Langmuir 2008, 24, 8427-8430
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Site-Specific Dense Immobilization of Antibody Fragments on Polymer Brushes Supported by Silicone Nanofilaments Yasuhiko Iwasaki,*,† Yuki Omichi,‡ and Ryoko Iwata§ Departments of Chemistry and Materials Engineering, and Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai UniVersity, 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680, Japan, and Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental UniVersity, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan ReceiVed April 28, 2008. ReVised Manuscript ReceiVed June 10, 2008 For site-specific dense immobilization of antibodies on a solid support, we prepared phosphorylcholine copolymer brushes on silicone nanofilaments. The nanofilaments were prepared on a silicon wafer by treatment with trichloromethylsilane (MeSiCl3). To generate Si-OH groups on the nanofilaments, O2 plasma was irradiated on the surface. Initiators for atom transfer radical polymerization (ATRP) were then coupled on the filaments. Phosphorylcholine copolymer brushes were prepared by a “grafting from” process, and pyridyl disulfide groups were introduced into the polymer chains. F(ab′) fragments were then specifically immobilized onto these surfaces via a thiol-disulfide interchange reaction. The amount of antibodies immobilized on the nanofilament-supported copolymer brushes was approximately 65 times greater than that on smooth wafer-supported copolymer brushes.
Protein immobilization onto solid supports is an important aspect of proteomic and diagnostic assays in obtaining information about protein functions and interactions, and for screening complex protein samples.1,2 For these applications, proteins are employed as molecular recognition elements because of their specific affinity. The control of the surface properties of the solid supports, which can enrich specific proteins, maintain native protein structure, and control ordered orientation, must therefore be a key technology. Protein immobilization has been studied both covalently and noncovalently. Customarily, immobilized proteins on solid supports generally have random orientations.3,4 In contrast, some tests of the site-specific immobilization of proteins have been found to optimize the interfacial function of proteins. Molecular affinity,5,6 tag-mediated complex formation,7 and selective chemical reactions8–11 are effective for site-specific immobilization. As solid supports for proteins, various types of surfaces have been demonstrated such as self-assembled monolayers,10,12,13 * To whom correspondence should be addressed. Fax: +81-6-6368-0090. Telephone: +81-6-6368-0090. E-mail:
[email protected]. † Department of Chemistry and Materials Engineering, Kansai University. ‡ Department of Life Science and Biotechnology, Kansai University. § Tokyo Medical and Dental University.
(1) Rusmini, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2007, 8, 1775–1789. (2) Angenendt, P. Drug DiscoVery Today 2005, 10, 503–511. (3) Lahiri, J. I. L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777–790. (4) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760–1763. (5) Lue, R. Y.; Chen, G. Y.; Zhu, Q.; Lesaicherre, M. L.; Yao, S. Q. Methods Mol. Biol. 2004, 264, 85–100. (6) Andresen, H.; Gro¨tzinger, C.; Zarse, K.; Kreuzer, O. J.; Ehrentreich-Fo¨rster, E.; Bier, F. F. Proteomics 2006, 6, 1376–1384. (7) Nieba, L.; Nieba-Axmann, S. E.; Persson, A.; Ha¨ma¨la¨inen, M.; Edebratt, F.; Hansson, A.; Lidholm, J.; Magnusson, K.; Karlsson, A. F.; Plu¨ckthun, A. Anal. Biochem. 1997, 252, 217–228. (8) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 11790–11791. (9) Watzke, A.; Ko¨hn, M.; Gutierrez-Rodriguez, M.; Wacker, R.; Schro¨der, H.; Breinbauer, R.; Kuhlmann, J.; Alexandrov, K.; Niemeyer, C. M.; Goody, R. S.; Waldmann, H. Angew. Chem., Int. Ed. 2006, 45, 1408–1412. (10) Kalia, J.; Abbott, N. L.; Raines, R. T. Bioconjugate Chem. 2007, 18, 1064–1069. (11) Decre´au, R. A.; Collman, J. P.; Yang, Y.; Yan, Y.; Devaraj, N. K. J. Org. Chem. 2007, 72, 2794–2802.
