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Formation, Characterization, and Chemistry of Undecanoic Acid-Terminated Silicon Surfaces: Patterning and Immobilization of DNA Raluca Voicu,†,§ Rabah Boukherroub,‡ Vasiliki Bartzoka,† Tim Ward,† James T. C. Wojtyk,† and Danial D. M. Wayner*,† Steacie Institute for Molecular Sciences, National Research Council, 100 Sussex Drive, Ottawa, Ontario, K1A 0R6 Canada, and Interdisciplinary Research Institute, IRI - IEMN, Avenue Poincare´ - BP. 69, 59652 Villeneuve d’Ascq, France Received August 24, 2004 This paper describes a simple strategy for DNA immobilization on chemically modified and patterned silicon surfaces. The photochemical modification of hydrogen-terminated Si(111) with undecylenic acid leads to the formation of an organic monolayer covalently attached to the surface through Si-C bonds without detectable reaction of the carboxylic acid group, providing indirect support of a free radical mechanism. Chemical activation of the acid function was achieved by a simple chemical route using N-hydroxysuccinimide (NHS) in the presence of N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride. Single strand DNA with a 5′-dodecylamine group was then coupled to the NHS-activated surface by amide bond formation. Using a previously reported chemical patterning approach, we have shown that DNA can be immobilized on silicon surfaces in spatially well-resolved domains. Methoxytetraethyleneglycolamine was used to inhibit nonspecific adsorption. The resulting DNA-modified surfaces have shown good specificity and chemical and thermal stability under hybridization conditions. The sequential reactions on the surface were monitored by ATR-FTIR, X-ray Photoelectron Spectroscopy, and fluorescence spectroscopy.
Introduction There is increasing focus on the development of new biochip technologies1 for monitoring biologically active molecules for expression profiling,2 genotyping,3 DNA sequencing,4 diagnostics,5,6 immunology,7 and drug discovery and development.8 However, there are still technical hurdles to overcome before biomolecular-based diagnostic devices will become robust and reliable. The improvement of DNA- and protein-based biosensors and microchips relies on the reproducible and effective immobilization of biomolecules onto solid substrates.4,9,10 Several methods are used to attach DNA molecules to surfaces including (a) covalent binding of DNA to self* Author to whom correspondence should be addressed. † National Research Council. ‡ Interdisciplinary Research Institute. § Current address: Institute for Microstructural Sciences, National Research Council, 1200 Montreal Road, Ottawa, Ontario, K1A 0R6 Canada. (1) Call, D. R.; Chandler, D. P.; Brockman, F. BioTechniques 2001, 30, 368. (2) Schena, M.; Shalon, D.; Heller, R.; Chai, A.; Brown, P. O.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10614. (3) Guo, Z.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.; Smith, L. M. Nucleic Acid Res. 1994, 22, 5456. (4) Caviani-Pease, A.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022. (5) Drmanac, S.; Kita, D.; Labat, I.; Hauser, B.; Schmidt, C.; Burczak, J. D.; Drmanac, R. Nat. Biotechnol. 1998, 16, 54. (6) Yershov, G.; Barsky, V.; Belgovskiy, A.; Kirillov, E.; Kreindlin, E.; Ivanov, I.; Parinov, S.; Guschin, D.; Drobishev, A.; Dubiley, S.; Mirzabekov, A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4913. (7) Heller, R. A.; Schena, M.; Chai, A.; Shalon, D.; Bedilion, T.; Gilmore, J.; Woolley, D. E.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2150. (8) Debouck, C.; Goodfellow, P. N. Nat. Genet. 1999, 21, 48. (9) Yang, M.; McGovern, M. E.; Thompson, M. Anal. Chim. Acta 1997, 346, 259. (10) Lipshutz, R. J.; Morris, D.; Chee, M.; Hubbell, E.; Kozal, M. J.; Shah, N.; Shen, N.; Yang, R.; Fodor, S. P. A. Biotechniques 1995, 19, 442.
