Anchor-Lipid Monolayers at the Air−Water Interface; Prearranging of

Jun 9, 2007 - Köper, I.; Schiller, S. M.; Giess, F.; Naumann, R.; Knoll, W. Chapter 2 Functional Tethered Bimolecular Lipid Membranes (tBLMs). In Adv...
1 downloads 0 Views 141KB Size
Download PDF
7672

Langmuir 2007, 23, 7672-7678

Anchor-Lipid Monolayers at the Air-Water Interface; Prearranging of Model Membrane Systems Petia P. Atanasova, Vladimir Atanasov, and Ingo Ko¨per* Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany ReceiVed February 1, 2007. In Final Form: April 27, 2007 Model membrane systems are gaining more and more interest both for basic studies of membrane-related processes as well as for biotechnological applications. Several different model systems have been reported among which the tethered bilayer lipid membranes (tBLMs) form a very attractive and powerful architecture. In all the proposed architectures, a control of the lateral organization of the structures at a molecular level is of great importance for an optimized preparation. For tBLMs, a homogeneous and not too dense monolayer is required to allow for the functional incorporation of complex membrane proteins. We present here an alternative approach to the commonly used selfassembly preparation. Lipids are spread on the air-water interface of a Langmuir film balance and form a monomolecular film. This allows for a better control of the lateral pressure and distribution for subsequent transfer to solid substrates. In this paper, we describe the properties of the surface monolayer, in terms of surface pressure, structure of the lipid molecule, content of lipid mixtures, temperature, and relaxations features. It is shown that a complete mixing of anchor-lipids and free lipids can be achieved. Furthermore, an increase of the spacer lengths and a decrease of the temperature lead to more compact films. This approach is a first step toward the fully controlled assembly of a model membrane system.

Introduction Biological membranes are highly complex architectures, which ensure the proper function of any cell. They ensure controlled transport in and out of the cell through well-defined barrier properties. Several model systems have been proposed in order to establish a simplified platform for systematic investigations of the related processes and for possible applications.1-3 Among these systems, solid supported membranes and especially tethered bilayer lipid membranes (tBLMs) gained growing interest during the last years.4-11 In principle, these architectures consist of a lipid bilayer, the proximal leaflet of which is anchored via a spacer unit to a solid support.12 In a typical configuration, this support can be used as an electrode, and electrical characterization * Corresponding author. E-mail: [email protected] (1) Bell, G. M.; Combs, L. L.; Dunne, L. J. Theory of cooperative phenomena in lipid systems. Chem. ReV. 1981, 81, 15-48. (2) Ottova, A.; Tvarozek, V.; Racek, J.; Sabo, J.; Ziegler, W.; Hianik, T.; Tien, H. Self-assembled BLMs: Biomembrane models and biosensor applications. Supramol. Sci. 1997, 4, 101-112. (3) Winterhalter, M. Black lipid membranes. Curr. Opin. Colloid Interface Sci. 2000, 5, 250-255. (4) Naumann, R.; Schmidt, E. K.; Jonczyk, A.; Fendler, K.; Kadenbach, B.; Liebermann, T.; Offenha¨usser, A.; Knoll, W. The peptide-tethered lipid membrane as a biomimetic system to incorporate cytochrome c oxidase in a functionally active form. Biosens. Bioelectron. 1999, 14, 651-662. (5) Stora, T.; Lakey, J. H.; Vogel, H. Ion-channel gating in transmembrane receptor proteins: Functional activity in tethered lipid membranes. Angew. Chem. Int. Ed. 1999, 38 (3), 389-392. (6) Knoll, W.; Frank, C. W.; Heibel, C.; Naumann, R.; Offenha¨usser, A.; Ru¨he, J.; Schmidt, E. K.; Shen, W. W.; Sinner, A.; Functional tethered lipid bilayers. ReV. Mol. Biotechnol. 2000, 74, 137-158. (7) Sinner, E. K.; Knoll, W. Functional tethered membranes. Curr. Opin. Chem. Biol. 2001, 5, 705-711. (8) Naumann, R.; Baumgart, T.; Gra¨ber, P.; Jonczyk, A.; Offenha¨usser, A.; Knoll, W. Proton transport through a peptide-tethered bilayer lipid membrane by the H+-ATP synthase from chloroplasts measured by impedance spectroscopy. Biosens. Bioelectron. 2002, 17, 25-34. (9) Terrettaz, S.; Mayer, M.; Vogel, H. Highly electrically insulating tethered lipid bilayers for probing the function of ion channel proteins. Langmuir 2003, 19, 5567-5569. (10) Sackmann, E. Supported membranes: Scientific and practical applications. Science 1996, 271, 43-48. (11) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. A biosensor that uses ion-channel switches. Nature 1997, 387, 580-583.