polymer brushes,14–16 and lipid bilayers.17,18 In particular, polymer brushes have great capability for surface modification on solid supports because of their well-defined structure, stability, and multifunctionality.19–21 “Grafting from” systems have recently been used to prepare polymer brushes and enable the preparation of dense polymer brushes. Atom transfer radical polymerization (ATRP) is one of the well-studied methods of living radical polymerization because a wide range of monomers can be used in the process.22 Accumulating proteins on a limited surface is an effective means of obtaining definite responses. As one of the challenges in accomplishing this, polymer brushes have been studied because the number of binding sites for proteins can be increased in the polymer chain.15,23,24 We have also been studying site-specific immobilization of antibody fragments on polymer brushes.25 Although the surface density of the immobilized antibody fragments on the surface increased, the amount of antibody fragments was still significantly low as compared with the fraction of binding sites on the polymer brushes. The enrichment of protein (12) Wang, H.; Castner, D. G.; Ratner, B. D.; Jiang, S. Langmuir 2004, 20, 1877–1887. (13) Cheng, F.; Gamble, L. J.; Castner, D. G. Anal. Chem. 2008, 80, 2564– 2573. (14) Dong, R.; Krishnan, S.; Baird, B. A.; Lindau, M.; Ober, C. K. Biomacromolecules 2007, 8, 3082–3092. (15) Xu, F. J.; Cai, Q. J.; Li, Y. L.; Kang, E. T.; Neoh, K. G. Biomacromolecules 2005, 6, 1012–1020. (16) Tugulu, S.; Arnold, A.; Sielaff, I.; Johnsson, K.; Klok, H. A. Biomacromolecules 2005, 6, 1602–1607. (17) Vockenroth, I. K.; Atanasova, P. P.; Jenkins, A. T.; Ko¨per, I. Langmuir 2008, 24, 496–502. (18) Ross, E. E.; Joubert, J. R.; Wysocki, R. J., Jr.; Nebesny, K.; Spratt, T.; O’Brien, D. F.; Saavedra, S. S. Biomacromolecules 2006, 7, 1393–1398. (19) Ohno, K.; Morinaga, T.; Koh, K.; Tsujii, Y.; Fukuda, T. Macromolecules 2005, 38, 2137–2142. (20) Matyjaszewski, K.; Dong, H.; Jakubowski, W.; Pietrasik, J.; Kusumo, A. Langmuir 2007, 23, 4528–4531. (21) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. ReV. 2004, 33, 14–22. (22) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921–2990. (23) Kurosawa, S.; Aizawa, H.; Talib, Z. A.; Atthoff, B.; Hilborn, J. Biosens. Bioelectron. 2004, 20, 1165–1176. (24) Pirri, G.; Chiari, M.; Damin, F.; Meo, A. Anal. Chem. 2006, 78, 3118– 3124. (25) Iwata, R.; Suk-In, P.; Hoven, V. P.; Takahara, A.; Akiyoshi, K.; Iwasaki, Y. Biomacromolecules 2004, 5, 2308–2314.
10.1021/la801327a CCC: $40.75 2008 American Chemical Society Published on Web 07/16/2008
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Figure 1. SEM picture of silicone nanofilaments.
