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Bivalent-Ion-Mediated Vesicle Adsorption and Controlled Supported Phospholipid Bilayer Formation on Molecular Phosphate and Sulfate Layers on Gold Johan Ekeroth and Peter Konradsson* Department of Chemistry, Linko¨ ping University, SE-581 83 Linko¨ ping, Sweden
Fredrik Ho¨o¨k* Department of Applied Physics, Chalmers University of Technology and Go¨ teborg University, SE-412 96 Go¨ teborg, Sweden Received June 26, 2002 Strategies to form supported lipid assemblies on organophosphate- and organosulfate-monolayer-modified gold surfaces are described. By varying surface treatment and the Mg2+ (Ca2+) content in a solution containing phosphatidylcholine vesicles, we demonstrate (i) efficient formation of supported phosphatidylcholine bilayers (SPBs), (ii) formation of supported nonruptured phosphatidylcholine vesicles, and (iii) reduced phosphatidylcholine vesicle adsorption. Thus, by simply varying the solution conditions, the system can be tuned to controlled formation of either a SPB, supported nonruptured vesicles, or a surface with fairly low coverage of nonruptured vesicles. The profound effects induced on the system by Mg2+ and Ca2+ are assigned to a combination of ion-coordination to the surface, ion-association to the lipid headgroups, and osmotic pressure.
Introduction Supported phospholipid bilayers (SPBs) and nonruptured surface-bound vesicles are growing in importance as model systems for cell membranes,1 as templates for biosensing devices,2-4 and, of course, for the scientific challenge of understanding the physical and chemical properties of these types of assemblies, with1,5 or without6 reconstituted biological functionalities. It has long been well established that spontaneous rupture of vesicles into SPBs occurs efficiently under a wide range of solution conditions on SiO2 and mica.1,7-10 In contrast, vesicles have a tendency to adsorb as nonruptured vesicles on various metals/metal oxides (TiO2, Pt, Au),8,11 whereas lipid monolayers, with the hydrophilic headgroup facing toward the solution, are formed on highly hydrophobic surfaces.8,12 From this it is obvious that an improved understanding of, as well as the * To whom correspondence should be addressed. Konradsson: phone, +46 13 281728; fax, +46 13 281399; e-mail,
[email protected]. Ho¨o¨k: phone, +46 31 7723464; fax, +46 31 7723134; e-mail,
[email protected]. (1) Sackmann, E. Science 1996, 271, 43-48. (2) Vikholm, I.; Albers, W. M. Langmuir 1998, 14, 3865-3872. (3) Tien, H. T.; Barish, R. H.; Gu, L. Q.; Ottova, A. L. Anal. Sci. 1998, 14, 3-18. (4) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. Rev. Phys. Chem. 2000, 51, 41-63. (5) Liebau, M.; Hildebrand, A.; Neubert, R. H. H. Eur. Biophys. J. Biophys. Lett. 2001, 30, 42-52. (6) Mouritsen, O. G. Curr. Opin. Colloid Interface Sci. 1998, 3, 7887. (7) Radler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 45394548. (8) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397-1402. (9) Keller, C. A.; Glasmastar, K.; Zhdanov, V. P.; Kasemo, B. Phys. Rev. Lett. 2000, 84, 5443-5446. (10) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 25542559. (11) Csucs, G.; Ramsden, J. J. Biochim. Biophys. Acta-Biomembr. 1998, 1369, 61-70. (12) Heyse, S.; Stora, T.; Schmid, E.; Lakey, J. H.; Vogel, H. Biochim. Biophys. Acta-Rev. Biomembr. 1998, 1376, 319-338.