of the system is possible.13 tBLMs have been shown to form highly insulating barriers that allow for the functional incorporation of small membrane proteins or peptides. This opens the possibility of systematic investigations of membrane-related processes or can even lead to practical biosensing applications.11,14-16 However, the dense inner layer of a tBLM can form a barrier that hinders the incorporation of complex membrane proteins in a functional form.13,17 This is, at least partially, due to the restricted mobility of the anchored lipids. Deverall et al. have recently shown that a high tether density, however, in a slightly different architecture, leads to a reduction of the lipid mobility.18 In biological membranes, lipids are densely packed and form a homogeneous but very dynamic system. The problem of the reduced diffusion in highly tethered systems can be overcome by dilution of the inner leaflet with small, surfaceactive molecules, which can intercalate between the anchorlipids.17,19 This approach might yet lead to a phase-separation of the anchor-lipids from the lateral spacer, which would lower the reproducibility and the quality of the membrane, especially (12) Ko¨per, I.; Schiller, S. M.; Giess, F.; Naumann, R.; Knoll, W. Chapter 2 Functional Tethered Bimolecular Lipid Membranes (tBLMs). In AdVances in Planar Lipid Bilayers and Liposomes; Academic Press: New York, 2006; Vol. 3, pp 37-53. (13) Terretaz, S.; Vogel, H. Investigating the function of ion channels in tethered lipid membranes by impedance spectroscopy. MRS Bull. 2005, 30, 207-210. (14) Atanasov, V.; Atanasova, P.; Vockenroth, I.; Knorr, N.; Ko¨per, I. Highly insulating tethered bilayer membranes. A generic approach for various substrates. Bioconjugate Chem. 2006, 17, 631-637. (15) Atanasov, V.; Knorr, N.; Duran, R. S.; Ingebrandt, S.; Offenha¨user, A.; Knoll, W.; Ko¨per, I. Membrane on a chip. A functional tethered lipid bilayer membrane on silicon oxide surfaces. Biophys. J. 2005, 89, (3), 1780-1788. (16) Naumann, R.; Schiller, S. M.; Giess, F.; Grohe, B.; Hartman, K. B.; Ka¨rcher, I.; Ko¨per, I.; Lu¨bben, J.; Vasilev, K.; Knoll, W. Tethered lipid bilayers on ultraflat gold surfaces. Langmuir 2003, 19, 5435-5443. (17) Valincius, G.; McGillivray, D. J.; Febo-Ayala, W.; Vanderah, D. J.; Kasianowicz, J. J.; Losche, M. Enzyme activity to augment the characterization of tethered bilayer membranes. J. Phys. Chem. B 2006, 110, (21), 10213-10216. (18) Deverall, M. A.; Gindl, E.; Sinner, E.-K.; Besir, H.; Ru¨he, J.; Saxton, M. J.; Naumann, C. A. Membrane lateral mobility obstructed by polymer-tethered lipis studied at the single molecule level. Biophys. J. 2005, 88, 1875-1886. (19) He, L. H.; Robertson, J. W. F.; Li, J.; Ka¨rcher, I.; Schiller, S. M.; Knoll, W.; Naumann, R. Tethered bilayer lipid membranes based on monolayers of thiolipids mixed with a complementary dilution molecule. 1. Incorporation of channel peptides. Langmuir 2005, 21 (25), 11666-11672.

