Molecular Recognition at the Exterior Surface of a Zwitterionic Telomer

Jan 20, 2010 - Surface Modification of a Biodegradable Magnesium Alloy with Phosphorylcholine (PC) and Sulfobetaine (SB) Functional Macromolecules for...
0 downloads 4 Views 2MB Size
Download PDF
pubs.acs.org/Langmuir © 2010 American Chemical Society

Molecular Recognition at the Exterior Surface of a Zwitterionic Telomer Brush Hiromi Kitano,*,† Hisatomo Suzuki,† Kazuhiro Matsuura,† and Kohji Ohno‡ †

Department of Applied Chemistry, Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Japan and ‡Institute for Chemical Research, Kyoto University, Uji 611-0011, Japan Received October 29, 2009. Revised Manuscript Received December 26, 2009

3-Sulfo-N,N-dimethyl-N-(20 -methacryloyloxyethyl)propanaminium inner salt (SPB) was polymerized on a glass plate with a surface-confined initiator of atom transfer radical polymerization (ATRP) having a 2-bromoisobutyryl group. The glass plate modified with a brush of sulfobetaine telomer (PSPB) was highly hydrophilic and showed a strong resistance against nonspecific adsorption of proteins such as lysozyme and albumin. Through the polymerization from the free surface of PSPB chain by ATRP, furthermore, N-methacryloyloxysuccinimide (MAOSu) residues were introduced, and the incubation of the telomer (PSPB-b-PMAOSu)-modified glass chip with a lectin (concanavalin A, Con A) gave a glass chip covered with the Con-A-modified PSPB brush. The Con A fixed to the zwitterionic telomer brush pursued specific binding of mannose residues accumulated on the surface of Au colloidal particles, resulting in the increase in absorbance at 550 nm ascribable to localized surface plasmon resonance, while the nonspecific adsorption of proteins to the surface of the glass chip was still largely suppressed. The present results indicate usefulness of the zwitterionic telomer surface with antibiofouling properties as a scaffold for specific sensing devices.

Introduction Modification of solid surfaces with a functional moiety drastically increases usefulness of the solid materials. In recent years, various kinds of polymer brushes that are polymer chains accumulated on solid surfaces have been extensively investigated, and it has been clarified that, by the introduction of polymer brushes, changes in surface properties such as wetness, adhesiveness, and so on can easily be made.1 The reason for these properties has been attributed to the condensed structure of the well-defined brush. Furthermore, polymer brushes which resist nonspecific adsorption of proteins and cells are expected to be biocompatible materials.2-6 There are mainly two strategies for constructing polymer brushes on the surface of solid materials. One is surface-initiated polymerization, which is the so-called “grafting-from” method.7-15 *To whom correspondence should be addressed. E-mail: kitano@ eng.u-toyama.ac.jp. (1) Tsujii, Y.; Ohno, K.; Yamamoto, S.; Goto, A.; Fukuda, T. Adv. Polym. Sci. 2006, 197, 1. (2) Zhang, Z.; Chao, T.; Chen, S.; Jiang, S. Langmuir 2006, 22, 10072. (3) Zhang, Z.; Chao, T.; Chen, S.; Jiang, S. Biomacromolecules 2006, 7, 3311. (4) Feng, W.; Brash, J. L.; Zhu, S. Biomaterials 2006, 27, 847. (5) Iwata, R.; Suk-In, P.; Hoven, V. P.; Takahara, A.; Akiyoshi, K.; Iwasaki, Y. Biomacromolecules 2004, 5, 2308. (6) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Langmuir 2005, 21, 5980. (7) Ohno, K.; Koh, K.; Tsujii, Y.; Fukuda, T. Macromolecules 2002, 35, 8989. (8) Ohno, K.; Koh, K.; Tsujii, Y.; Fukuda, T. Angew. Chem., Int. Ed. 2003, 115, 2857. (9) Matyjaszewski, K.; Miller, P. J.; Shukla, N.; Immaraporn, B.; Gelman, A.; Luokala, B. B.; Siclovan, T. M.; Kickelcik, G.; Vallant, T.; Hoffman, H.; Pakula, T. Macromolecules 1999, 32, 8716–8724. (10) Mandal, T. K.; Fleming, M. S.; Walt, R. D. Nano Lett. 2002, 2, 3. (11) Jordan, R.; West, N.; Ulman, A.; Chou, Y.-M.; Nuyken, O. Macromolecules 2001, 34, 1606. (12) Raula, J.; Shan, J.; Nuopponen, M.; Niskanen, A.; Jiang, H.; Kauppinen, E.; Tenhu, H. Langmuir 2003, 19, 3499. (13) Nuss, S.; B€ottcher, H.; Wurm, H.; Hallensleben, M. L. Angew. Chem., Int. Ed. 2001, 40, 4016. (14) Rusa, M.; Whitesell, J. K.; Fox, M. A. Macromolecules 2004, 37, 2766. (15) Shan, J.; Nuopponen, M.; Jiang, H.; Viitala, T.; Kauppinen, E.; Kontturi, K.; Tenhu, H. Macromolecules 2005, 38, 2918.

