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Heterogeneous Surfaces of Structured Hairy-Rod Polymer Films: Preparation and Methods of Functionalization G. Wiegand,*,† T. Jaworek,‡ G. Wegner,‡ and E. Sackmann† Physik Department (E22), Biophysics Group, Technische Universita¨ t Mu¨ nchen, James-Franck-Strasse, D-85748 Garching, Germany, and Max-Planck-Institut fu¨ r Polymerforschung, Postfach 3148, D-55021 Mainz, Germany Received January 2, 1997X We report a versatile technique for the formation of a biofunctionalized micrometer scale pattern on solids which is based on Langmuir-Blodgett multilayers of hairy-rod polymers stabilized by partial photochemical cross-linking. Three types of polymer patterns consisting of hydrophobic and hydrophilic domains are generated. These structured films are locally functionalized in two ways: (1) by activating the hairy-rod cushions with active esters and (2) by the deposition of a lipid monolayer onto the hydrophobic domains. For the latter procedure use is made of the fact that lipid monolayers are only assembled over domains with matched polarity. The fluidity and continuity of the monolayer domains was analyzed by lateral diffusion measurements on the basis of photobleaching techniques. The polarity of the domains is monitored by the interference contrast microscopic observation of water microdrops deposited on the structured hairy-rod films.
1. Introduction The present paper is motivated by efforts to functionalize solid surfaces by deposition of ultrathin polymer films. Such films are expected to exhibit several advantages. One ultimate goal is to separate biogenic membranes from the solid by soft polymer films. The lipid membranes can slide freely over hydrated surfaces1 which allows the surface to maintain a high lateral pressure, enabling the self-healing of local defects in membranes. This is a basic requirement for the deposition of high electrical resistance membranes suitable for the design of biosensors on the basis of electrooptical detection.2 Fully hydrated polymer cushions could provide a quasi-natural environment for membrane-spanning functional proteins (enzymes, receptors). Defect free supported membranes would drastically reduce the nonspecific binding of proteins to solid surfaces in particular by incorporating lipopolymers providing an artificial glycocalix.3 A major problem encountered during the design of polymer-lipid composite films is dewetting caused by the van der Waals attraction between the lipid layer and the solid surface4 or by incompatibility of the polarity of the surfaces of the bilayer and the polymer film. Three successful strategies to prepare suitable polymer cushions have been followed: (1) the anchoring of dextran films,5 (2) the separation of the bilayer and solid by stealths provided by lipopolymers,6,7 and (3) the deposition of multilayers of rodlike macromolecules with substituted alkyl chains8 (so-called hairy-rod-type polymers). * To whom correspondence should be addressed. Fax: (49) (89) 289 12469. Tel: (49) (89) 289 12478. E-mail:
[email protected]. † Technische Universita ¨ t Mu¨nchen. ‡ Max-Planck-Institut fu ¨ r Polymerforschung. X Abstract published in Advance ACS Abstracts, May 15, 1997. (1) Tamm, L. K.; McConnell, H. M. Biophys. J. 1985, 47, 105113. (2) Sackmann, E. Science 1996, 271, 43-48. (3) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann, E. Langmuir 1995, 11, 3975-3987. (4) Elender, G.; Sackmann, E. J. Phys. II France 1994, 4, 455-479. (5) Elender, G.; Ku¨hner, M.; Sackmann, E. Biosens. Bioelectron. 1996, 11, 565-577. (6) Spinke, J.; Yang, J.; Wolf, J.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 167. (7) Lang, H.; Duschl, C.; Gratzel, H.; Vogel, H. Thin Solid Films 1992, 210, 818.
