Langmuir 1996, 12, 1997-2006
1997
Immobilization of Antibodies on a Photoactive Self-Assembled Monolayer on Gold E. Delamarche,† G. Sundarababu,‡ H. Biebuyck,† B. Michel,*,† Ch. Gerber,† H. Sigrist,‡ H. Wolf,§ H. Ringsdorf,§ N. Xanthopoulos,| and H. J. Mathieu| IBM Research Division, Zurich Research Laboratory, CH-8803, Ru¨ chlikon, Switzerland, Institut fu¨ r Biochemie, Universita¨ t Bern, CH-3012 Bern, Switzerland, Institut fu¨ r Organische Chemie, Johan-Gutenberg-Universita¨ t, D-55099 Mainz, Germany, and De´ partement des Mate´ riaux, EPFL, CH-1015 Lausanne, Switzerland Received October 5, 1995. In Final Form: January 4, 1996X This paper presents a strategy for immobilizing biomolecules on a photoactivable surface. A selfassembled monolayer is prepared by adsorbing an ω-functionalized dialkyl disulfide on gold. Functional groups of this monolayer are converted in two steps into a benzophenone derivative with an overall yield of 50 ( 10%. Several independent techniques (ellipsometry, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, radiolabel assay, and autoradiography) characterize the reaction and photoimmobilization of antibodies on this surface. The photoimmobilized antibodies cover the surface as a homogeneous and dense monolayer that could not be disrupted by vigorous washing with the detergent Tween 20. Immunoassays demonstrated specific recognition of the immobilized immunoglobulins as measured by their complexation with alkaline phosphatase-linked antibodies. The method of photoimmobilization used here leads to a homogeneous single layer of IgGs, in which the proteins maximize their contact with the surface. Residual adsorption of IgG on the nonirradiated surface of benzophenone remains one limitation of this approach. Progressively higher coverages of IgGs on the surface did not lead to strictly proportional changes of the biological activity of these surfaces, probably because of interactions between the IgGs in the film. This method of photoimmobilization is nonetheless useful as an experimental system to immobilize other proteins because it is simple, flexible, and efficient.
I. Introduction The present contribution develops sequentially a “model” system for the covalent attachment of proteins. The method used follows a versatile approach introduced by Rozsnyai et al.:1 The generation of a photoactive surface by its reaction with a benzophenone derivative. A principal goal of this work was to establish conditions for the formation of a well-characterized photoactivable surface on gold and to explore possible limitations and pitfalls of this approach. Thin gold, a surface having a simple topography with a well-defined chemistry, is functionalized by a dialkyl disulfide.2-4 Conversion of reactive ester groups at the termini of the resulting self-assembled monolayer (SAM) into photoactivable groups based on benzophenone occurred in two steps (Scheme 1). Reaction on the surface was desirable as part of an overall design giving maximum control and flexibility over the state of the surface, although the benzophenone derivative could also be prepared by synthesis in solution followed by adsorption of its disulfide to form the SAM. Coupling takes place at a dense organic interface 1.5 nm away from the gold substrate preventing adventitious attachment of proteins to the gold via disrupted disulfide bridges. Benzophenone is a well-known and widely used cross-linking agent having * Corresponding author: e-mail,
[email protected]. † IBM Research Division, Zurich Research Laboratory. ‡ Universita ¨ t Bern. § Johan-Gutenberg-Universita ¨ t. | De ´ partement des Mate´riaux, EPFL. X Abstract published in Advance ACS Abstracts, March 15, 1996. (1) Rozsnyai, L. F.; Benson, D. R.; Fodor, S. P. A.; Schultz, P. G. Angew. Chem., Int. Ed. Engl. 1992, 31, 759-761. (2) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 44814483. (3) Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc. 1987, 109, 733-740. (4) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. J. Am. Chem. Soc. 1987, 109, 2358-2368.
0743-7463/96/2412-1997$12.00/0
Scheme 1. Derivatization of a Gold Substrate by a Functionalized Disulfide and the Subsequent Conversion of the Resulting Monolayer into a photoactive surface used to attach proteins
several advantages over other photosensitive groups:5-7 It is more stable than diazo esters, aryl azides, and diazirines. It is inert under ambient light and is activated by near-UV (λ > 350 nm) thus avoiding lower, proteindamaging, wavelengths while remaining convenient to use. UV irradiation causes biradical formation at the ketyl center of benzophenone. The O radical abstracts a proton, preferentially from C-H groups R to an electronwithdrawing group (an abundant target in proteins but not water), followed by C-C bond formation on radical recombination.8-10 Protein immobilization can, therefore, proceed in a liquid buffer. (5) Breslow, R.; Baldwin, S.; Flechtner, T.; Kalicky, P.; Liu, S.; Washburn, W. J. Am. Chem. Soc. 1973, 95, 3251-3262. (6) Galardy, R. E.; Craig, L. C.; Jamieson, J. D.; Printz, M. P. J. Biol. Chem. 1974, 249, 3510-3518. (7) Baley, H. Photogenerated Reagents in Biochemistry and Molecular Biology; Laboratory Techniques in Biochemistry and Molecular Biology; Work, T. S., Burdon, R. H., Eds.; Elsevier: Amsterdam, 1983. (8) Dorma´n, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661-5673. (9) Brunner, J. Annu. Rev. Biochem. 1993, 62, 483-574.
