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Miscibility of Lipids in Monolayers Investigated through Adsorption Studies of Antibodies P. Ihalainen* and J. Peltonen Department of Physical Chemistry, Åbo Akademi University, Porthaninkatu 3-5, FIN-20500 Turku, Finland Received September 17, 2002. In Final Form: November 18, 2002 The miscibility and reactivity of binary monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycolipoate (DPPGL) and 1-palmitoyl-2-(16-(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine (DSDPPC) have been studied. The morphology of the binary monolayers transferred on a gold substrate at different surface pressures was determined by scanning probe microscopy (SPM) and compared with the interfacial properties of the films at the air/water interface. Specific immobilization of pepsin-cleaved antibody Fab′ fragments from a Langmuir film onto a solid-supported DSDPPC-DPPGL (95:5 mol %) monolayer was utilized in the visualization of the miscibility and reactivity of the mixed lipid monolayers. The protein immobilization was shown to be dependent on the degree of condensation of the DSDPPC-DPPGL film. Furthermore, the linker molecules were shown to be favorably orientated for cross-linking reaction when amidst liquidcondensed domains of the matrix molecules. A closer look at the Fab′ domains revealed, however, that the domains were not fully covered with antibodies. The lower than expected relative surface density (RSD) of the Fab′ fragments was believed to be mainly due to random orientation of the Fab′ fragments at the air-water interface, which prevented part of them from participating in a thiol-disulfide interchange reaction between the antibodies and the linker molecules.

Introduction Langmuir-Blodgett (LB) or Langmuir-Schaefer (LS) techniques have been used to fabricate functional surfaces for controlled immobilization (orientation, surface density, and specific binding) of biomolecules.1,2 LB/LS film techniques offer several ways to obtain protein layers. Proteins can be spread directly at the air-water interface and transferred onto a solid substrate,3-7 adsorbed from the subphase onto a preformed monolayer,8-12 or covalently coupled to biofunctional molecules, such as linker lipids, included in a floating lipid matrix monolayer or solid-supported LB/LS film.13-16 Thiol-reactive crosslinking reagents, such as maleimides and disulfides, are generally employed for binding of Fab′ fragments in a highly oriented manner.15,16 The careful selection of the components in a mixed film to ensure a homogeneous distribution of the biofunctional lipids within the mono* To whom correspondence should be addressed. Telephone: +358-2-215-4616. Fax: +358-2-215-4706. E-mail: [email protected]. (1) Petty, M. C. J. Biomed. Eng. 1991, 13, 209. (2) Wang, H.; Brennan, J. D.; Gene, A.; Krull, U. J. Appl. Biochem. Biotechnol. 1995, 53, 163. (3) Ahluwalia, A.; De Rossi, D.; Monici, M.; Schirone, A. Biosens. Bioelectron. 1991, 6, 133. (4) Ahluwalia, A.; De Rossi, D.; Rwastori, C.; Schirone, A. Biosens. Bioelectron. 1992, 7, 207. (5) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1991, 205, 113. (6) Dubrovsky, T. B.; Demcheva, A. P.; Savitsky, A. P.; Mantrova, E. Y.; Yaropolov, A. I.; Savransky, V. V.; Belovolova, L. V. Biosens. Bioelectron. 1993, 8, 377. (7) Tronin, A.; Dubrovsky, T.; de Nitti, C.; Gussoni, A.; Erokhin, V.; Nicolini, C. Thin Solid Films 1994, 238, 127. (8) Tomoaia-Cotisel, M.; Candenhead, A. D. Langmuir 1991, 7, 964. (9) Sugawara, M.; Sazawa, H.; Umezawa, Y. Langmuir 1992, 8, 609. (10) Ebara, Y.; Okahata, Y. Langmuir 1993, 9, 574. (11) Fujiwara, I.; Ohnishi, M.; Seto, J. Langmuir 1992, 8, 2219. (12) Heckl, W. M.; Thompson, M.; Mo¨hwald, H. Langmuir 1989, 5, 390. (13) Egger, M.; Heyn, S. P.; Gaub, H. E. Biophys. J. 1990, 57, 669. (14) Ahluwalia, A.; Carra, M.; De Rossi, D.; Ristori, C.; Tundo, P.; Bomben, A. Thin Solid Films 1994, 247, 244. (15) Viitala, T.; Vikholm, I.; Peltonen, J. Langmuir 2000, 16, 4953. (16) Ihalainen, P.; Peltonen, J. Langmuir 2002, 18, 4953.