S0743-7463(97)00005-X CCC: $14.00
Deposition of hairy-rod multilayers by the LangmuirBlodgett (LB) technique offers a variety of advantages. The surface of the films can be rendered hydrophobic or hydrophilic by the choice of appropriate hairy-rod-type polymers.9,10 The hydrophobic surface with the alkyl chains forming a brush provides the same environment as the hydrophobic surface of a lipid monolayer, which enables the deposition of lipid monolayers and renders them highly stable. The polymer cushions can be stabilized by partial cross-linking of the hairy rods by photochemically active cross-linkers without destroying the lateral homogeneity by phase separation. This occurs if one tries to cross-link ultrathin layers of flexible polymers.11 Therefore lithographical techniques can be applied for the patterning of the polymer films into regions of different properties. In the field of heterogeneous functionalization of solids, different approaches have already been published. For example light-directed synthesis has been used to generate spatially addressable combinatorial libraries of DNA and protein sequences.12 Patterns of self-assembled monolayers produced by microcontact printing13 or LangmuirBlodgett transfer of structured monolayers14 and substrateinduced patterning of fluid lipid bilayers have been described recently.15 In the present paper we report two techniques that enable the heterogeneous functionalization of solids on micrometer scales on the basis of supported hairy-rod multilayers. In the first case the surface of the patterned hairy-rod films is directly functionalized by replacement of some of the alkyl hairs by functional groups (e.g., active esters) to which proteins can be coupled. A second way is the deposition of lipid monolayers on (8) Wegner, G. Thin Solid Films 1992, 216, 105-116. (9) Schaub, M.; Wenz, G.; Wegner, G.; Stein, A.; Klemm, D. Adv. Mater. 1993, 5, 919-922. (10) Sigl, H.; Brink, G.; Schulze, M.; Wegner, G.; Sackmann, E. Eur. Biophys. J., in press. (11) Ku¨hner, M.; Tampe´, R.; Sackmann, E. Biophys. J. 1994, 67, 217-226. (12) Fodor, S. P. A.; Leighton Read, J.; Pirrung, M. C.; Stryer, L.; Tsai Lu, A.; Solas, D. Science 1991, 251, 767-773. (13) Lo´pez, G. P.; Biebuyck, H. A.; Ha¨rter, R.; Kumar, A.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 23, 10774-10781. Jackman, R. J.; Wilbur, J. L.; Whitesides, G. M. Science 1995, 269, 664-666. (14) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229-1237. (15) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651653.
© 1997 American Chemical Society
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Wiegand et al. Table 1. Molecular Data of the Used Hairy-Rod Molecules Giving the Molar Fractions of Comonomers x, y, and z of the Copolyglutamates and the Degree of Substitution of the Side Chains x, y, and z of the Cellulose Derivative Shown in Figure 1 and Also the Degree of Polymerization and the Molar Weight of all Hairy Rods
hairy-rod polymer COM10 COM15 OM20 OM40 OM60 THXC
molar fraction [%] (THXC: degree of substition [%]) x y z 60 64 81 60 44 40
30 22 19 40 56 50
10 14
10
degree of polymerization
mol wt Mw [g/mol]
350 1600 1640 1010 200 160
342 244 183 239 283 409
fluidity depends on the density of the alkyl chains of the hairy-rod molecules. 2. Materials and Methods
Figure 1. Structure of the hairy-rod molecules: (top) copolyglutamate glutamates with methyl, octadecyl, and cinnamoylundecyl side chains (COM); (middle) cellulose derivative substituted by thexyldimethylsilyl and cinnamoyl groups (THXC); (bottom) schematic view of cross-linking reaction by photocycloaddition.
