymer cushion have been reviewed.3 Unfortunately, little lateral mobility information has been reported, and there is a shortage of kinetic information in the literature due to the limitation of the techniques used. To explore the mobility of solid supported biomembranes cushioned by a hydrated polymer, we focused on a model membrane system of lipid mixtures including an anionic phospholipid and a zwitterionic phospholipid. Negatively charged and zwitterionic phospholipids are major components of natural biomembranes.12 In this work, 1-stearoyl-2-oleoylphosphatidylserine (SOPS) and 1-palm(9) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316. (10) Walter, A.; Steer, C. J.; Blumental, R. Biochim. Biophys. Acta 1986, 861, 319. (11) Oku, N.; Yamaguchi, Na.; Yamaguchi, No.; Shibamoto, S.; Ito, F.; Nango, M. J. Biochemistry 1986, 100, 935.

10.1021/la9913405 CCC: $19.00 © 2000 American Chemical Society Published on Web 05/05/2000

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20, to our membrane system. This short (20 amino acids) amphiphilic peptide takes a large role in the initiation of viral fusion21,22 by being inserted into the neighboring endosomal membrane.23,24 Previous work with free bilayers showed that the synthesized wt-20 peptide inserts rapidly into the membrane (to a larger extent at low pH than high pH) and that insertion can mainly be reversed by rinsing the membrane with buffer.25,26 Therefore, we monitored both the adsorption and desorption of the peptide from our supported membrane system. Experimental Section

Figure 1. Schematic representation of the model membrane system. The alkylthiol (MUA) layer self-assembled on a gold surface. The negatively charged headgroups of MUA adsorb a cationic polymer (PDDA) layer. A lipid bilayer with negative charges is then deposited on the PDDA/MUA layer pair.

itoyl-2-oleoylphosphatidylcholine (POPC) were tested. Poly(diallyldimethylammonium chloride) (PDDA) was used as the polymer to support the lipid bilayer. Extensive research has been done on PDDA adsorption on solid substrates to form a stable layer, and it has been shown that the PDDA layer thickness depends on the ionic strength of the buffer solution that dissolves the polycation.13,14 Surface plasmon resonance (SPR) was used in this work to detect the adsorption of the polymer layer and the lipid layers. The SPR technique is a sensitive method to monitor the growth of organic layers on noble metal surfaces.15-17 We also used fluorescence recovery after photobleaching (FRAP) to measure the lateral mobility of the lipid membranes formed on the polymer layer. It is well-known that self-assembled monolayers (SAMs) of alkylthiols are able to form on gold by the chemisorption of the thiol group to gold.18 A hybrid bilayer membrane composed of a hydrophobic SAM of alkylthiol formed on gold and a phospholipid monolayer has been studied as cell membrane mimics.19,20 In our system, we used an alkylthiol with hydrophilic acidic headgroups, 11-mercaptoundecanoic acid (MUA), to form a homogeneous negatively charged surface for the adsorption of a PDDA layer. The PDDA/MUA layer pair lifts the biomembrane away from the solid gold surface. It should be noted here that both sides of the lipid membrane are in hydrated environments. A schematic drawing of this system is shown in Figure 1. Supported biomembranes that retain fluidity may be useful as model systems for sensing molecules that interact with cell membranes, e.g., peptides and proteins. As a means to investigate sensing abilities and biological function, we introduced the influenza fusion peptide, wt(12) Vance, D. E.; Vance, J. E. Biochemistry of Lipids, Lipoproteins and Membranes; Elsevier Science B. V.: Dordrecht, The Netherlands, 1996; Chapter 1. (13) Kleinfeld, E. R.; Ferguson, G. S. Science 1994, 265, 370-373. (14) Decher, G. In Comprehensive Supramolecular Chemistry; Sauvage, J. P., Ed.; Pegamon Press: New York, 1996; Vol. 9, Chapter 14. (15) Knoll, W. Annu. Rev. Phys. Chem. 1998, 49, 569-638. (16) Knoll, W. MRS Bull. 1991, 16, 29-39. (17) Aust, E. F.; Ito, S.; Sawodny, M.; Knoll, W. Trends Polym. Sci. 1994, 2, 313-323. (18) Ulman, A. An Introduction to Ultrathin Organic Films, from Langmuir-Blogett to Self-Assembly; Academic Press: San Diego, 1996; Chapter 3. (19) Plant, A. L. Langmuir 1999, 15, 5128-5135. (20) Rao, N. M.; Plant, A. L.; Silin, V.; Wight, S.; Hui, S. W. Biophys. J. 1997, 73, 3066-3077.