possibility to control the outcome of, vesicle-surface interactions set strong requirements on the ability to control and vary the properties of the underlying support. Adsorption of a single molecular layer of organothiols on gold is one approach to obtain surfaces with desired properties.13,14 There are a number of previous examples of the use of this surface-modification strategy for investigations of vesicle adsorption behavior.8,15,16 However, in none of these studies has the surface charge/ polarity been directly tunable for the purpose of controlling the SPB formation process. Furthermore, previous studies of lipid bilayer formation on modified gold surfaces have been based on vesicles with a fraction of lipids carrying a net charge (negative or positive),17-19 in addition to the common neutral lipid phosphatidylcholine, to promote the vesicle-surface interaction and the rupture process. We have recently shown that Ca2+ and Mg2+ significantly lower the polarity of an organophosphate layer on gold.20 In addition to affecting the polarity and charge of phosphates, calcium and magnesium ions are known to strongly promote fusion of vesicles and cells,17,19,21,22 through coordination to the lipid headgroup. Taking this into account, a phosphate-terminated surface in combina(13) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (14) Jung, C.; Dannenberger, O.; Xu, Y.; Buck, M.; Grunze, M. Langmuir 1998, 14, 1103-1107. (15) Stora, T.; Dienes, Z.; Vogel, H.; Duschl, C. Langmuir 2000, 16, 5471-5478. (16) Steinem, C.; Janshoff, A.; Ulrich, W. P.; Sieber, M.; Galla, H. J. Biochim. Biophys. Acta-Biomembr. 1996, 1279, 169-180. (17) Puu, G.; Gustafson, I. Biochim. Biophys. Acta-Biomembr. 1997, 1327, 149-161. (18) Steinem, C.; Janshoff, A.; Galla, H. J.; Sieber, M. Bioelectrochem. Bioenerg. 1997, 42, 213-220. (19) Ross, M.; Steinem, C.; Galla, H. J.; Janshoff, A. Langmuir 2001, 17, 2437-2445. (20) Ekeroth, J.; Konradsson, P.; Bjo¨refors, F.; Lundstro¨m, I.; Liedberg, B. Anal. Chem. 2002, 74, 1979-1985. (21) Matsumura, H.; Verbich, S. V.; Dirnitrova, M. N. Colloid Surf., A 2001, 192, 331-336. (22) Morillo, M.; Sagrista, M. L.; de Madariaga, M. A. Lipids 1998, 33, 607-616.
10.1021/la026131q CCC: $22.00 © 2002 American Chemical Society Published on Web 08/31/2002
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Figure 1. Structure of thiols used for gold surface modification.
tion with various amounts of Mg2+ and/or Ca2+ in solution arises as an interesting candidate system for a detailed investigation of vesicle-adsorption behavior, in general, and the vesicle-to-SPB formation process in particular. In addition to an improved understanding of the SPB formation process, success in such an approach would increase the arsenal of applicable surface analytical tools (for example, electrochemical, optical, and scanning probe techniques). It would also open up new strategies to develop surfaces patterned with SPBs, which have recently been proven valuable for precise control of lateral displacement of either charged lipids and/or biomacromolecules reconstituted in or on the SPBs.23 To analyze the interaction between egg-phosphatidylcholine (egg-PC) vesicles and organophosphate layers as a function of Mg2+ and Ca2+ surface treatment and solution concentration, we have used the QCM-D technique.24 This technique simultaneously measures changes in resonant frequency, f (related to coupled mass), and energy dissipation, D (related to viscous losses in adsorbed films). It is established that adsorbed nonruptured vesicles contribute to both f and D, while a flat SPB contributes to f only; that is, SPBs do not induce significant viscous losses and thus changes in D.9 The QCM-D response can thus be very efficiently used to discriminate between these two states of adsorbed lipid assemblies. In this work we have investigated the influence of Mg2+ (Ca2+) concentration on vesicle adsorption to a phosphate (-OPO3)-modified gold surface and how the bivalent ion concentration influences the transition into complete SPBs or intermediate states. To investigate the role of Mg2+ coordination to the -OPO3-modified surface, these results are further compared with identical measurements on sulfate (-OSO3)- and hydroxyl (-OH)-modified gold surfaces, whose interactions with Mg2+ differ from those of -OPO3. In addition, all these results are compared with data on vesicle-to-bilayer formation on SiO2, where QCM-D8,9 and fluorescence recovery after photobleaching (FRAP)25 have been previously used to monitor and verify the process. Materials and Methods Thiols for Assembly. Structures of the compounds used for the surface modification are depicted in Figure 1. Syntheses of the -OPO3 and -OH derivatives have been described previously.26 The -OSO3 analogue was prepared by reacting the S-acetyl-protected -OH derivative26 with pyridine/SO3, followed by flash column chromatography (EtOAc/MeOH 2:1). The acetyl was cleaved using 0.1 M NaOMe in MeOH, and the product was purified using flash column chromatography (EtOAc/MeOH 4:1 + 1% NEt3). 1H NMR (MeOH-d4) δ 4.04 (t, J ) 5.4 Hz, 2H), 3.45 (t, J ) 5.4 Hz, 2H), 2.74 (t, J ) 6.9 Hz, 2H), 2.52 (t, J ) 6.9 Hz, 2H). 13C NMR (MeOH-d4) δ 173.9, 67.3, 41.0, 40.1, 21.0. Surface Preparation. Polished AT-cut quartz crystals (Maxtek Inc., Santa Fe Springs) with a fundamental frequency of 5 MHz, coated on both sides with a 3 nm adhesion layer of Cr and a 200 nm layer of gold, were used. The crystals were cleaned in an UV/ozone chamber for 2 × 10 min, followed by cleaning in (23) Cremer, P. S.; Yang, T. L. J. Am. Chem. Soc. 1999, 121, 81308131. (24) Rodahl, M.; Hook, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. Rev. Sci. Instrum. 1995, 66, 3924-3930. (25) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307-316. (26) Ekeroth, J.; Borgh, A.; Konradsson, P.; Liedberg, B. Accepted for publication in J. Colloid Interface Sci.