assembled monolayers (SAMs) of aminosilanes using heterobifunctional cross-linkers,11 (b) attachment of thiolmodified oligonucleotides to thiol-functionalized silicon wafers using bifunctional alkyltrichlorosilanes,12 (c) tethering of DNA to glass slides via an epoxysilane-amine covalent linkage,13 (d) chemoselective coupling of oligonucleotides to alkanethiol SAMs on gold,14 and (e) crosslinking to poly-L-lysine-coated microscope slides.15 In fact, most of the reported work for covalent immobilization of DNA on solid substrates relies on surface chemistry developed for glass slides. This approach has some limitations related to the nature of the substrate and the surface chemistry. Indeed, the amorphous character of silica and SiO2/Si makes very difficult to control the density of Si-OH groups generated on the surface that are necessary for chemical linkages. The silanisation reaction itself is complicated by the difficult-to-control polymerization occurring during the reaction. Crystalline silicon is a very attractive alternative substrate for biomolecule immobilization because of its well-defined structure, as well as for the ease and reproducibility of the existing method for the preparation of hydrogen-terminated surfaces.16-19 Moreover, modifica(11) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24, 3031. (12) McGovern, M. E.; Thompson, M. Can. J. Chem. 1999, 77, 1678. (13) Beattie, W. G.; Meng, L.; Turner, S. L.; Varma, R. S.; Dao, D. D.; Beattie, K. L. Mol. Biotechnol. 1995, 4, 213. (14) Boncheva, M.; Scheibler, L.; Lincoln, P.; Vogel, H.; Akerman, B. Langmuir 1999, 15, 4317. (15) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467. (16) Higashi, G. S.; Chabal, Y. J.; Trucks, G. W.; Raghavachari, K. Appl. Phys. Lett. 1990, 56, 656. (17) Jakob, P.; Chabal, Y. J.; Raghavachari, K.; Dumas, P.; Christman, S. B. Surf. Sci. 1993, 285, 251. (18) Linford, M. R.; Fenter, P.; Eisenberger, P. M.; Chidsey, C. E. D. J. Am. Chem. Soc. 1995, 117, 3145. (19) Allongue, P.; Villeneuve, C. Henry de; Morin, S.; Boukherroub, R.; Wayner, D. D. M. Electrochim. Acta 2000, 45, 4591.
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tion of crystalline silicon offers the possibility of using well-established microfabrication methods for the integration of diverse chemical and biochemical functionality into microelectronic platforms and the use of intrinsic properties of silicon to detect molecular events occurring on the surface.20,21 In the past few years, there have been significant advances in the chemical functionalization of crystalline silicon surfaces. It has been demonstrated that organic monolayers covalently attached to hydrogen-terminated silicon surfaces could be formed under various conditions.22 These organically modified silicon surfaces were found to be chemically robust and to display good electrical properties (low density of electrically active defects).23-26 Recently, different strategies were developed for chemical functionalization and passivation of hydrogen-terminated silicon Si(111) surfaces under various conditions using 1-alkenes and aldehydes to form organic monolayers covalently attached to the semiconductor surface through Si-C and Si-O-C bonds.27-32 For example, reaction of ester-terminated alkenes with Si(111)-H led to the formation of an organic monolayer bearing an ester terminal group.29 The ester functional group remained chemically accessible and could be displaced with a variety of standard chemical reagents.33 In the present work, monolayers are covalently attached by irradiation of an alkene at 300 nm. While Zuilhof et al.34,35 have shown that these reactions proceed by irradiation at longer wavelengths (450 nm), irradiation at 300 nm is more convenient, as the reactions are completed in about 3 h rather than the 15 h required at longer wavelengths. Zuilhof’s work does, however, raise interesting questions about the mechanism of the formation of the Si-C bonds. A number of approaches for the covalent attachment of DNA have been reported. Houlton et al. outlined a method that allows the combination of molecular self-assembly and automated synthesis with silicon and have developed approaches for the on-chip synthesis of DNA oligomers.36-39 Hamers et al. have explored the chemical derivatization (20) Vo-Dinh, T.; Cullum, B. Fresenius J. Anal. Chem. 2000, 366, 540. (21) Vo-Dinh, T.; Alarie, J. P.; Isola, N.; Landis, D.; Wintenberg, A. L.; Ericson, M. N. Anal. Chem. 1999, 71, 358. (22) Buriak, J. M. Chem. Rev. 2002, 102, 1272. (23) Bansal, A.; Lewis, N. S. J. Phys. Chem. B 1998, 102, 1067. (24) Haber, J. A.; Lauermann, I.; Michalak, D.; Vaid, T. P.; Lewis, N. S. J. Phys. Chem. B 2000, 104, 9947. (25) Webb, L. J.; Lewis, N. S. J. Phys. Chem. B 2003, 107, 5404. (26) Yu, H.