layer is desirable to obtain a good sensing surface. The unfavorable orientation of the biofunctional groups in the monolayer may decrease the reactivity of the surface toward the desired protein.17 Miscibility and phase behavior in floating Langmuir films can be detected by, e.g., Brewster angle microscopy (BAM)18 and in LB/LS films on solid substrates by scanning probe microscopy (SPM), especially by, e.g., using phase-contrast imaging in tapping mode.19-21 However, the visual investigation of the miscibility with these methods is quite difficult, especially when the linker concentration is very low. Also, it is possible to study the miscibility of the binary monolayers with surface pressure isotherms, surface phase rule, and excess area criterion, but with these methods it is often difficult to make a clear distinction between complete miscibility and immiscibility.18,22-24 Covalent coupling of pepsin-cleaved antibody Fab’ fragments via a thiol-disulfide interchange reaction to 1,2-dipalmitoyl-sn-glycero-3-phosphoglycolipoate (DPPGL) embedded in a host monolayer matrix of 1-palmitoyl2-(16-(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine (DSDPPC) has been recently demonstrated.16 The excess area criterion was tested for this binary system, but it did not fully explain the miscibility or immiscibility of the components in the monolayers. Large deviations from the additivity rule indicated partial miscibility at high surface pressures for a system with over 20 mol % (17) Vikholm, I.; Albers, W. M. Langmuir 1998, 14, 3865. (18) Gyo¨rvary, E.; Albers, W. M.; Peltonen, J. Langmuir 1999, 15, 2516. (19) Solletti, J. M.; Botreau, M.; Sommer, F.; Tran Minh Duc; Celio, M. R. J. Vac. Sci. Technol., B 1996, 14 (2), 1492. (20) Whangbo, M.-H.; Magonov, S. N.; Bengel, H. Probe Microsc. 1997, 1, 23. (21) Burnham, N. A.; Behrend, O. P.; Oulevey, F.; Gremaud, G.; Gallo, P.-J.; Gourdon, D.; Dupas, E.; Kulik, A. J.; Pollock, H. M.; Briggs, G. A. D. Nanotechnology 1997, 8, 67. (22) Viitala, T.; Albers, W. M.; Vikholm, I.; Peltonen, J. Langmuir 1998, 14, 1272. (23) Deleu, M.; Paqout, M.; Jacques, P.; Thonart, P.; Adriaensen, Y.; Dufreˆne, Y. F. Biophys. J. 1999, 77, 2304. (24) Do¨rfler, H.-D. Adv. Colloid Interface Sci. 1990, 31, 1.

10.1021/la020791d CCC: $25.00 © 2003 American Chemical Society Published on Web 01/31/2003