Ekeroth et al. a mixture of water (Milli-Q), ammonia (25% in water, Merck), and hydrogen peroxide (30% in water, Merck), 5:1:1 (v/v), at 70° for 10 min. The crystals were rinsed with water and ethanol (99.5%, Kemetyl, Haninge, Sweden) and immersed into a solution of the thiol (0.5 mM) in ethanol overnight. The crystals were subsequently rinsed with ethanol and subjected to ultrasonication in ethanol for 10 min. Prior to use, the crystals were dried under a stream of nitrogen. The SiO2 surface was prepared by cleaning a gold-coated QCM crystal with brief etching in an oxygen plasma (reactive ion etching) and evaporating a 3 nm layer of Ti as an adhesion layer, followed by 100 nm of SiO2, onto the crystal. XPS analysis shows that this procedure produces a clean SiO2 surface. The SiO2 crystals were cleaned between lipid measurements by placing them in 10 mM SDS for several hours, followed by rinsing with water and drying under N2. Immediately before each use, the surface was cleaned with two cycles of the following procedure: exposure to UV-light and UV-produced ozone in air at atmospheric pressure for 10 min, rinsing with water, and drying under N2. Phospholipid Vesicle Preparation. The buffer used throughout this work was 10 mM Tris, pH ) 8, containing 100 mM NaCl. Small unilamellar vesicles were prepared by extrusion of a 10 mg/mL solution of egg-phosphatidylcholine (egg-PC) (Sigma) in buffer through a 0.03 µm carbonate membrane (Avanti lipids) 11 times. Photocorrelation Spectroscopy Measurements. PCS measurements were performed with a Brookhaven BI-90 particle sizer, equipped with a Lexel 2W Ar laser operated at 50-300 mW. The vesicle size was calculated by the instrument software. Solutions used for measurements were 0.1 mg/mL vesicles in buffer. Addition of 5 mM MgCl2 (CaCl2) was followed by a size determination every 30 min for 2 h. QCM-D Measurements. Prior to each experiment, surfaces were cleaned by treatment twice with a solution of SDS (10 mM) and EDTA (1 M) for 1 min, followed by rinsing with buffer. This produced a Na+-equilibrated and Mg2+ (Ca2+)-depleted surface. For obtaining an Mg2+-equilibrated surface, this procedure was followed by treatment with 10 mM MgCl2 in buffer for 5 min and rinsing with buffer three times. The solution for subjection to the surface was prepared as follows: 20 µL of the solution with extruded vesicles (10 mg/mL lipids) was diluted with buffer to a final volume of 2 mL. In cases of solution Mg2+ (Ca2+), the correct amount of a 1 M solution of MgCl2 (CaCl2) (10 µL w 5 mM) was added to the buffer before adding the vesicles. The concentration of vesicles was 0.1 mg/mL in all experiments. The solution was subjected to the surface within 1 min. Combined frequency and energy dissipation measurements were performed on a system (Q-Sense AB, Go¨teborg, Sweden) described elsewhere.24,27-29 The temperature was kept constant at 22 °C. Since the resonant frequency of the QCM depends on the total oscillating mass, a mass adsorbed on the surface can be detected as a decrease in f. Under ideal conditions, there is a linear relation between the change in frequency and the adsorbed mass:30
C ∆m ) ∆f n
(1)