© 1996 American Chemical Society
1998 Langmuir, Vol. 12, No. 8, 1996
Control over the deposition of native biomolecules is important in many areas of technology. Proteins deposited on a solid substrate find applications in enzyme-based biosensors,11 the separation of proteins by chromatography,12,13 tissue cultures,14 diagnostic immunoassays,15-17 and biomolecule device architectures,18 for example. Prevention of protein deposition helps prepare cardiovascular and artificial kidney devices and keeps them biocompatible,19 is essential to ensure the accurate dosing of proteins for clinical applications, and is useful in the food-processing industry. The apparent simplicity of methods used to deposit proteins does not immediately reveal a formalism that models it qualitatively. Fifteen years ago, Norde and Lyklema initiated a rationalization of the processes that govern the deposition of proteins at liquid-solid interfaces.20 These authors described adsorption of protein onto a surface using a sequential colloidchemical approach:21 Transport of protein toward the surface is followed by its attachment at the interface. Protein can become irreversibly attached, leading to possible changes of its structure, or desorb.22 Desorption from the surface is particularly important when the protein is dilute in the solution, although specificity and enhanced interaction between the protein and surface may result if the surface offers ligands with an affinity for that protein. Detection of protein on surfaces remains an experimental challenge, more so when the composition and the topology of the surface are not both well-known. Measurements typically average over proteins with different conformation and render the description of the factors that generate the rich behavior of proteins on solid substrates difficult and often speculative. Whitesides and co-workers introduced an experimental system based on SAMs with well-defined structures and chemical composition as a model to study the adsorption of proteins on surfaces.23,24 They concluded that nonspecific adsorption of several representative proteins was enhanced by methyl termination and suppressed by ethylene glycol termination of the SAMs. SAMs with a mixture of these two components showed intermediate amounts of adsorption proportional to the mole fraction of the hydrophobic component in the monolayer. Their results emphasize the utility of molecular-scale control over the interface to influence the placement of proteins by nonspecific adsorption. Specifically linking proteins to surfaces poses several additional challenges. Immobilization of proteins of SAMs provides an opportunity to investigate their structure at surfaces and their relationship with the substrate.25,26 The (10) Sigrist, H.; Collioud, A.; Cle´mence, J. F.; Gao, H.; Luginbu¨hl, R.; Sa¨nger, M.; Sundarababu, G. Opt. Eng. 1995, 34, 2339-2348. (11) Cass, A. E. G., Ed. Biosensors. A Practical Approach; Oxford University Press: New York, 1990. (12) Cooper, T. G. The Tools of Biochemistry; Wiley-Interscience: New York, 1977. (13) Porath, J. Biotechnol. Prog. 1987, 3, 14-21. (14) Maroudas, N. G. Nature 1973, 244, 353-354. (15) Engvall, E.; Perlmann, P. J. Immunol. 1972, 109, 129-135. (16) Hendry, R. M.; Herrmann, J. E. J. Immunol. Methods 1980, 35, 285-296. (17) Gao, H.; Sa¨nger, M.; Luginbu¨hl, R.; Sigrist, H. Biosens. Bioelectron. 1995, 10, 317-328. (18) Koyama, K.; Yamaguchi, N.; Miyasaka, T. Science 1994, 265, 762-765. (19) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 79, 1-63. (20) Norde, W.; Lykleman, J. J. Colloid Interface Sci. 1979, 71, 350366. (21) Lyklema, J. Colloids Surf. 1984, 10, 33-42. (22) Norde, W. Adv. Colloid Interface Sci. 1986, 25, 267-340. (23) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (24) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (25) Karrasch, S.; Dolder, M.; Schabert, F.; Ramsden, J.; Engel, A. Biophys. J. 1993, 65, 2437-2446.
Delamarche et al.
fragile structure of proteins and their susceptibility to irreversible denaturation limit the scope and conditions for coupling reactions. Nonspecific deposition of protein may also occur on nonactivated regions, thus diminishing the utility of photoattachment methods. Analysis of the activity of photoimmobilized proteins is further complicated by intrinsic factors such as protein orientation or tertiary structure and extrinsic effects caused by intermolecular interactions between proteins in high-density films. A combination of these factors may not be easily distinguishable, so that structure-activity relationships may remain uncertain. How biological activity changes with surface coverage, for example, is a question posed by current immobilization strategies. The covalent coupling strategy used in this paper allowed control over the composition of the interface and maintained the biological function of attached proteins. Each step of the conversion of the monolayer and the attachment of a protein (immunoglobulin G, IgG) was followed by a variety of characterization techniques (ellipsometry, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, radiography). Immobilization of IgG provided a useful test of the cross-linking strategy. IgGs are fairly large and complex proteins that are also robust (15 disulfide linkages per IgG). IgGs are widely available from commercial sources in a variety of forms. Immunoglobulins are well characterized at the sequence and structural level, capable of high-affinity binding (