Materials. 11-Mercaptoundecanoic acid (MUA) was purchased from Aldrich (Milwaukee, WI). The polymer poly(diallyldimethylammonium chloride) (PDDA) was purchased from Polysciences, Inc. (Warrington, PA). The lipids 1-stearoyl-2-oleoylphosphatidylserine (SOPS) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) as well as the fluorescence probe (16: 0-6:0 NBD-PS) were purchased from Avanti Polar Lipids (Alabaster, AL). The buffer tris(hydroxymethyl)aminomethane (Tris) was obtained from Sigma (St. Louis, MO) while 4-morpholineethanesulfonic acid (MES) was from Aldrich. Influenza wt-20 peptide (NH2-GLFGAIAGFIENGWEGMIDG-COOH) was obtained from Dr. Alan Waring (see ref 25 for the synthesis procedure) as a synthesized material. HPLC-grade dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific. The refractive index matching fluid 1-iodonaphthalene was obtained from Cargille Laboratories Inc. (Cedar Grove, NJ). All chemicals were used as received without further purification. In all experiments, water was purified by a Nanopure reverse osmosis purification system from Barnstead (Dubuque, IA). The resistivity was better than 17.7 MΩ‚cm. Three buffers were used throughout the experiments: Tris 8.5 (Tris buffer, pH ) 8.5), Tris 7.2 (Tris buffer, pH ) 7.2), and MES 5.0 (MES buffer, pH ) 5.0). All three buffers were at a concentration of 5 mM. When a system was prepared, the PDDA, the vesicle solution, and the peptide solution were all prepared with the same buffer. The percentages mentioned throughout the article indicate molar fraction unless otherwise stated. Preparation of Gold Slides with Functionalized Alkylthiol Monolayers. Gold slides covered by a close-packed selfassembled monolayer of alkylthiol MUA were used as substrates for subsequent deposition of a PDDA layer and lipid bilayers. For SPR experiments, high index glass LaSFN9 slides (n ) 1.85 at λ ) 633 nm, Schott, Germany) were used as substrates for gold deposition. About 50 nm of gold was deposited on LaSFN9 at a rate of 0.2 Å/s by vacuum evaporation in an electron beam chamber (pressure below 5 × 10-6 mbar). The sample slides used for FRAP experiments were prepared from cleaned glass microscope slides from Fisher Scientific. In this case, a 3 nm thick titanium layer was sputtered first on the glass slides to improve the adhesion of gold to glass. The thickness and the deposition rate of the gold layer were the same as for the LaSFN9 glass slides. Alkylthiol monolayers were deposited on gold surfaces by immersing the gold-coated slides in a 5 mM solution of MUA in pure ethanol for at least 18 h at room temperature. The slides were removed from the solution, rinsed with ethanol, and dried thoroughly with N2 right before use. Vesicle Preparation. Approximately 100 µL from 10 mg/mL lipid solutions in chloroform was added to a precleaned glass vial. When lipid mixtures were involved, the solutions were vortexed for 2 min to mix the lipids. A small N2 stream was then (21) Daniels, R. S.; Downie, J. C.; Hay, A. J.; Knossow, M.; Skehel, J. J.; Wang, M. L.; Wiley, D. C. Cell 1985, 40, 431-439. (22) Gething, M. J.; Domes, R. W.; York, D.; White, J. J. Cell Biol. 1996, 102, 11-23. (23) Stegmann, T.; Delfino, J. M.; Richards, F. M.; Helenius, A. J. Biol. Chem. 1991, 266, 18404-18410. (24) Weber, T.; Paesold, G.; Galli, C.; Mischler, R.; Semenza, G.; Brunner, J. J. Biol. Chem. 1994, 269, 18353-18358. (25) Longo, M. L.; Waring, A. J.; Gordon, L. M.; Hammer, D. A. Langmuir 1998, 14, 2385-2395. (26) Longo, M. L.; Waring, A. J.; Hammer, D. A. Biophys. J. 1997, 73, 1430-1439.