prestructured hairy-rod layers. The lateral distribution of the lipid is controlled by the pattern of the hairy-rod multilayer since the hydrophobicity of the polymer surface and the lipid layer has to be matched to form stable lipidpolymer composites below water. Therefore the lipid is displaced from areas exhibiting opposite polarity. Two types of hairy-rod molecules were used: derivatives of poly(γ-alkyl glutamates) and of cellulose silanes. Multilayers of the latter can be rendered hydrophilic if the “hairs” consisting of alkylsilyl groups are cleaved by a short exposure to HCl vapor. This treatment regenerates the cellulose from its derivative. The supported lipid layers were controlled by fluorescence microscopy, and their fluidity was determined by lateral diffusion measurements. It is shown that the
2.1. Polymers, Lipids, and Chemicals. Hairy-Rod Polymers. Poly{(γ-methyl L-glutamate)-co-(γ-octadecyl L-glutamate)co-(γ-[11-cinnamoylundec-1-yl] L-glutamate)} (abbreviated as COM; Figure 1 (top)), with different molar fraction x, y, z of the comonomers were synthesized as described elsewhere.16 The composition of the polymers investigated here and their degree of polymerization are summarized in Table 1. The synthesis of poly{(γ-methyl L-glutamate)-co-(γ-octadecyl L-glutamate)} (abbreviated as OM) with different molar fractions x and y of the comonomers (Table 1) was described by Mathy et al.17 The synthesis of thexyldimethylsilyl-cellulose cinnamate (abbreviated as THXC; Figure (middle)) was performed according to the general procedure developed by Klemm and Stein18 using thexyldimethylsilyl chloride as the reagent and lithium chloride/ dimethylacetamide as the solvent. In a second step the desired fraction of cinnamate groups was attached by reacting the thexyldimethylsilyl-cellulose with the acid chloride of the cinnamic acid in tetrahydrofuran (THF) with pyridine as the base.19 Purification of the crude product was achieved by repeated precipitation from mixtures of THF/water and dichloromethane/ methanol. The degree of substitution of the different side groups x, y, and z and the degree of polymerization are contained in Table 1. The lipid monolayers were prepared from a mixture of 99% L-R-dimyristoyl phosphatidylcholine (DMPC) and 1% N-(7-nitro2,1,3-benzoxadiazol-4-yl)-L-R-dimyristoylphosphatidylethanolamine (NBD-DMPE). Both lipids were purchased from Avanti PolarLipids (Alabaster, AL). All chemicals used in the functionalization procedures were commercial products and were used as purchased without further purification. N-Hydroxysuccinimide (NHS), iodoacetic acid (IAA), and N,N′-dicyclohexylcarbodiimide (DCC) were obtained from Fluka (Neu-Ulm, Germany). The fluorescent dye Lissamine rhodamine B sulfonylethylenediamine (LRSED) was obtained from Molecular Probes (Eugene, OR) and octadecyltrichlorosilane (OTS) was supplied from Sigma (Deisenhofen, Germany). Chloroform, N,N′-dimethylformamide (DMF) (both from Fluka, Neu-Ulm, Germany), and n-hexadecane (Sigma, Deisenhofen, Germany) were HPLC-grade quality. Water was ion-exchanged and Millipore filtered (Millipore Milli-Q-System, Molsheim, France, R > 18 MΩ cm-1, pH 5.5). Glass cover slides of size 24 × 24 mm2 (AL, Germany) were used as substrates. They were carefully cleaned by successive ultrasonifications first twice in a 2% (v/v) aqueous Hellmanex (Hella, Mu¨hlheim, Germany) solution (30 min) and subsequently (16) Mathauer, K.; Mathy, A.; Bubeck, C.; Wegner, G. Thin Solid Films 1992, 210/211, 449-451. (17) Mathy, A.; Mathauer, K.; Wegner, G.; Bubeck, C. Thin Solid Films 1992, 215, 98-102. (18) Klemm, D.; Stein, A. Wiss. Z. - Friedrich-Schiller-Univ. Jena: Naturwiss. Reihe 1987, 36, 675-679. (19) Klemm, D.; Schnabelrauch, M.; Stein, A.; Niemann, M.; Ritter, H. Makromol. Chem. 1990, 191, 2985-2991.