-Z.; Morin, S.; Wayner, D. D. M.; Allongue, P.; Henry de Villeneuve, C. J. Phys. Chem. B 2000, 104, 11157. (27) Sieval, A. B.; Demirel, A. L.; Nissink, J. W. M.; Linford, M. R.; van der Maas, J. H.; de Jeu, W. H.; Zuilhof, H.; Sudho¨lter, E. J. R. Langmuir 1998, 14, 1759. (28) Boukherroub, R.; Bensebaa, F.; Morin, S.; Wayner, D. D. M. Langmuir 1999, 15, 3831. (29) Boukherroub, R.; Wayner, D. D. M. J. Am. Chem. Soc. 1999, 121, 11513. (30) Boukherroub, R.; Morin, S.; Sharpe, P.; Wayner, D. D. M.; Allongue, P. Langmuir 2000, 16, 7429. (31) Wojtyk, J. T. C.; Tomietto, M.; Boukherroub, R.; Wayner, D. D. M. J. Am. Chem. Soc. 2001, 123, 1535. (32) Liu, Y.-J.; Navasero, N. M.; Yu, H.-Z. Langmuir 2004, 20, 4039. (33) Seneci, P. Solid-Phase Synthesis and Combinatorial Techniques; John Wiley and Sons: New York, 2000. (34) 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.; Sudho¨lter, E. J. R. J. Am. Chem. Soc. 2003, 125, 13916. (35) Sun, Q.-Y.; de Smet, L. C. P. M.; van Lagen, B.; Wright A.; Zuilhof, H.; Sudho¨lter, E. J. R. Angew. Chem., Int. Ed. 2004, 43, 1352. (36) Pike, A. R.; Lie, L. H.; Eagling, R. A.; Ryder, L. C.; Patole, S. N.; Connolly, B. A.; Horrocks, B. R.; Houlton, A. Angew. Chem., Int. Ed. 2002, 41, 615. (37) Pike, A.; Horrocks, B.; Connolly, B.; Houlton, A. Aust. J. Chem. 2002, 55, 191. (38) Patole, S. N.; Pike, A. R.; Connolly, B. A.; Horrocks, B. R.; Houlton, A. Langmuir 2003, 19, 5457.
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of hydrogen-terminated silicon surfaces for direct attachment of DNA. They showed that the DNA-modified surfaces exhibit a high density of binding sites and a high specificity and stability to the hybridization conditions.40,41 These strategies are based on the attachment of monolayers with protected hydroxyl or carboxyl groups on the terminus of the monolayers on silicon surfaces and their subsequent deprotection by acid or base hydrolysis. In many cases, the somewhat harsh reaction condition for deprotection results in surfaces of lower chemical and/or electrical quality. From this perspective, more simple approaches are desirable for the immobilization of chemical and biological species on the semiconductor surfaces without the need to build up thick organic layers, postmodification strategies to limit nonspecific adsorption of the target on the surface, and chemical robustness of the monolayer under physiological or wet chemical conditions. This paper describes a direct method for anchoring a terminal carboxylic acid function, representing a significant advance in preparation of silicon surfaces suitable for the incorporation of biomolecules. In particular, we present a simplified chemical approach for the attachment and hybridization of single-strand DNA. The functionalized surfaces and the different steps leading to biomolecule immobilization are characterized using infrared, X-ray photoelectron, and fluorescence spectroscopies. In combination with photopatterning techniques, these modified surfaces are shown to undergo a number of hybridization/denaturation cycles without significant loss of material and with limited nonspecific adsorption. Experimental Section Materials. ATR silicon crystals (25 × 4.5 × 1 mm3) were purchased from Harrick and the silicon wafers from Virginia Semiconductor. All cleaning and etching reagents were cleanroom grade. Sulfuric acid, 96% (H2SO4), hydrogen peroxide, 30% (H2O2), ammonium floride, 40% (NH4F), and hydrofluoric acid, 48% (HF) were supplied by Amplex. All other chemicals were reagent grade or higher and were used as received unless otherwise specified. MilliQ water (18 MΩ) was used for all experiments. Undecylenic acid, ethanolamine, N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), triethylamine (TEA), tetraethyleneglycol monomethyl ether, sodium dodecyl sulfate (SDS), ammonia (28%), 1,1,1-trichloroethane (TCE), and dichloromethane (DCM) were all available from Aldrich. All other solvents were obtained from VWR and distilled under an inert atmosphere. Tetrahydrofuran (THF) was passed through an activated column of alumina and refluxed over Na for 24 h prior to distillation. N,N-dimethylformamide (DMF) was distilled under reduced pressure over CaH2. Phosphate-buffered saline (PBS) solutions (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4; pH ) 7.2) were obtained from Aldrich, and the pH was adjusted with a 0.1 M KOH solution. Tween 20 (polyoxyethylene sorbitan monolaurate) was purchased from BioRad Laboratories. The single-strand DNA (Escherichia coli probe 16S), 5′-H2NC12H24-TT-CCT-GTT-ACC-GTT-CGA-CTT-G-3′, and amino homopolymeric deoxythymidine 5′-H2N-C6-TTT-TTTTTT-TTT-TTT-TTT-TT-3′ (H2N-dT20) and labeled deoxyadenosine (5′-Cy3-AAA-AAA-AAA-AAA-AAA-AAA-AA-3′ (Cy3-dA20) were acquired from Dr. Roland Brousseau at BRINRC and BioCorp, Inc., respectively. Synthesis of Methoxytetraethyleneglycolamine H3C(OCH2CH2)4NH2 (TEGamine). Methyltetraethyleneglycol Meth(39) Lie, L. H.; Patole, S. N.; Pike, A. R.; Ryder, L. C.; Connolly, B. A.; Ward, A. D.; Tuite, E. M.; Houlton, A.; Horrocks, B. R. Faraday Discuss. 2004, 125, 235. (40) Strother, T.; Cai, W.; Zhao, X.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205. (41) Strother, T.; Hamers, R. J.; Smith, L. M. Nucleic Acids Res. 2000, 28, 3535.
