Langmuir 1998, 14, 7213-7216
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Ultrathin Polymer Films on Gold Supports: LB-Transfer versus Self Assembly Marcus Hausch,† Dierk Beyer,‡ Wolfgang Knoll,§ and Rudolf Zentel* BUGH Wuppertal, Fachbereich Chemie und Institut f. Materialwissenschaften, Gauss Strasse 20, D-42097 Wuppertal, Germany, Clariant Germany GmbH, Division CP, F+E PM I, G 832, D-65926 Frankfurt am Main, Germany, and Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany Received May 5, 1998. In Final Form: September 7, 1998 Lipopolymers comprised of an N-isopropyl acrylamide backbone modified with lipid side chains and a disulfide moiety can be used for the preparation of thin films by chemisorption from solution and by LB-transfer as well. The resulting thin polymer layers were characterized using surface plasmon resonance, contact angle measurements, and grazing incidence FTIR spectroscopy. With both methods thin polymer films of a comparable thickness can be prepared (about 20 Å). But while chemisorption is the easier technique, LB-transfer has the advantage that a transfer at different surface areas allows an additional variation of the film thickness and the contact angle.
Introduction The preparation of ultrathin amphiphilic polymer films on solid supports provides one possibility to stabilize lipid bilayers on solid substrates1,2 and allows the utilization of a large number of analytical methods, which were originally developed for surface analysis. Such combined systems may ultimately lead to a novel generation of biosensors3 which use artificial membranes with natural membrane proteins as analytical devices. Particularly intriguing examples for such systems combining biological principles and polymer materials are the application of self-assembled monolayers in ELISA-type applications4 and the concept of investigating the signal transduction of neuronal cells on a silicon-based field effect transistor (FET).5 Water-swollen amphiphilic polymer films, as intermediate layers, are interesting in this context to decouple the lipid bilayer from the inorganic substrate (especially the proximal lipid layer).6 This contact can lead to differences in the lateral diffusion of the lipids in contact with the substrate, to complete lysis of a membrane, and/or the denaturation of incorporated membrane proteins. To eliminate such effects, ideally a polymer “cushion layer” is introduced via self-assembly of a thin polymer film,2 which insulates the lipid bilayer from the influence of the substrate7 and on the other hand stabilizes the lipid bilayer via a specific lipid-polymer layer interaction. This stabilizing effect of the polymer layer * To whom correspondence should be addressed. Phone: +49202-439-2493. Fax: +49-202-439-3880. † BUGH Wuppertal. ‡ Clariant Germany GmbH. § Max Planck Institute for Polymer Research. (1) Sackmann, E. Science 1996, 271, 43. (2) Beyer, D.; Elender, G.; Knoll, W.; Ku¨hner, M.; Maus, S.; Ringsdorf, H.; Sackmann, E. Angew. Chem. 1996, 108, 1791. (3) Cornell, B. A.; Braac-Maksvytis, V. L. B.; King, L.; Raguse, P. D. J.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580. (4) Mu¨ller, W.; Ringsdorf, H.; Rump, E.; Wildburg, G.; Zhang, X.; Andermaier, L.; Knoll, W.; Liley, M.; Spinke, J. Science 1993, 262, 1706. (5) Fromhertz, P.; Offenha¨user, A.; Vetter, T.; Weis, J. Science 1991, 252, 1290. (6) Thompson, N.; Poglitsch, C.; Timbs, M.; Pisarchick, M. Acc. Chem. Res. 1993, 26, 567. (7) Bohanon, T. M.; Elender, G.; Knoll, W.; Koeberle, P.; Lee, J.-S.; Offenha¨usser, A.; Ringsdorf, H.; Sackmann, E.; Tovar, J.; Winnik, F. J. Biomat. Sci., Polym. Ed. 1996, 8, 19.