where C ()17.7 ng cm-2 Hz-1 at f ) 5 MHz) is the mass-sensitivity constant and n ()1, 3, ...) is the overtone number. Combined frequency and energy dissipation measurements, as used in the present work, give essential information about both the adsorbed amount (∆f) and the viscoelastic properties (rigidity) (∆D) of the adsorbed film.31-33 Data were collected using Q-soft (Q-Sense AB, Go¨teborg, Sweden). All data shown are measured on the third harmonic, n ) 3; that is, at 15 MHz. All measurements were repeated at least twice, with the same result. The estimated 95%-confidence intervals for the bilayer formation on -OPO3 were ∆f ) -86 ( 3 Hz and ∆D ) 0.5 ( 0.1 (n ) 5). (27) Hook, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14, 729-734. (28) Rodahl, M.; Kasemo, B. Rev. Sci. Instrum. 1996, 67, 3238-3241. (29) Rodahl, M.; Hook, F.; Kasemo, B. Anal. Chem. 1996, 68, 22192227. (30) Sauerbrey, G. Z. Phys. 1959, 155, 206-222.
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Figure 2. Changes in ∆f and ∆D versus time upon exposure of (A) Na+ equilibrated -OPO3, -OSO3, and -OH and (B) SiO2 surfaces to a vesicle solution (0.1 mg/mL lipids). In part A a buffer containing 5 mM MgCl2 was used, while in part B pure buffer was used during the experiment.
Results We have studied the adsorption behavior of lipid vesicles on fully phosphorylated (-OPO3), hydroxylated (-OH), and sulfated (-OSO3) surfaces (Figure 1) under different Mg2+ treatment and solution conditions and compared these results with spontaneous bilayer formation on SiO2.34 Initially, we emphasize the conditions that appear to lead to spontaneous SPB formation on -OSO3, -OPO3, and SiO2 surfaces but not on -OH. In the following sections, these results will be used as reference and guide the analysis of how different Mg2+ concentrations and Mg2+ surface treatments influence the vesicle adsorption and rupture process on primarily the -OSO3 and the -OPO3 surfaces. Spontaneous SPB Formation. Figure 2A shows changes in f and D versus time upon exposure of NaEDTA-treated, that is Mg2+-depleted, -OPO3, -OSO3, and -OH surfaces to a vesicle solution containing 5 mM Mg2+. Also shown is the QCM-D response upon exposure of the same vesicle solution to SiO2 (Figure 2B), on which a planar bilayer is known to form spontaneously.8,9,35 On all surfaces, f and D initially decrease and increase, respec(31) If the adsorption-induced ∆D is large, the Sauerbrey relation might no longer be valid. In such situations, a Voight-Kelvin-based model,32,33 in which the adsorbed film is represented by a homogeneous thickness, viscosity, and complex shear modulus, should be applied. In the present work the Sauerbrey relation holds for the complete SPBs (low ∆D), while the mass estimated using eq 1 is underestimated by 10-20% for the adsorbed nonruptured vesicles (high ∆D). Since the conclusions drawn from this work are not dependent on very precise quantification of the amount of adsorbed nonruptured vesicles, this type of modeling is, however, not explicitly included. (32) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391-396. (33) Hook, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H. Anal. Chem. 2001, 73, 5796-5804. (34) Measurements presented in this work are based on organophosphate (-OPO3) interaction with magnesium ions; however, calcium ions show similar results.