Langmuir 2010, 26(9), 6767–6774

The other is the grafting of preformed polymers on the surface of solid materials via covalent bonding, which is the so-called “grafting-to” method.15-23 These grafting procedures can easily be carried out by application of the preparation procedure of self-assembled monolayers which conjugate organic and inorganic components.1-23 Organosilane compounds such as alkyl silane, for example, form a selfassembled monolayer (SAM) on inorganic material surfaces (silicon and glass) via covalent Si-O bonds,24-26 while organosulfur compounds such as alkyl or aromatic thiols and disulfides form a SAM on noble metal surfaces via chemisorptive Au-S and Ag-S bonds.27-30 Atom transfer radical polymerization (ATRP) is categorized as living radical polymerization, which can be applied to a wide variety of monomers, varying the topology of the polymer (linear, (16) Lowe, A. B.; Sumerlin, B. S.; Donovan, M. S.; McCormick, C. L. J. Am. Chem. Soc. 2002, 124, 11562. (17) Wuelfing, W. P.; Gross, S. M.; Miles, D. T.; Murray, R. W. J. Am. Chem. Soc. 1998, 120, 12696. (18) Corbierre, M. K.; Cameron, N. S.; Lennox, R. B. Langmuir 2004, 20, 2867. (19) Sakura, T.; Takahashi, T.; Kataoka, K.; Nagasaki, Y. Colloid Polym. Sci. 2005, 284, 97. (20) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226. (21) Takase, S.; Akiyama, Y.; Otsuka, H.; Nakamura, T.; Nagasaki, Y.; Kataoka, K. Biomacromolecules 2005, 6, 818. (22) Shimmin, R. G.; Schoch, A. B.; Braun, P. V. Langmuir 2004, 20, 5613. (23) Shan, J.; Nuopponen, M.; Jiang, H.; Kauppinen, E.; Tenhu, H. Macromolecules 2003, 36, 4526. (24) Ostaci, R.-V.; Damiron, D.; Capponi, S.; Vignaud, G.; Leger, L.; Grohens, Y.; Drockenmuller, E. Langmuir 2008, 24, 2732. (25) Seitz, O.; B€ocking, T.; Salomon, A.; Gooding, J. J.; Cahen, D. Langmuir 2006, 22, 6915. (26) Joseph, K. W.; Alexander, F. Y. Langmuir 2006, 22, 8271. (27) Nuzzo, R. G.; Fusco, F. A.; Allara, D L. J. Am. Chem. Soc. 1987, 109, 2358. (28) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (29) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (30) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103.

Published on Web 01/20/2010

DOI: 10.1021/la904111r

6767

Article

branched, etc.) and the composition of polymeric chains (block or graft copolymers, etc.).31-37 The ATRP method has also been applied to the polymerization of various zwitterionic monomers.2-6,38-48 Zwitterionic polymers have been designed to mimic phosphatidylcholine that is abundant in cell membranes, and their applicability to biomedical fields has extensively been investigated. For example, polymer films composed of n-butyl methacrylate (BMA) and a zwitterionic monomer such as 2-methacryloyloxyethyl phosphorylcholine (MPC, phosphobetaine), 3-sulfo-N,N-dimethylN-(30 -methacrylamidopropyl)propanaminium inner salt (SPB, sulfopropylbetaine with an amide form), and 1-carboxy-N,Ndimethyl-N-(20 -methacryloyloxyethyl)methanaminium inner salt (CMB, caboxymethylbetaine) were found to be highly biocompatible.49-53 We have reported that the number of platelets adhered onto a film of random copolymer of CMB and BMA was much less than that to PBMA.54-56 In addition, the solution behavior of zwitterionic polymers has received much attention due to unique properties to typical polyelectrolytes.57-59 Raman and infrared spectroscopies indicated that the hydrogen-bonded network structure of water in the vicinity of zwitterionic polymers is not largely disturbed.54-56,60-62 Based on these findings, we have been insisting that the small perturbation effect of zwitterionic polymers on the structure of water at polymer-water interfaces is one of the important factors for their excellent biocompatibility. (31) Wang, J.-S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614. (32) Patten, T. E.; Xia, J.; Abermathy, T.; Matyjaszewski, K. Science 1996, 272, 866. (33) Kamigaito, M.; Ando, T.; Sawamoto, M. Chem. Rev. 2001, 101, 3689. (34) Matyjaszewski, K.; Xia, J. Chem. Rev. 2001, 101, 2921. (35) Boyes, S. G.; Akgun, B.; Brittain, W. J.; Foster, M. D. Macromolecules 2003, 36, 9539. (36) Yu, K.; Wang, H.; Han, Y. Langmuir 2007, 23, 8957. € (37) Lindqvist, J.; Nystr€om, D.; Ostmark, E.; Antoni, P.; Carlmark, A.; Johansson, M.; Hult, A.; Malmstr€om, E. Biomacromolecules 2008, 9, 2139. (38) Matsuura, K.; Kitano, H. Polym. Prepr. Jpn. 2005, 54, 1900. (39) Matsuura, K.; Ohno, K.; Kagaya, S.; Kitano, H. Macromol. Chem. Phys. 2007, 208, 862. (40) Lobb, E. J.; Ma, I.; Billingham, N. C.; Armes, S. P.; Lewis, A. L. J. Am. Chem. Soc. 2001, 123, 7913. (41) Ma, I. Y.; Lobb, E. J.; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salvage, J. Macromolecules 2002, 35, 9306. (42) Ma, I. Y.; Tang, Y.; Billingham, N. C.; Armes, S. P.; Lewis, A. L.; Lloyd, A. W.; Salvage, J. P. Macromolecules 2003, 36, 3475. (43) Mu, Q. S.; Lu, J. R.; Ma, Y. H.; Paz de Banez, M. V.; Robinson, K. L.; Armes, S. P.; Lewis, A. L.; Thomas, R. K. Langmuir 2006, 22, 6153. (44) Li, Y.; Tang, Y.; Narain, R.; Lewis, A. L.; Armes, S. P. Langmuir 2005, 21, 9946. (45) Li, Y.; Armes, S. P.; Jin, X.; Zhu, S. Macromolecules 2003, 36, 8268. (46) Chang, Y.; Chen, S.; Zheng, Z.; Jiang, S. Langmuir 2006, 22, 2222. (47) Iwasaki, Y.; Akiyoshi, K. Macromolecules 2004, 37, 7637. (48) Iwata, R.; Suk-In, P.; Hoven, V. P.; Takahara, A.; Akiyoshi, K.; Iwasaki, Y. Biomacromolecules 2004, 5, 2308. (49) Ishihara, K.; Aragaki, R.; Ueda, T.; Watanabe, A.; Nakabayashi, N. J. Biomed. Mater. Res. 1990, 24, 1069. (50) Ishihara, K. Sci. Technol. Adv. Mater. 2000, 1, 131. (51) Yuan, J.; Mao, C.; Zhou, J.; Shen, J.; Lin, S. C.; Zhu, W.; Fang, J. F. Polym. Int. 2003, 52, 1869. (52) Yuan, Y.; Zang, X.; Ai, F.; Zhou, J.; Shen, J.; Lin, S. Polym. Int. 2004, 53, 121. (53) Kitano, H.; Mori, T.; Tada, S.; Takeuchi, Y.; Gemmei-Ide, M.; Tanaka, M. Macromol. Biosci. 2005, 5, 314. (54) Kitano, H.; Tada, S.; Mori, T.; Takaha, K.; Gemmei-Ide, M.; Tanaka, M.; Fukuda, M.; Yokoyama, Y. Langmuir 2005, 21, 11932. (55) Tada, S.; Inaba, C.; Mizukami, K.; Fujishita, S.; Gemmei-Ide, M.; Kitano, H.; Tanaka, M.; Mochizuki, A.; Matsunaga, T. Macromol. Biosci. 2009, 9, 63. (56) Fujishita, S.; Tada, S.; Inaba, C.; Gemmei-Ide, M.; Kitano, H.; Saruwatari, Y. Biol. Pharm. Bull. 2008, 31, 2309. (57) Lowe, A. B.; Billingham, N. C.; Armes, S. P. Chem. Commum. 1996, 1555. (58) Laschewsky, A.; Touillaux, R.; Hedlinger, P.; Vierengel, A. Polymer 1995, 36, 3045. (59) Kudaibergenov, S.; Jaeger, W.; Laschewsky, A. Adv. Polym. Sci. 2006, 201, 157. (60) Kitano, H.; Sudo, K.; Ichikawa, K.; Ide, M.; Ishihara, K. J. Phys. Chem. B 2000, 104, 11425. (61) Kitano, H.; Imai, M.; Sudo, K.; Ide, M. J. Phys. Chem. B 2002, 106, 11391. (62) Kitano, H.; Imai, M.; Mori, T.; Gemmei-Ide, M.; Yokoyama, Y.; Ishihara, K. Langmuir 2003, 19, 10260.