may be obtained through an interaction of charged lipid headgroups with an inversely charged polymer film8 or through the fixation of “anchor” lipids on the surface of a highly functionalized polymer layer,9-11 which forms covalently attached “inserts” in the proximal lipid layer. In this contribution we investigate an amphiphilic copolymer with lipid side chains and a hydrophilic backbone, which has the ability to covalently attach to gold substrates and which may serve as a stabilizing support for lipid bilayers. This lipopolymer is unique in the fact that thin polymer films can be prepared using self-assembly12-15 from isotropic solution and by LBtransfer16-18 (see Scheme 1) as well. Therefore this novel system allows a direct comparison of both methods to study the influence of the polymer film preparation on the orientation of the lipid moieties of the polymer and the stability of the resulting covalently attached amphiphilic polymer layer. The use of such defined lipopolymer films for the preparation of polymer supported-bilayers will be described in a separate communication. Experimental Section Materials. The lipopolymer was prepared according to Scheme 2.19 Polymers prepared in this way (radical copolymerization of acrylamides and reactive esters) are random (8) Chi, L.; Johnston, R.; Ringsdorf, H.; Kimizuka, N.; Kunitake, T. Langmuir 1992, 8, 1360. (9) Beyer, D.; Bohanon, T. M.; Knoll, W.; Ringsdorf, H.; Elender, G.; Sackmann, E. Langmuir 1996, 12, 2514. (10) Beyer, D.; Nakao, A.; Matsuzawa, M.; Knoll, W. Langmuir 1998, 14, 3030. (11) Zhou, Y.; Bruening, M. L.; Bergbreiter, D. E.; Crooks, R. M.; Wells, M. J. Am. Chem. Soc. 1996, 118, 3773. (12) Bain, C.; Whitesides, G. Angew. Chem. 1989, 101, 522. (13) Sun, F.; Grainger, D. J. Polym. Sci.: Polym. Chem. 1993, 31, 1729-1740. (14) Erdelen, C.; Ha¨ussling, L.; Naumann, R.; Ringsdorf, H.; Wolf, H.; Yang, J.; Liley, M.; Spinke, J.; Knoll, W. Langmuir 1994, 10, 1246. (15) Lenk, T.; Hallmark, V. M.; Rabolt, J. F.; Ha¨ussling, L.; Ringsdorf, H. Macromolecules 1993, 26, 1230. (16) Mo¨hwald, H. Angew. Chem., Int. Ed. Engl. 1998, 27, 728. (17) Embs, F.; Funhoff, D.; Laschewsky, A.; Licht, U.; Ohst, H.; Prass, W.; Ringsdorf, H.; Wegner, G.; Wehrmann, R. Adv. Mater. 1991, 3, 25. (18) Baekmark, T.; Elender, G.; Lasic, D.; Sackmann, E. Langmuir 1995, 11, 3975. (19) Hausch, M.; Zentel, R.; Knoll, W. Accepted in Macromol. Chem. Phys.
10.1021/la9805239 CCC: $15.00 © 1998 American Chemical Society Published on Web 11/14/1998
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Scheme 1. Transfer Modes for the Buildup of Ultrathin Polymer Films on Gold Substrates
Scheme 2. Chemical Structure of the Lipopolymer
copolymers.20 The conversion of the reactive polymer with DMPE (dimyristoyl L-R-phosphatidylethanolamine) and 1-aminoethyl methyl disulfide was checked to be quantitative by thin layer chromatography. For the lipopolymer the copolymer composition could be roughly verified by 1H NMR. A determination of the molecular weight by GPC for the lipopolymer was not possible, because of aggregate formation. For a similiar polymer (the (20) Pollak, A.; Blumenfeld, H.; Wax, M.; Baughn, R.; Whitesides, G. J. Am. Chem. Soc. 1980, 102, 6324.