tively, with very similar rates signaling formation of a water rich viscous film, interpreted as an initial adsorption of nonruptured vesicles.9 However, at a certain surface coverage, the adsorption behavior changes abruptly in qualitatively the same manner on -OPO3, -OSO3 (Figure 2A), and SiO2 (Figure 2B). This is reflected in an increase and a decrease in f and D, respectively, signaling loss of coupled mass and a decrease in viscous losses within the layer. In contrast, this behavior is completely absent on the -OH surface, where a monotonic decrease and increase in f and D, respectively, is observed toward saturated adsorption. The decrease in mass (increase in f) after the observed break point on -OPO3, -OSO3, and SiO2 is interpreted as release of water from the interior of the nonruptured vesicles during rupture into a SPB,9 whereas on the -OH surface (where this behavior is not observed) there is a continuous adsorption of nonruptured vesicles. This interpretation is further supported by the D data, which after the characteristic break point reflect a transition from a system inducing high viscous losses (nonruptured vesicles) to a system inducing low viscous losses (flat bilayer). Furthermore, the qualitative adsorption behavior and the ∆f and ∆D values at saturation for the phosphate, sulfate, and the SiO2 measurements in Figure 2 are similar and in good agreement with previously obtained data for the vesicle-to-SPB formation process on SiO2,8,9 which in the latter case also has been verified through FRAP measurements.36 Except for an overall slower rate of the bilayer formation on the -OPO3 surface and larger magnitudes of the changes in ∆f and ∆D at the (35) We have chosen to present the experiment with no Mg2+; however, Mg2+ was found to slightly lower the barrier for the vesicle f SPB process on SiO2 but did not affect the qualitative appearance of the process or the final result. (36) Due to high quenching effects from the underlying gold surface on dye-labeled lipids, we were not able to conduct FRAP experiments on the phosphate-terminated surface.
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Figure 3. Same type of data and experiment as shown in Figure 1A on Na+ equilibrated -OPO3 (A) and -OSO3 (B) surfaces with varying solution concentrations of MgCl2 (0, 1, 5, and 10 mM).
break point compared with the cases of the SiO2 and the -OSO3 surfaces, the only differences observed are a 5 Hz larger decrease in ∆f and a somewhat higher ∆D value at saturation on the -OPO3. The observed difference can be due to several factors, such as the difference in the properties of the underlying surface, including its influence on water and ion accumulation, or a not fully complete SPB formation on the -OPO3 surface. Regarding the latter, we emphasize, though, that the SPB formation is complete to at least 95%37 and was found to be stable over several hours. It is also interesting to note that, for the -OSO3 surface, the vesicle rupture process starts earlier than that on the -OPO3 surface and induces a response that more resembles that on the SiO2 surface. Taken together, these data thus show that it is not only the influence from the bivalent ions on the vesicles properties that is critical for bilayer formation, but also how the nature of the surface interaction is influenced for different surfaces by the presence of Mg2+. Dependence on Mg2+ Concentration and Mg2+ Surface Treatment. The three surfaces (-OPO3, -OSO3, and SiO2) that promote spontaneous bilayer formation under the conditions presented above have in common that they are negatively charged at pH 8. However, -OPO3 hosts two negative charges, while -OSO3 and SiO2 host one negative charge per functional unit. This, in turn, has a profound influence on the interaction with bivalent ions. While -OPO3 associates bivalent ions such as Mg2+ and Ca2+ irreversibly upon rinsing,26 the association to -OSO3 and SiO2 appears reversible upon rinsing (see further below). In particular, this was shown in previous electrochemical investigations of the particular -OPO3 surface used in the present work, signaling a strong coordination of Mg2+ to the -OPO3, not reversible upon rinsing.20 This, combined with the fact that spontaneous bilayer formation (37) Estimated from the relative response in f.
on SiO2 does not require the presence of bivalent ions, motivated us to extend the set of data presented in Figure 2 to investigate the influence on the vesicle adsorption behavior of bivalent-ion concentration and different pretreatment of the surfaces. In Figure 3 the effect of varying the solution Mg2+ concentration (0, 1, 5, and 10 mM) under otherwise identical measurement conditions to those for the experiment in Figure 2A is shown for the -OPO3 (Figure 3A) and -OSO3 (Figure 3B) surfaces.38 Both increased and decreased Mg2+ concentrations were found to have a significant influence on the outcome of the adsorption process. Note first that, at no Mg2+, there is very low adsorption on both surfaces, whereas, at higher concentrations, the overall qualitative response is relatively similar to those shown in Figure 2, including rapid adsorption of nonruptured vesicles (initial decrease and increase in f and D, respectively) and rupture (increase and decrease in f and D, respectively) at a certain coverage. Concerning the efficiency of SPB formation, complete SPB formation is observed at 5 mM solution Mg2+ while, upon both increased and decreased Mg2+ concentrations, some kinds of intermediate states are formed at saturation. Thus, while bilayer formation occurs efficiently on SiO2, independent of the concentration of bivalent ions, vesicle adsorption on both -OPO3 and -OSO3 surfaces is (i) suppressed without Mg2+, (ii) promoted at 1 mM but does not lead to spontaneous SPB formation, (iii) efficient at 5 mM and results, in this case, in spontaneous SPB formation, and (iv) still efficient at 10 mM.But, apparently, for option iv some kind of structural rearrangement hampers the bilayer formation, as reflected in a continuous decrease and increase in f and D toward saturation. It is also interesting to note that, at 10 mM Mg2+, the efficiency of the bilayer formation is higher on the -OSO3 surface (38) The adsorption behavior on the -OH surface was not significantly influenced by the Mg2+ concentration.