Mobile Phospholipid Bilayers used to evaporate chloroform. Caution was taken so that the lipids were evenly distributed on the wall of the vial after drying. Two milliliters of 5 mM buffer solution was added to the vial to redisperse the lipids to get a final lipid concentration of 0.5 mg/ mL. The lipid solution was then incubated in a 50 °C water bath for 15 min with several vortexing periods of 15 s in between. The solution was sonicated in a small plastic tube with an ultrasonic tip (1/8 in. diameter, Branson 250, 10 W output) for 1 min followed by three pulsed periods of 10 s each. The clear lipid solution was used after equilibrating at room temperature for 30 min. For FRAP experiments, 2 mol % of NBD-PS was included in the lipid solution in chloroform before evaporation. All the other steps of vesicle preparation remained the same. Preparation of Peptide Solution. The influenza HA fusion active peptide wt-20 was stored frozen in DMSO with a concentration of 2 mM. Immediately before the insertion experiment, 10 µL of the solution was mixed with 2 mL of the desired buffer to make a 10 µM aqueous solution. Insertion of the peptide into the lipid bilayer was tested at pH 7.2 and pH 5.0. Refractive Index Measurements. Refractive indices of PDDA solutions and vesicle solutions were measured on an Abbe 60 refractometer (Bellingham and Stanley, Ltd., England) using a sodium spectral lamp with a wavelength of 589.3 nm. Adsorption Experiments. The adsorption experiments were monitored with a SPR setup using a laser beam with a wavelength of 633 nm. The SPR was set up according to the Kretschmann configuration.27 The angular position of the minimum angle, θm, i.e., the matching condition between the evanescent electromagnetic waves and surface plasmons, is a function of the thickness and refractive indices of the layers adsorbed on the gold surface and that of the bulk solution.5 From the shift of θm, the thickness of the organic layers can be calculated using the Fresnel equations (SPR software: WASPLAS version 2.1, MaxPlanck-Institute for Polymer Research, Mainz, Germany) if the refractive index information on adsorbed organic layers is known. Kinetic information on adsorption of each layer was obtained by setting the angle of incidence 0.5° below θm. A Teflon cell was used to hold 0.8 mL of fluid on which a gold-coated glass slide covered with a MUA monolayer was mounted. Solutions were exchanged by injecting and withdrawing simultaneously using two syringes through two holes in the Teflon cell. A LaSFN9 glass prism was mounted on the glass slide, and 1-iodonaphthalene was used as the refractive index matching fluid between the prism and the glass slide. A desired buffer was injected into the Teflon cell and was allowed to incubate for approximately 15 min to equilibrate with the surrounding room temperature before SPR measurements were taken. Then a 0.2 M PDDA solution dissolved in the same buffer was exchanged into the cell for at least 20 min before it was rinsed with buffer. The cell was rinsed until there was no further decrease of reflectivity. The freshly prepared vesicle solutions were then injected into the cell. The growth of the bilayer, including a rinsing step, was again monitored by a SPR kinetic measurement. Fluorescence Recovery after Photobleaching (FRAP). The mobility of the supported bilayers was tested at room temperature by FRAP experiments on a Nikon Diaphot 300 fluorescence microscope. Two half-circular Teflon spacers with a thickness of 60 µm were placed on a cleaned microscope cover slide. About 60 µL of a 0.2 M PDDA solution in Tris 8.5 buffer was added in the middle of the two spacers. A gold-coated glass slide covered by a self-assembled MUA monolayer was then mounted on the spacers. The setup was allowed to incubate in a humid environment for about 30 min. The polymer solution captured between the slides was exchanged with a buffer solution, and subsequently a lipid vesicle solution was introduced to the space by exchanging solutions. The setup was then allowed to incubate for at least 3 h. The vesicle solution was then exchanged with Tris 8.5 buffer solution for at least 10 times. The NBD fluorescent probe was excited by blue light filtered from a Mercury lamp and emitted bright green light. Using a 20× objective lens on the microscope, a spot of 60 µm in diameter was bleached for 3 min with the diaphragm closed and with both neutral density filters out. The bleached spot was then viewed (27) Aust, E. F.; Sawodny, M.; Ito, S.; Knoll, W. Scanning 1994, 16, 353-361.