6768 DOI: 10.1021/la904111r

Kitano et al. Scheme 1. Chemical Structures of (a) 3-Sulfo-N,N-dimethyl-N-(20 -methacryloyloxyethyl) Propanaminium Inner Salt (SPB), (b) N-Methacryloyloxysuccinimide (MAOSu), (c) Dithiolated Poly(2-methacryloyloxyethyl D-mannopyranoside) (DT-PMEMan), (d) (11-(2-Bromo-2-methyl)propionyloxy) Undecyltrichlorosilane (Br-PUCS), and (e) Methoxytri(ethylene glycol) 2-Bromo-2-methylpropionate (TEG-Br)

Besides, it has been found that zwitterionic telomer brushes (PMPC, PSPB, and PCMB) constructed on a gold surface via Au-S bonds resist against nonspecific adsorption of proteins by using an electrochemical method (cyclic voltammetry) and localized surface plasmon resonance spectroscopy.38,39,63 In a recent study, it was reported that a carboxybetaine polymer grafted on a glass substrate highly resists protein adsorption and cell adhesion.2 A dispersion of gold nanoparticles possesses a strong color,64 and the optoelectronic properties of these particles are attributed to the collective oscillation of the conduction band electrons on the particle (localized surface plasmon resonance, LSPR).65 The absorption is strongly dependent on particle size and shape, interparticle distance, properties of the surface-passivating compound and surrounding medium.65,66 Hybrids of gold nanoparticles and biological molecules have extensively been studied as colloidal biosensors for lectin,20,21 DNA,67 sugar,68 and antibodies.69 In this report, a zwitterionic telomer-protected glass substrate has been prepared by ATRP of 3-sulfo-N,N-dimethyl-N-(20 methacryloyloxyethyl)propanaminium inner salt (SPB, sulfobetaine with ester form, Scheme 1a) initiated by the surface-confined ATRP initiator, and resistance against nonspecific adsorption of (63) Kitano, H.; Kawasaki, A.; Kawasaki, H.; Morokoshi, S. J. Colloid Interface Sci. 2005, 282, 340. (64) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293. (65) Hutter, E.; Fendler, J. H. Adv. Mater. 2004, 16, 1685. (66) Brust, M.; Kiely, C. J. Colloids Surf., A 2002, 202, 175. (67) Cao, Y. C.; Jin, R.; Mirkin, C.-A. Science 2002, 297, 1536. (68) Aslan, K.; Lakowicz, J. R.; Geddes, C. D. Anal. Chem. 2005, 77, 2007. (69) Zang, C.; Zhang, Z.; Yu, B.; Shi, J.; Zhang, X. Anal. Chem. 2002, 74, 96.

Langmuir 2010, 26(9), 6767–6774

Kitano et al.

Article ultrapure water (18 MΩ cm-1, Millipore System). Other reagents used were commercially available.

