© Copyright 2007 American Chemical Society
FEBRUARY 27, 2007 VOLUME 23, NUMBER 5
Letters Stable Immobilization of an Oligonucleotide Probe on a Gold Substrate Using Tripodal Thiol Derivatives Toshiya Sakata,†,‡ Sumio Maruyama,† Aiko Ueda,† Hidenori Otsuka,† and Yuji Miyahara*,†,‡ Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan, and Center for NanoBio Integration, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ReceiVed June 6, 2006. In Final Form: NoVember 23, 2006 We proposed an interface molecule for immobilization of DNA probes on solid substrates of DNA chips. We have designed and synthesized tripodal thiol derivatives for stable immobilization of oligonucleotide probes on a gold surface. On the basis of the tetrahedral structure of tripod, the tripodal thiol derivatives were bonded upright to the gold substrate, which would control the orientation of oligonucleotide probes. When the gold substrate with oligonucleotide probes tethered using the thiol derivatives was exposed to deionized water at higher temperatures, the tripodal interface molecules were attached to the gold surface more stably than the single contact molecules. The DNA chip platform combined with the functional interface molecule is suitable for a reproducible, inexpensive, and high-throughput detection system for genetic analyses in clinical diagnostics.
Introduction The study of bio/solid substrate interface has been used to develop devices for biochemical analyses using biochips/ biosensors.1 Several materials such as gold, glass, mica, and silicon oxide are often used for immobilization of biomolecules such as DNA, because their surface properties are easily modified by forming self-assembled monolayers (SAMs) with different functionalities.2 Functional interface molecules used to prepare SAMs contain two terminated groups. In the case of immobilization on a gold surface, one is a thiol group, -SH, which forms a -S-Au covalent bond on gold. The other terminated * Yuji Miyahara Ph.D., National Institute for Materials Science, Biomaterials Center, Bioelectronics Group, 1-1 Namiki, Tsukuba 305-0044 Japan. Tel.:+81-29-860-4506. Fax: +81-29-860-4714. E-mail:
[email protected]. † National Institute for Materials Science. ‡ The University of Tokyo. (1) (a) Lenigk, R.; Carles, M.; Ip, N.Y.; Sucher, N. J. Langmuir 2001, 17, 2497-2501. (b) Jain, K. K. Science 2001, 294, 621-623. (c) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192-1199. (2) (a) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (b) Vargo, T. G.; Gardella, J. A.; Calvert, J. M.; Chen, M. S. Science 1993, 262, 1711.
group is designed for DNA immobilization, based on the electrostatic interaction or chemical bonds with the chemically modified oligonucleotides.3 Control of the surface density and orientation of the DNA probes is important for efficient and specific hybridization on the substrate. Improvement of adhesion strength of immobilized DNA probes would allow us to realize precise and reliable genetic analysis as well as repeated use of the DNA chip which leads to reduction of cost per assay. In the present study, we designed and synthesized functional interface molecules for stable immobilization of oligonucleotide probes on a gold surface, and fundamental characteristics such as immobilization stability of the functional interface molecules were investigated.
Experimental Section Immobilization of Oligonucleotide Probes. The tripodal and single-contact molecules as functional interface derivatives were synthesized according to the Supporting Information.4 Both thiol (3) (a) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24, 3031-3039. (b) Edman, C. F.; Raymond, D. E.; Wu, D. J.; Tu, E.; Sosnowski, R. G.; Butler, W. F.; Nerenberg, M.; Heller, M. J. Nucleic Acids Res. 1997, 25, 4907-4914.
10.1021/la0616193 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/02/2007
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was 5′-CCACTACCAGGGCACGT-3′ (17-mer), which was used in our previous works.5 XPS Analysis. The thiol derivatives tethered on the gold surface were analyzed in XPS measurements using parallel angle-resolved XPS (Thermo Electron Corporation). All of the data were collected using a 400-µm spot of Al KR X-rays from a microfocusing monochromator. XPS spectra were collected from the C 1s, O 1s, S 2p, and Au 4f regions, which were obtained by acquiring 16 angle channels (each 3.75° wide) simultaneously in each scan. A nondestructive depth profile was constructed from the angleresolved XPS data using a technique involving maximum entropy methods.6,7 Contact Angle Measurements. The contact angles were determined optically with the Automatic Contact Angle Meter system (Kyowa Interface Science Co., Ltd.) using deionized water (filtered Millipore water).