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Figure 2. Part of the SPR curves near the minimum angles, where the reflectivity is at its lowest value. The curves shift to the right with successive deposition of PDDA and a lipid bilayer. A SPR curve measured after the deposition of a SOPS bilayer is shown as an example. Inset: complete SPR curves measured from 45° to 70°. The parts near the minimum angles are enlarged for better resolution. using the same objective lens with the diaphragm open. A digital camera captured images every 15 min until the spot was totally diffused and could not be observed on the screen. On-line software, Scion Imaging (www.scioncorp.com), was used to analyze the images. For each image, an optical intensity profile was generated. The data were smoothed, and the differences of maximum and minimum values of the curves were normalized and plotted versus time. The lateral diffusion coefficient (D) was calculated from D (cm2/s) ) 0.224ω2 (cm)/t1/2 (s), where ω is the radius of the bleached spot and t1/2 is the half-life of the fluorescence recovery.28,29

Results Refractive Indices. To calculate the equilibrium thickness of each adsorbed layer from the SPR measurements (Figure 2), the refractive index of the adsorbed layer is needed. The typical literature value for the refractive index for long chain lipids, namely 1.49,30 was used in our calculations. The refractive index values used for lipid layers in the literature vary from 1.45 to 1.50. A refractive index value of 1.45 is assumed at a wavelength of 750 nm in an SPR study of lipid monolayer deposited on hydrophobic thiol.31 Stelzle and co-workers4 assumed the same value for supported lipid bilayers. Salamon et al.32 and Knoll16 both used 1.50 as the refractive index of their lipid layers, and Huang and Thompson33 measured the refractive index of a POPC bilayer to be 1.493. The refractive indices of the bulk lipid vesicle (0.5 mg/mL) and peptide solutions (10 µM) in buffer were measured by us to be 1.332 and 1.333, respectively. For the PDDA layer, a refractive index of 1.41 was assumed which corresponds to a dielectric constant of 2 and water content of 63%. This corresponding water content was determined by measuring the refractive indices of a series of PDDA solutions in 5 mM Tris 8.5 buffer. By fitting a linear equation using least-squares (28) Mercel, R.; Sackmann, E.; Evans, E. J. Phys. (Paris) 1989, 50, 1535-1555. (29) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055-1069. (30) Schouten, S.; Stroeve, P.; Longo, M. Langmuir 1999, 15, 81338139. (31) Hubbard, J. B.; Silin, V.; Plant, A. L. Biophys. Chem. 1998, 75, 163-176. (32) Salamon, Z.; Huang, D.; Cramer, W. A.; Tollin, G. Biophys. J. 1998, 75, 1874-1885. (33) Huang, C.; Thompson, T. E. J. Mol. Biol. 1965, 13, 183-193.

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Figure 3. SPR kinetic profiles of PDDA adsorption on MUA monolayer on gold at pH 8.5. The first arrow indicates injection of a 0.2 M PDDA solution. The initial fast increase in reflectivity is due to the refractive index increase of the bulk solution as a result of the injection of PDDA solution. The other three arrows correspond to the rinses of the Teflon cell with 5 mM Tris 8.5.

