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Langmuir 1996, 12, 3276-3281
Incorporation of Lipid-Tagged Single-Chain Antibodies into Lipid Monolayers and the Interaction with Antigen Inger Vikholm,*,† Erika Gyo¨rvary,‡ and Jouko Peltonen‡ Chemical Technology, Technical Research Centre of Finland, P.O. Box 14021, FIN-33101 Tampere, Finland, and Department of Physical Chemistry, A° bo Akademi University, Turku, Finland Received October 30, 1995X Various amounts of a bacterially produced lipid-tagged single-chain antibody were incorporated into phospholipid monolayers preformed at the air-water interface. The mixed layers were transferred onto octadecyl mercaptan-treated gold films, and the binding of hapten, which was used as antigen, was determined by surface plasmon resonance. Incorporation of the single-chain antibody, transfer of the layer onto solid slides, amount of nonspecific adsorption, and thus the amount of specific binding depended on the composition of the lipid matrix. Studies by atomic force microscopy revealed that the film consisted of antibody-enriched lipid domains and that the films were not stable when stored long times in aqueous solution.
Introduction The main problem in the design of a sensing surface for immunoassay is the production of homogeneously oriented and stable antibody layers. The Langmuir-Blodgett (LB) technique makes it possible to produce uniform and molecularly ordered organic films on solid supports.1 The technique for preparing monolayers of proteins is, however, not as straightforward as that of conventional insoluble monolayers. Different approaches have been employed to construct artificial membranes by the LB technique; monoclonal antibodies have been spread directly onto the air-water interface,2-4 adsorbed from the subphase to a charged lipid matrix,5,6 or bound to preformed phospholipid LB layers.7 The antibodies have a random distribution at the air-water interface2-4 and site-directed immobilization could not be obtained by adsorption.5-7 Fab′ fragments have been used in order to improve orientation and surface density.8,9 Fab′ fragments covalently bound to phospholipids have been embedded into a monolayer by fusion from vesicles.9 Membrane proteins have been reconstituted into lipid monolayers by allowing vesicles to fuse or by spreading the proteins from a suitable detergent solution onto a preformed lipid layer at the air-water interface.9-12 In order to obtain an oriented and sensitive layer of antibodies, our approach has been to incorporate a lipid* Author to whom correspondence should be addressed. † Technical Research Centre of Finland. ‡ A ° bo Akademi University. X Abstract published in Advance ACS Abstracts, May 15, 1996. (1) Hann, R. A. Langmuir-Blodgett Films; Roberts, G., Ed.; Plenum Press: New York, 1990; Chapter 2. (2) Ahluwalia, A.; Rossi, D. D.; Rwastori, C.; Schirone, A.; Serra, G. Biosensors Bioelectron. 1992, 7, 207. (3) Turko, I. V.; Yurkevich, I. S.; Chashchin, V. L. Thin Solid Films 1991, 205, 113. (4) Dubrovsky, T. B.; Demcheva, M. V.; Savitsky, A. P.; Mantrova, E. Y.; Yaropolov, A. I.; Savransky, V. V.; Belovolova, L. V. Biosensors Bioelectron. 1993, 8, 377. (5) Vikholm, I.; Teleman, O. J. Colloid Interface Sci. 1994, 168, 125. (6) Barraud, A.; Perrot, H.; Billard, V.; Martelet, C.; Therasse, J. Biosensors Bioelectron. 1993, 8, 39. (7) Wright, L. L.; Palmer, A. G.; Thompson, N. Biophys. J. Biophys. Soc. 1988, 54, 463. (8) Ahluwalia, A.; Carra, M.; Rossi, D. D.; Rwastori, C. Thin Solid Films 1994, 247, 244. (9) Fischer, B.; Heyn, S. P.; Egger, M.; Gaub, H. E. Langmuir 1993, 9, 136. (10) Scho¨nhoff, M.; Lo¨sche, M.; Meyer, M.; Wilhelm, C. Prog. Colloid Polym. Sci. 1992, 89, 243. (11) Krull, U. J.; Brown, R. S.; Vandenberg, E. T.; Heckl, W. M. J. Electron Microsc. Tech. 1991, 18, 212.