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Figure 2. Polymer patterning by UV lithography and photoablation. twice in pure Millipore water (30 min). Each sonification was followed by the mixture being rinsed with Millipore water. Finally the substrates were dried at 75 °C for at least 3 h. 2.2. Preparation of Hairy-Rod Multilayers. Multilayers of various numbers of layers, usually 6-12, were deposited by the Langmuir-Blodgett technique onto hydrophobized substrates. For silanization the cover slides were first kept for 2 min in a 0.1% (v/v) solution of OTS dissolved in a 1:4 (v/v) chloroform/n-hexadecane solution and were subsequently washed twice for 2 min in pure chloroform. To built-up multilayer assemblies, a solution of 0.4 mg of polymer per milliliter of chloroform was spread on the pure water surface of a Langmuir film balance (Lauda FW 1). The transfer was performed at a surface pressure of π ) 19 ( 1 mN/m and a temperature of T ) 19 ( 1 °C. The deposition rate was 200 µm/s and the transfer ratio was 1 ( 0.1 for both dipping and lifting. Film formation and transfer conditions as well as general properties of the hairyrod multilayer films were found to be in agreement with the published results for similar polymers.8,16,20,21 2.3. Regeneration of Cellulose. The regeneration procedure for obtaining cellulose films from cross-linked THXC multilayers was modified to that published by Schaub et al.9 To cleave the thexyldimethylsilyl side groups, the samples were exposed twice for 10 min to the saturated HCl atmosphere above a concentrated HCl solution (37%). Each step was followed by 2 min of washing with chloroform. The surface properties of the thin films change drastically during this conversion which can be shown by measuring the contact angle of small water droplets deposited onto the coated substrates. It changes from 85° on THXC to 12 degrees on regenerated cellulose.22 The regeneration of cellulose from THXC films does not affect the homogeneity of the films. The increase of the surface roughness is negligible although the complete film thickness is reduced to nearly half of its original value.9 2.4. UV-lithography and Local Photoablation. Using hairy rods with photoreactive cinnamoyl moieties in the side chains (COM, THXC) the LB films can be mechanically and thermally stabilized by cross-linking without changing the quality of the multilayers. UV irradiation induces a cycloaddition between cinnamate groups (Figure 1 (bottom)). Cross-linked multilayers are insoluble in common solvents. Two different methods were used successfully for pattern formation. The first one was based on a lithographic process described by Mathauer et al.16 The deposited films were exposed for a few minutes (1-4 min) to the light of a Hg(Xe) lamp (200 W; distance, (20) Duda, G.; Schouten, A. J.; Arndt, G.; Lieser, G.; Schmidt, F.; Bubeck, C.; Wegner, G. Thin Solid Films 1988, 159, 221-230. (21) Kawaguchi, T.; Nakahara, H.; Kiyoshige, F. Thin Solid Films 1985, 133, 29-38. (22) Wiegand, G.; Jaworek, T.; Wegner, G.; Sackmann, E. In preparation.
50 cm) through a mask in order to cross-link the material within the irradiated domains. The incident light from the source was reflected by an Al mirror in order to cut off wavelengths λ < 200 nm. Finally the (negative) patterns were developed by being rinsed with chloroform for at least 2 min (Figure 2) in order to remove the non-cross-linked hairy-rod domains. Note that the short irradiation time of 1-5 min was sufficient to cross-link the hairy rods but still short enough to avoid substantial decomposition. The second method made use of local photoablation. As in the first method the films were irradiated for a short time to render them insoluble by cross-linking. In this first step the whole film was irradiated without a mask. Afterward the multilayers were once again partially exposed to UV light through a mask for at least 15 min/layer. The wavelengths between 200 and 300 nm, especially the intense 254 nm line, cause photo (oxidative) decomposition during the prolonged irradiation, leading to low molecular weight fragments.23 Residual fragments were removed by a final rinsing in chloroform, leading to an inverted (positive) pattern. As masks we used electron microscopy grids (Plano, Marburg, Germany) of two different mesh sizes, namely 12.5 µm (width of bars, 5 µm, side length of square holes, 7.5 µm) and 62 µm (width of bars, 20 µm, side length of square holes, 42 µm). 2.5. Fluorescence Microscopy and Fluorescence Recovery after Photobleaching (FRAP). The experimental setup for the fluorescence microscope and the FRAP experiments was used was described elsewhere.5,24 The fluorescence intensity was measured by a photomultiplier. For the diffusion measurements, the conventional spot-bleaching technique was applied using a uniform circular laser beam.25 The time dependence of the fluorescence intensity F(t) was analyzed following Soumpasis.26 Two parameters were fitted to the F(t) versus-t curves: the fluorescence intensity F(∞) at infinite time after bleaching (t ) 0) where saturation is reached and the half-time of fluorescence recovery T1/2. The recovery R is defined by
R)
F(∞) - F(0) F0 - F(0)
(1)
where F0 is the fluorescence intensity before bleaching and F(0) is the intensity at the end of the bleaching pulse. The diffusion (23) Sawodny, M.; Embs, F.; Miller, R. D.; Aussenegg, F.; Stumpe, J.; Wegner, G.; Knoll, W. Makromol. Chem. Makromol. Symp. 1991, 46, 235-239. (24) Merkel, R.; Sackmann, E.; Evans, E. J. Phys. II France 1989, 50. (25) Axelrod, D.; Koppel, D. E.; Schlessinger, E.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055-1069. (26) Soumpasis, D. M. Biophys. J. 1983, 16, 95-97.