regression to the measurements (n ) 0.2217w + 1.3322, R2 ) 0.9989; n ) refractive index; w ) weight fraction of PDDA) and interpolating to a refractive index of 1.41, the concentration of PDDA can be estimated to be 37 wt %. The refractive index of the 0.2 M bulk PDDA solution was measured to be 1.339. PDDA Adsorption on Supported Monolayers of MUA on Gold. PDDA, a positively charged polymer, was deposited onto a negatively charged surface formed by a self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) on evaporated gold. A polymer concentration of 0.2 M (based on its monomer MW ) 161.7) was used. At PDDA concentrations below 0.2 M, the adsorbed PDDA layer was not sufficiently thick to overcome the negative charges of the MUA surface and thus prevented vesicle fusion (data not shown). As shown by the kinetic profile measured by SPR (Figure 3), the reflectivity increased significantly in seconds upon injection of the 0.2 M PDDA solution. This fast and significant increase of reflectivity is due to the higher refractive index of the 0.2 M PDDA solution injected (1.339) than the original buffer solution (1.332). A relatively slower kinetic process on the order of a couple of minutes before a flat equilibrium stage was most probably due to the adsorption of the PDDA layer. After several rinses with buffer, the reflectivity dropped considerably. The final increase of reflectivity compared to that before PDDA injection was about 0.02. Simulation gave a thickness increase of 10 ( 1 Å assuming a refractive index of 1.41 for the adsorbed PDDA layer. No significant difference was observed under the three pH conditions 8.5, 7.2, and 5.0. In some of the experiments, SPR measurements were taken before the buffer rinses to find out the thickness of PDDA layer at that stage. Using the measured refractive index of 1.339 for the bulk PDDA solution, it was found that the thickness of the adsorbed PDDA layer was also 10 Å, identical to the PDDA thickness after rinsing. Deposition of Lipids on PDDA. Vesicle fusion has been used extensively as an effective method to deposit lipid layers on solid substrates.1,4,6,30,34,35 Lipid layers were deposited on the PDDA covered substrates by vesicle fusion at pH 8.5. The pH 8.5 is used here because it ensures that the acidic headgroups of the alkylthiol render negative charges at this pH, and this ensures the strong adsorption of the PDDA layer to the substrate. The same buffer was used throughout the multilayer formation for consistency. The depositions of the negatively charged lipid, SOPS,

Figure 4. SPR kinetic profiles of lipid adsorption with different compositions of SOPS and POPC (a: 100% SOPS, 87.5% SOPS/ 12.5% POPC, 75% SOPS/25% POPC; b: 50% SOPS/50% POPC, 10% SOPS/90% POPC, 100% POPC) on PDDA/MUA covered gold surfaces at pH 8.5. Note that the reflectivity scales are different in (a) and (b). For clarity purposes, the kinetic profiles shown in the figures connect the bilayer adsorption kinetics and the rinsing kinetics. The perpendicular arrows indicate rinses with buffer to eliminate unbound vesicles. The reflectivity changes were negligible before and after rinsing.

and zwitterionic lipid, POPC, as well as lipid mixtures made out of the combination of these two lipids are shown in Figure 4a,b. The reflectivity changes were minor before and after rinsing. As shown by SPR kinetic profiles, pure SOPS followed a sharp and fast deposition, and the reflectivity increased to reach an equilibration within an hour. In Figure 5, the thickness values are obtained from the simulation of the SPR curves, before and after adsorption, with the software WASPLAS 2.1. The standard deviations in our results were obtained by repeating each experiment at least three times. Thickness measurements were made both before and after rinsing and did not give significant difference. The thickness data presented are those measured after rinsing. Simulation gave a thickness of 32 ( 2 Å of the adsorbed lipid membrane with a refractive index of 1.49 for the lipid layer. If a refractive index of 1.45 is used, the average simulated thickness is 43 Å. Vesicle fusion of pure POPC showed a different kinetic

Mobile Phospholipid Bilayers

Figure 5. Lipid layer thickness changes versus percentage of POPC in SOPS/POPC vesicles used for vesicular deposition on PDDA/MUA covered gold surfaces at pH 8.5. Each data point represents the average of at least three measurements. Similar thickness data were obtained before and after rinsing. The data presented here are those after rinsing. The error bars represent the standard deviations of the experimental data. The thickness of the lipid layers increases from a single bilayer thickness to multilayers at about 50% POPC in the mixture.