immobilization on dense polymer brushes is limited because of the high chain density. Many applications of immunoglobulin G (IgG) have been found in biotechnology and clinical medicine. An IgG molecule is “Y” shaped and consists of three domains. The arms of the Y-shaped IgG are F(ab′) fragments, each containing an antigenbinding site.26 Therefore, the orientation of surface-immobilized IgG with its F(ab′)-localized antigen-binding sites accessible to antigens is crucial for the performance of immunoassay techniques.27 In this study, site-specific dense immobilization of antibodies on polymer brushes supported with silicone nanofilaments was first performed. Gao and McCarthy reported their preparation of silicone nanofilaments on a silicon wafer surface, called Lichao’s surface.28 Absolutely clean silicon wafers were soaked in toluene solution containing trichloromethylsilane (MeSiCl3) under moderate humidity. The surface morphology as observed by scanning electron microscope (SEM) is shown in Figure 1. Highly dense nanofilaments were formed on the surface. Both the advancing (θA) and receding (θR) water contact angles were above 165°, and superhydrophobic surfaces were obtained (Figure 2a). Seeger et al. prepared an ultrahydrophobic surface generated by silicone nanofilaments of MeSiCl3 formed by vapor phase deposition.29 The nanofilaments were obtained not only on the silicon surface but also on the polymer and metal substrates. Furthermore, the nanofilaments had full optical transparency and thus could be used for optical sensing devices. The stability of the nanofilaments (26) Karyakin, A. A.; Presnova, G. V.; Rubtsova, M. Y.; Egorov, A. M. Anal. Chem. 2000, 72, 3805–3811. (27) Rao, S. V.; Anderson, K. W.; Bachas, L. G. Mikrochim. Acta 1998, 128, 127–143. (28) Gao, L.; McCarthy, T. J. J. Am. Chem. Soc. 2006, 128, 9052–9053. (29) Artus, G. R. J.; Jung, S.; Zimmermann, J.; Gautschi, H.-P.; Marquardt, K.; Seeger, S. AdV. Mater. 2006, 18, 2758–2762.
Figure 2. Pictures of a water droplet on sample surfaces: (a) silicone nanofilaments, (b) nanofilaments treated with O2 plasma, (c) BDCS immobilized on nanofilaments, and (d) nanofilament-supported PMG brushes.
was effectively improved by high-temperature annealing. Very recently, the same research group demonstrated that the functionalized silicone nanofilaments had good potential for selective protein enrichment through electrostatic interaction.30 The formation of the nanofilaments was very sensitive to moisture. We adjusted the humidity to 50-60% of a reaction atmosphere. The ultrahydrophobic nanofilament surfaces were treated with O2 plasma to produce Si-OH groups.25 The surface wettability of the nanofilaments treated with O2 plasma is shown in Figure 2b. A water droplet spread out on the surface completely. After the wafers were placed in a clean oven at 120 °C for 2 h, silanization with 3-(2-bromoisobutyryl)propyl dimethylchlorosilane (BDCS) was immediately carried out according to previous reports.31 After BDCS treatment of the surface, the (30) Zimmermann, J.; Rabe, M.; Verdes, D.; Seeger, S. Langmuir 2008, 24, 1053–1057. (31) Iwata, R.; Satoh, R.; Iwasaki, Y.; Akiyoshi, K. Colloids Surf., B 2008, 62, 288–298.
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Langmuir, Vol. 24, No. 16, 2008 8429 Scheme 1. Site-Specific Immobilization of F(ab′) on Random Polymer Brushes Supported by Silicone Nanofilaments
water contact angles (θA/θR) again increased to 102°/99°. When smooth silicon wafers were treated with BDCS, θA/θR values were 73°/65°.31 The surface features influenced the surface wettability because of the differences in the surface contact phenomena of a water droplet.32 The lower hysteresis of the water contact angles (θA - θR) on the nanofilaments treated with BDCS is also notable. Random copolymer brushes of 2-methacryloyloxyethy phosphorylcholine (MPC) and glycidyl methacrylate (GMA), namely, PMG brushes, were prepared on silicone nanofilaments by surface-initiated ATRP using Cu(I)Br and bipyridine as polymerization catalysts. The mole fraction of MPC/GMA was adjusted to 0.5/0.5 in the feed because MPC polymers effectively reduce nonspecific protein adsorption and cell adhesion when the MPC mole fraction of the random copolymers is more than 0.3.33 The polymerization kinetics was determined by gel permeation chromatography and 1H NMR; the semilogarithmic plot of monomer