Synthesis of (11-(2-Bromo-2-methyl)propionyloxy)undecyltrichlorosilane (Br-PUCS, Silane ATRP Initiator Scheme 1d). Br-PUCS was synthesized from 10-undecen-1-ol by the two-step reaction as described in the Supporting Information (Schemes S-2 and S-3).9

Synthesis of Water-Soluble Free ATRP Initiator, Methoxy Tri(ethylene glycol) 2-Bromo-2-methylpropionate (TEG-Br) (Scheme 1e). TEG-Br was synthesized from tri(ethylene glycol) monomethylether by the reaction described in the Supporting Information (Scheme S-4).

Preparation of Initiator-Coated Glass via Silane-Coupling (Scheme 2). To toluene (40.0 mL) in a sample vial was

Figure 1. Schematic of the antibiofouling property and the specific recognition of the Con-A-modified PSPB brush.

proteins to the surfaces of PSPB brush has been examined. Furthermore, monomer residues with an active ester as a side chain (N-methacryloyloxysuccinimide, MAOSu, Scheme 1b) have been introduced to the free surface of the PSPB brush by ATRP. A sugar-binding protein, concanavalin A (Con A), has been covalently immobilized to the MAOSu residues of the block copolymer brush, and the recognition of sugar residues on the surface of Au nanoparticle (colloid) by the surfaceconfined Con A has been examined with the help of LSPR spectroscopy (Figure 1).

Experimental Section Materials and Methods. 3-Sulfo-N,N-dimethyl-N-(20 -methacryloyloxyethyl)propanaminium inner salt (SPB, ester form) was prepared by the coupling of 2-dimethylaminoethyl methacrylate and 1,3-propanesultone in acetone at room temperature (r.t.) for 24 h as described in the Supporting Information (Scheme S-1).57 Ethyl 2-bromo-2-methylpropionate (ET-Br, 98%) and 2,20 -bipyridine (Bpy, 99.5%) were purchased from Merck. Tri(ethylene glycol) monomethylether (98%), 2-bromoisobutyryl bromide (98%), copper(I) bromide (99.999%), tetrahydrofuran (THF, 99.5%), and N-methyl-2-pyrrolidinone (NMP, 99.0%) were purchased from Wako Pure Chemicals, Osaka, Japan. Hydrogen tetrachloroaurate(III) trihydrate (99.9%), concanavalin A (Con A) from Canavalia ensiformis, bovine serum albumin (BSA), and lysozyme (LYS) from egg white were obtained from SigmaAldrich. N-Methacryloyloxysuccinimide (MAOSu) was prepared by a similar method reported previously for the preparation of acryloxy succinimide.70,71 A disulfide-carrying glycopolymer, dithiolated poly(2-methacryloyloxyethyl D-mannopyranoside) (DT-PMEMan, Mw =9.35  103, Mn =7.78  103, DP (based on Mn)=26.6, Mw/Mn =1.20, Scheme 1c) was prepared by the ATRP method as previously reported.72 Slide glass and micro cover glass from Matsunami Glass (Kishiwada, Osaka, Japan) were cut into the most suitable size (38 mm  26 mm and 24 mm  14 mm, respectively). Silicon wafers (N(100), having 0.001-0.005 Ω cm-1 resistivity and 0.525 ( 0.025 mm thickness) from Furuya Metal Co., Ltd., Tokyo, Japan, were cut into the most suitable size (20 mm  10 mm). All aqueous solutions were prepared with

(70) Pollak, A.; Blumenfeld, H.; Wax, M.; Baughm, R. L.; Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 6324. (71) Kitano, H.; Akatsuka, Y.; Ise, N. Macromolecules 1991, 24, 42. (72) Kitano, H.; Takahashi, Y.; Mizukami, K.; Matsuura, K. Colloids Surf., B 2009, 70, 91.

Langmuir 2010, 26(9), 6767–6774

added (11-(2-bromo-2-methyl)propionyloxy)undecyltrichlorosilane (Br-PUCS) at 4.0 mM. A glass plate (38 mm  26 mm or 24 mm  14 mm), which had been washed with water, methanol, and acetone and subsequently cleaned by a UV/ozone method (UV/ozone cleaner UV253E, Filgen, Nagoya, Japan), was immersed into the solution, and after replacing the atmosphere with Ar gas the vial was tightly sealed. After 18 h at r.t., the plate was washed with pure toluene two times. Furthermore, the plate was immersed in toluene and washed by ultrasonication for 1 min, and after further rinsing with toluene the plate was dried in N2 and stored in a sample vial filled with Ar. Preparation of PSPB by ATRP on Glass (Scheme 2). To a sample vial (50 mL) was added a magnetic stirrer chip and a homemade substrate stand made of Teflon. After degassing, a solution of SPB (2.23 g, 8.0 mmol) dissolved in a mixture of methanol and water (1/1 (v/v), 40.0 mL), which had been bubbled with Ar beforehand, was put into the vial. While Ar was continuously flowed, the glass plate was put into the vial. Cu(I)Br (184 mg, 1.28 mmol), 2,20 -bipyridine (400 mg, 2.56 mmol), and TEG-Br (50.1 mg, 0.160 mmol) were added, and the vial was tightly sealed. After the reaction, the glass plate was recovered and washed with ethanol, NMP, water, methanol, and chloroform. The plate was immersed in water for 4 days at 30 °C.73 The reaction conversion was determined by the analysis of a small aliquot using 1H NMR. The reaction reagents were precipitated by acetone, and Cu(I) in the solution mixture was convered to Cu(II) by bubbling air. After drying in vacuo, the product mixture was dissolved in water and passed through a chelate resin column (IRC748 Amberlite, Organo Ltd., Tokyo, Japan). The solution obtained was purified by ultrafiltration (Amicon; membrane, MWCO 1000) and lyophilized to give a white powder (PSPB). The molecular weight and dispersity of PSPB were determined by gel-permeation chromatography (GPC) using pullulan standards (column, Shodex OHpak SB-803HQ (Showa Denko, Tokyo, Japan); mobile phase, 0.1 M aqueous NaBr solution at 30 °C).