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Figure 4. Same type of data as in Figures 1 and 2, upon exposure of a Mg2+-equilibrated -OPO3 (A) surface and the corresponding nonphosphorylated (-OH) (B) surface to a vesicle solution containing no MgCl2. At 20.4 and 34.0 min for the -OPO3 Mg and -OH surface measurements, respectively, the vesicle exposure is interrupted with (i) an exchange to a pure buffer, followed by addition of (ii) buffer containing Na-EDTA (1 mM), (iii) pure buffer, (iv) buffer containing MgCl2 (5 mM), and (v) pure buffer. Each step is marked with an arrow and the corresponding number (i-v).
than on the -OPO3 surface, which further strengthens the hypothesis that the nature of the association of Mg2+ to the surface and/or the Mg2+-mediated coordination of vesicles to the surface is critical for the outcome of the process. To further analyze the role of Mg2+ for the interaction between vesicles and the phosphate and sulfate surfaces, a set of experiments were performed in which Mg2+ preequilibrated -OPO3 and -OSO3 surfaces were exposed to a vesicle solution with and without Mg2+. For the -OSO3 surface, the adsorption behavior was essentially identical to the results shown in Figure 3B at the same Mg2+ concentration (not shown), signaling that the association of Mg2+ to the -OSO3 surface is reversible upon rinsing. In contrast, for the -OPO3 surface at no Mg2+, there is, instead of very low adsorption (cf. Figure 4A), substantial vesicle adsorption, but no signs of vesicle rupture are observed (not shown). In contrast, in the presence of 5 mM Mg2+, the adsorption behavior is similar to that observed on the Na-EDTA depleted surface at high (10 mM) Mg2+ concentration (cf. Figure 3A); that is instead of spontaneous bilayer formation, the process includes a rearrangement, reflected in a slow decrease and increase in f and D, respectively. It is also worthwhile to point out that these results for the -OPO3 surface were relatively independent of solution Mg2+ concentration between 1 mM and 10 mM, and at all concentrations qualitatively similar to the results presented in Figure 3 at 10 mM Mg2+. Preequilibration of the -OSO3 surface was, however, shown not to significantly influence the adsorption and rupture behavior. Thus, whereas the -OPO3 surface has a clear memory of a past preequilibration with Mg2+, the -OSO3 surface has not, signaling that the nature of the coordination of Mg2+ to at least the -OPO3 surface is critical for the outcome of the process. In summary, for Na-EDTA-equilibrated phosphate and sulfate surfaces, 1 mM Mg2+ is sufficient to promote vesicle adsorption, but not vesicle rupture and SPB formation. At 5 mM, SPB formation is efficient on both surfaces, whereas, at 10 mM, initial vesicle rupture is observed but subsequently hampered, though more on the -OPO3 surface, by slow structural rearrangements of surfaceassociated vesicles. This quite surprising observation is attributed to superimposed effects from the nature by which the surface coordinates Mg2+ and the influence from the bivalent ions on the vesicles, including direct Mg2+ association to the lipid headgroups as well as an increase in osmotic pressure as the ion-concentration difference
between the inside and the outside of the vesicles increases.39 To test whether the kinetics of the latter effects may influence the adsorption behavior, vesicles were equilibrated with Mg2+ for extended times prior to exposure to the Mg2+-depleted -OPO3 surface (not shown). This was found to slightly increase the kinetics of the vesicle adsorption and bilayer formation process, but it did not affect the final result or the critical Mg2+ concentration (∼5 mM) required for efficient SPB formation (not shown). To further investigate the influence from Mg2+ on the vesicle properties and surface interaction, the fate of adsorbed intact vesicles on the Mg2+equilibrated phosphate surface (Figure 4A) was followed by removal of Mg2+ (by Na-EDTA treatment) after complete vesicle adsorption. To ascribe the effect to the -OPO3 surface, the experiment was compared with the same type of treatment for nonruptured vesicles adsorbed on the -OH surface (cf. Figure 2A). Significantly, removal of Mg2+ has a profound effect on D (+8 × 10-6) for the -OPO3 surface, signaling a significant increase in viscous losses, whereas essentially no changes are observed upon the same treatment