reactive polymer N-isopropylacrylamide-N-acryloxysuccinimide with the composition 5 mol % N-acryloxysuccinimide and 95 mol % N-isopropylacrylamide) the number average molecular weight could be determined by GPC in THF against a polystyrene standard to be 27 000 g/mol.21 Preparation of Gold Substrates. (a) for SPS (Surface Plasmon Resonance Spectroscopy). Gold (purity at least 99.99%) was evaporated onto clean glass slides (BK-270) using a Balzers (Bae250) vapor deposition apparatus. The glass slides (78 × 26 mm2) were cleaned by ultrasonification in a methanol detergent solution (Hellmanex, Hellma, Mu¨llheim, Germany) and copious amounts of Milli-Q water. For the SPS measurements chromium and gold films (thicknesses 20 and 480 Å, respectively) were consecutively evaporated on a glass slide in a vacuum chamber at 5 × 10-6 mbar. (b) for FTIR Measurements. For the FTIR measurements the layer thickness was increased to 50 Å for the chromium and 800 Å for the gold layer. After preparation, the gold substrates were stored under argon. Preparation of the Self-Assembled Films on Gold Substrates. Self-assembled monolayers of the copolymers were prepared by immersing the freshly prepared gold-coated substrate for 60 min into a solution of copolymer (1 mg/1 mL) in ethanol. The substrate was then thoroughly washed using ethanol and for the contact angle measurements dried in an argon stream. Preparation of the LB-Transferred Films on Gold Substrates. After the gold substrate was immersed vertically into the water subphase, the polymer solution was gently (by use of a microsyringe) deposited on the water surface. After relaxation for 5 min, the polymer film was compressed until the desired surface pressure was obtained. Hysteresis experiments show that at the surface pressures 20 and 35 mN m-1 the polymer monolayer is stable, whereas at surface pressures higher than 40 mN m-1 the surface pressure is lowered by 2-3 mN m-1 if the film is held for several minutes at a constant trough area. This could be due to reorientation processes. The substrate was pulled out of the subphase with a speed of 2 mm/min. Transfer ratios were measured using glass slides which were covered with a thin gold layer on both sides. A transfer ratio (reduction of the area on the trough during transfer divided by the area of the gold slides which were immersed) of approximately 1.2 independent of the applied surface pressure was measured. Monolayers at the Air-Water Interface. Chloroform was used as spreading solvent, and the concentration of the polymer solutions was usually 1 mg/1 mL. A computer-controlled film balance with a Wilhelmy plate pressure pickup system (Nima 611 D) was used to examine the behavior of the spread (21) Simon, J. Dissertation, Mainz, 1994.
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Table 1. Film Thickness and Advancing Contact Angle for Films of the Lipopolymer, Prepared by Self-Assembly or LB-Transfer at Different Surface Areasa film thickness film advancing contact before washing thickness angleb (deg) (Å) (Å) after washing self assembly LB-transfer at 20 mN m-1 LB-transfer at 35 mN m-1
70 ( 0.6
23 23 24 20
23 23 24 16
76 ( 0.6
18 16 41
15 13 35
96 ( 0.8
40 38
34 33
a For each experiment three different samples were prepared. The standard deviation is determined from five measurements at different locations on the same sample.
b
monolayers22 at the air-water interface. The isotherms were recorded at 25 °C on purified water (Milli-Q). Contact Angle Measurements. Contact angle measurements with water were performed using a Kru¨ss G1 system (Kru¨ss GmbH, Germany). The advancing contact angles reported in this paper are average values obtained by measuring at five different spots on the substrate. Surface Plasmon Spectroscopy. The glass slide with the gold film and self-assembled monolayer on top of it was refractive index matched to a 90° glass prism used as a surface plasmon coupler in the Kretschmann configuration.23 Resonant excitation of the surface mode, which is very sensitive to the actual interfacial architecture,24,25 was then monitored by recording the total internally reflected light from a He-Ne laser (λ ) 633 nm) as a function of the angle of incidence. First the bare gold substrates were measured at three different spots in water. After LB transfer, the film thickness was measured again at three different spots. To characterize the stability of the transferred film, it was washed extensively with ethanol and water, dried in an argon stream, and measured again. For the self-assembled films, first the gold substrate was measured in water. After self-assembly from ethanol for 60 min, the film thickness was measured in water again. After washing with ethanol and water, the final film thickness was measured again in water. The results for different samples, prepared under identical conditions, are summarized in Table 1. Differences between these samples show the reproducibility of the preparation method. Therefore the uncertainty in comparing samples prepared by different transfer modes is greater than that for the comparison before and after washing, for which identical samples are compared. FTIR Spectroscopy. All FTIR spectra were measured using a Nicolet 5DXC FTIR spectrometer, equipped with an “EverGlo” light source and a nitrogen-cooled Hg-Cd-Te narrow-band detector. The samples were measured in the dry state using a grazing incidence (80°) reflection on a FT80 setup. The spectra were taken with 2000 scans at 4 cm-1 resolution and a HappGenzel apodization.