S0743-7463(95)00949-8 CCC: $12.00
tagged single-chain antibody into phospholipid monolayers at the air-water interface and to transfer this layer onto the sensor surface. We have used surface plasmon resonance, SPR, to determine nonspecific and specific binding of protein. SPR is sensitive to changes in the refractive index and the thickness of the film on the metal surface. SPR has been applied to study molecular layers deposited on silver and gold surfaces,13 protein binding to supported lipid membranes,14 and interaction between antigens and antibodies15 and between streptavidin and biotin.16 Furthermore, we have employed atomic force microscopy, AFM, to measure both the topography and frictional forces of the film. In recent years AFM has been used to image the topography of lipid layers and proteins.17-19 The ability to image in liquid and, in addition, to measure frictional forces further increases the interest toward this technique.20,21 As a model protein we have used a single-chain antibody of an anti-2-phenyloxazolone IgG1 (Ox scFv) consisting of the variable domains of the heavy chain and light chain, joined together by a short fungal cellulose linker, which had been expressed and secreted in Escherichia coli.22 Ox scFv was fused with the major lipoprotein of E. coli by genetic engineering, in order to produce a biosynthetically lipid-tagged antibody.23 The lipid-tagged single-chain antibody designed as Ox lpp-scFv displayed membrane protein properties and specific hapten-binding activity both after solubilization with nonionic detergents and after (12) Heyn, S. P.; Egger, M.; Gaub, H. E. J. Phys. Chem. 1990, 94, 5073. (13) Rothenhausler, B.; Duschl, C.; Knoll, W. Thin Solid Films 1988, 159, 323. (14) Errettaz, S.; Stora, T.; Duschl, C., Vogel, H. Langmuir 1993, 9, 1361. (15) Sadana, A.; Sii, D. J. Colloid Interface Sci. 1992, 151, 166. (16) Ha¨ussling, L.; Ringsdorf, H.; Schmidt, F.; Knoll, W. Langmuir 1991, 7, 1837. (17) Weisenhorn, A. L.; Drake, B.; Prater, C. B.; Gould, S. A. C.; Hansma, P. K.; Ohnesorge, F.; Egger, M.; Heyn, S.-P.; Gaub, H. E. Biophys. J. 1990, 58, 1251. (18) Radmacher, M.; Tillman, R. W.; Fritz, M.; Gaub, H. E. Science 1992, 257, 1900. (19) Overney, R. M.; Meyer, E.; Frommer, J.; Gu¨ntherodt, H.-J. Langmuir 1993, 9, 341. (20) Green, J.-B.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys. Chem. 1995, 99, 10960. (21) Hoh, J. H.; Engel, A. Langmuir 1993, 9, 3310. (22) Takkinen, K.; Laukkanen, M.-L.; Sizmann, D.; Alfthan, K.; Immonen, T.; Vanne, L.; Kaartinen, M.; Knowles, K. C.; Teeri, T. Protein Eng. 1991, 4, 837. (23) Laukkanen, M.-L.; Teeri, T.; Keina¨nen, K. Protein Eng. 1993, 6, 449.