10.1021/la7002854 CCC: $37.00 © 2007 American Chemical Society Published on Web 06/09/2007

Prearranging of Model Membrane Systems

Langmuir, Vol. 23, No. 14, 2007 7673

Figure 1. Chemical structures of the investigated lipids. The anchor-lipids DPhyTT, DPhyHT, and DPhyOT as well as DPhyG were synthesized as described previously.14 The lipids were mixed with DPhyPE as “free” lipid.

in terms of its electrical sealing properties. Furthermore, the molecular assembly of the system in a self-assembly process is rather difficult to control. As an alternative way to achieve a better control of the lipid distribution, we chose to preorganize a lipid layer at the airwater interface of a Langmuir film-balance. The dilution of the anchor-lipids is achieved by mixing with free (nonanchored) phospholipids. This allows controlling the distribution and the concentration of the anchor-lipids. Subsequent transfer of the film onto a solid substrate can then lead to the formation of a tBLM. In this paper, we will concentrate on the characterization of the monomolecular film at the air-water interface. The characteristic of the surface films of lipids and also of lipids containing long hydrophilic spacers have been studied extensively.20-22 However, in most of the cases, the studies have been restricted to the description of the film properties in terms of compressibility and phase behavior. Our approach is motivated by the fact that the films will later on be used as monolayers in tethered bilayer membranes, which have already shown practical application in the biosensing field.10,11 The characterization of pure and of diluted surface films will have a direct impact on the formation of solid supported membranes, when the film is transferred to a substrate. The anchor-lipids used in this study consist of a lipophilic (hydrophobic) and a lipophobic (hydrophilic) part and thus they act as surfactants on the water surface. Their use as pure layers in tBLMs has been shown previously.12,14-16,23 The lipid head group consists of a short oligo(ethylene glycol), which on one side is terminated by a thiol functionality. The other end is coupled to hydrophobic alkyl chains. Films have been prepared using mixtures of these anchor lipids with free lipids (DPhyPE). Therefore, we can control the concentration of the individual components in the film and thus later on control the inner membrane architecture of a tBLM. (20) Minamikawa, H.; Hato, M. Phase behavior of synthetic phytanyl-chained glycolipid/water systems. Langmuir 1997, 13 (9), 2564-2571. (21) Mitchell, D. J.; Tiddy, G. J. T.; Waring, L.; Bostock, T.; McDonald, M. P. Phase-behavior of polyoxyethylene surfactants with water-mesophase structures and partial miscibility (cloud points). J. Chem. Soc. Faraday Trans. 1 1983, 79, 975-1000. (22) Smith, R. D.; Berg, J. C. The collapse of surfactant monolayers at the air-water interface. J. Colloid Interface Sci. 1980, 74 (1), 273-286. (23) Schiller, S. M.; Naumann, R.; Lovejoy, K.; Kunz, H.; Knoll, W. Archaea analogue thiolipids for tethered bilayer lipid membranes on ultrasmooth gold surfaces. Angew. Chem., Int. Ed. 2003, 42 (2), 208-211.