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linker component in the mixture. The DSDPPC-DPPGL monolayer seemed to be miscible only when the linker lipid was a clear minority.16 The aim of the present study was to gain more information on the miscibility and reactivity of DSDPPCDPPGL monolayers as a function of deposition surface pressure. The morphology of the mixed monolayers transferred on a gold substrate was determined by SPM and compared with the interfacial properties of the films at the air-water interface. Successful immobilization of Fab′ fragments deposited from a Langmuir film onto DSDPPC-DPPGL (95:5 mol %) monolayers on gold was demonstrated by SPM. The covalently immobilized Fab′ fragments were furthermore used as labels to localize the linker lipids in the matrix and thereby to visually investigate not only the biofunctional reactivity but also the miscibility within the DSDPPC-DPPGL monolayers. Experimental Section Lipids and Proteins. The linker lipid DPPGL and the host matrix lipid DSDPPC were synthesized as previously described.16,25,26 DPPGL was prepared using 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) as a starting compound, and DSDPPC was synthesized using 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine as a starting compound. Both lipids were obtained from Avanti Polar Lipids (purity >99%). HEPES-EDTA buffer (10 mM HEPES (Sigma), 150 mM NaCl (Fluka), 5 mM EDTA (Sigma), pH 6.8) was used as a buffer solution for all the protein measurements. The model antibody was polyclonal goat anti-human F(ab′)2 (Jackson ImmunoResearch) with minimized cross reaction to bovine, horse, and mouse serum proteins. F(ab′)2 was cleaved into Fab′ fragments with dithiothreitol (DTT, Acros Organics) under argon overnight in a microdialysis tube prior to use.27 According to a previous report, more than 95% of the F(ab′)2 fragments is converted to Fab′ during such a reduction.28 Monolayer Formation and Deposition. A commercially available computer-controlled KSV LB-5000 Langmuir minitrough (KSV-Instruments, Helsinki, Finland) with a Wilhelmy balance was used. The MilliQ filtration system (Millipore Corp.) was used to purify the water (18 MΩ cm) for the subphase and the buffer solution. Phospholipids were dissolved in chloroform (J. T. Baker). DSDPPC was mixed with DPPGL in a molar ratio of 95:5 and spread onto the surface of the subphase using a microsyringe. Films of this material were compressed at 5 mm min-1 (0.02 nm2 molecule-1 min-1), to produce surface pressure vs mean molecular area isotherms. The experiments were carried out at 20 ( 0.5 °C. Fab′ isotherms were obtained at 23 ( 0.5 °C by spreading 70 µL of a 0.7 mg/mL HEPES/EDTA buffer solution onto the subphase (HEPES/EDTA) degassed with argon. The monolayer was compressed at a velocity of 5 mm min-1 (0.6 nm2 molecule-1 min-1). Ultraflat gold surfaces as substrates for horizontal deposition of lipid and Fab′ films were prepared following the procedure described by Wagner et al.29 The mixed film of DSDPPC and DPPGL was compressed to a predetermined pressure, and the gold substrate was then brought into contact with the floating monolayer for 2 h.16 After lifting, the lipidcoated substrate was washed with high-purity water and absolute ethanol and dried with nitrogen. The Fab′ film was then deposited onto the lipid-coated substrate. The Langmuir-Schaefer deposition was performed at a surface pressure of 20 mN/m, and a deposition time of 30 min was used to ensure sufficient reaction. After deposition the substrate was again washed with high-purity water and dried with nitrogen. All samples were stored dry at room temperature until further analyses were performed. Scanning Probe Microscopy (SPM). A Nanoscope IIIa (Digital Instruments, Inc., Santa Barbara, CA) SPM in tapping mode was used for imaging the sample surfaces in ambient air. (25) Pax, H.; Blume, A. Chem. Phys. Lipids 1993, 66, 63. (26) Ihalainen, P.; Peltonen, J. Langmuir 2000, 16, 9571. (27) Ishikawa, E. J. Immunoassay 1983, 4, 209. (28) Martin, F. J.; Hubbell, W. L.; Papahadjopoulos, D. Biochemistry 1981, 20, 4229. (29) Wagner, P.; Hegner, M.; Gu¨ntherodt, H.-J.; Semenza, G. Langmuir 1995, 11, 3867.

Langmuir, Vol. 19, No. 6, 2003 2227 A J-scanner (150 × 150 µm2 scan range) and silicon cantilevers (TESP, resonance frequency between 250 and 300 kHz) supplied by the manufacturer (Nanoprobes TM) were used for imaging. The free amplitude of the oscillating cantilever (off contact) was 60 ( 5 nm. The engage procedure caused a shift in the resonance frequency, which was taken into account. The new resonance frequency for the tip in contact was determined and used as the operating frequency. Light tapping with a damping ratio (contact amplitude/free amplitude) of about 0.7-0.8 was used for imaging. The images were analyzed using Scanning Probe Image Processor (SPIP) software.

Results and Discussion Compression Isotherm Studies. DSDPPC and DPPGL have essentially different phase transition surface pressures from the liquid-expanded (LE) to the liquidcondensed (LC) state.16 This must affect the condensing and miscibility properties of the binary monolayers of these lipids. The surface compressibility can be used to more carefully characterize the details of the LE-LC phase transition compared with conventional surface pressure isotherms.30 The compressibility coefficient β of a monolayer at any area A at constant temperature is defined as follows:

β ) -1/A (δA/δπ)T

(1)