response on PDDA than SOPS. The reflectivity continued to increase and was still changing slowly when the experiments were stopped at about 10 h after the vesicle solution was injected. Thickness calculation gave a result of 140 ( 15 Å of the POPC layers. When mixtures with different compositions of SOPS and POPC were used for deposition, the final thickness of lipid adsorbed varied (Figure 5). The thickness increased from a single bilayer thickness to that of a multilayer when the ratio of POPC was beyond 25%. When the POPC composition in the lipid mixtures increases to 50%, a “kink” in the kinetic curve was often observed when the reflectivity approached 0.2, and the final thickness was more than that of a single bilayer. Mobility of Lipid Bilayers Tested by FRAP. Quantitative analysis was performed on the lateral mobility of single lipid layers adsorbed on PDDA as described in the Experimental Section. In all cases, the fluorescence intensity of the bleached spot recovered about 90% at 90 min after bleaching. The bilayer formed with 25% POPC/ 75% SOPS had a faster recovery than that formed by pure SOPS (Figure 6). The calculations for the lateral diffusion coefficients (D) showed that for the mixture with 75% SOPS and 25% POPC on PDDA D ∼ 2 × 10-9 cm2/s, whereas for the bilayer formed from pure SOPS bilayer D ∼ 1 × 10-9 cm2/s. Peptide Insertion. Peptide insertion experiments were monitored by SPR on single bilayers formed by 80% SOPS/ 20% POPC. The thickness of the bilayer formed at pH 5.0 was larger than for pH 7.2. Two milliliters of 10 µM solution of peptide wt-20 in buffer was injected into the cell. The reflectivity increased upon injection and decreased upon rinsing. The cell was rinsed with buffer until there was no further decrease in reflectivity. The final reflectivity after rinsing was always higher than that before peptide injection, indicating that a certain amount of peptide was bound to the bilayer (Figure 7). The thickness results after rinsing are listed in Figure 7. At pH 5.0, the bilayer thickness increased from 49 to 53 Å. The thickness change at pH 7.2 was from 38 to 42 Å. Therefore, the net thickness change at both pH 5 and 7 due to peptide adsorption was approximately 4 Å.

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Figure 6. FRAP data curves obtained for bilayers (100% SOPS; 75% SOPS/25% POPC) formed on PDDA/MUA covered gold surfaces at pH 8.5 under room temperature. The fluorescence intensities of the two bilayers were normalized independently. Data were collected every 15 min. The time interval for taking images was restricted within 3 s. The shutter was closed between each datum collection to prevent excessive bleaching.

Figure 7. SPR kinetic profiles of peptide (wt-20) insertion into bilayers composed of 80% SOPS/20% POPC formed on PDDA/MUA covered gold surfaces. Experiments were conducted with two buffers: MES 5.0 and Tris 7.2. The small perpendicular arrows indicate rinses with buffer solutions. Data of thickness measurements are listed with the curves for bilayer thickness before and after peptide insertion (after rinsing): /, pH 5.0; //, pH 7.2. Thickness data are in angstroms.

Discussion Deposition of PDDA. PDDA adsorbed to the MUA monolayer covered gold surface within minutes at all three pH conditions tested, 8.5, 7.2, and 5.0. The Coulombic interaction of the positively charged PDDA and the negatively charged substrate induced the adsorption of PDDA layers that were approximately 10 Å thick assuming a water content of 63%. The relatively small thickness of the adsorbed PDDA layer can be explained by the low ion concentration in the buffer solutions. In the presence of few counterions, most of the charges of the PDDA are not screened, and thus the charged groups on the polymer chains repel each other to maximize interchain distance, leading to a relatively flat conformation onto the solid surface.14,36 We determined PDDA layer thicknesses using (34) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 25, 651653. (35) Oudenaarden, A. V.; Boxer, S. G. Science 1999, 285, 1046-1048. (36) Decher, G.; Lehr, B.; Lowack, K.; Lvov, Y.; Schmitt, J. Biosen. Bioelectron. 1994, 9, 677-684.