Experimental Part Lipids. The structures of the investigated lipids are shown in Figure 1. The anchor-lipids (DPhyTT, DPhyHT, and DPhyOT) and diphytanyl glycerol (DPhyG) were synthesized as described previously.14,24 1,2-Diphytanoyl-sn-glycero-3-phosphatidylethanolamine (DPhyPE) was obtained from Avanti Polar Lipids and used without further purification. Lipid mixtures were prepared by dissolving the respective compounds in chloroform (2 mg/mL), followed by 10 min sonication prior to use. Langmuir-Blodgett Setup and Procedure. The Langmuir film balance technique is a well-established method to study films at the air-water interface.25,26 When amphiphiles spread on the water surface, they can organize in a monomolecular film. The hydrophilic parts of the molecules enter the water subphase, while the hydrophobic parts point toward the air. Through movable barriers, the area available for the film can be varied. The surface pressure can be measured with a Wilhelmy balance. By compression of the film while the surface pressure is monitored, pressure-area isotherms (Π-A) can be recorded. The latter describe the phase-behavior of these films. The study of the lateral organization of the amphiphiles can indicate eventual phase separation, interaction forces, and surface properties of the films. The film balance was carefully calibrated and each experiment was performed at least three times. Usually, the pressure and area values for different experiments show a very good agreement. The anchor-lipids were spread on the water surface of a Langmuir film balance. This method can be used to study the assembly of amphiphilic molecules at the air-water interface. It ensures a homogeneous deposition of the organic molecules onto the water surface, and by using movable barriers, the surface films can be compressed, while the surface pressure (Π) is recorded as a function of the mean molecular area (A). The resulting isotherms can provide useful information, for example, about the collapse pressure (Π*) and the corresponding mean molecular area (A*), where the film is compressed to a maximum and starts to collapse, i.e., molecules enter the subphase and three-dimensional aggregates are formed. Furthermore, the behavior and the interactions of the amphiphilic molecules at the air-water interface can be investigated. A KSV 5000 (KSV, Helsinki) instrument equipped with a Wilhelmy film balance was used. The subphase consisted of ultrapure water (Millipore Milli-Q system, R > 18 mΩ cm). If not otherwise (24) In contrast to previously publications, we will use the abbreviation “Phy” for phytanyl throughout the paper. (25) Petty, M. C. Langmuir-Blodgett Films: An Introduction; Cambridge University Press: New York, 1996. (26) Auch, M.; Fischer, B.; Mo¨hwald, H. Lateral lipid diffusion in phospholipid monolayers coupled to polyelectrolyte films. Colloids Surf. A 2000, 164, 39-45.

7674 Langmuir, Vol. 23, No. 14, 2007

Figure 2. The field-desorption mass spectra (FD-MS) of DPhyTT, DPhyHT, and DPhyOT show the presence of molecule dimers, formed by an S-S formation. stated, experiments were carried out at 20 ( 1 °C. The operational area of the trough was 81 000 mm2. The speed of the barriers during the compression was held constant at 10 mm/min. Monolayers were obtained by spreading 25 µL of the corresponding lipid solution on the water surface. The organic solvent was allowed to evaporate for 20 min before compression of the film.

Results and Discussion The anchor-lipids DPhyTT, DPhyHT, and DPhyOT were synthesized according to a procedure developed initially for DPhyTT.14 The structures are shown in Figure 1. The hydrophobic part of these amphiphiles consists of two isoprenoidic phytanyl chains. Phytanyl-based lipids form the main part of the archaea membranes. Archaea are known to withstand hot and acidic conditions.27 In model systems, phytanyl-based lipids have been shown to form extremely stable and impermeable and insulating membranes.23 As can be seen in Figure 1, the phytanyl chains are connected to a functionalized spacer part via a glycerol moiety. Ethylene glycols as spacers were chosen for their hydrophilicity and biocompatibility;28 tetra-, hexa-, and octaethylene glycols are commercially available. The use of even longer ethylene glycols requires synthetic efforts and seems to not form very insulating membranes.29 Due to the synthetic pathway via a precursor molecule, a thiol anchor can be easily added.14 Moreover, the alternatively used lipoic anchor has shown some instability due to oxidation. A similar reaction is observed when a thiol is used as an anchor.30,31 However, the dithio-linked lipid dimers are still soluble and surface active and can form dense monolayer when reacting with a solid support, e.g., mercury or gold.14 The thiol coupling is a rather fast reaction and thus only dimers can be seen in the field desorption mass spectrogram, as shown in Figure 2. In order to prove that the dimerization is due to the thiol coupling, reduction with tris(2-carboxyethyl)phosphine hydrochloride led to monomer signals in the FD-mass spectrogram (data not shown). Pure Lipid Films. In order to investigate the influence of the spacer length of the anchor-lipid, the Π-A isotherms of DPhyTT, (27) Woese, C. R.; Fox, G. E. Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc. Natl. Acad. Sci. U.S.A. 1977, 74 (11), 5088-5090. (28) Harris, J. M. Poly(ethyleneglycol) chemistry: Biotechnical and biomedical Applications; Plenum Press: New York, 1992. (29) Breffa, C. New synthetic strategies to tethered bilayer lipid membranes. Johannes Gutenberg Universita¨t, Mainz, 2006. (30) Wagner, A. F.; Walton, E.; Boxer, G. E.; Pruss, M. P.; Holly, F. W.; Folkers, K. Properties and Derivatives of Alpha-Lipoic Acid. J. Am. Chem. Soc. 1956, 78 (19), 5079-5081. (31) Padeken, H.-G.; Klamann, D. Methoden der Organischen Chemie; Georg Thieme Verlag Stuttgart: New York, 1985; Vol. E11, pp 38-62.