The monolayer of DSDPPC was chosen to demonstrate the different nature of information that is observed from a conventional compression isotherm and a compressibility curve (Figure 1A). In the isotherm, two weak kink points are observed at surface pressures 2.2 and 7.8 mN/m but otherwise the curve is pretty monotonic. The β-π curve, in contrast, shows that (a) the onset of the LE-LC phase transition takes place already before the kink point (7.8 mN/m) of the isotherm and (b) the maximal compressibility indicating maximal intermolecular cooperativeness occurs at 8.7 mN/m. The β-π curve also reveals the elevated level of surface pressure, but also somewhat unstable cooperativeness already within surface pressures π ) 2.27.8 mN/m. The β-π curve is hence more sensitive to intermolecular interactions, and especially to changes in them. Figure 1B shows the compressibility curves for DPPC, DSDPPC, DPPGL, and DSDPPC-DPPGL (95:5 mol %) monolayers as a function of surface pressure (π) at 20.0 ( 0.5 °C. The LE-LC phase transition in a DPPC monolayer is indicated by a sharp peak in the β-π curve between surface pressures 2.0 and 8.0 mN/m, with a maximum compressibility of 0.3 m/mN at 3.0 mN/m. The peak appears quite symmetric and, in contrary to an earlier report,30 refers to a pure one-step transition. The surface pressure range of the LE-LC phase transition is comparable with previous phase studies.31 The behavior of the DSDPPC monolayer was discussed already above. The main phase transition occurring around the compressibility maximum seems to be preceded by a pre-transition regime. Assuming that the pre-transition is part of the LE-LC transition, the onset pressures of the phase transition of the DPPC and DSDPPC monolayers coincide nicely. The β-π curve hence clearly shows that the unequal length of the hydrophobic alkyl chains of DSDPPC prolongs the completion of, and makes the LE-LC phase transition of the DSDPPC film clearly more complex compared to DPPC. A peak in the β-π curve between surface pressures 21 and 33 mN/m was observed for the DPPGL monolayer. (30) Yu, Z.-W.; Jin, J.; Cao, Y. Langmuir 2002, 18, 4530. (31) Kane, A. S.; Floyd, S. D. Phys. Rev. E 2000, 62 (6), 8400.

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Figure 2. Surface pressure-molecular area isotherm of Fab′ film on a HEPES-EDTA buffer subphase at 23 ( 0.5 °C.

Figure 1. (A) Comparison between the surface pressuremolecular area isotherm curve (solid line) and the β-π curve (dotted line) of pure DSDPPC monolayer. (B) Compressibility plotted as a function of surface pressure for DPPC, DSDPPC, DPPGL, and DSDPPC-DPPGL (95:5 mol %) monolayers at 20.0 ( 0.5 °C.

Again comparing with the reference monolayer of DPPC, the transition of DPPGL is clearly broader and it takes place at significantly higher pressures. This implies that the polar glycolipoate headgroup of the molecule has a dominating effect on the phase behavior of DPPGL. The β-π curve of the binary monolayer of DSDPPCDPPGL (95:5 mol %) shows that the introduction of the linker molecule even at a low concentration has an expanding effect on the DSDPPC film. The LE-LC phase transition can be seen in the β-π curve between surface pressures 3 and 29 mN/m. Although the shape of the curve is similar to that of the pure DSDPPC film, the peak presenting the main LE to LC phase transition is much more subtle with the maximum at 10.4 mN/m. There is also a weak local maximum around 20 mN/m, indicating an additional phase transition. According to the β-π curve the behavior of the DSDPPC-DPPGL mixed monolayer is not a direct superposition of the behavior of the pure film components because none of the local maxima in the curve coincide with the maxima observed for the pure lipid films. This is the first indication of at least partly mixed components in the DSDPPC-DPPGL monolayer. The second indication of a miscible film is the low intensity of the maxima in the β-π curve being a measure of the intermolecular cooperativeness. The highest cooperativeness can be observed for the reference monolayer of DPPC, with the highest peak intensity of the transition. The modified structure of DSDPPC as well as DPPGL de-