© 1996 American Chemical Society
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incorporation to liposomes.23 In preliminary studies we have, furthermore, showed that Ox lpp-scFv can be incorporated into a phospholipid monolayer matrix at the air-water interface and transferred onto solid supports with remaining functional activity.24 Different requirements have, however, to be taken into account when selecting a proper lipid matrix for the incorporation of membrane proteins. The lipid environment has to enable solubilization of the protein but, on the other hand, transfer of the monolayer onto a solid substrate requires a highly ordered film, that preferably exists in a condensed-like state.10 The main purpose of this study has been to compare the influence of different lipid matrices on the antibody incorporation in order to increase the activity of the layer and reduce nonspecific binding. Experimental Section Materials. 1,2-Dimyristoyl phosphatidylcholine (DMPC), 1,2dipalmitoyl phosphatidylcholine (DPPC), 1,2-dimyristoyl phosphatidylethanolamine (DMPE), 1,2-dipalmitoyl phosphatidylethanolamine (DPPE), 1,2-dipalmitoyl phosphatidic acid (DPPA), cholesterol (CHOL), and arachidic acid were obtained from Fluka. Bovine serum albumin (BSA) was purchased from Sigma. The single-chain lipid-tagged antibody (Ox lpp-scFv) solubilized in 1% n-octyl β-D-glucopyranoside and BSA conjugated with approximately 16 groups of 2-phenyloxazolone, Ox16BSA was a kind gift from Dr. M.-L. Laukkanen.23,25 Monolayer Formation and Deposition. A KSV-2200 LB system was used for monolayer formation and deposition. Three different lipid matrices were prepared: DMPC/DMPE was mixed in a molar ratio of 9/1, DPPC/DPPE/DPPA in a molar ratio of 3/2/5, and DPPC/DPPE/DPPA/CHOL in a molar ratio of 3/3/ 1.5/2.5. The lipids were dissolved in chloroform and spread onto a 0.25 mM CaCl2 subphase buffered at pH 7.4 with NaH2PO4NaOH. The lipids were compressed to a surface pressure of 10 mN/m, and hereafter Ox lpp-scFv was added by droplets onto the interface. The initial surface pressure was maintained constant. The mixed layer was compressed to a surface pressure of 30 mN/m and transferred onto solid slides. The layers were transferred by horizontal deposition by moving the slides through the monolayer-covered interface into the subphase. The slides were kept in the subphase until further analysis was performed, in order to avoid protein denaturation. Glass slides were cleaned in a Piranha solution (hydrogen peroxide/sulfuric acid (1:4)) at 70 °C for 1 h, rinsed with water, and stored in sodium hydroxide at pH 10 for 12 h. The glass slides were hereafter coated with five layers of cadmium arachidate at a surface pressure of 30 mN/m by conventional vertical deposition in order to render the surfaces hydrophobic.26 Glass slides onto which a thin gold film had been e-beam evaporated were used for SPR measurements. These slides were, prior to deposition of the antibody/lipid layer, soaked in a 1 mM solution of octadecyl mercaptan in ethanol for 24 h and rinsed with ethanol. Atomic Force Microscopy. A Nanoscope III (Digital Instruments, Inc., Santa Barbara, CA) AFM in the contact mode was used for the sample surface imaging in a liquid environment. The scanner head J (150 µm exact scan range) was applied with a liquid cell. Cantilevers with different spring constants (k) and lengths were tested for imaging: 0.58 N/m (100 µm), 0.38 N/m (100 µm), 0.12N/m (200 µm), and 0.06 N/m (200 µm). The cantilevers of 0.38 and 0.12 N/m were found to be the most suitable ones, resulting in the most stable imaging. To further prevent the cantilever from modifying the sample surface, the applied force was first minimized on a small scanning area. The samples were stored in a NaH2PO4-NaOH buffer at pH 7.4 prior to transfer to the liquid cell. This was done by placing an O-ring seal onto the sample, thus preventing the layer from being (24) Vikholm, I.; Peltonen, J. Thin Solid Films, in press. (25) Laukkanen, M.-L.; Alfthan, K.; Keina¨nen, K. Biochemistry 1994, 33, 11664. (26) Vikholm, I.; Peltonen, J.; Teleman, O. Biochim. Biophys. Acta 1995, 1233, 111.