AtanasoVa et al.

DPhyHT, and DPhyOT were recorded. The experimental data is depicted in Figure 3. All isotherms show a continuous transition from the gaseous phase to a condensed phase. No explicit phase transitions, e.g., no plateau region, can be seen. However, the anchor-lipids undergo a phase transition, and kinks in the isotherms at 31, 37, and 42 mN/m respectively are observed. These kinks correspond to the collapse of the monolayer and the formation of three-dimensional structures in the water subphase. Similar and relatively low collapse pressures have already been reported for similar phytanyl-based (archaeal-type) lipid monolayers.32 Kitano et al. have compared the collapse pressures of 32-37 mN/m for archaea lipids with values of 54-56 mN/m for conventional lipids with straight, saturated hydrophobic chains. It has been proposed that the decrease in collapse pressure of archaeal phytanyl-based lipids is due to the exceptionally high conformational disorder of the branched chains.33 Nevertheless, membranes formed of archaeal lipids have been shown to provide a high bilayer stability and low ion leakage.34,35 Additionally, a very smooth isothermal line was observed after the collapse kink. This is a characteristic feature for similar phytanyl-based lipids.32 It has been attributed to the formation of amorphous monolayers, which are softer than straight-chain lipid monolayers. The latter tend to form stiff crystalline phases, which usually show rough isothermal shapes after the collapse. Furthermore, the reversibility of the collapse process has been proven for a similar molecule by recording a hysteresis with the upper limit above the collapse pressure.36 Under such conditions, the hysteresis should register significant material loss. However, such a loss has not been observed. Thus, it has been proposed that, during the decompression of the already collapsed film, the lipid molecules in the water phase efficiently recovered to the released surface area. In Figure 3, the isotherm of DPhyG, which possesses two phytanyl chains and glycerol as hydrophilic unit, is compared to the isotherms of DPhyTT, DPhyHT, and DPhyOT. One can see that the isotherms of the anchor-lipids are shifted to higher areas and are broader. Compared to DPhyG, the larger hydrophilic parts (tetra-, hexa-, and octaethylene glycol) cause interactions between the anchor-lipids at a much higher area per molecule. However, DPhyPE shows an opposite effect. Even if DPhyPE is a larger molecule than DPhyG, the isotherm is narrower and shifted to smaller areas. This effect is due to the higher hydrophilicity of the phosphoethanolamine group in DPhyPE compared to the hydroxyl group of DPhyG.33 Thus, DPhyPE is immersed more deeply into the water subphase than DPhyG and oriented more perpendicular to the water surface. This reduces the average area per molecule and allows for compression to much higher surface pressures, as shown in Figure 3. The experimental area per molecule for DPhyPE collapse is 72 Å,2 which is in a good agreement with literature values.37 Similarly, the increase of the collapse surface pressure of the anchor-lipids can be explained by the increase in length of the ethylene oxide (EO) chains that stabilize the monolayer. Hence, (32) Kitano, T.; Onoue, T.; Yamauchi, K. Archaeal lipids forming a low energysurface on air-water interface. Chem. Phys. Lipids 2003, 126 (2), 225-232. (33) Gauger, D. R.; Binder, H.; Vogel, A.; Selle, C.; Pohle, W. Comparative FTIR-spectroscopic studies of the hydration of diphytanoylphosphatidylcholine and -ethanolamine. J. Mol. Struct. 2002, 614 (1-3), 211-220. (34) Lindsey, H.; Petersen, N. O.; Chan, S. I. Physicochemical Characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems. Biochim. Biophys. Acta 1979, 555 (1), 147-167. (35) Braach-Maksvytis, V.; Raguse, B. Highly impermeable “soft” selfassembled monolayers. J. Am. Chem. Soc. 2000, 122, 9544-9545. (36) Kunze, J.; Leitch, J.; Schwan, A. L.; Faragher, R. J.; Naumann, R.; Schiller, S.; Knoll, W.; Dutcher, J. R.; Lipkowski, J. New method to measure packing densities of self-assembled thiolipid monolayers. Langmuir 2006, 22 (12), 55095519.