creases the intermolecular cooperativeness, which, however, appears on a clearly higher level than that of the mixed film. The surface pressure-molecular area isotherm for the Fab′ on a HEPES-EDTA buffer subphase at 23.0 °C is shown in Figure 2. Although the protein was surface active as evident from the isotherm, the monolayer remained in the LE state throughout the compression. The isotherm is comparable to the F(ab′)2 isotherms compressed at 2025 °C on a phosphate buffered subphase.32 In agreement with an earlier report,3 minimum compressibility (maximum compressional modulus) was observed at about 20 mN/m, and this was also found to be the optimal pressure for film deposition. The mean molecular area of the Fab′ fragments at 20 mN/m was 21 nm2 molecule-1. The dimensions of a Fab′ fragment, 7 × 5 × 4 nm3, measured by X-ray diffraction33 indicate that the larger cross section of the Fab′ molecule was oriented parallel to the interface plane in the floating monolayer. Thus the height of the Fab′ monolayer was expected to fall between 4 and 5 nm. The binding area of Fab′ estimated from the compression isotherm is large relative to the mean lipid area in the matrix monolayer (0.42 nm2 molecule-1). This means that about 50 lipid molecules become covered by one immobilized Fab′ fragment. This further means that in an ideal case only 2 mol % of linker lipids in the mixed monolayer is enough to fill the surface with bound Fab′ fragments, assuming that their orientation is preserved after deposition. SPM. Figure 3A-F shows typical SPM height images of samples with Fab′ fragments immobilized from the Langmuir film at 20 mN/m onto solid-supported binary lipid monolayers (DSDPPC-DPPGL 95:5 mol %). The deposition surface pressures (πd) of the lipid monolayers represented both the LE-LC coexistence region ((A) 5, (B) 10, (C) 15, (D) 21, and (E) 25 mN/m) and the LC state ((F) 30 mN/m) according to the β-π curve. Clear domains of Fab′ molecules are visible in samples being deposited at 5, 10, 15, and 21 mN/m. The domain size increases steeply between 5 and 10 mN/m, but the increase slows down with further increase of the deposition pressure. It rather seems that it is the number of Fab′ domains that increases. The domains exhibit an almost uniform shape, although they become more fused together as the deposition pressure increases. When the deposition pressure of (32) Ahluwalia, A.; De Rossi, D.; Schirone, A. Thin Solid Films 1992, 210/211, 726. (33) Sarma, V. R.; Silverton, E. W.; Davies, D. R.; Terry, W. D. J. Biol. Chem. 1971, 246, 3753.

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Figure 4. Relative surface coverage of Fab′ domains plotted as a function of deposition surface pressure (πd) of DSDPPCDPPGL (95:5) monolayer.

Figure 3. Typical SPM height images of samples with Fab′ fragments immobilized from the Langmuir film at 20 mN/m onto solid-supported binary lipid monolayers deposited at (A) 5, (B) 10, (C) 15, (D) 21, (E) 25, and (F) 30 mN/m. The height scale is 10 nm, and the image size is 50 × 50 µm2. (inset) Crosssection profile over typical Fab′ domains in the sample deposited at 21 mN/m.

the lipid monolayer was increased to 25 mN/m (Figure 3E), only the boundaries were left between the Fab′-rich domains. Finally, Figure 3F, representing the condensed state of the binary monolayer, reveals a homogeneous distribution of proteins at the surface. The surface coverage of the domains was calculated to be 4% (5 mN/m), 21% (10 mN/m), 38% (15 mN/m), 58% (21 mN/m), and over 95% (25 mN/m) of the total surface area.

Nonspecific binding of antibodies was tested by depositing Fab′ fragments onto a pure DSDPPC monolayer (data not shown). The amount of immobilized proteins was found to be negligible, not to speak of any domain formation. This indicates indirectly that the reaction of Fab′ molecules with the linker molecule DPPGL (Figure 3) is of a specific nature. There is a sharp height contrast of about 3.5-5 nm between the Fab′-rich domains and the Fab′-free lipid surface as demonstrated in the cross-section profile (inset of Figure 3). The result indicates that in the lipid films deposited at 5, 10, 15, and 21 mN/m there is little or no Fab′ bound outside the domains. The height value of the Fab′ domains shows that the majority of the proteins are lying side-on on the surface, which was expected from the mean molecular area value of the isotherm of the antibody film at 20 mN/m. This also suggests that the orientation of the antibodies was preserved after deposition. The appearance of the Fab′ domains is direct evidence for the existence of domains containing linker lipids in the binary lipid film. The Fab′ fragments hence act as labels revealing, however, the location of only functional linker lipids. The term “functional” should here be understood as “properly oriented for binding of Fab′ fragments”. When the polar headgroup of the linker lipid is oriented parallel to the film surface, it is more difficult for the terminal thiol group of the Fab′ fragments to “reach” the disulfide group of the linker unit. In other words, the thiol-disulfide interchange reaction between DPPGL and a Fab′ molecule is sterically hindered. As the surface pressure of the binary lipid monolayer increases, an increasing fraction of DPPGL molecules orient themselves parallel to the surface normal, thus increasing the amount of Fab′ fragments specifically bound to the surface. Figure 4 shows the relative surface coverage of the Fab′ domains plotted as a function of πd of the DSDPPC-DPPGL (95:5 mol %) monolayers. Extrapolation of the linear portion of the data points to zero coverage gave a value of 2.9 mN/m for the onset surface pressure of the Fab′ domain formation. This value is close to the value of 3.0 mN/m for the LE-LC phase transition onset surface pressure observed from the β-π curve of the mixed lipid monolayer. A similar kind of analysis for the epifluorescence microscopic data published by Kane and Floyd31 for DPPC gives an extrapolated value of 1.9 mN/m for the onset surface pressure of the LC domain formation. This value again coincides fairly well with the value of 2.0 mN/m from the β-π curve of DPPC in Figure 1.