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a refractive index of 1.41 that corresponded to a water content of 63 wt %. Changing the refractive index in our calculations from 1.40 (water content 69.4 wt %) to 1.45 (water content 46.8 wt %) results in a change of calculated thickness from 11 to 7 Å. In all these cases the PDDA layer provides an aqueous cushion for the deposition of a lipid bilayer. Thickness of aqueous polyelectrolyte layers adsorbed from low ionic strength solutions has been reported to be in a similar range as measured here. Smallangle X-ray reflectivity study of a polyethylenimine (PEI) layer (adsorbed from a PEI solution of 0.03 mol/L in water) between a quartz substrate and a lipid bilayer revealed a thickness of 11-12 Å, including the lipid headgroup thickness.2 It should be noted here that thicker aqueous polymer layers can be achieved by employing the layerby-layer polyion adsorption technique.14 Additional layers of PDDA can be adsorbed by interleaving with a polyanion such as PSS (polystyrene sulfonate).14 Deposition of Lipids on PDDA. Lipid layers were deposited on the positively charged PDDA layers. Vesicle fusion of the negatively charged lipid SOPS versus the zwitterionic lipid POPC showed very different results. Negatively charged SOPS vesicles were attracted to the PDDA layer by strong Coulombic interaction and were disrupted to form a single bilayer. Possibly, the negatively charged lipid surface repelled further adsorption of other negatively charged lipid molecules, and thus the lipid system equilibrated as a single bilayer structure. The fluorescence experiments showed a homogeneous distribution of the fluorescent probe in the bilayer. It should be noted that the thickness of the SOPS bilayer is 32 Å, lower than the usual bilayer thickness ∼40 Å for long chain phospholipids in the fluid phase.37 This is possibly due to two reasons. The first possibility is that there was interdigitation between the two lipid monolayers. Second, it is possible that there were very small defects in the bilayer that are beyond the resolution ability of the fluorescence microscope. In the case of the zwitterionic lipid POPC, vesicle fusion led to multilayer thicknesses on PDDA. Possibly the interaction of the POPC lipid layers and the PDDA layer was not strong enough so that the van der Waals interaction between the lipid layers dominated, leading to the formation of multilayer aggregates or the presence of undisrupted vesicles on the surface. When mixtures of the two lipids were used (POPC composition > 25%), the electrostatic interaction and van der Waals interaction were in competition and resulted in some intermediate thickness. The lipid mixtures with 12.5% POPC and 25% POPC followed similar kinetics in comparison to pure SOPS, i.e., a quick steep growth period and an almost flat equilibrium stage. The thickness increased slightly with an increase in POPC content. In these systems, it appears that the electrostatic attraction from the polymer layer was the driving force for bilayer adsorption, and then the electrostatic repulsion between the lipid bilayer and the vesicles prevented further deposition of lipid molecules. The somewhat larger bilayer thickness is possibly due to the fact that the addition of zwitterionic lipids diluted the charge density of the lipid membrane. The weaker electrostatic attraction due to the charge dilution effect by zwitterionic POPC molecules may result in less interdigitation between the two lipid monolayer pairs. It is also possible that zwitterionic lipid molecules filled in the defects and made a more complete bilayer. The increasing diffusion coefficient of the membrane with increasing percentage of POPC supports this hypothesis.

The kinetics and thickness for the lipid mixture with 50% POPC indicated an intermediate stage for adsorption. The “kink” in the kinetic profiles observed at the reflectivity of ∼0.2 is possibly due to the fact that a bilayer was formed before other lipid molecules were deposited as multilayers or as undisrupted vesicles on the surface. Another possibility is SPR line broadening and sharpening at the fixed angle due to a highly inhomogeneous layer formation.38 It is interesting that the kinks only show up when POPC composition is at or above 50%. Zwitterionic POPC molecules reduced the charge density of the membrane so that there was not sufficient repulsion to prevent further vesicle deposition. The reflectivity continued to increase until a flat equilibrium was reached. The slope of the bilayer growth, as shown in Figure 4b, was not as steep as those with lower POPC percentages. In the case of pure POPC adsorption, the lipid bilayers kept growing and did not reach equilibration even when 9-10 h of adsorption time was allowed. This slower adsorption kinetics with more POPC composition can be an indication of weaker electrostatic attraction between the polymer layer and the lipid layer. It is also possible that vesicles of the charged lipid SOPS and the zwitterionic lipid POPC render different sizes, which might influence the process of vesicle fusion. However, the driving force of curvature difference between the nanoscale vesicles and a planar surface is so large that the curvature difference of two nanoscale vesicles may be negligible. Kalb and co-workers reported a similar difference between the adsorption/fusion behavior of pure POPC vesicles and vesicles composed of 80% POPC and 20% anionic lipid on a hydrophobic lipid monolayer.9 The vesicles with different compositions were prepared by the extrusion method, and their sizes were measured to be the same (80-90 nm in diameter). Despite the same sizes, the same kind of difference in the kinetic behavior of zwitterionic vesicles and anionic vesicles was reported.9 The lateral diffusion coefficients of lipid bilayers measured for both SOPS and 75% SOPC/25% POPC mixture in this system are within the range of those of fluid bilayers. It should also be pointed out here that the lateral diffusivity values are well above the values characteristic for lipid membranes in the rigid Lβ phase (partly ordered, untilted, D < 10-11 cm2/s).28 Peptide Insertion. As shown in Figure 7, the injection of the wt-20 peptide solution at pH 5.0 and 7.2 caused an instantaneous increase in reflectivity followed by a slow increase for about 60 min and finally equilibration. The instantaneous increase is caused by the refractive index change of the pure buffer solution (1.332) to the peptide solution with 0.5% v/v DMSO (1.333). Experiments with 0.5% v/v DMSO in the same buffer but without peptide showed the same instantaneous increase in reflectivity (figure not shown). The slow increase in reflection in the presence of the peptide indicates an addition of peptide to the supported bilayer. It should be noted that with SPR or other optical techniques it is not easy to distinguish the exact position or orientation of the peptide molecules, i.e., whether the peptide molecules were inserted into the lipid membrane or were adsorbed on the membrane surface. Electrochemical experiments may be able to distinguish the difference since the insertion of peptide molecules can make the lipid membrane less insulating.19 Although previous reports found that the insertion kinetics of the peptide in “free” giant vesicles measured by area expansion is dependent on pH (keeps increasing at pH 5 while flattens out at a lower level at pH 7),25,26 it should be noted that

