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Patterned Adsorption of Protein onto a Carbohydrate Monolayer Immobilized on Si Naoto Shirahata,*,† Tetsu Yonezawa,‡,§ Yoshiko Miura,| Kazukiyo Kobayashi,| and Kunihito Koumoto† Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8603, Japan; Department Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; and Department of Molecular Design & Department of Biotechnology, Graduate School of Engineering, Nagoya University, Japan Received May 26, 2003. In Final Form: August 14, 2003 We propose a technique for fabricating a carbohydrate-terminated monolayer (CM) surface, which is extremely flat at the angstrom level. The CM was immobilized onto a Si surface by Si-C covalent bond. Specific adsorption of the protein molecule from a contacting solution was observed on the CM area of a patterned CM substrate surface, while its nonspecific adsorption was observed on the Si-O area.
Designing and controlling the surface chemical properties of hydrogen-terminated Si substrates (Si:H) through the immobilization of biomolecules is essential for the developmentofadvancedbiochipandbioarraytechnologies.1-3 This would allow us to take advantage of the benefits of Si surfaces, which include their angstrom-level surface flatness, surface homogeneity, thermal and chemical stability, reproducibility, and ease of biochemical manipulation.1,4 Carbohydrates form the major constituents of living organisms and play many important roles in, for example, cell differentiation, cell adherence, immune response, infection from viruses, and cancer metastasis.5 Materials modified with carbohydrates have recently attracted great interest. Two strategies have been focused on in the field of glycomaterials. The first is to develop carbohydrate chips and microarrays for a better understanding of the molecular basis for specific protein-carbohydrate (and carbohydrate-carbohydrate) interactions.6 This is extremely important to elucidate intercellular signaling pathways.5,7 The second strategy involves the building of biological and electrical devices containing organicinorganic hybrid materials,8 such as carbohydrate* To whom correspondence should be addressed. E-mail:
[email protected]. Phone: +81-52-736-7175. Fax: +81-52736-7182. Present address: National Institute of Advanced Industrial Science and Technology (AIST), 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan. † Department of Applied Chemistry, Graduate School of Engineering, Nagoya University. ‡ The University of Tokyo. § Japan Science and Technology Corporation. | Department of Molecular Design & Department of Biotechnology, Graduate School of Engineering, Nagoya University. (1) Strother, T.; Hamers, R. J.; Smith, L. M. Nucleic Acids Res. 2000, 28, 3535. (2) Gurtner, G.; Edman, C. F.; Formosa, R. E.; Heller, M. J. J. Am. Chem. Soc. 2000, 122, 8589. (3) Lin, Z.; Strother, T.; Cai, W.; Cao, X.; Smith, L. M.; Hamers, R. J.; Langmuir 2002, 18, 788. (4) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205. (5) Dwek, R. A. Chem. Rev. 1996, 96, 683. (6) Houseman, B. T.; Mrksich, M. Chem. Biol. 2002, 9, 443. (7) Huels, C.; Muellner, S.; Meyer, H. M.; Cahill, D. J. DDT 2002, 7. (18), S119. (8) Walcarius, A. Chem. Mater. 2001, 13, 3351.
conjugated metallic and semiconducting nanoparticles,9,10 which are frequently required to immobilize onto a highly flat inorganic substrate surface.10 To satisfy the various requirements6,10,11 for advancing both strategies, it is critical to develop a technique to selectively immobilize a carbohydrate monolayer onto predifined sites on a Si substrate without an intervening oxide layer. Furthermore, well-developed Si technology would allow us to construct more complicated biological integrated circuit systems. Few investigations, however, have been reported for immobilizing a carbohydrate monolayer onto a Si surface. Linford et al.12 have reported a new immobilization method for the organic functionalization of a Si surface through the formation of a Si-C linkage. Because of the high stability of the Si-C linkage against chemicals, sunlight, and humidity, the Si-C linked carbohydrate monolayer is superior for its commercial applicability, its ease of handling, and its reuse in the advancement of both strategies. In our research presented here, we applied 1-alkenemodified carbohydrates for the first time to form a uniform monolayer directly onto a Si surface. Such a surface has the potential to act as a tool for evaluating specific interactions in fundamental biological science. In addition, we demonstrate here the site-selective specific adsorption of carbohydrate-binding proteins (lectins) onto patterned carbohydrate monolayers fabricated using our previously reported ultraviolet (UV) lithography technique.13 First, a Si:H surface was obtained by removal of the SiO2 layer covering a Si (100) surface. Figure 1 illustrates the mechanism of the sugar monolayer formation on the Si:H surface. A silicon-hydrogen bond is homolytically cleaved by thermal activation to give a silicon-centered radical.12,14 Subsequently, 1-allyl R-D-galactopyranoside (9) Lin, C. C.; Yeh, Y. C.; Yang, C. Y.; Chen, C. L.; Chen, G. F.; Chen, C. C.; Wu, Y. C. J. Am. Chem. Soc. 2002, 124, 3508. (10) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128. (11) Love, K. R.; Seeberger, P. H. Angew. Chem., Int. Ed. 2002, 41, 3583. (12) Linford, M. R.; Fenter, P.; Eisenberger, P. M.; Chidsey, C. E. D. J. Am. Chem. Soc. 1995, 117, 3145. (13) Shirahata, N.; Yonezawa, T.; Seo, W. S.; Koumoto, K. Langmuir (submitted). (14) Sieval, A. B.; Linke, R.; Zuilhof, H.; Sudho¨lter J. R. Adv. Mater. 2000, 12, 1457.