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The results indicate that the Fab′-rich domains correspond not only to areas where the linker molecules are oriented vertically but also to areas where the DSDPPC matrix has reached the condensed state. This again means that the linker molecules are miscible in the DSDPPC matrix and do not become pushed out from the LC domains during compression. The data of Figure 4 also show a steep rise in the relative surface coverage of the Fab′ domains between surface pressures 21 and 25 mN/m. Why this accelerated completion of the film condensation takes place is not clear. It seems obvious, however, that the phase transition of the mixed film levels off at a lower surface pressure compared to the monolayer of pure DPPGL. A closer look at the Fab′ domains revealed that the surface inside the domains was not fully covered with proteins. The SPM images in Figure 5A,B have been taken inside the domains for (A) 15 and (B) 25 mN/m samples. The maximum peak-to-peak height value varied between 6.0 and 8.9 nm depending on the sample. A typical crosssection profile is shown in the inset of Figure 5. The height values indicate that not all the Fab′ fragments were oriented side-on on the surface, although the grain size analysis revealed that the height of most of the Fab′ molecules (∼95%) was between 4 and 5 nm (cf. Figure 3). Thus the height threshold was chosen according to the height value of 4.0 nm of a single Fab′ molecule lying side-on. As a result, the images of Figure 5 show only the morphologically highest components (appearing as light objects), which are included in the relative surface density (RSD) calculation by grain size analysis. The choice of the threshold minimizes, but does not completely cancel out, the tip-sample convolution effect. A RSD value of 35 ( 9% of Fab′ fragments was observed for all the samples. This is far below an ideal case where a linker concentration of only 2 mol % is enough to bind a complete monolayer of Fab′. The relatively small variations in the RSD values for different samples suggest that the lower than expected binding of antibodies inside the domains was not related to the reactivity of DPPGL but rather to the random orientation of Fab′ molecules at the air-water interface.32 This causes the reactive thiol groups of some of the Fab′ fragments to be unable to participate in the thiol-disulfide interchange reaction with DPPGL. The unfolding of the proteins at the air/water interface and during the deposition process could also lower the amount of the Fab′ fragments bound onto the surface.34 Conclusions It was shown through the compressibility studies that the introduction of a small amount of DPPGL has an expanding effect on the DSDPPC monolayer. Successful immobilization of Fab′ fragments from a Langmuir film onto a DSDPPC-DPPGL (95:5 mol %) monolayer was demonstrated by SPM. The formation of Fab′ domains was dependent on the state of the mixed lipid monolayer. Comparison of the relative surface coverage of Fab′ domains with the interfacial properties of the mixed Langmuir films showed that the Fab′-rich domains corresponded not only to areas where the linker molecules were oriented favorably for binding but also to areas where the DSDPPC matrix had reached the condensed state. This indicated that DPPGL molecules are miscible in DSDPPC matrix. The orientation of the Fab′ fragments were preserved after deposition, and the degree of unspecific binding was found to be negligible. A closer look at the Fab′ domains revealed, however, that the (34) Sun, S.; Ho-Si, P. H.; Harrison, J. D. Langmuir 1991, 7, 727.

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Figure 5. Typical SPM images taken inside the domains shown in Figure 3 for (A) 15 and (B) 25 mN/m samples. The images show only the morphologically highest components (appearing as light objects), which are included in the relative surface density (RSD) calculation by grain size analysis.The height scale is 10 nm, and the image size is 1 × 1 µm2. (inset) Crosssection profile over typical Fab′ fragments in 15 mN/m sample.

surface was not fully covered with antibodies. The lower than expected RSD values of Fab′ fragments were believed to be mainly due to the random orientation of the Fab′ molecules at the air-water interface, which prevented some of the antibodies from participating in a thioldisulfide interchange reaction with the linker molecules. The results demonstrate that Fab′ fragments can be used as labeling agents for identification of biofunctional molecules in binary monolayers, enabling the visualization of condensed domains in the solid-supported monolayers. Acknowledgment. Financial support from the National Technology Agency of Finland (Grant 40141/00) is gratefully acknowledged. LA020791D