Prearranging of Model Membrane Systems

Langmuir, Vol. 23, No. 14, 2007 7675

Figure 3. Pressure-area isotherms for different pure lipids at 20 °C. DPhyPE shows the steepest isotherm, the highest collapse pressure Π*, and the lowest corresponding area per molecule. The three anchor-lipids show smooth curves; the collapse pressure increases with increasing spacer length.

Figure 4. Pressure-area isotherms for different DPhyTT/DPhyPE mixtures, measured at 20 °C. The legend displays the percentage of the anchor-lipid (DPhyTT) in the mixture. With increasing amount of DPhyPE, the curves shift to lower mean areas, the collapse pressure increases, and a second kink at higher surface pressure can bee seen.

increasing the number of EO units in the spacer of the anchorlipid allows for the formation of a denser monolayer. However, even if the area per molecule at the collapse point of the lipid monolayer decreased from ∼200 Å2 for DPhyTT to ∼180 Å2 for DPhyOT, the monolayer is still very diluted compared to 51 Å2 measured using reductive desorption for a monolayer of similar lipid prepared by a self-assembly procedure.16 However, one has to take into account that the reported values for area per molecule in Figure 3 were obtained for the lipid-dimers. Once those dimers are transferred to a gold substrate, they become monomers that bind to the gold. Therefore, the actual area per monomeric molecule is ∼100 Å2 for DPhyTT and ∼90 Å2 for DPhyOT. However, the self-assembly technique still seems to lead to even

higher packing densities, a fact that also has been reported by Kunze et al.36 Mixed Lipids Layer. Dense layers, however, might hinder the functional incorporation of complex membrane proteins. One approach might be the dilution of the anchor-lipids with free, nonbound lipids. This approach would also be a further step toward more complex membrane architectures that resemble more the natural analogues, which are composed of various lipids and other compounds. Isotherms of mixed anchor-lipid and a free lipid (DPhyTT/DPhyPE) for various mixing ratios are shown in Figure 4. In contrast to the isotherms of the pure lipids, the mixtures show two kinks instead of one, as has been reported for similar systems.38 With an increasing amount of the anchor-

(37) Shinoda, K.; Shinoda, W.; Baba, T.; Mikami, M. Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers. J. Chem. Phys. 2004, 121 (19), 9648-9654.

(38) Kim, K.; Shin, K.; Kim, H.; Kim, C.; Byun, Y. In situ photopolymerization of a polymerizable poly(ethylene glycol)-covered phospholipid monolayer on a methaeryloyl-terminated substrate. Langmuir 2004, 20 (13), 5396-5402.

7676 Langmuir, Vol. 23, No. 14, 2007

AtanasoVa et al.

Figure 5. In order to investigate the mixing behavior of DPhyTT and DPhyPE, the mean molecular area is plotted for different surface pressures as a function of the molar fraction of DPhyTT in the mixture. The linear relationship indicates a complete miscibility over the whole experimental parameter range.