Figure 1. Surface pressure-area isotherm of (a) DPPC/DPPE/ DPPA/CHOL in a molar ratio of 3/3/1.5/2.5, (b) the lipid mixture on addition of Ox lpp-scFv, (c) DPPC/DPPE/DPPA in a molar ratio of 3/2/5, and (d) on addition of Ox lpp-scFv. The molar fraction of Ox lpp-scFv incorporated into the lipid matrix was 0.027. The molecular surface area was calculated from the total amount of lipids and Ox lpp-scFv spread. exposed to air. The same buffer was used during imaging. The lateral forces (friction) and normal forces (topography) acting on the tip were measured simultaneously. The Quartz Crystal Microbalance. The amount of film transferred by horizontal deposition was measured with a 10 MHz quartz crystal microbalance, QCM, purchased from Universal Sensors, Inc. (USA).26 The quartz crystal was connected to a custom-made oscillator and driven at 5 V dc. The frequency of the vibrating quartz was measured in air by a universal frequency counter (HP 5316B) as previously described.5 Surface Plasmon Resonance. The configuration of the optical experiment has been described by Sadowski et al.27 Briefly, linearly p-polarized light of a wavelength 632.8 nm from a He-Ne laser is directed through a prism onto a slide, that is coated with a thin gold film, positioned according to the so-called Kretschmann configuration.28 The intensity of the reflected light is measured as a function of the angle of incidence, θ, using a photodiode with a chopper/lock-in amplifier technique. At the angle of excitation of surface plasmons, a minimum in intensity of the reflected light is observed. The angle position of this minimum depends on the thickness and optical properties of the layer on the gold film.27 The coated slide was taken from the 1 mM NaH2PO4-NaOH buffer solution and the lipid/Ox lpp-scFv layer was exposed to air while the slide was placed on the prism with index matching oil. A liquid cell was placed over the measurement area of the film and was filled with buffer. The SPR resonance curve was recorded and the angle of incidence was driven to a point slightly above the resonance minimum. BSA at a concentration of 0.1 mg/mL was injected into the cell, and changes in the intensity of the light were detected. BSA was added several times until no additional change in intensity was observed, in order to block nonspecific protein-binding sites. Hereafter various concentrations of Ox16BSA, which was used as the antigen, were injected into the cell. An additional increase in intensity was observed in the case that Ox lpp-scFv had been incorporated into the lipid layer.24 Part of the protein causing the intensity change could, however, be removed from the surface by rinsing with buffer.24 The concentration of Ox16BSA injected into the measuring cell was gradually raised and the surface was rinsed with buffer between every injection of protein in order to determine the amount of antigen specifically bound. The intensity was allowed to stabilize and the difference between the levels was recorded. The SPR measurements were performed immediately after deposition of the antibody/lipid film, and storage of the film was reduced to a few hours.
Results and Discussion Monolayer Formation and Deposition. In Figure 1 the surface pressure-area isotherm shows the mean (27) Sadowski, J.; Korhonen, I.; Peltonen, J. Opt. Eng. 1995, 34, 2583. (28) Kretschmann, E.; Raether, H. Z. Naturforsch., A 1968, 23, 2135.
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Vikholm et al. Table 2. Surface Density of the Antibody, DΛOx lpp-scFv Obtained from the Amount of Film Transferred onto the QCM, and the Transfer Ratio, Tr, for the Different Lipid Matricesa lipid matrix
∂ΛOx lpp-scFv (%)
Tr
DMPC/DMPE DPPC/DPPE/DPPA DPPC/DPPE/DPPA/CHOL
36 52 25
0.7 0.9 0.4
a T was calculated as a ratio between the mass of the layer r transferred onto the QCM and the amount of material spread onto the interface. The antibody surface area was taken to be 1200 Å2. The mole fraction of Ox lpp-scFv in the film was 0.03.
Figure 2. Increase of the molecular area of DMPC/DMPE (b), DPPC/DPPE/DPPA (2), and DPPC/DPPE/DPPA/CHOL (9) after addition of antibody as a function of mole fraction of Ox lpp-scFv at a surface pressure of 30 mN/m. Table 1. Surface Area Characteristics of Ox-lpp scFv for the Different Lipid Matrices lipid matrix
XOx lpp-scFv
∂Aa (%)
AOx lpp-scFvb (Å2/molecule)
DMPC/DMPE DPPC/DPPE/DPPA DPPC/DPPE/DPPA/CHOL
0.03 0.05 0.06
40 50 60
700 1200 1300
a The relative increase in molecular surface area, ∂A, on addition of Ox lpp-scFv to saturation. b The molecular surface area of Ox lpp-scFv at a surface pressure of 30 mN/m, calculated from the surface area increase assuming an ideal mixing behavior.