Preparation of Con-A-Modified PSPB Brush on Glass via ATRP and Amide-Coupling. The fixation of lectin to the surface of the brush was a two-step reaction. The first step was the preparation of the diblock copolymer brush (P(SPB-b-MAOSu)) by ATRP on glass (Scheme 3). The PMAOSu block was prepared in a way similar to that of the PSPB brush. MAOSu (1.47 g, 8.0 mmol) was dissolved in NMP (40.0 mL), which had been degassed and bubbled with Ar beforehand. While Ar was continuously flowed, the PSBP-modified glass was put into the vial. Cu(I)Br (57.4 mg, 0.40 mmol), 2,20 -bipyridine (125 mg, 0.80 mmol), and ET-Br (78.0 mg, 0.40 mmol) were added, and the vial was tightly sealed. After the reaction at 30 °C, the glass plate was recovered and washed with NMP, and Cu(I) in the solution mixture was converted to Cu(II) by bubbling air. The glass plate was immersed in NMP until use. The reaction conversion was determined by the analysis of a small aliquot using 1H NMR. The second step was the amide-coupling of Con A with the MAOSu residues at the MAOSu block of the brush (Scheme 4). (73) Cho, K. W.; Kong, B.; Choi, S. I. Langmuir 2007, 23, 5678.

DOI: 10.1021/la904111r

6769

Article

Kitano et al. Scheme 2. Preparation of Silane-Carrying PSPB Using ATRP

Scheme 3. Preparation of Silane-Carrying PSPB-b-PMAOSu Using ATRP

Scheme 4. Preparation of Con A-Fixed PSPB Brush

Con A (40.0 mg, 1.0 mg/mL) was dissolved in a 5 mM HEPES buffer (40.0 mL, pH 7.5, 0.1 mM Mn2þ and Ca2þ). The glass chip modified with the diblock copolymer brush (P(SPB-b-MAOSu)) was immersed in 5 mM HEPES buffer for several minutes for priming and subsequently incubated in the Con A solution at 6770 DOI: 10.1021/la904111r

25 °C for 24 h. The glass chip was finally washed well with HEPES and PBS buffer solutions. Measurement of Contact Angles. Static contact angles, θ, of a droplet of water (3-4 μL) on the surface of various polymer brushes constructed on the glass substrates were determined Langmuir 2010, 26(9), 6767–6774

Kitano et al.

Article

Table 1. Contact Angles for Polymer Brushes on the Glass Substrate Determined by the Sessile Drop and Air-in-Water Methodsa contact angle (deg) sample

sessile drop

air-in-water

total

glass 8.2 ((0.8) 155.3 ((2.8) 163.5 BrPUCS 86.6 ((2.0) 97.3 ((2.2) 183.9 PSPB 9.4 ((2.0) 159.8 ((2.8) 167.2 PSPB-b-MAOSu 70.9 ((1.0) 151.2 ((1.0) 222.1 PSPB-Con A 47.5 ((2.7) 151.3 ((1.5) 198.7 PMAOSu 70.6 ((1.3) 115.5 ((4.8) 186.1 a Standard deviation is shown in parentheses. Measurement conditions: temperature of air, 25.0 °C; relative humidity, 45.5%; temperature of water, 25.0 °C.

15 times to obtain a reliable average value (sessile drop method). Similarly, the θ values of air bubbles (10 μL) attached to the surface of the polymer brush immersed in water were also determined (air-in-water method). Measurement of Brush Thickness. The thicknesses of initiator SAM and polymer brushes on a silicon wafer were determined by using a spectroscopic ellipsometer (M-2000U, J. A. Woollam Co., Inc.). Measurements were taken at an incident angle of 70°. The ellipsometric angles were recorded in a wavelength range from 242 to 999 nm. The thickness was calculated from the ellipsometric angles assuming that the refractive index of the graft layer is 1.49, which is the experimented value for a poly(methyl methacrylate) film.74,75 All measurements were conducted in air at room temperature.

Adsorption of Protein to the Surface of the Polymer Brush. Two pieces of the PSPB-modified glass plates were attached to a U-shaped silicon spacer to give a glass cell. The polymer-modified surface of each glass chip was facing inside. Various kinds of protein solutions (BSA and LYS; 4.5 mg/mL in PBS) were put into the cell at 37 °C, and the cell was incubated for 90 min. The protein solution was discarded, and the cell was rinsed with PBS. A 5 wt % sodium dodecyl sulfate (SDS) solution was put into the cell, and after sonication of the cell for 60 min (at 28 and 40 kHz alternately every 1 min) the solution mixture was recovered and mixed with a solution of bicinchoninic acid (BCA). The absorbance at 560 nm was observed using a microplate reader (see Supporting Information Figure S-1).55,56

Localized Surface Plasmon Resonance (LSPR) Spectroscopy. Gold colloids were prepared by the reduction of hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 3 3H2O) with sodium citrate in water at 100 °C as previously reported.39,76,77 The hydrodynamic diameter of the gold colloids was determined to be 20 nm on average using the dynamic light scattering technique (DLS-7000, Otsuka Electronics, Hirakata, Osaka, Japan; light source, He-Ne laser at 632.8 nm). The gold colloids were modified with disulfidecarrying poly(2-methacryloyloxyethyl D-mannopyranoside) (DTPMEMan) (12.5 μg/mL) and purified by centrifugation at 1.0  104 G for 60 min four times (Supporting Information Scheme S-6).72 The Con-A-carrying brush-modified glass plate (24  14 mm2, thickness 0.12-0.17 mm) was incubated with the PMEMancarrying gold colloid, and the absorbance at 550 nm was followed using a spectrophotometer (Lambda 19 UV/vis/NIR spectrometer, Perkin-Elmer).