Figure 6. The excess free mixing energy (∆Gex) is shown for different surface pressures as a function of the molar fraction of DPhyTT. The low values indicate a complete miscibility of the two lipids. Solid lines are guides to the eyes.

lipid in the mixture, the Π-A isotherms shift to higher mean molecular areas and a nearly constant collapse pressure can be observed. The kink at low surface pressure (30-35 mN/m) correlates very well with the collapse pressure of pure DPhyTT and the kinks at higher surface pressure (45-47 mN/m) match exactly the collapse pressure of pure DPhyPE. We propose that once the mixed monolayer reaches the collapse pressure of DPhyTT, the anchor-lipids collapse and are transferred to the subphase until a pure DPhyPE film is formed. For increasing amounts of DPhyTT in the mixed layer, the kink at lower surface pressure becomes more pronounced and shifts to higher area per molecule. Similar isotherms were recorded for mixtures of DPhyHT/DPhyPE and DPhyOT/DPhyPE (data not shown). In these cases, both the kinks were more difficult to distinguish due to the proximity of the collapse pressure of DPhyHT and DPhyOT to that of DPhyPE.

Isotherms with double-kink shape have already been reported for mixtures of lipids with and without PEG chains attatched.38 However, the kinks have been observed at significantly lower surface pressure (∼10 mN/m) and have been attributed to conformational changes of the much longer PEG chains underneath the water surface. Such a low surface pressure kink was observed neither for pure anchor-lipids (Figure 3) nor for mixed lipids (Figure 4). The only kink at 30-35 mN/m corresponds to the collapse pressure of DPhyTT. Similarly, there was also no kink visible in the isotherms of DPhyHT and DPhyOT that would correspond to conformational changes of the PEG unit. We propose that this absence is due to the much shorter PEG chains (four, six, and eight ethylene glycol units for DPhyTT, DPhyHT, and DphyOT, respectively). Conformational changes do not dominate the interactions of the hydrophobic (phytanyl) chains.

Prearranging of Model Membrane Systems

Langmuir, Vol. 23, No. 14, 2007 7677

Figure 7. Hysteresis experiments performed on a pure DPhyTT film at 20 °C. The film was compressed and released at a compression rate of 10 mm/min. The inset shows the high-pressure range. The step width between the compression cycles is almost constant and might be due either to a reorganization of the film or due to partial loss of material to the subphase.

Figure 8. Isobaric creep experiment. A pure DPhyTT film has been compressed to 31 mN/m and the decrease in the area per molecule has been recorded over time.

When proposing lipid mixtures as membrane systems, one is interested in a homogeneous distribution of the individual components, i.e., a complete miscibility. To create a stable and versatile tBLM, we aim for a uniform distribution of the anchorlipids throughout the whole membrane. The DPhyTT/DPhyPE mixed films were analyzed according to an additive rule.25 Basically, ideal mixtures of two components should fulfill an additive rule, where the molecular area of the mixture (Amixture) for a given pressure is equal to the sum of the area of both components with respect to their molar fractions. In Figure 5, the mean area per molecule recorded at different pressures is plotted as a function of the composition. A linear dependence over the whole concentration and pressure range indicates either ideal mixing or complete segregation of DPhyTT and DPhyPE. In order to distinguish between the two extreme cases, a thermodynamic analysis of the DPhyTT/DPhyPE isotherms was performed. As can be seen from Figure 6, the ∆Gex values were determined by integration of the DPhyTT/

DPhyPE isotherms from Π ) 0 mN/m to a certain surface pressure.25 The relatively low values for ∆Gex indicate a good miscibility. The tendency for positive deviations from ideal mixing with increasing the surface pressure might induce the lateral phase separation at high surface pressures. Solely, the monolayer composed of 25% DPhyTT has negative ∆Gex values, indicating attractive interactions between the different types of molecules at all surface pressures. Thus, relatively low, positive ∆Gex values and fluctuation of the function ∆Gex(xDPhyTT) around zero proposed a good miscibility of the mixed lipids. The homogeneous mixing of the lipids is also a result of the lack of nonspecific intermolecular interactions. This statement is supported by the results obtained by recording the hysteresis of DphyTT, as depicted in Figure 7. The shape of the hysteresis does not show any irregularity during the compression-expansion cycles. A decrease of ∼2 Å in molecular area upon the hysteresis cycle can be observed. This may be due to partial destruction

7678 Langmuir, Vol. 23, No. 14, 2007

AtanasoVa et al.