molecular surface area of the total amount of lipids and the antibody fragment spread onto the air-water interface. The molecular surface area increased on addition of Ox lpp-scFv. The packing of the lipids might, however, be affected by incorporation of antibodies. At a higher surface pressure the antibodies seemed to be squeezed out of the DPPC/DPPE/DPPA/CHOL matrix. This could be due to the condensing effect of CHOL on the monolayer.29 Membrane proteins cannot be embedded into monolayers that are too rigid.10 A sufficient evaporation time of the solvent turned out to be crucial for film formation. If the delay before spreading and compression was less than 15 min, the film collapsed at a surface pressure much lower than otherwise observed. The added antibody might be influenced by solvent remaining in the lipid matrix. If the monolayer was expanded to a surface pressure below 5 mN/m, after addition of the antibody, a sudden increase in surface pressure was observed. This could be due to the adsorption of detergent from the subphase. The detergent molecules do not have monolayer-forming properties but should be transferred to the subphase as micelles. It can be assumed that most of the detergent was compressed out of the layer at the surface pressure of 30 mN/m used for deposition. In Figure 2 the relative increase in surface area at a surface pressure of 30 mN/m is plotted as a function of mole fraction of Ox lpp-scFv. Antibodies could be incorporated with a scattering in ∂A of about 6%. This scattering was most affected by the spreading method used. The amount of antibody that was incorporated depended on the lipid matrix and increased in the order of DMPC/DMPE < DPPC/DPPE/DPPA < DPPC/DPPE/ DPPA/CHOL. A maximum number of molecules could be incorporated as indicated by the plateau value (Table 1). The molecular area of Ox lpp-scFv can be estimated from the increase in surface area, if an ideal mixing behavior (29) Chapman, D. Langmuir 1993, 9, 39.
was assumed. A value quite close to the theoretical surface area for an antibody fragment (3 × 4 nm2) was obtained for antibodies incorporated into the DPPC/DPPE/DPPA and DPPC/DPPE/DPPA/CHOL matrix, whereas the antibody incorporated into the DMPC/DMPE matrix gave a molecular surface area of about 7 nm2 (Table 1). The molecular area did not considerably depend on the antibody mole fraction ((2 nm2). In order to evaluate the transferability of the layers, a QCM was employed. The lowest surface mass density was obtained for the DPPC/DPPE/DPPA/CHOL matrix, although the increase in surface area had been the largest (Table 2). The transfer ratio for this layer was very poor, whereas it was considerably better for the other matrices, especially for DPPC/DPPE/DPPA (Table 2). The interaction between the lipid-tagged antibody fragment and the phospholipid matrix can be expected to be electrostatic at the polar head group region and hydrophobic in the hydrocarbon core. Incorporation of antibodies should thus be affected by the charge density at the membrane surface and, on the other hand, the lipid density should determine the magnitude of the hydrophobic interaction. The high amount of antibodies incorporated and the good transfer of DPPC/DPPE/DPPA/Ox lpp-scFv could be due to the fact that the ratio between zwitterionic and anionic lipids was chosen to be the same as in cells of Escherichia coli,30 the natural surrounding of the antibody. Atomic Force Microscopy. The height image of DMPC/DMPE/Ox lpp-scFv in Figure 3a (left) reveals that the film is quite rough and nonhomogeneous. Typical characteristics of the underlying cadmium arachidate layers were clearly visible in the image. The layers were also nonhomogeneous as revealed by a stepwise structure, most often corresponding to a bilayer (5.6 nm) or even twice a bilayer thickness of cadmium arachidate.26 Domains of different height could be observed on top of the fatty acid layer. The friction force measurement mode in Figure 3a (right) shows a dark/bright contrast, which indicates that the surface consists of two components of low (dark) and high (bright) friction. The scale in the friction images is in relative units, not corresponding to absolute values of friction. This is due to the fact that we did not calibrate the friction with respect to the total normal force between the sample and the tip. The average height of the domains with low friction was 6.4 nm and corresponds well to the expected length of the lipid-tagged antibody (about 6 nm). These dark areas of low relative friction are suggested to correspond to antibody-enriched lipid areas. Segregation into protein-rich domains has also been observed from the respective AFM measurements in air.24 The domains were not homogeneous with respect to the height within a domain. Histograms revealed height levels of about 2.3 nm (data not shown), relative to the underlying fatty acid layer, indicating the existence of the lipid matrix layer. The poor transfer ratio (30) Rilfors, L.; Lindblom, B.; Weislander, A° .; Christiansson, A. In Biomembranes; Kates, M., Manson, L. A., Eds.; Plenum Press: New York and London, 1984; Vol. 2, p 205.