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coefficient, D, is defined as
D ) 0.224(r2/T1/2)
(2)
where r is the radius of the bleaching and observation spot. In the case of very slow molecular motion in the lipid layer (D < 0.01 µm2/s), the fitting of both R and D was not possible under normal measuring conditions (observation time e 30 min). Here R was arbitrarily set to a low value of 0.7 to estimate an upper limit for D. All measurements took place at 27 ( 2 °C.
3. Results and Discussion 3.1. Preparation of Heterogenous Hairy-Rod Multilayer Structures. Combinations of different types of hairy-rod multilayers and structuring techniques are applied to prepare structured surfaces with heterogenous surface properties. In the following, the procedures to prepare three different types of heterogeneous surfaces are presented. Subsequently, the local functionalization of these polymer patterns by two different methods is described (sections 3.2 and 3.3). Type I. Several layers of THXC were transferred onto the hydrophobized OTS substrates and structured by UV lithography using EM grids as masks. Thus a gridlike pattern consisting of hydrophobic squares of cross-linked THXC and bars of OTS-covered glass was obtained. Afterward the conversion of the THXC to a cellulose pattern (section 2.3) was performed, changing the polarity of the polymer-covered areas. The conversion is possible without any observable perturbation of the quality of the lateral precision of the pattern. The result was a pattern consisting of hydrophilic squares (cellulose) and hydrophobic bars (OTS). Type II. The same pattern with regard to polarity was also realized as follows. Hairy-rod multilayers (COM or THXC) were structured by photoablation (section 2.4). Both the hairy-rod and the silane layer were locally removed, resulting in a pattern of hydrophilic squares (bare SiO2) separated by hydrophobic bars (hairy rods). Type III. In order to prepare a structure with the opposite distribution of polarity as in the case of type I or II, we deposited a composite multilayer from two different types of hairy rods. First a film consisting of several layers of THXC was deposited which was cross-linked homogeneously. Subsequently, a second set of COM layers was transferred onto this THXC film which was structured by UV lithography. In the last step the THXC multilayer was converted into cellulose by exposure to HCl vapor. Again this treatment did not influence the structure of the COM cushions superimposed on this film. In this way a pattern with hydrophobic squares (COM) and hydrophilic bars (cellulose) was obtained. The chemical heterogeneity of the surfaces was monitored by observation of the wetting of the substrates by small water droplets. Two examples are shown in Figure 3. The contact line is deformed in such a way that it follows the boundaries between the attractive hydrophilic and repulsive hydrophobic domains of the pattern. The hydrophilic parts act as pinning centers in the case of a moving contact line. The quantitative characterization of structure and surface properties of the pattern by microinterferometry will be described in detail elsewhere.22 3.2. Chemical Functionalization. The possibility of regenerating thin films of cellulose from THXC multilayers opens the possibility of functionalizing the films, further applying concepts found in polysaccharide chemistry. In the following we present a procedure to functionalize cellulose layers by reaction with an active ester
Figure 3. Reflection interference micrograph of partial wetting of the heterogeneous substrate by small liquid droplets. The top image shows a water droplet receding by evaporation on a type I structure (hydrophilic fields/ hydrophobic bars) on a glass/ MgF2/SiO2 substrate. The bottom image shows a water droplet on a type III structure (hydrophobic fields/hydrophilic bars). The mesh size is both times 12.5 µm (bars, 5 µm; fields, 7.5 µm). The dark spots in the bottom figure are residual microdroplets from spraying the liquid on the sample.22
(N-hydroxysuccinimide (NHS)) in a two-step process as described by Lo¨fås and Johnson:27 Step 1. Carboxylation of the cellulose film by incubation of the samples in a 0.1 M aqueous solution of iodoacetic acid (at an unaltered pH of 2.5) for 48 h at room temperature. Step 2. Functionalization of the carboxylated samples by incubation for 10 min in a DMF solution of 0.2 M DCC and 0.05 M NHS. In literature27 this step is also performed alternatively in an aqueous solution of 0.2 M N′-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.05 M NHS (pH 6-7). The succinimidyl group can now be reacted for example with any molecule of interest having a primary amino group as illustrated in Figure 4 (top). To verify this approach, the functionalized cellulose was marked with (27) Lo¨fås, S.; Johnson, B. J. Chem. Soc., Chem. Commun. 1990, 1526-1528. Osa, T.; Anzai, J. Hyomen 1992, 30, 985-990.
