Langmuir 1992,8, 1413-1416
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Specific Recognition-Induced Self -Assembly of a Biotin Lipid/Streptavidin/Fab Fragment Triple Layer at the Air/Water Interface: Ellipsometric and Fluorescence Microscopy Investigations James N. Herron,?Wolfgang Muller,$Michaela Paudler,s Hans Riegler,g Helmut Ringsdorf,’ and Peter A. Suci*J Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112, Insitut fiir Organische Chemie, Universitat Mainz, J.- J.-Becherweg 18-20, 6500 Mainz, Germany, Jakob- Welder- Weg 11, Insitut fiir Physikalische Chemie, Universitiit Mainz, J.-J.6500 Mainz, Germany, and Center for Interfacial Microbial Process Engineering, Montana State University, Bozeman, Montana 5971 7 Received July 29, 1991. I n Final Form: February 12, 1992 Self-assembled biotin lipid/streptavidin/Fab triple layers at the aidwater interface were investigated by ellipsometry and fluorescence microscopy. The triple layer was prepared by spreading a biotin lipid monolayer at the aidwater interface and then by sequentiallyinjecting solutions of first streptavidin and then biotinylated Fab into the subphase. Between the injections the subphase was flushed with pure buffer. The preparation method was designed to promote the formation of a close-packed monolayer of confluent streptavidin (crystalline)domains which served as a template for Fab binding. Ellipsometric measurements indicated a dense attachment of the biotinylatedFab to this streptavidin template through a specific interaction with the streptavidin binding sites.
Introduction The methods of constructing lamellar, molecular-scale, multilayer films are conventionally classifiedas either “LB deposition-, or as ‘self-assembly”.l A variant of the selfassembly technique employs specific protein/ligand interactions as a self-organizingdriving force, using proteins as fundamental building block~.~a Proteins offer many built-in functional possibilities, and thus efforts directed toward their incorporation into ultrathin films have been inten~ive.~ Construction of specific-interaction-directed, selfassembled protein films has typically been performed at the aidwater interface. In-plane order can be greatly enhanced by exploiting the combination of planar orientation and mobility which this interface provides. With respect to biomolecules, this is demonstrated by the spontaneous formation of two-dimensional (2-D) crystalline protein domains upon binding to the appropriately functionalized lipid m ~ n o l a y e r . One ~ ~ ~ of the proteins which can be induced to crystallize in this manner is streptavidin. Streptavidin is an exceptionally resilient tetrameric protein, the four subunits of which all bind the biotin ligand with a free energy of binding which is comparable to that of a covalent bond.7 Streptavidin serves as a molecular linker between biotin-derivatized units for various + University of Utah.
Institut fih Organische Chemie, Universitit Mainz. Institut ftir Physikalische Chemie, Universitit Mainz. 1 Montana State University. (1) Ulman, A. Anlntroduction to Ultrathin OrganicFilmsfromLangmuirdlodgett to Self-assembly;Academic Press, Inc.: New York, 1991. (2) Ahlers, M.; Miiller, W.; Reichert, A.; Ringsdorf, H.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1990,29, 1269. (3) Hoffmann, M.; Miiller, W.; Reichert, A.; Ringsdorf, H.; Rump, E. Makromol. Chem., Macromol. Symp. 1991,48149,435. (4) Lvov, Y. M.; Erokhin, V. V.; Zaitaev, S. Y. Biol. Membr. 1990, 7, 917. ( 5 ) Uzgiris, E.E.; Kornberg, R. D. Nature 1983, 301, 125. (6) Uzgiris, E. E. Biochem. J. 1987, 242, 293. (7) Green, N. M. Adu. Protein Chem. 1975,29, 85. t
biotechnological applications.8 Particularly relevant in the present context is that the four biotin binding sites have been shown to be arranged in two opposing pairs in each molecule of the oriented 2-D crystals such that two sites per streptavidin molecule are bound to the lipid layer and the remaining two sites are unoccupied.+ll Astreptavidin 2-D crystal thus presents a matrix of well-ordered binding sites to the adjacent subphase which may be employed as a template for further construction or functionalization using biotinylated molecules. In principle, surfaces with binding specificity for almost any molecule of choice could be prepared using thin films which incorporated monoclonal antibodies.12 In this paper a well-characterizedmonoclonal (antifluorescyl) antibody, Fab,13 was biotinylated and attached to a streptavidin template at the aidwater interface. The resulting triplelayer structure (biotin lipid/streptavidin/Fab) was investigated using ellipsometry. The Fab can be described globally by a relatively simple geometric shape (an ellipsoid), and therefore it might be expected to integrate in a well-ordered manner into a self-organized multilayer structure. In support of this prediction results presented here indicate that the adsorbed Fab molecules preferentially orient themselves on the streptavidin template to form a densely packed monolayer in which Fab molecules have an axial orientation (long axis of the ellipsoid perpendicular to the plane of the interface). A preliminary investigation of the extent of availability of the templatebound Fab antigen binding sites to subphasic (fluorescein) haptens was made. (8)Wilchek, M.; Bayer, E. A. In Methods in Enzymology; Abelaon, J. N., Simon, M. I., Eds.; Academic Press, Inc.: New York, 1990; Vol. 184. (9) Blankenburg,R.;Meller,P.;Ringsdorf,H.; Salesse, C. Biochemistry 1989,28,8214.