Figure 9. Pressure-area isotherms for a pure DPhyTT film at different temperatures. The collapse pressure increases with decreasing temperature.

of the monolayer (dissolution and collapse of the monolayer) and relaxation of the film. Additionally, isobaric creep experiments were performed at a pressure of 31 mN/m, as shown in Figure 8. The isobaric curve is relatively steep during the first 1.5-2 h (1.5% at 2 h). Later on it follows a slow and constant decrease of A/A0 with time. Similar behavior has been observed for lipids as well as for surfactant molecules.22,39 The effect was called “initial area loss” and was attributed to a structural rearrangement in terms of relieving surface inhomogenities. They appear as a result of pushing together of condensed film islands as described by the nucleation theory for growth in supersaturated monolayers.40 It has been found that the magnitude of the “initial area loss” depends directly on the compression rate. Thus, the effect is considerable at a high rate of compression and is insignificant at lower one. This fact has to be taken into account when the surface films are transferred to solid substrate as a basis for a tBLM. Preferentially one would allow the film to relay completely before a transfer. In Figure 9, the temperature dependence of the DPhyTT isotherm is shown. Measurements were conducted at 4, 20, and 40 °C for a given DPhyTT concentration (25 µL, 2 mg/mL). As reported for similar lipids,20,32 higher temperatures lead to a decrease of the collapse pressure and an increase of the corresponding area per molecule. At higher temperatures, Brownian motions increase and the hydration of the EO chain decreases,21 leading to collapse of the monolayer at lower surface pressure. At lower temperatures, more densely packed monolayers can be prepared. A similar but smaller effect could be seen for DPhyOT (data not shown). (39) Feria, J. d. l. F.; Patino, J. M. R. Destabilization of monoglyceride monolayers at the air-aqueous subphase interface. 1. Kinetics. Langmuir 1994, 10, 2317-2324. (40) Vollhardt, D. Nucleation and growth on supersaturated monolayers. AdV. Colloid Interface Sci. 1993, 47, 1-23.

Conclusions The synthesis of three new thiolated anchor-lipids and their characterization at the air-water interface has been reported. Monomolecular films on a Langmuir film balance can be used as a model system for membrane architectures. The results give useful information for the preparation of tBLMs, when the monolayer is transferred to a solid substrate. In this study, we investigated the packing and mixing behavior at the air-water interface. Mixing of anchor-lipid with free lipids might result in a decreased packing density, which might allow for the incorporation of complex membrane proteins. The results indicated that an increase in the hydrophilicity (PEG chain length) of the anchor-lipids leads to a higher packing density. A decrease in the temperature results in a similar trend. However, self-assembly seems to form even more dense layers.16,36 Furthermore, mixing the anchor-lipids with free lipids can homogeneously dilute the anchor lipid monolayers. Thus, full control over the lateral arrangement of the lipid monolayers is possible and should allow for a controlled transfer to solid substrates, leading to an adjustable platform for solid supported membranes. The presented approach can also have an impact on more fundamental studies of various membrane parameters. For example, Garg et al. have studied domain formation in and coupling of the two leaflets of a lipid bilayer in a similar system.41 Our studies show how the composition of a tethered membrane can be controlled and designed architectures can be created. LA7002854 (41) Garg, S.; Ruhe, J.; Ludtke, K.; Jordan, R.; Naumann, C. A.; Domain, registration in raft-mimicking lipid mixtures studied using polymer-tethered lipid bilayers. Biophys. J. 2007, 92 (4), 1263-1270.