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Langmuir, Vol. 13, No. 13, 1997 3567 Table 2. Measurement of the Fluorescence Intensity Ratio of LRSED Bound to Polymer-Covered and OTS-Covered Domains polymer
IF(polymer)/IF(OTS)
functionalized cellulose cellulose
7.6 ( 0.7 2.1 ( 0.6
a Pure cellulose (note that the polymer and the OTS domains are on the same sample).
Figure 4. (Top) Functionalization of structured hairy-rod multilayers (type I) by coupling of succinimide groups to a cellulose multilayer generated by cleaving the alkylsilyl chains from the THXC (Figure 1b) in HCl vapor. (Middle) Demonstration of successful heterogenous functionalization with succinimide groups by coupling of the fluorescent probe (LRSED). Fluorescence image of the NH2-Rhodamin marked sample structured with a mesh size of 12.5 µm (square size, 7.5 × 7.5 µm). (Bottom) Fluorescence image of the LRSED-labeled sample structured with a mesh size of 62 µm (square size, 42 × 42 µm).
a fluorescent dye exhibiting a primary amino group (LRSED) by chemisorption from a dilute solution of the dye in DMF. After intensive washing, the samples were monitored by fluorescence microscopy and the fluorescence intensity was measured. Figure 4 (middle and bottom) shows the fluorescence images of hairy-rod patterns of type I functionalized and fluorescence marked as described. These heterogeneously functionalized hairy-rod films enable differential measurements of ligand binding by a comparison of the signals from functionalized cellulose and nonfunctionalized OTS areas of the same sample. In addition reference samples of nonfunctionalized cellulose and OTS domains were investigated. This combined method yields information on the background signal due to nonspecific binding of the fluorescent species. The fluorescence intensity ratios measured on polymer-covered domains and on areas of OTS-covered glass are presented in Table 2. A significant increase in the magnitude of this ratio was observed by a comparison of the functionalized cellulose samples with the unfunctionalized ones. This demonstrates the increased affinity of the succinimide-functionalized cellulose towards primary amino groups. 3.3. Functionalization by Local Deposition of SelfAssembled Lipid Monolayers. The second method of functionalization of hairy-rod multilayers consists of the transfer of a lipid monolayer containing functional lipids such as a chelator lipid,28 biotinylated lipids,29 or lipidcoupled antigens. In the present work we only studied the self-assembly, the continuity, and the fluidity of pure lipid monolayers containing fluorescent probes on hairyrod multilayers. As a result of the Y-type deposition of the hairy rods,8 the polymer film offers a hydrophobic surface similar to that of a supported monolayer. Therefore the lipid monolayer was deposited by horizontal transfer of the substrate through the lipid monolayer at the air/water interface. With the exception of the experiment shown in Figure 3 (bottom), the lipid monolayers were deposited at a surface pressure of 30 mN/m and at room temperatures. To keep the lipid monolayer stable, the sample surface was kept in a small glass chamber filled with a constant volume of water. Applying the horizontal dipping technique does not allow measurements of the transfer ratio to check the quality of the transfer process. For this reason all samples were checked on the micrometer scale by fluorescence microscopy and lateral diffusion measurements. The lipid monolayers transferred onto homogenous hairy-rod films were homogeneous in accordance with earlier results.10 On a heterogeneous polymer pattern the lipid monolayer deposition depends on the local surface polarity of the sample. As demonstrated in Figure 5 physisorption of the lipid molecules takes place predominantly on the areas with a hydrophobic polymer surface. In Figure 5 (top left) a schematic drawing of the composite film and in Figure 5 (bottom left) the fluorescence image of a lipid monolayer transferred onto (28) Schmitt, L.; Dietrich, C.; Tampe´, R. J. Am. Chem. Soc. 1994, 116, 8485-8491. (29) Blankenburg, R.; Meller, P.; Ringsdorf, H.; Salesse, C. Biochemistry 1989, 28, 8214-8221.