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Figure 3. Simultaneously measured topography (left) and friction (right) AFM images of (a, top) DMPC/DMPE/Ox lpp-scFv and (b, bottom) DPPC/DPPE/DPPA/Ox lpp-scFv in buffer. In the topography images the color contrast is as follows: dark ) low and bright ) higher areas. Friction images show dark ) lower friction and bright ) higher friction. The mole fraction of Ox lpp-scFv was 0.03. The layers were transferred onto glass slides made hydrophobic with five layers of cadmium arachidate. The different surface characteristics explained in the text have been indicated by arrows: the steplike structure of cadmium arachidate (arrow 1), antibody-enriched areas (arrow 2), and higher domains (arrow 3).
and storage of the film in buffer for some days before imaging may have caused some peeling off or reorganization of the film. Reorganization of LB films in liquids and the growth of holes have been reported by different groups.19,31,32 Storage of the films in buffer for 1 week showed that the size and the number of holes increased (data not shown). Films stored and imaged in an air environment were much more homogeneous.24 Both topography and friction images of the DPPC/DPPE/ DPPA/Ox lpp-scFv film in Figure 3b consist of three different height levels. The lowest height level in the (31) Schwartz, D. K.; Viswanathan, R.; Zasadzinski, J. A. N. J. Phys. Chem. 1992, 96, 10444. (32) Gyo¨rvary, E.; Peltonen, J.; Linde´n, M.; Rosenholm, J. Thin Solid Films, in press.
image was supposed to be the surface of the pure lipid layer. This conclusion comes from the fact that no features characteristic of the underlying cadmium arachidate multilayer structure were visible in the image.26 This also indicated that the transfer of this lipid matrix was much better than that of DMPC/DMPE/Ox lpp-scFv. The small domains in the image had an average height of 3 nm and appeared as areas of low friction. These domains are assumed to consist of single-chain antibodies, protruding from the layer, as the height corresponds well to a single-chain antibody. Ox lpp-scFv was aggregated into smaller domains and they were more homogeneously distributed in the DPPC/ DPPE/DPPA matrix than in the DMPC/DMPE matrix. Furthermore, there was a greater amount of domains in
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Figure 4. Binding isotherms of Ox16BSA to layers of DMPC/ DMPE (b) and DPPC/DPPE/DPPA (2) incorporating Ox lppscFv at a mole fraction of 0.03. The intensity change caused by BSA was reduced. The layers were transferred onto Au made hydrophobic with octadecyl mercaptan.
the DPPC/DPPE/DPPA matrix. Larger domains of height 6-10 nm are visible in both matrices. The origin of these domains is not known and they were not visible in all images. One possible explanation is that they are detergent micelles from the subphase adsorbing to the layer, but they may also be due to artifacts. The regular horizontal lines especially visible in Figure 3 might be due to a deformation of the surface during scanning, showing that the tip-sample interaction forces should be even further reduced. This would be possible by applying the tapping mode AFM, but this option is not yet included in our microscope. Imaging of the films after storage in buffer for 1 week indicated a much higher stability of the DPPC/DPPE/ DPPA/Ox lpp-scFv layer, with only a partial peeling off the lipid layer. This could explain the difference in roughness of the layers. On the other hand, it was found out that the binding between cadmium arachidate and the pure DPPC/DPPE/DPPA layer without antibodies was not stable. The AFM images showed a clear peeling off of the phospholipid layer after 1 week storage in buffer. Incorporation of Ox lpp-scFv to the matrix made the film more stable. This agrees with earlier results on proteins having a stabilizing effect on monolayers.5,11 Protein-Binding Measurements. In a previous study we have shown that when Ox16BSA was allowed to interact with a layer of DMPC/DMPE/Ox lpp-scFv, previously blocked with BSA, there was an additional shift in the intensity of the SPR signal.24 A similar change in the intensity of light was observed when Ox lpp-scFv had been incorporated into a monolayer of DPPC/DPPE/DPPA. Figure 4 shows that the intensity change was dependent on the concentration of Ox16BSA. The change was higher for the DPPC/DPPE/DPPA matrix, which can be explained by both a higher incorporation of antibodies and a better transfer ratio of the layer (Table 2). The total change in intensity obtained from the SPR curve after interaction of BSA and Ox16BSA to saturation for the different lipid matrices employed is compared in Figure 5. BSA has no special affinity for lipid membranes, although it does bind to fatty acids and adsorb to almost any surface.33 Under physiological conditions most proteins adsorb to a larger extent to hydrophobic than to hydrophilic surfaces.33,34 Nonspecific binding of BSA to the DPPC/DPPE/DPPA/CHOL/Ox lpp-scFv layer was (33) Go¨lander, C.-G.; Kiss, E. J. Colloid Interface Sci. 1988, 121, 240. (34) Wright, L. L.; Palmer, A. G.; Thompson, N. L. Biophys. J. 1988, 54, 463.