10.1021/la0349020 CCC: $25.00 © 2003 American Chemical Society Published on Web 10/01/2003
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Figure 1. (a) Representative illustration of the formation model of the Allyl-Gal monolayer onto the H:Si surface by a thermal radical reaction. The preparation process for the GM immobilized on the Si surface is described in detail in the Supporting Information. (b) Time dependence of the water drop contact angle (θc) on the Si:H sample surfaces in a DMSO solution containing Allyl-Gal molecules of 0.008 M at 189.4 °C under a N2 atmosphere.
(Allyl-Gal) adds to the silicon-centered radical in an antiMarkovnikov fashion14 to form a Gal-terminated monolayer (GM) covalently bonded to the Si atoms (Si-C bond) of the Si substrate surface. In Figure 1b, the water drop contact angles (θc) on the Si:H surfaces decreased with increasing reaction time and almost saturated after 3.5 h at 184.9 °C, indicating that the surface was modified from hydrophobic to hydrophilic due to sugar moieties. Figure 2 shows two representative XP spectra of a GM substrate prepared with a reaction time of 4.0 h. The C 1s narrow-scan spectrum seen in Figure 2a was resolved by a pseudo-Voigt function to three components. The main peak at 284.8 eV is attributable to the C-C bonds15 of the immobilized GM. The other two weaker components, at 285.9 and 283.8 eV, can be assigned respectively to C-O bonds of the carbohydrate rings15-17 and Si-C bonds at the interface between the terminated carbon atoms of the GM and the Si substrate surface.18 This is because, compared to carbon atoms, oxygen is more electronegative and silicon is more electropositive. Similarly, the Si 2p narrow-scan spectrum seen in Figure 2b was resolved to four components: Si-Si bonds, which appear as Si 2p3/2 and Si 2p1/2 with 0.6 eV energy separations,19 Si-C bonds20 (at 100.5 eV), and Si-O bonds21,22 (at 103.1 eV). These XP spectra indicate that Gal can be displayed at the reducing end and is immobilized to the Si surface by Si-C covalent bonds. The surface roughness of the GM on the Si surface was measured by AFM to be rmsGal ) 0.46 ( 0.32 nm, and (15) Yan, J.; Tender, L. M.; Hampton, P. D.; Lo´pez, G. P. J. Phys. Chem. B 2001, 105, 8905. (16) Ying, L.; Yin, C.; Zhuo, R. X.; Leong, K. W.; Mao, H. Q.; Kang, E. T.; Neoh, K. G. Biomolecules 2003, 4, 157. (17) Quirk, R. A.; Davies, M. C.; Tendler, S. J. B.; Chan, W. C.; Shakesheff, K. M. Langmuir 2001, 17, 2817. (18) Terry, J.; Linford, M. R.; Wigren, C.; Cao, R.; Pianetta, P.; Chidsey, C. E. D. Appl. Phys. Lett. 1997, 71, 1056. (19) Tufts, B. J.; Kumar, A.; Bansal, A.; Lewis, N. S. J. Phys. Chem. 1992, 96, 4581. (20) Boo, J. H.; Lee, S. B.; Yu, K. S.; Sung, S. S.; Kim, Y. Surf. Sci. Coat. Technol. 2000, 131, 147. (21) Kennou, S.; Ladas, S.; Paloura, E. C.; Kalomiros, J. A. Appl. Surf. Sci. 1995, 90, 283. (22) Zhou, X.; Ishida, M.; Imanishi, A.; Nakato, Y. J. Phys. Chem. B 2001, 105, 156.
Figure 2. X-ray photoelectron spectra of (a) the C 1s and (b) the Si 2p regions of GMs immobilized onto Si(100) surfaces in a DMSO solution at 189.4 °C for 4.0 h. The solid lines with open circles (O) indicate the experimental data; the dashed lines are deconvolutions; and the completely solid lines are the resulting fits to the spectra. The inset shows an enlargement of the Si 2p spectrum.