Results and Discussion
Figure 1. Structure of interface molecules and immobilization of oligonucleotide probes: (a) tripodal thiol derivative; (b) singlecontact thiol derivative. Both derivatives can be attached to gold surfaces forming -S-Au bonds. The Cy5 fluorescent molecules are labeled at the 3′ end of oligonucleotide probes, while the amino group modified at the 5′ end of probes is bonded with carboxyl group of derivatives.
derivatives were used for the investigation of the immobilization stability of biochemically modified gold substrates and designed to functionally utilize carboxyl group for immobilization of oligonucleotide probes, as shown in Figure 1. They were dissolved in tetrahydrofuran at a concentration of 2-20 mM. The gold substrates were then soaked in that solvent for about 15 h at room temperature. The Au/Cr thin film was deposited by sputtering or vacuum evaporation. The thickness of the Au/Cr film was designed to be 100 nm/50 nm, respectively. No surface treatment was specifically performed before adding thiol derivatives on the gold substrates. The 5′ end of oligonucleotide probe is modified with an amino group which can combine with carboxyl group of thiol derivatives (Figure 1). The formation of an amide bond was achieved in deionized water including the amino-modified oligonucleotide probes (100 µM) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, in which the gold substrate with thiol derivatives was soaked at room temperature for 15 h. Moreover, the 3′ end of oligonucleotide probe was labeled with fluorescent dyes (Cy5) in order to evaluate the immobilization strength of interface molecules. The base sequence of oligonucleotide probe (4) (a) Davis, G. L.; Hey, D. Y.; Williams, G. H. J. Chem. Soc. 1956, 43974408. (b) Heim, C.; Affeld, A.; Nieger, M.; Vo¨gtle, F. HelV. Chim. Acta 1999, 82, 746-759. (c) Zhu, Z.; Moore, J. S. J. Org. Chem. 2000, 65, 116-123. (d) Hirayama, D.; Takimiya, K.; Aso, Y.; Otsubo, T.; Hasobe, T.; Yamada, H.; Imahori, H.; Fukuzumi, S.; Sakata, Y. J. Am. Chem. Soc. 2002, 124, 532-533.
Figure 1 shows the chemical structures of the synthesized interface molecules. Details of synthesis process is described in the Supporting Information.4 Since the tripodal molecule can be bound to a gold surface through three thiol groups (Figure 1a), adhesion strength to the substrate is expected to be improved in comparison with interface molecules with a single thiol group such as alkanethiol. In addition, the chemical structure of tripodal molecule is based on regular tetrahedron structure so that oligonucleotide probes can be immobilized perpendicular to the surface of the substrate. It is therefore possible to control orientation of the immobilized oligonucleotide probes. The interface molecule with single point contact as shown in Figure 1b was also synthesized for comparison. The scheme for immobilization of oligonucleotide probes based on the synthesized thiol derivatives is also shown in Figure 1. The 5′ end of oligonucleotide probe is modified with amino group, which can be combined with carboxyl group of thiol derivatives. Immobilization strength of interface molecules were evaluated using oligonucleotide probes labeled with fluorescent dyes (Cy5). After the immobilization of oligonucleotide probes with Cy5 dyes by use of each thiol derivative, the fluorescent intensities of the probes with tripodal molecules were lower than those of the probes with single contact molecules. This result indicates that the densities of the single contact molecules would be higher than those of the tripodal molecules, as expected from the structural difference. The quantitative characterization of tripodal thiol derivatives tethered on a gold surface was evaluated with a X-ray photoelectron spectroscopy (XPS) measurement using parallel angleresolved XPS (Figure 2). This method is well-suited to the analysis of such materials as synthesized in this study, because the depth profile of immobilized molecules on a substrate can be easily calculated.6,7 The XPS profile is obtained from a spot of 400 µm2 (5) (a) Sakata, T.; Kamahori, M.; Miyahara, Y. Mater. Sci. Eng., C 2004, 24, 827-832. (b) Sakata, T.; Kamahori, M.; Miyahara, Jpn. J. Appl. Phys. 2005, 44 (4B), 2854-2859. (c) Sakata, T.; Matsumoto, S.; Nakajima, Y.; Miyahara, Y. Jpn. J. Appl. Phys. 2005, 44 (4B), 2860-2863. (d) Sakata, T.; Miyahara, Y. ChemBioChem 2005, 6, 703-710. (e) Sakata, T.; Miyahara, Y. Biosens. Bioelectron. 