concentration versus time was linear. The linearity of the first-order plot of the monomer concentration suggested that the polymer radical concentration remained constant and that polymerization could be controlled on a polymerization time scale. The polymerization ability and monomer reactivity of MPC corresponded to those of GMA. The mole fraction was stable during the polymerization periods. Following this study, polymer brushes that were prepared for 3 h were used. Random polymer brushes were also prepared on a smooth wafer by previously reported methods.31 The apparent thickness and density of the polymer chains determined using smooth wafer surfaces were 15.6 nm and 0.32 chains/nm2, respectively. The chain density of a random polymer brush nearly corresponded to that of the poly(MPC) brush (0.35 chains/nm2)25 and was slightly lower than that of the poly(GMA) brush (0.43 chains/nm2).25 The bulky MPC influenced the brush formation. θA/θR values of the copolymer brushes supported by nanofilaments were effectively reduced to 25°/19° due to the hydrophilic MPC unit, and water adsorbed well onto the surface. PMG brushes having pyridyl disulfide moieties (PMG-SS) were ¨ ner, D. Langmuir 2000, 16, 7777–7782. (32) McCarthy, T. J.; O (33) Ishihara, K.; Oshida, H.; Endo, Y.; Ueda, T.; Watanabe, A.; Nakabayashi, N. J. Biomed. Mater. Res. 1992, 26, 1543–1552.
prepared via a reaction of epoxy groups in GMA units31 (Scheme 1); the reaction was confirmed through the detection of a sulfur signal determined by X-ray photoelectron spectroscopy. Figure 3 shows fluorescence micrographs and quantitative data for fluorescence isothiocyanate (FITC)-labeled F(ab′) fragments immobilized on the polymer brushes supported by the nanofilaments. F(ab′) fragments were obtained by reduction of F(ab′)2 fragments and used immediately. On the negative control poly(MPC-co-GMA) (PMG) surface (without pyridyl disulfide groups), the fluorescence intensity was extremely low (Figure 3a). Although epoxide groups of the copolymer have reactivity to protein amino groups, the reactive condition using a phosphate buffer (pH 6.0) was not suitable for coupling. This result also indicates that nonspecific physical adsorption of proteins on the
Figure 3. Fluorescence signals after FITC-labeled F(ab′) immobilization: (a) PMG brush and (b) PMG-SS brush.
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copolymer brushes occurs with difficulty. In our previous study, the homopolymer brushes of GMA were more effective for enriching F(ab′) fragments compared with the copolymer brushes with MPC.32 However, the amount of nonspecifically adsorbed proteins increased with an increase in the thickness of the poly(GMA) brush chain. Introducing MPC units into the polymer brushes effectively reduced nonspecific biofouling. The fluorescence image of the nanofilament-supported PMGSS brush in contact with F(ab′) fragments was remarkably bright, as shown in Figure 3b. The fluorescence intensity of the surface was significantly (p < 0.005) greater than that on the PMG brush surfaces. The reason for this result is that the thiol-disulfide interchange reaction occurred preferentially. In a comparison of surface morphologies, the fluorescence intensity on the copolymer brushes having the pyridyl disulfide moieties prepared on the nanofilaments was 65 times greater than that on a smooth surface. The nanofilament surfaces were very effective in increasing the capacity for protein immobilization. MPC units in the polymer
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brushes acted to reduce protein denaturation and preserve their function.34 The surface modification with silicone nanofilaments can be performed for a variety of substrates. Subsequently, nanofilament-supported MPC copolymer brush surfaces might prove to be useful as diagnostic substrates. Acknowledgment. We thank the Japan Society for the Promotion of Science for its financial support (#18681018). We also thank Dr. Thomas J. McCarthy for kindly information about nanofilaments and Dr. Kazunari Akiyoshi for use of his fluorescence plate reader. Supporting Information Available: Experimental details and data for nanofilament-supported polymer brushes. This material is available free of charge via the Internet at http://pubs.acs.org. LA801327A (34) Sakaki, S.; Nakabayashi, N.; Ishihara, K. J. Biomed. Mater. Res. 1999, 47, 523–528.