after completed vesicle adsorption on the -OH surface. The fact that this treatment results in essentially no changes in f and D for vesicles adsorbed on the -OH surface also rules out the possibility that the observed effect from Na-EDTA addition results from osmotic swelling of adsorbed vesicles.40 This, together with the low adsorption obtained when no Mg2+ was present prior to and during adsorption, indicates that Na-EDTA treatment (absence of Mg2+) has a drastic influence on the Mg2+-mediated coupling of vesicles to the -OPO3 surface. Discussion We have demonstrated two chemical-surface modifications whose charge and polarity can be varied to control phospholipid-vesicle adsorption in general and supported phospholipid bilayer formation in particular. This was achieved by varying the charge and polarity of single molecular layers of organophosphates and organosulfates on gold using different bivalent cations. The most im(39) Osmotic pressure changes induced by varying the concentration of NaCl during vesicle adsorption on SiO2 have, indeed, shown that both increased and decreased ion concentrations outside the vesicles facilitate the bilayer formation process (E. Reimhult, personal communication). (40) No change in vesicle size upon Mg2+ addition was observed over several hours, using photocorrelation spectroscopy (PCS).
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Figure 5. Schematic illustration of the two most extreme scenarios observed upon vesicle adsorption on the -OPO3 surface: (a) pretreatment with a solution of 10 mM SDS and 1 mM Na-EDTA efficiently cleaning and eliminating Mg2+ and Ca2+ from the -OPO3 surface; (b) treatment with 10 mM MgCl2 followed by rinsing with buffer three times, leading to efficient coordination of Mg2+ to the surface; (c) addition of vesicles equilibrated with 5 mM Mg2+, leading to SPB formation; (d) addition of vesicles without Mg2+, leading to adsorption of nonbroken vesicles. Magnifications illustrate the proposed coordination of Mg2+ in each case.
portant findings are that introducing a vesicle solution to (i) Na-EDTA equilibrated, that is Mg2+ (Ca2+)-depleted, phosphate and sulfate surfaces produces supported phospholipid bilayers, provided that the vesicle solution contains 5 mM Mg2+ (Ca2+), (ii) Na-EDTA equilibrated, that is Mg2+ (Ca2+)-depleted phosphate and sulfate surfaces, results in weak vesicle adsorption, provided that no Mg2+ (Ca2+) is present in the vesicle solution (Figure 2), and (iii) a Mg2+ (Ca2+) preequilibrated phosphate surface results in substantial adsorption of nonruptured vesicles, independent of whether Mg2+ (Ca2+) is present in the vesicle solution, whereas identical treatment of the sulfate surface results in essentially the same adsorption as that without Mg2+ preequilibration of the surface, that is, weak adsorption without Mg2+ (Ca2+) but substantial adsorption with Mg2+ and even bilayer formation at 5 mM Mg2+. The outcomes of (i) and (iii) for the phosphate surface are schematically depicted in Figure 5. Considering the spontaneous SPB formation on the Na+equilibrated -OPO3 and -OSO3 surfaces (Figure 2A), it is clear that the rupture is not exclusively mediated by the influence from Mg2+ on the vesicle properties or the role of surface-associated Mg2+ as a mediator between the surface and the phospholipids alone. Rather, the role of Mg2+ (Ca2+) seems to be a combination between its action as a mediator between the surface and the vesicles (as suggested from the absence of adsorption without Mg2+) and its influence on the vesicle properties, like lipid headgroup coordination and osmotic pressure (as suggested from the strong concentration dependence of vesicle rupture behavior). Even if a detailed molecular and kinetic description about the role of the bivalent ions on the investigated systems cannot be presented on the basis of these data, the fact that the bilayer formation at 5 mM Mg2+ is hampered on the -OPO3 surface but not on the