no particles of impurities were observed. This rms value is only slightly larger than that measured for the Si:H surface (rmsSi:H ) 0.15 ( 0.03 nm). Next, by preparing patterned GM substrates through UV lithography, we simultaneously examined the effective specific affinity of the protein-sugar interaction and whether the nonspecific adsorption of proteins onto unintended areas could be prevented. Figure 3a shows an optical spectroscopic image of the TEM copper grid photomask we used to pattern GM (nonirradiated) and Si-O (irradiated) areas on the Si substrate. The Si-O areas were formed by the UV-induced cleavage of Si-C bonds13 at the interface between the GM and the Si surface. RCA120 was selected as a monitoring protein because the lectin can bind to galactose and N-acetylgalactosamine, specifically.23 As shown in Figure 3b, RCA120 molecules specifically and site-selectively adsorbed only onto the immobilized Gal moieties of the patterned GM substrate. No nonspecific adsorption onto the Si-O areas was observed. Without the UV lithography, RCA120 molecules specifically adsorbed onto the entire surface of the GM substrate, as shown in Figure 3c. There are two reasons for this successful prevention of nonspecific adsorption of RCA120 molecules onto the Si-O surface from the phosphate-buffered saline (PBS) solution employed. The first is attributable to the hydrophobic character of RCA120s the Si-O surface is hydrophilic with a water contact angle of ca. 5°. The second is the electrostatic repulsion between RCA120 molecules and the Si-O surface. A Si-O surface was found to be negatively charged in pH 7.2 solutions (23) Sweeney, E. C.; Tonevitsky, A. G.; Temiacov, D. E.; Agapov, I. I.; Saward, S.; Palmer, R. A. Proteins: Struct., Funct., Genet. 1997, 28, 586.
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Figure 3. Optical spectroscopic images of (a) the photomask (Cu-grid mesh for TEM observation) employed for UV lithography of the GM substrate; (b) RCA120 molecules specifically adsorbed only onto the Gal surface of a patterned GM substrate after UV lithography; and (c) RCA120 molecules specifically adsorbed onto the entire surface of a GM substrate unpatterned by UV lithography. In image a, the black circles show holes (areas to be UV-irradiated) in the Cu-grid mesh.
containing 1 mM KCl as a supporting electrolyte.24 RCA120 is also negatively charged in a PBS solution of pH 7.2. Therefore, the technique we propose here, including the lithographic process, is very useful for detecting lectin. The peak assignable to Si-O was also observed in the as-formed GM substrate in Figure 2b, suggesting a low coverage of GM on Si due to the incomplete replacement of Si:H by Si-C bonds. There are three reasons for explaining this phenomenon. First of all, not all Si atoms at the surface make the Si-C bond. According to a molecular modeling study on a monolayer derived from 1-octadecene reported by Sieval,25 ca. 61% of the SiH2 sites on a H:Si(100) surface are substituted with alkyl groups. (24) Hozumi, A.; Sugimura, H.; Yokogawa, Y.; Kameyama, T.; Takai, O. Colloids Surf. 2001, 182, 257.
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Second is a correlation between the tilt angle of the monolayer and its chain length. The tilt angle of the monolayer from the surface normal generally increases with shortening chain length (in particular, n < 6).26 Third is a larger molecular size of the terminated carbohydrate ring than the terminated-methyl group of all-trans alkyl monolayer. Thus, the coverage of the GMsespecially an Allyl-Gal with a very short alkene groupswould be lower than that of the 1-octadecene monolayer. The remaining Si:H groups would be transformed into Si-O groups after the monolayer formation. Due to the Si-O formation, the surface roughness of the GM (rmsGal) became slightly larger than that of the Si:H surface (rmsSi:H). As shown in Figure 3b and c, the specifically adsorbed protein appears as black dots on the GM surface, and its density seems low. This is probably due to the low density of Gal on the Si surface, as well as to the shorter alkyl chain of Allyl-Gal, since lectins are quite large and no free conformational change promoting effective adsorption can be generated by this relatively small sugar. The larger black dots can be attributed to the aggregation of RCA120 molecules by nonspecific interaction with each other. In conclusion, we have succeeded in immobilizing a sugar monolayer onto specific sites on a Si surface and, additionally, have fabricated microscale structures composed of a protein by utilizing carbohydrate-protein interactions. We discovered the intriguing phenomenon that Allyl-Gal precipitates, prepared as a solid-state material under ambient conditions, could be transformed into a GM. This basic finding implies that solid materials, including those with exceedingly high melting points or no melting points, have the potential to be transformed into monolayers directly bonded to inorganic substrates, with the only limitation being that the material must be soluble into any kind of solvent. Acknowledgment. The authors thank Dr. A. Hozumi of AIST for his warm hospitality. This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS). Supporting Information Available: Detailed experimental procedures for the synthesis of 1-allyl R-D-galactopyranoside (Allyl-Gal) precipitates; the formation of the Galterminated monolayer (GM) onto the Si surface; the UV lithography and the carbohydrate-protein specific interaction on the GM substrate before and after the UV lithography; and data analyses for the XPS, water contact angle measurements, AFM, and ζ-potential measurements of the RCA120 molecules in the PBS solution. These materials are available free of charge via the Internet at http://pubs.acs.org. LA0349020 (25) Sieval, A. B.; Hout, B.; Zuilhof, H.; Sudho¨lter, E. J. R. Langmuir 2000, 16, 2987. (26) Kluth, G. J.; Sung, M. M.; Maboudian, R. Langmuir 1997, 13, 3775. (b) Wassermann, S. R.; Tao, Y.-T.; Whitesides, G. M. Langmuir 1989, 5, 1074. (27) Shirahata, N.; Masuda, Y.; Yonezawa, T.; Koumoto, K. Langmuir 2002, 18, 10379.