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Figure 5. Functionalization of a supported hairy-rod multilayer by deposition of a lipid monolayer. The heterogeneous surface polarity leads to localized deposition of lipid monolayers: (top left) schematic view of a functionalized sample of type III. The DMPC/NBD-DMPE lipid monolayer predominantly deposited on the hydrophobic domains of the sample. (Bottom left) Fluorescence image of the localized deposition of the DMPC/NBD-DMPE lipid monolayer on the hydrophobic squares. Mesh size 12.5 µm (square size, 7.5 × 7.5 µm). (Top right) Schematic view of a sample of type II with a transferred DMPC/NBD-DMPE lipid monolayer. (Top right) Fluorescence image of the localized deposition of DMPC/NBD-DMPE lipid monolayer (deposition at 9 mN/m transfer pressure) on the hydrophobic COM bars. Mesh size 12.5 µm (bar width, 5 µm). In Figure 5 (bottom left and bottom right) the weak background intensity of the darker regions in the fluorescence images is partly due to reflections from the substrate and a small amount of residual lipid molecules remaining in these regions.
a heterogeneous polymer film of type III is depicted. The selective deposition of the lipid monolayer on the hydrophobic surface areas is apparent. Consequently, the lipid monolayers on the different polymer domains are separated from each other, and therefore the possibility arises to treat them individually. Similar techniques have been used by Groves et al.15 to pattern lipid bilayers and thus create isolated bilayer patches. As shown in Figure 5 (top right and bottom right), the same result was obtained when a lipid monolayer was transferred onto a type II polymer film with an inverted distribution of polarity. This demonstrates that heterogenous surfaces can be used to achieve a heterogeneous functionalization by localized lipid monolayer deposition. 3.4. Lipid Monolayer Stability and Lateral Diffusion. In the context of applications, the stability of the lipid monolayers on the hairy-rod cushions and the mobility of the lipid molecules in the monolayer are of main interest. Observation of both homogeneous and laterally structured samples by fluorescence microscopy revealed that the monolayers were stable for weeks if
stored below water and at room temperature. During this time no significant change in lipid diffusivity could be detected. The diffusion measurements by FRAP (section 2.5) were performed at temperatures above 24 °C, where DMPC is in the fluid phase. First, homogeneous systems were investigated to obtain more accurate data about the molecular mobility in lipid monolayers deposited on hairyrod films. Previous studies24 have shown that the diffusivity of the lipid depends on the frictional coupling between the monolayer and the substrate and on the possible alteration of the structure of the transferred monolayer induced by the structure of the substrate. Therefore the chain length and packing density of the alkyl side chains of the hairy-rod molecules play an important role. The lipid diffusivity on a series of copolyglutamate multilayers (COM10, -15 and OM20, -40, -60) with different packing densities of the long (CH2)18 side chains of the top hairy-rod layer was investigated (Figure 6). It is known from mechanical studies of these films30-32that, depending on the composition, the side
Structured Hairy-Rod Polymer Films
Figure 6. Dependence of average lateral diffusion coefficients of fluorescent probes in the DMPC/NBD-DMPE lipid monolayers deposited on a homogeneous multilayer of copolyglutamates, as function of the lateral density of the octadecyl side chains. The two sets of data correspond to two series of measurements performed with different samples.