(37) Marsh, D. CRC Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990.

(38) Silin, V.; Plant, A. Trends Biotechnol. 1997, 15, 353-359.

Mobile Phospholipid Bilayers

the lipid membrane studied here was supported on a solid substrate. The solid substrate may exert constraint to the expansion of the lipid membrane and set a limit for the maximum amount of peptide insertion. The reduction in reflectivity upon rinsing is mainly due to the refractive index change of the bulk solution from 1.333 (with DMSO) back to 1.332, the refractive index of the pure buffer solution without DMSO. However, the reflectivity goes back to the value before DMSO solution was injected when no peptide is involved, while there is still about 0.03 net increase of reflectivity when peptide is included. This net increase of reflectivity corresponds to a thickness change of about 4 Å at both pH 5.0 and pH 7.2. It should be pointed out here that the same value of refractive index has been used throughout the experiments for bilayers before and after peptide insertion. Therefore, the thickness change mentioned here should be regarded as an increase of materials in the lipid membrane structure. The observation that the presence of the peptide can be monitored at the pH values employed in this study suggests that this lipid bilayer structure can be used for detection of molecules that are retained by the membrane. Conclusions A supported lipid membrane system cushioned by a hydrated polycation/alkylthiol layer pair was investigated by SPR and FRAP. An acidic alkylthiol layer was selfassembled on a gold surface to create the first organic layer with a well-defined negatively charged surface. A thin hydrated PDDA layer was then deposited on the alkylthiol-covered solid substrate. Using the method of

Langmuir, Vol. 16, No. 11, 2000 5099

vesicle fusion, pure anionic lipid SOPS formed a single bilayer through electrostatic interaction with the positively charged polymer layer. Mixtures of SOPS and the zwitterionic lipid POPC (composition < 25%) also adsorbed on the layer pair as a single bilayer. Mobility experiments by FRAP showed that the lateral diffusion coefficient of the single bilayers fell in the range of those for mobile membranes. When the composition of POPC in the mixture exceeded 25%, however, multilayers of lipid molecules built up on the surface. SPR measurements suggest that the membrane fusion peptide wt-20 from influenza virus added to a bilayer formed by a SOPS/POPC mixture under physiological pH values. This demonstrates the potential of this supported membrane system to be used in biosensor research and applications. Acknowledgment. This work was supported by the MRSEC Program of the National Science Foundation (NSF) under Award DMR-9808677. M. L. acknowledges funding from NSF through the CAREER program (BES9733764). The authors give special thanks to Dr. Alan Waring for providing the synthesized wt-20 peptide (Department of Pediatrics, Martin Luther King Jr./Drew University Medical Center and Perinatal Laboratories, Harbor-UCLA). We also thank B. Argo and Dr. S. Schouten for their help with the surface plasmon resonance. Dr. A. McKiernan is thanked for help with fluorescence microscopy and useful discussions. We are also very grateful to Dr. Z. Hou for assistance with gold evaporation on glass slides. LA9913405