Results and Discussion To compare the different transfer modes, a lipopolymer was synthesized according to Scheme 2. It was the synthetic concept to incorporate all three structural units, which are necessary for the fixation of lipid bilayers on a solid support, into one polymer: lipid chains to stabilize the lipid bilayer, a hydrophilic polymer cushion to reduce substrate effects from the solid support, (22) Albrecht, O. Thin Solid Films 1983, 99, 227. (23) Kretschmann, E. Opt. Commun. 1983, 6, 185. (24) Raether, H. Surface Plasmons; Springer Tracts in Modern Physics; Springer: Berlin, 1988; Vol. 3. (25) Knoll, W. MRS Bull. 1991, 16, 29.
Figure 1. Isotherm of the lipopolymer at 25 °C.
and disulfide moieties for the fixation of the polymer film on the gold substrate. According to the synthetic route, these groups are distributed randomly along the polymer chain. Figure 1 shows the isotherms of the lipopolymer at 25 °C. The area per lipid chain was calculated on the basis of the copolymer composition given in Scheme 2. For this lipopolymer the isotherms can be divided into three regimes (see Figure 1; 50-120 Å2, 120-300 Å2, and >300 Å2). At large surface areas (regimes 2 and 3), the isotherms are similar to that of N-isopropylacrylamide.26 During this stage of the compression the polymer coils interact with each other. Below 120 Å2 (regime 1) the isotherm corresponds to a dense packing of double-chain lipids with a high collapse pressure (60 mN m-1). The collapse areas for the double-chain polymeric lipids (50 Å2) almost correspond to the collapse areas of normal double-chain lipids (42 Å2). Therefore it is reasonables for this case of high surface pressuressto assume an almost tight monolayer of pendant lipid chains on top of a hydrophilic polymer cushion. This behavior is as expected for a decoupling of the lipid chains by a hydrophilic main chain spacer.27 Film Thickness and Contact Angles. The film thickness of the polymer was measured in dependence of the transfer mode using surface plasmon spectroscopy on gold supports. The data are summarized in Table 1. Theoretically it should be possible to control the thickness of an LB-transferred film by transferring the polymer at different pressures. As can be seen from Figure 1 the copolymer forms a monolayer with a high collapse pressure, which means that the polymer is confined to the air-water interface. The film thickness increase at the air-water interface can therefore be assumed to be linear with the reduction of the area per lipid (respectively the trough area). In our measurements we transferred the polymer films at the surface pressures 20 and 35 mN m-1. At the surface pressure 35 mN m-1 the area per lipid (120 Å2) is reduced by a factor of 2.4 in comparison to the area per lipid (290 Å2) for a surface pressure of 20 mNm-1, which should therefore result in a film 2,4 times as thick. As described in the Experimental Section the transfer ratio for the LB-transfer is 1.2, indicating the transfer of more than a monolayer of the polymer to the substrate. The reason for the large transfer ratio is not known but could result through formation of polymeric micelles during transfer. This assumption is based on the result that in contrast to the self-assembled film the LB film is not perfectly stable against washing with ethanol and (26) Kawaguchi, M.; Saito, W.; Kato, T. Macromolecules 27 1994, 5882. (27) Laschewsky, A.; Ringsdorf, H.; Schmidt, G.; Schneider, J. J. Am. Chem. Soc. 1982, 109, 788.