Results and Discussion Preparation of Initiator-Coated Glass via Silane-Coupling. The construction of a SAM of ATRP initiator conjugated (74) Brandrup, J.; Immergut, E. H.; Grulke, E. A.; Grulke, E. A.; Bloch, D. Polymer Handbook, 4th ed.; Wiley-Interscience: New York, 2003. (75) Ohno, K.; Morinaga, T.; Koh, K.; Tsujii, Y.; Fukuda, T. Macromolecules 2005, 38, 2137. (76) Kanayama, N.; Kitano, H. Langmuir 2000, 16, 577. (77) Kitano, H.; Kago, H.; Matsuura, K. J. Colloid Interface Sci. 2009, 331, 343.

Langmuir 2010, 26(9), 6767–6774

Figure 2. Plot of Mw/Mn value (O), brush thickness (determined by ellipsometry, b), and graft density (9) versus the value Mn of free PSPB. [SPB]:[TEG-Br]:[Cu(I)Br]:[Bpy] = x:1:8:16 (x = 20, 30, 50, 70, 100, 150). Solvent, methanol, and water (1:1 (v/v)) at 30 °C. Graft density was obtained via eq 1.

with a silane coupling reagent onto a silicon substrate was previously reported using the same or similar compounds.9 The silane coupling reaction was pursued with 4 mM Br-PUCS in toluene at r.t. for 18 h. The progress of the modification reaction was confirmed by the increase in contact angle of the glass plate (sessile drop method) from 8.2° to 88.6° (Table 1) due to the changes from OH groups to 2-bromoisobutyroylundecyl groups on the glass plate. Preparation of PSPB Brush by ATRP. Previously, it was reported that the usage of oligomeric methoxy poly(ethylene glycol) 2-bromoisobutyrate (OEG-Br) initiator for polymerization of MPC by ATRP was successful.4-6 It was also reported that the polymerization of SPB using the 2-bromoisobutyryl group-carrying initiator Cu(I)Br and Bpy catalyst system at r.t. in methanol or a methanol-water mixture had a good correlation between the theoretical and experimental molecular weights.38-48 Based on these previous results, in this report, SPB was polymerized in a mixture of methanol and water (1/1 (v/v), 40.0 mL) at 30 °C using the surface-confined ATRP initiator with the initial molar ratio of [SPB]:[TEG-Br]:[Cu(I)Br]:[Bpy] = 50:1:8:16. The evaluation of both the living behavior and the correlation of thickness and molecular weight of the brush was carried out under the conditions of [SPB]:[TEG-Br]:[Cu(I)Br]:[Bpy] = x:1:8:16 (x = 20, 30, 50, 70, 100, and 150). Previously, it was reported that when the polymerization was pursued both in solution and at the solid surface simultaneously, the Mw/Mn value at the solid surface was similar to that in the solution phase.75 Figure 2 shows the plots of Mw/Mn and brush thickness (determined by ellipsometry) versus evolution of Mn for the ATRP of SPB. These results were similar to the tendency for the solution-phase polymerization of SPB (Figure 2). The Mw/Mn ratios of yielded polymers ranged from 1.20 to 1.40 and decreased with the increase in molecular weight, indicating the progress of living polymerization. The figure also suggests that the thickness of the PSPB brush on the glass substrate linearly increased with the evolution of Mn and the graft density changes of the brush are small (Figure 2), which supports the living behavior of the polymerization reaction, too. The graft density of the brush, σ, was determined using eq 1.5 σ ¼

dFNA Mn

ð1Þ

where d is the layer thickness determined by ellipsometry, F is the density of the dry polymer layer (1.30 g/cm3 for PMPC was DOI: 10.1021/la904111r

6771

Article

adopted),5 NA is the Avogadro number, and Mn is the numberaverage molecular weight of polymer chains on the surface. The Mn value of the polymer brush was assumed to be equal to that for the free PSPB produced in the solution phase as mentioned above. Since the graft density was almost constant irrespective of the Mn value and the thickness of the polymer brush (Figure 2), the graft density was affected only by the surface density of starting points, and the brush grew equally above the glass plate. Taking into account that the graft density of PSPB was 0.35 chains/nm2 and that of the ethynyldimethylchlorosilane SAM, which is analogous to the Br-PUCS SAM, was about 1.8 residues/nm2,24 the effectiveness of the initiation was estimated to be 19%. The graft density of the PSPB brush was greater than 0.1 chains/nm2. This value reaches the region where the introduction rate of the polymer chain to the introduction rate of the initiator residue is balanced.1 Therefore, it is thought that a polymer brush of satisfactorily high density could be constructed. Preparation of Con-A-Modified PSPB Brush on Glass via ATRP and Amide-Coupling. Previously, it was very often reported that polymerization of block copolymer (A-b-B) by ATRP was successful.35-37 Based on this knowledge, MAOSu was polymerized in NMP at 30 °C with the molar ratio of [MAOSu]:[Et-Br]:[Cu(I)Br]:[Bpy] = 20:1:2:4 in the presence of PSPB-modified glass here. Fixation of protein molecules to the solid surface has been widely carried out using condensation reagents or active esters.71 In this report, the amide-coupling of Con A with the MAOSu residues in the polymer brush was carried out. The unreacted active ester seemed to be deactivated (from MAOSu to methacrylic acid (MA)) during the reaction for 24 h and the subsequent storage in water. In fact, the contact angle of the PMAOSu brush by the air-in-water method leveled off at 25 °C in around 3 h, suggesting the formation of MA residues (see Supporting Information Figure S-4). Characterization of Brush Surface. a. Contact Angles. The contact angle measurement was carried out to confirm the formation of polymer brushes on glass substrates (Table 1). Both the sessile drop method and the air-in-water method indicated that the modification with Br-PUCS (initiator) largely decreased the hydrophilicity of the glass substrate in comparison with the bare glass, whereas that with the PSPB brush largely increased, indicating that the zwitterionic polymer brush was very hydrophilic. The contact angle for the hybrid brush, PSPB-b-PMAOSu, showed the same tendency as that for the PSPB brush by the air-in-water method, whereas the decrease in hydrophilicity was observed for the brush in the dry state (sessile drop method), giving the sum of larger than 180° due to the responsiveness to the surrounding medium.53-56 This result might be consistent with the appearance of the hydrophobic PMAOSu block at the exterior surface of the brush upon contact with air and that of hydrophilic PSPB block at the lower part of the brush upon contact with water, minimizing the difference in free energy of the interfaces. For comparison, the contact angle of the PMAOSu brush (composed only with the MASOu residues) was measured. The responsiveness to the surrounding medium was not observed (Table 1), indicating that the responsiveness was solely ascribed to the composite block copolymer brush (PSPB-b-PMAOSu). b. Brush Thickness. The ellipsometric measurement was carried out to confirm the formation of various kinds of polymer brushes on solid substrates. The polymer brushes in Table 2 (footnote a) were prepared under the same conditions as those for the measurement of protein adsorption and LSPR. The data of 6772 DOI: 10.1021/la904111r