2005, 21, 827-832. (f) Sakata, T.; Miyahara, Y. Angew. Chem., Int. Ed. 2006, 45, 2225-2228. (6) (a) Skilling. J. Classic maximum entropy. In Maximum Entropy and Bayesian Methods; Skilling, J., Ed.; Kluwer Academic: Norwell, MA, 1989; pp 45-52. (b) Opila, R. L.; Eng, J., Jr. Prog. Surf. Sci. 2002, 69, 125-163. (c) Watts, J. F.; Wolstenhomes, J. An Introduction to Surface Analysis by XPS and AES; Wiley: New York, 2003; pp 87-89. (d) de Smet, L. C. P. M.; Stork, G. A.; Hurenkamp, G. H. F.; Sun, Q.-Y.; Topal, H.; Vronen, P. J. E.; Sieval, A. B.; Wright, A.; Visser, G. M.; Zuilhof, H.; Sudholter, E. J. R. J. Am. Chem. Soc. 2003, 125, 1391613917. (e) Jakubowicz, A.; Jia, H.; Wallace, R. M.; Gnade, B. E. Langmuir 2005, 21, 950-955. (7) http://www.thermo.com/com/cda/product/detail/1,1055,15914,00.html.
Letters
Figure 2. Depth profile of tripodal thiol derivatives on gold surface constructed from angle-resolved XPS data using maximum entropy techniques.6
on a gold substrate and averaged for depth in the measured spot. From Figure 2, all of the tripodal derivatives were found not to be perpendicular to the substrate, because the oxygen was not localized near the surface only. However, more oxygen was detected near the surface due to the carboxyl group as shown in Figure 2; the tripodal derivatives were found to be oriented in average. The tripodal derivatives were found to be immobilized with orientation to the surface of the substrate, because the O 1s peak and OsCdO bond spectra of the carboxyl group of the immobilized thiol tripod were detected with strong intensity away from the gold surface, while the bonding between the thiol group and gold was localized at the surface, estimated from the distribution of the S 2p peak intensity. A nondestructive depth profile was constructed from the angle-resolved XPS data using a technique involving maximum entropy methods,6 and the thickness of the layers was calculated using the multilayer thickness calculator part of the Advantage Data System.7 The thickness of the tripodal thiol derivative layer was found to be about 1.7 nm as shown in Figure 2, which was in good agreement with the computed length of the tripodal molecule at about 1.8 nm. The small difference between the analyzed and calculated values seems to be due to the roughness of the gold substrate surface as well as the tilt of tethered molecules from the surface normal. The orientation analysis indicates the tridentate binding of the tripodal thiol derivatives at the gold substrate without the monodentate or bidentate contact after the reaction for about 15 h at room temperature. Therefore, the orientation of oligonucleotide probes can be controlled using the tripodal derivatives. Figure 3 shows the measurement of contact angle for the tripodal and single derivative-modified substrates. The contact
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angle for the single derivatives on the gold substrate was smaller than that for the tripodal derivatives. This result indicates that the single derivatives were immobilized in a more tightly packed arrangement and that carboxyl groups of single derivatives caused a more hydrophilic surface than the tripodal derivatives. Temperature stress was applied to the oligonucleotide probes immobilized using the interface molecules, and fluorescent emission was evaluated for both tripodal and single-contact derivatives. Figure 4 shows a comparison of immobilization strength between the tripodal derivative and the single-contact derivative. The gold substrates with oligonucleotide probes were soaked in deionized water at 60 °C (Figure 4a). Fluorescent intensity was normalized on the basis of the initial fluorescent intensity at time 0 min. Normalized fluorescent intensity was evaluated as a function of time. The lifetime of the fluorescent intensity of the Cy5 molecule was confirmed to be stable and did not change even after incubation at 60 °C for 120 min. The fluorescent intensity of oligonucleotide probes bound to the tripodal derivatives was two or three times bigger than that of those bonded with the single-contact derivative at the beginning of the stability test. As shown in Figure 4a, the tripodal derivative was attached to the gold surface more stably and strongly than that of the single-contact derivative, because of the difference of the number of chemical bonding between thiol derivatives and gold substrate. Although immobilization strength of the tripodal derivative was