-OSO3 surface, if preequilibrated with Mg2+ prior to the addition of vesicles, shows that the outcome of the process depends on whether the surface association of Mg2+ competes with vesicle binding or not. Thus, even if Mg2+induced vesicle destabilization certainly is important, the nature of the Mg2+ coordination to the surface seems critical for the outcome of the process. Furthermore, even if this association is irreversible on the -OPO3 surface, one should keep in mind that, in the presence of Mg2+, changes in the interfacial properties of the -OSO3 surface, and thus the nature of its association with lipid vesicles, are expected. We speculate further that one important contribution to the Mg2+-induced increase in interaction between the -OPO3 as well as the -OSO3 surfaces and the lipid structures might be mediated by Mg2+ association to the outer hydrophilic part of the vesicle. In such a scenario, we propose that Mg2+ coordinates to the phosphodiester in the phosphocholine structure and thereby shields the intramolecular electrostatic interaction (see enlargements in Figure 5): an interpretation that is supported by IR measurements, which reveal a conformational change within the lipid headgroup upon interaction with Ca2+.41 The quarternary amine may then be more available for intermolecular interaction with a negative surface, in agreement with previous results from SPB formation on negatively charged surfaces.11,16,17 The important role of Mg2+ as a mediator for increased adhesion strength is also supported by the slightly more rapid kinetics observed upon prolonged Mg2+ treatment of vesicles prior to surface exposure and the results from removing and adding Mg2+ (Figure 4A). Furthermore, this interpretation is also in agreement with the strong (41) Casal, H. L.; Mantsch, H. H.; Hauser, H. Biochemistry 1987, 26, 4408-4416.
Supported Lipid Assemblies on Modified Au Surfaces
dependence on Mg2+ concentration for the Mg2+-depleted surfaces (Figures 2A and 3). At low concentration, the vesicle adsorption is mediated by Mg2+, but the Mg2+induced (osmotic) destabilization of the vesicles is not sufficient to promote rupture. At increased concentrations, the process passes an optimum (∼5 mM) after which an additional factor that hampers complete SPB formation gains in importance. The fact that this additional effect is more pronounced on the -OPO3 surface than the -OSO3 surface indicates that the interfacial ionic structure at the -OPO3 and -OSO3 surfaces plays a central role. This is also supported by the fact that prolonged exposure of vesicles to Mg2+ does not influence the concentration dependence and the fact that Mg2+ pretreatment (surface Mg2+ saturation) prevents effective SPB formation on -OPO3 but not on -OSO3. Still, the influence from osmotic pressure at elevated bivalent-ion concentration and the associated vesicle destabilization seems important, presumably also at the highest concentration at which some kind of structural rearrangement interferes with the rupture and the autocatalytic bilayer formation process. In conclusion, this investigation has improved the understanding of vesicle-surface interaction, in general, and of the role of Mg2+ (and Ca2+) to promote vesicle adsorption and surface-induced rupture to SPBs, in particular. The developed system also provides a range of applications. For example, it allows for precise control and tuning of SPB formation on a flat metal surface,
Langmuir, Vol. 18, No. 21, 2002 7929
without incorporation of a fraction of lipids designed to increase the strength of the vesicle-surface interaction, as previously presented,17,19 but mediated by the phospholipids (phosphatidylcholine) themselves. Our system thus opens up the possibility of introducing both electrical and optical analytical techniques for analysis of supported natural phospholipid membranes, where impedance spectroscopy and surface plasmon resonance are the two most obvious ones not being straightforward to apply on SiO2 or mica. In addition, since the thiol chemistry is gold specific, patterns with alternating gold and other materials will allow construction of various patterns of lipid structures, where the conductive properties of gold will open up the possibility of various combined optical and electrochemical sensing applications. These and similar extensions of this work are at present under investigation in our laboratories. Acknowledgment. We would like to thank Erik Reimhult for fruitful discussions. This work was financed by the Swedish Research Council for Engineering Sciences (TFR), the Swedish National Science Research Council (NFR), and the Biomimetic Material Science program funded by the Swedish Foundation for Strategic Research (SSF). J.E. would like to thank the interdisciplinary graduate school Forum Scientum, financed by SSF. LA026131Q