Vikholm et al.
Figure 5. Total change in intensity caused by the interaction of BSA and Ox16BSA to saturation with the different Ox lppscFv incorporating phospholipid layers transferred onto Au treated with octadecyl mercaptan.
Figure 6. Schematic view of the self-assembled layer of octadecyl mercaptan and the lipid matrix incorporating lipidtagged single-chain antibodies built up onto a gold film and the binding of BSA and Ox16BSA.
remarkably high. This was expected as the transfer ratio was very poor. Holes in the layer act as nonspecific adsorption sites, because BSA interacts with the hydrophobic chains of the layer. The interaction of Ox16BSA with the membrane was, in addition, influenced by the low incorporation of protein. The transfer ratio also affected the adsorption of BSA to the DMPC/DMPE/Ox lpp-scFv layer. The amount of nonspecific and specific binding was almost equal. The nonspecific binding may also have been affected by defects in the octadecyl mercaptan layer. Nanometer scale defects similar to those in LB films, but originating from dissolution of gold, have been reported in self-assembled films.35-38 The gold film used in this study was, furthermore, much more rough than the glass slide onto which the layers of cadmium arachidate were transferred.27 The nonspecific binding of BSA to the DPPC/DPPE/DPPA/Ox lpp-scFv layer was very low. This could be explained by a good transfer ratio, giving a homogeneous layer with a minimum of pinholes. Furthermore, PC layers tightly packed do not readily bind proteins.29 The highest specific interaction was obtained with this matrix. The attempted molecular arrangement is shown in Figure 6. (35) Kim, Y. T.; Bard, A. J. Langmuir 1992, 8, 1096. (36) Edinger, K.; Go¨lzha¨user, A.; Demota, K.; Wo¨ll, Ch.; Grunze, M. Langmuir 1993, 9, 4. (37) McCarley, R. L.; Dunaway, D. J.; Willicut, R. J. Langmuir 1993, 9, 2775. (38) Geddes, N. J.; Paschinger, E. M.; Furlong, D. N.; Caruso, F.; Hoffmann, C. L.; Rabolt, J. F. Thin Solid Films 1995, 260, 192.
Single-Chain Antibody/Lipid Monolayers
Conclusions The results presented in this paper show that the incorporation of a biosynthetically lipid-tagged singlechain antibody into lipid monolayers at the air-water interface and the transfer onto solid supports is dependent on the lipid matrix. The increase in molecular area of the DPPC/DPPE/DPPA/CHOL matrix on the incorporation of antibodies was the highest of the matrices studied, but a poor transfer ratio led to a high nonspecific adsorption of protein and subsequently to a low specific binding. The antibodies were segregated into protein-rich domains, that were smaller and more homogeneously distributed if DPPC/DPPE/DPPA was used as a layer matrix, instead of DMPC/DMPE. The binding of hapten, which was used as antigen, was also higher to DPPC/DPPE/DPPA/Ox lppscFv. AFM measurements indicated that the peeling off
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lipids, when the layer was stored in aqueous solution, could turn out to be a crucial problem, when using the layer for immunoassay. Further studies are therefore going to deal with the long-term stability of the layer. In the context of a future practical immunosensor, lipidtagged single-chain antibodies of clinical interest have, however, to be produced. Acknowledgment. We thank Drs. Marja-Leena Laukkanen and Kari Keina¨nen (Technical Research Centre of Finland, Biotechnology and Food Research) for providing the Ox lpp-scFv antibody and for helpful comments on the manuscript. This work has been supported by the Academy of Finland. LA950949C