improved as compared with that of the single-contact derivative, its fluorescent intensity decreased gradually over the period of the stability test. Adhesion strength of the tripodal derivative should be improved further. Adhesion strength of oligonucleotide probes tethered using thiol derivatives on gold substrates was evaluated at various temperatures. Figure 4b shows the change of fluorescent intensity for oligonucleotide probes after immersing in deionized water for 30 min at various temperatures. In this case, fluorescent intensity was normalized on the basis of that before temperature stress. The evaluated parameter was the ratio of the fluorescent intensities before and after exposure into deionized water at each temperature. The decrease of fluorescent intensity of the Cy5 molecule itself was not found even when exposed at 90 °C for 30 min. In this study, we estimated the stability on the basis of the number of remaining oligonucleotide probes under the temperature stress. The oligonucleotide probes with tripodal thiol derivatives were more tightly bound to the gold surface than those with single-contact thiol derivatives at temperatures lower than 71 °C. The amount of immobilized DNA probes with tripodal thiol derivatives, however, decreased gradually at temperatures higher than 59 °C and was similar to that of the single-point
Figure 3. Drop of water resting on the tripodal derivative-modified gold surface (a), and the single-contact derivative-modified gold surface (b).
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contact derivatives peeled off from the gold substrate when singlepoint contacts were broken. At temperatures higher than 80 °C, almost all S-Au bonds of both thiol derivatives would be broken within 30 min. An increase of the probe stability has also been reported using the trithiol molecules at the specific conditions (in 10 mM DTT and 0.3 M NaCl solutions at 40 °C).9 By using the tripodal derivatives, the immobilization stability of DNA probes on the solid substrate can be controlled and improved, and efficient hybridization and highly sensitive and quantitative detection of target DNA molecules is expected.
Conclusions
Figure 4. Comparison of immobilization strength between tripodal thiol derivatives and single-contact thiol derivatives. The immobilization stability was evaluated at 60 °C (a) and for 30 min at 21-90 °C (b).
derivative at temperatures higher than 80 °C. This is because the gold-sulfur bond was broken under oxidative conditions where the gold substrates with thiol derivatives were exposed for 30 min at higher temperatures.8 The immobilization strength is dependent on the number of chemical bonding between thiol derivatives and gold substrate. Tripodal derivatives remained connected to the gold surface at one or two point contacts, even if one or two S-Au bonds were broken under the thermal stress conditions. On the other hand, oligonucleotide probes with single(8) (a) Schlenoff, J. B.; Li, M.; Ly, H. J. Am. Chem. Soc. 1995, 117, 1252812536. (b) Ulman, A. Chem. ReV. 1996, 96, 1533-1554.
In summary, the stable immobilization of oligonucleotide probes was demonstrated by the use of synthesized tripodal thiol derivatives, because they could be bound to the gold surface through three thiol groups. In addition, oligonucleotide probes could be immobilized perpendicular to the surface of the substrate based on the regular tetrahedron structure of tripodal derivatives. The stable immobilization method controlling orientation of the oligonucleotide probes is necessary for realizing a reliable DNA chip. The DNA chip platform combined with the functional interface molecule is suitable for a reproducible, inexpensive, and high-throughput detection system for genetic analyses in clinical diagnostics. Acknowledgment. The authors wish to thank Drs. Y. Horiike and T. Tateishi of National Institute for Materials Science in Japan, Profs. K. Kataoka and K. Ishihara of the University of Tokyo, Mr. Y. Nakajima of Ryokusei M.E.S. Co., Ltd., in Japan, Dr. M. Kamahori of Hitachi Ltd. in Japan and Prof. P. Fortina of Thomas Jefferson University in U.S.A. for their help and useful discussion. Supporting Information Available: Synthesis of interface derivatives. Materials, instrumentation, and analytical data. This material is available free of charge via the Internet at http://pubs.acs.org. LA0616193 (9) Li, Z.; Jin, R.; Mirkin, C. A.; Letsinger, R. L. Nucleic Acids Res. 2002, 30, 1558-1562.