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water. The decrease in film thickness after washing can be explained by assuming that the amount of polymer which exceeds a transfer ratio of 1.0 is washed away during rinsing. The data in Table 1 show a decrease of 15% in the film thickness for the LB films, which would result in a “corrected” transfer ratio of about 1. If the experimental film thicknesses of the LB films transferred at the surface pressures 20 and 35 mN m-1 are compared, one finds that the film transferred at 35 mN m-1 is 2.3 times as thick as the film transferred at 20 mN m-1, which is in good agreement with the expected value of 2.4. In comparison to the LB films, the self-assembled film from water is completely stable against washing with water and ethanol. The film thickness of the self-assembled film is slightly higher than that for the film transferred at 20 mN m-1 (within experimental error, they may be identical). It would correspond to an area of 190 Å2 per lipid for a film on the film balance. An advantage of LB-transfer over self-assembly of amphiphilic polymers on gold substrates is not only that the film thickness can be easily varied but also that the amphiphilic structure of the lipopolymer is preoriented. At least at high surface pressures the hydrophilic part of the polymer will be mostly in the subphase, and the covalently attached lipids will be more or less densely packed at the air-water interface. This architecture produces a more clearly defined boundary between lipid chains and polymer support than that in an entirely chemisorbed system. For these self-assembled systems the packing and the arrangements of the lipids cannot be controlled. One property of the polymer support which is strongly influenced by the packing of the lipids is the contact angle. It is known that for self-assembled films the contact angle increases with increasing lipid content.18 Using the LB technique in conjunction with such lipopolymers, it is possible to increase the contact angle not only by using lipopolymers with higher lipid content but also by transferring at higher surface pressures, because a densely packed lipid monolayer should show a higher contact angle than one in which hydrophilic polymer parts still influence the surface properties. This behavior can be seen from the data in Table 1. The self-assembled polymer support exhibits the lowest contact angle. In the case of LBtransfer the contact angle increases dramatically with an increase in transfer pressure. FTIR Spectroscopy. The orientation of the lipid chains relative to the surface can be characterized using the infrared dichroism of the methyl and methylene groups. For p-polarized light the peak height of the CH3 vibration is independent of the number of methylene groups.28 On the other hand the peak heights for the νa(CH2) and νs(CH2) modes are dependent on the relative orientation of the respective groups with respect to the surface. For an all-trans alkyl chain oriented perpendicular to the surface, the νa(CH2) and νs(CH2) modes show only very low absorbances with p-polarized light. Since the number of methyl groups in comparison to the number of methylene groups is constant for LB or self-assembly films, the peak heights of the νa(CH2) and νs(CH2) modes may directly be correlated to the orientation of the lipid (28) Allara, D.; Swalen, J. J. Phys. Chem. 1982, 86, 2700.
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Figure 2. FTIR spectra of an LB film (35 mN/m) (2), a selfassembled film (9), and the bulk (b). The spectra are normalized by multiplication of the intensities by the peak height of the νa(CH3) mode.
chains relative to the surface. With a smaller peak height of the νa(CH2) and νs(CH2) modes in comparison to that for the νa(CH3) mode, the orientation of the lipid chains increases. In Figure 2 different FTIR spectra are displayed as normalized on the νa(CH3) mode. For films transferred at 20 mN m-1 the intensity of the FTIR signal was too low to be evaluated. As can be seen in Figure 2 the intensities for the νa(CH2) and νs(CH2) modes are much smaller for the LB film transferred at a surface pressure of 35 mN/m than for the self-assembled film, or the bulk spectra. This shows that for the transfer at 35 mN/m the polymeric lipids not only are more densely packed than those in self-assembled films but also have a greater orientation relative to the surface. The spectra of the self-assembled film is almost identical with that of the bulk sample, showing that through the self-assembly process no orientation of the lipids is induced and that the lipid chains are rather isotropically distributed. Conclusion The lipopolymer used in this work was found to be unique in the fact that thin chemisorbed polymer films, of comparable thickness, can be prepared by self-assembly and by LB-transfer as well. Both types of films are covalently linked to the gold substate. LB-transfer allows control of the film thickness, the contact angle, and the orientation of the lipid part in comparison to the case for purely self-assembled systems. The self-assembled film exhibits probably a rather isotropic orientation of the lipid side chains, whereas the orientation of the lipid chains in the polymer film transferred by the LB technique at higher surface pressure (35 mN/m-1) is more pronounced. We suggest for the lipopolymer transferred at 35 mN m-1 a structure on the substrate which is composed of two layers: directly on the surface, the hydrophilic polymer backbone and, on top of this covalently substrate-bound polymer, the covalently attached lipids. In contrast to this the self-assembled film should be composed of rather randomly absorbed polymer coils with no pronounced structure. LA9805239