chains between two hairy-rod layers in a multilayer system may be in a crystalline, partly crystalline, or amorphous state. In Figure 6 the logarithmic averages of the measured diffusion coefficients, D, are plotted versus the area per (CH2)18 side chain, AC18. The values of AC18 were estimated from the area per monomer, AM, of the hairy-rod monolayer on the LB trough at the transfer pressure chosen and the molar fraction of (CH2)18 per monomer Y (Table 1) by AC18 ) AM/Y. The fluorescence recoveries obtained by fitting the fluorescence recovery curves varied between 85% and 98%. With the set of polymers mentioned above, two series of measurements of averaged values for the diffusivity, D, were performed which both show the following behavior of the diffusivity: the decrease of the area per (CH2)18 side chain leads to a reduced mobility of the lipid molecules. This can be explained in terms of an increase in the friction between the hairy-rod layer and the lipid monolayer or an induced crystallization of the lipid monolayer caused by an increase of the packing density of long alkyl side chains in the top hairy-rod layer. The difference in the diffusion (30) Schmidt, A.; Mathauer, K.; Reiter, G.; Foster, M. D.; Stamm, M.; Wegner, G.; Knoll, W. Langmuir 1994, 10, 3820-3826. (31) Vierheller, T.; Foster, M.; Schmidt, A.; Mathauer, K.; Knoll, W.; Wegner, G.; Satija, S.; Majkrzak, C. F. Macromolecules 1994, 27, 68936902. (32) Schmitt, F.-J.; Yoshizawa, H.; Schmidt, A.; Duda, G.; Knoll, W.; Wegner, G.; Israelachvili, J. Macromolecules 1995, 28, 3401-3410.
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constants of the two series of measurements shown are attributed to a variation of the ambient temperature during the FRAP measurements. By using an aperture of 11.6 µm, it was possible to obtian reliable measurements of the diffusion coefficients on the different areas of patterned lipid-polymer systems exhibiting a mesh size of 62 µm (width of bars, 20 µm; side length of squares, 42 µm). The position of the spot was located in the center of the squares or of the bar crossings and was controlled by fluorescence microscopy. Although the condition of an infinitely extended monolayer required for the validity of eq 2 is not given, we determined D by applying the same fitting procedure worked out for infinite layers. Within the limits of experimental accuracy, the diffusion coefficients at the lipid-covered domains of the pattern agreed well with the values found for infinitely large monolayers on homogeneous samples of the same material as in the domain regions. Figure 5 (bottom left and bottom right) shows that only one part of the heterogenous polymer film is continuously covered by lipid molecules. A drastic variation in the lipid mobility on a single sample was observed on structured but completely (bars and squares) hydrophobic samples. For these experiments the pattern was prepared by UV-lithographic structuring of COM15 multilayers on OTS, so both parts of the pattern were covered by the transferred lipid monolayer. On the COM15-covered squares the mean diffusion coefficient was D ) 0.7 ( 0.2 µm2/s (R ) 96 ( 2%) and on the OTS-covered bars it was D ) 10-3-10-4 µm2/s in good agreement with results found for homogeneous samples. Thus we were able to prepare a structured monolayer with a bimodular distribution of molecular mobility differing by 3 orders of magnitude. Unfortunately, however, the spot-bleaching technique is not suitable for analyzing the lipid diffusivity across the boundaries separating the different domains of the pattern. Nevertheless the OTS bars should act as a barrier for the diffusion of lipid molecules from one polymer domain to another. Acknowledgment. This work was funded by the Bundesministerium fu¨r Bildung und Forschung (BMBF Contract KFA 0310851 BEO). We also gratefully acknowledge the support by the Fonds der chemischen Industrie and the Bayerische Staatsministerium fu¨r Wirtschaft und Verkehr (Fo¨rderkennzeichen B31040B). LA970005I