Kitano et al. Table 2. Results of Ellipsometric Measurements of the Polymer Brushes on Silicon Wafersa sample

conversion thickness Δ (%) Mn,free (nm) (nm)c

σ (chains/ nm2)

Br-PUCS 1.72 99< 6.1  103 4.16 2.44 0.32 PSPB20a 87.4 3.4  103 5.31 1.15 0.28 PSPBa MAOSu PSPB-Con Aa 7.74 2.43 99< 1.5  104 7.94 6.22 0.33 PSPB50b 15.7 7.9  102 8.22 0.29 0.29 PSPBb MAOSu 10.28 2.06 PSPB-Con Ab a The target DP value (tDP) of PSPB was 20, and the polymerization time of MAOSu was 24 h. b The tDP value of PSPB was 50, and the polymerization time of MAOSu was 4 h. c The symbol Δ denotes the difference in thickness of the brushes.

Figure 3. Results of thickness and contact angles of the polymer brushes on the silicon wafers by ellipsometric measurements (bar graph; black and white bars) and contact angles of the sessile drop method (O) versus each sample ((black bar) Mn,PSPB = 6.1  103 and Mn,PMAOSu = 3.4  103; (white bar) Mn,PSPB = 1.5  104 and Mn,PMAOSu = 7.9  102).

Table 2 indicated by footnote a correspond to the PSPB with lower DP and the PMAOSu with higher DP than those of Table 2 indicated by footnote b which were examined to make the change in thickness of the MAOSu block upon modification. The thickness of the brush was in the order Br-PUCS SAM < PSPB brush < PSPB-b-PMAOSu brush < PSPB-b-PMAOSu brush coupled with Con A. From the changes in contact angle, the progress of the modification was definitely confirmed (Tables 1 and 2, and Figure 3). This result showed, even if the lengths of MAOSu blocks are different, there is no significant difference in the increase of thickness by the modification with Con A molecules. In addition, it seems that the long PMAOSu chain is not necessary for the introduction of the functional part to the exterior end of the brush, because there was not a strong correlation between the amount of immobilized Con A and the amount of MAOSu introduced. Adsorption of Proteins to the Polymer Brush on Glass Surface. Using the BCA method, the nonspecific adsorption of proteins (BSA and LYS) to the surfaces of PSPB-brush was observed. Figure 4 indicates that the PSPB brush showed a very slight nonspecific adsorption of proteins, which is in good contrast with the significant adsorption to a bare glass and the initiator-modified glass. These tendencies could be explained by the hydrophilicity of the substrate, the electric charges, and the freedom of the brushes Langmuir 2010, 26(9), 6767–6774

Kitano et al.

Figure 4. Protein adsorption to various polymer brushes at 37 °C. The y-axis expresses the amount of adsorbed BSA or LYS when the relative quantity of BSA adsorption to bare glass is 100% ((9) BSA and (0) lysozyme; 4.5 mg/mL phosphate buffer).

Article

Figure 5. Increase in absorbance at 550 nm of the Con-A-modified PSPB glass substrate upon immersion into various solutions: (A) DT-PMEMan-Au colloid, (B) DT-PMEMan-Au colloid þ R-MeMan (50 mM), and (C) bare glass substrate incubated in DT-PMEMan-Au colloid.