(10) Ahlers, M.; Blankenburg, D.; Grainger,D. W.; Meller, P.; Ringsdorf, H.; Salesse, C. Thin Solid Films 1989, 180, 93. (11) Darst,S. A.; Ahlers, M.; Meller, P. H.; Kukalek, E. W.; Blankenburg,R.;Ribi, H. 0.;Ringsdorf, H.; Komberg, R. D. Biophys. J. 1991, 59, 387. (12) Winter, G.;Milstein, C. Nature 1991,349, 293. (13) Herron,J.N.; He, X.; Mason, M. L.;Voss, E. W., Jr.; Edmundson, A. B. Proteim 1989,5, 271.
0743-746319212408-1413$03.00/0 0 1992 American Chemical Society
Herron et al.
1414 Langmuir, Vol. 8, No. 5, 1992
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Figure 1. Chemical structure of the biotin lipid N-biotinyl-S[1,2-bis(octadecyloxycarbonyl)ethyl]cysteamine.
Figure 2. Fluorescence micrograph of streptavidin domains bound to a biotin lipid monolayer at the air/water interface. For visualization the streptavidin has been labeled with fluorescein and is observed with polarized excitation light. The domains are optically anisotropic which is responsible for their different intensities. (The intensities vary when the plane of polarization of excitation light is rotated.) 1 cm represents 50 pm.
Materials and Methods The synthesis of the biotinylated lipid N-biotinyl-S-[ l,2-bis(octadecyloxycarbony1)ethyl]cysteamine as well as its monolayer behavior with and without streptavidin in the subphase is described el~ewhere.~ The lipid (Figure 1) was spread to the desired surface coverage (500 A2/molecule)from a 5 X 10-5 M CHCl3 solution. The aqueous subphase (Millipore water) contained 0.5 M NaC1. The method used to induce formation of (confluent) 2-D streptavidin crystals had two variants which produced in all cases identical results: (1)biotin lipid was spread, and then 300 pL of a lo+ M streptavidin solution (streptavidin from Boehringer, Mannheim, FRG) was injected at various points into the subphase (resulting subphase concentration 8 X M) or (2) biotin M lipid was spread onto a subphase already containing 8 X streptavidin. In either case the subphase was then heated to 36 "C, and after 2 h, cooled back to 20 "C within 30 min. Both variants described above produced confluent 2-D streptavidin crystals attached to the biotin lipid monolayer (Figure 2). The streptavidin used for the fluorescence microscopy studies was statistically labeled with two molecules of fluorescein isothiocyanate per protein according to standard ~r0cedures.l~ The Fab, derived from a monoconal (4-4-20) antifluorescyl immunoglobulin G antibody, has been sequenced,15and crystallized, and its three-dimensional structure has been determined.l3 The Fab was biotinylated by covalentbinding of a biotin active ester. For this purpose an active ester derivative of biotin (6-[64biotinoylamino)hexanoyl] aminohexanoic acid, succinimidyl ester, Molecular Probes) was added to a solution of Fab in phosphate buffer (0.05 M Na2HP04, pH 9) to achieve a 2:l molar ratio of biotin reagent:Fab. This solution was then stirred for 48 h at room temperature. The reaction results in binding to primary amines (lysine residues or peptide chain terminal amines). High molecularweight componentswere then separated on a PD-10 sephadex column and (by preequilibration of the (14) Nargessi, R. D.; Smith, D. S. Methods Enzymol. 1986, 122, 67. (15) Bedzyk, W. D.; Johnson, L. S.; Riordan, G.S.; Voss, E. W.,Jr. J. Biol. Chem. 1989, 264, 1565.
column) transferred into a second phosphate buffer (0.05 M NaH2POr, pH 5.6). By using Scatchard analysis,16 the activity of the Fab in the resulting fraction (i.e., binding affinity for fuorescein, K,)was measured as K, = 1.33 X 1Olo (20 "C, pH 8.4) which is 80% of the value published for unbiotinylated Fab a t 2 OC.17The total concentration of Fab, as estimated by UV spectroscopy, was 1.5 X lo+ M, but Scatchard analysis, assuming a valence of 1, indicated that only 22% of the Fab was active. No assay of the extent of biotinylation of the Fab was performed. However, comparison with the extent of labeling achieved with comparable reactive fluorescencelabeling reagents indicates that a t least some lysine residues on each molecule are deprotonated at this pH, and thus quite reactive. For the binding studies of the Fab to the streptavidin layer, quantities of the biotinylated fraction were injected directly into the subphase a t various points distributed along the length of the trough. The ellipsometric measurements were performed with a null ellipsometerin a conventionalPCSA canfiguration.laJg A heliumneon laser served as the light source. Its emission a t 632 nm was well away from any absorption bands of the substances investigated. The area of the incident laser beam on the surface ("footprint") was approximately 1 mm2. The measured data reflect an average of film characteristics taken over this footprint. The variation of the ellipsometric angles 6A and 6 9 relative to a clean subphase surface was used to characterize each of the thin layers (biotin lipid/streptavidin/Fab) in terms of a geometrical thickness and effective refractive index. With very thin films (