on the surfaces. The tendency that the proteins were extremely adsorptive to the bare glass and not to the PSPB brush is consistent with the hydrophobicity of the substrates. BSA and LYS have different pI values (BSA, 4.7-4.9; LYS, 11.0-11.4)78 and are negatively and positively charged at pH 7.0, respectively. The polymer brush reduced the adsorption of these proteins, indicating the decisive role of zwitterionic brushes irrespective of the charge of proteins. The proteins attached to the PSPB brush might be smoothly detached from the brush while keeping their native structures.42 Previously, it was revealed that PMPC brushes of high grafting density showed a dramatic reduction of the protein adsorption as compared to that of lower density.4 Therefore, it is understandable that the PSPB chains with a high graft density are also resistant against nonspecific adsorption of proteins. Further modification of the free surface of the zwitterionic brush with the protein molecule did not affect the adsorption behavior of proteins to the brush. The thickness of the Con A layer above the PSPB brush (2.1 nm) was much smaller than the actual size of Con A (diameter, about 8 nm),79 indicating that the degree of coverage of the surface with Con A was not complete. Therefore, the effectiveness of the zwitterionic groups, which could appear in the large hollow space between the Con A molecules, on the suppression of adsorption of the proteins was very clear. Characterization of PMEMan-Modified Au Colloids. The PMEMan-modified Au colloid was prepared by the simple mixing of the citrate-capped gold colloids with aqueous solution of the DT-PMEMan (DP = 26.6; the DP value for each block, 13.3) (see Supporting Information Scheme S-6). The dynamic light scattering (DLS) measurements showed the increase in diameter from 19.1 to 22.0 nm (2.9 nm) after modification.72 This value was equal to the stretched length of eight MEMan residues, suggesting that the grafted PMEMan chains on the gold colloids may have a somewhat coiled conformation. In addition, the DLS observation also shows that the prepared PMEMan-modified Au colloids were well dispersed, forming no aggregates. The dispersions of PMEMan-Au colloids in water were stable for at least 18 months or more at room temperature, and moreover, they were stable even at extremely high concentration. Molecular Recognition on the Surface of Polymer Brush. Next, we examined molecular recognition on the surface of the PSPB-b-PMAOSu brush on which Con A molecules had been

introduced. The PMEMan-modified gold colloid was adopted as a probe, because LSPR can sensitively detect increase and decrease in refractive index in the vicinity of the colloid. The significant increase of absorbance in Figure 5A shows that the Con A fixed to the surface of the copolymer brush on the glass chip could bind mannose residues on the PMEMan-carrying gold colloid. The much smaller absorbance change by the coexistence of a small sugar, R-methyl D-mannopyranoside (Figure 5B), indicated that the binding of Con A to the gold colloid corresponds to the specific recognition of mannose residues by Con A.72,79 The effect of other proteins on the binding of PMEManmodified Au colloids to the Con-A-modified PSPB brush was also examined. By the coexistence of bovine serum albumin (BSA), only a negligible small additional increase in absorbance at 550 nm was observed. By the presence of lysozyme, however, a much larger increase in absorbance was observed due to a nonspecific adsorption of positively charged lysozyme to a hollow space between the PMEMan chains on the negatively charged Au colloid.72 By the pretreatment of PMEMan-modified Au colloid with an aqueous solution of zwitterionic alkane thiol (3-[(6-mercaptohexyl)-N,N-dimethylamino]-propane-1-sulfonic acid, C6-SPB) (see Supporting Information Scheme S-7),80 however, such nonspecific adsorption of lysozyme to the Au colloid was drastically reduced, which enabled us to carry out the specific molecular recognition of mannose residues in the PMEMan brush on the Au colloid by the Con A molecule fixed to the exterior surface of PSPB brush. The advantage of the modification of proteins on the polymer brush examined in this work is the suppression of the nonspecific adsorption. This is because the zwitterionic polymer brush is known to show the excellent antibiofouling properties comparable to poly(ethylene glycol) brushes and SAMs. Especially the zwitterionic polymer brush with a high graft density prepared by the “grafting-from” method is expected to be more capable to suppress the nonspecific adsorption than that with a low density, which might result in a small background signal to give a highly sensitive detection. Based on the experimental results, it can be said that the proteins did not adsorb to the surface of the PSPB significantly. Further introduction of the active ester moiety at the free surface of the brush enabled proteins to fix to the exterior surface while keeping the ability to pursue their roles. The PSPB-modified glass

(78) Voet, D.; Voet, G. J. Biochemistry, 3rd ed.; John Wiley & Sons: New York, 2004. (79) Sharon, N.; Lis, H. In Lectins; Chapman and Hall Ltd.: London, 1989.

(80) Kitano, H.; Kondo, Y.; Saito, D.; Morita, H.; Kanayama, N. Submitted to Colloids Surfaces B: Biointerfaces.

Langmuir 2010, 26(9), 6767–6774

DOI: 10.1021/la904111r

6773

Article

Kitano et al.

substrate without biofouling may be highly useful for biomedical applications.

Conclusion The PSPB brush prepared with the surface-confined initiator for ATRP was resistant against the nonspecific adsorption of proteins (BSA and LYS) on the surface, which is consistent with the previously reported blood- and biocompatibilities of the PSPB polymers. Further ATRP of the active ester-carrying vinyl monomer made it possible to introduce protein molecules at the exterior surface of the brush, while the introduced protein soundly retained its biological role as indicated by the LSPR method. The PSPB brush spreading below the protein showed a suppression of the nonspecific adsorption of proteins. Therefore, the PSPB brush may be appropriate for diverse biomedical applications. Acknowledgment. This research was supported by a Grant-inAid for Scientific Research (19350055) from the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for Scientific

6774 DOI: 10.1021/la904111r

Research on Innovative Areas (20106007) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). We are grateful to Professors T. Fukuda and Y. Tsujii, Kyoto University, for allowing us to use the ellipsometer. Supporting Information Available: (1) Synthesis of sulfopropyl betaine monomer, (2) synthesis of (11-(2-bromo-2methyl)propionyloxy)undecyltrichlorosilane, (3) synthesis of water-soluble free ATRP initiator, methoxy tri(ethyleneglycol) 2-bromo-2-methylpropionate, (4) adsorption of protein to the surface of the polymer brush, (5) preparation of PSPB brush by ATRP, (6) characterization of brush surface with AFM, (7) preparation of Con-A-modified PSPB brush on glass via ATRP and amide-coupling, (8) measurement of brush thickness, (9) preparation of PMEMan-modified Au colloids, and (10) synthesis of 3-[(6-mercaptohexyl)-N,N-dimethylamino]-propane-1-sulfonic acid (C6-SPB). This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(9), 6767–6774