Undecanoic Acid-Terminated Silicon Surfaces anesulfonate. In a 250 mL round-bottom flask equipped with a magnetic stirrer and a nitrogen inlet were placed tetraethyleneglycol monomethyl ether (6.58 g, 31.6 mmol), TEA (7.4 mL), and dry DCM (55 mL). The mixture was cooled to 0 °C and a solution of methanesulfonyl chloride (3.8 mL) in dry DCM (20 mL) was added dropwise. After addition, the mixture was stirred 0.5 h at 0 °C, and water (30 mL) was added. The resulting mixture was acidified by adding aqueous sulfuric acid (2%). The mixture was then poured into a separatory funnel (500 mL) and mixed. After settling, the organic layer was separated and washed with water (4 × 40 mL). The aqueous phase was extracted with DCM (2 × 50 mL). The organic layers were combined, dried over MgSO4, and allowed to settle well. The supernatant was transferred by pipet into a flask and concentrated to leave the methanesulfonate as a pale yellow liquid, 7.85 g, 98%. 1H NMR (400 MHz, δ ppm, CDCl3): 4.36-4.31 (m, 2H); 3.80-3.76 (m, 2H); 3.71-3.62 (m, 10H); 3.58-3.53 (m, 2H); 3.39 (s, 3H); 3.08 (s, 3H). 13C NMR (100 MHz, δ ppm, CDCl3): 72.09, 70.80, 70.77, 70.73, 70.69, 70.69, 69.50, 69.18, 59.18, 37.89. MS(EI) [M + 1]: 287.2 for C10H22O7S. Methoxytetraethyleneglycolamine (TEGamine). Methyltetraethyleneglycol methanesulfonate (1.80 g, 7.08 mmol) was added to ammonia (concentrated 28%, 500 mL) and swirled to dissolve. The solution was allowed to stand for 8 days at room temperature. The mixture was then concentrated to leave a viscous pale salt. The residue was treated with 5 M NaOH (5 mL) and extracted with DCM (3 × 25 mL). The separated organic layer was dried over MgSO4 and allowed to settle well. The supernatant was transferred by pipet to another flask and concentrated to leave TEGamine as a colorless liquid, 1.44 g, 98%. 1H NMR(400 MHz, δ ppm, CDCl3): 3.70-3.61 (m, 10H); 3.57-3.53 (m, 2H); 3.533.49 (t, 2H); 3.38 (s, 3H); 2.89-2.85 (t, 2H); 1.95 (NH, deuterium exchangeable). 13C NMR(100 MHz, δ ppm, CDCl3): 73.32, 72.10, 70.76, 70.74, 70.74, 70.67, 70.46, 59.20, 41.90. MS(EI): [M + 1]: 208.2 for C9H21O4N. Surface Characterization. Attenuated Total Reflection Fourier Transform Infrared Spectrometry (ATR-FTIR). ATRFTIR spectra were recorded using a Nicolet MAGNA-IR 860 spectrometer at 2 cm-1 resolution. The ATR crystals were mounted in a purged sample chamber with the light focused normal to one of the 45° bevels. Background spectra were obtained using a hydrogen-terminated surface. X-Ray Photoelectron Spectroscopy (XPS). XPS spectra were recorded on a Kratos Axis instrument, using monochromated Al KR (1486 eV) radiation with detection on the surface normal. The pressure during analysis was ∼5 × 10-8 Torr. Spectra were fitted with Gaussian profiles using standard procedures. The positions of all peaks were normalized to C 1s at 285.0 eV. Confocal Microscopy. Array imaging was performed using a Virtek ChipReader. The system exhibits a number of 2 lasers. The laser is a solid-state semiconductor with internal optoelectronic beam stability monitoring system. The excitation wavelengths are 532 (Cy3) and 635 nm (Cy5). Monolayer Formation on Si(111)-H. Acid-Terminated Surfaces. Silicon crystals were cleaned with piranha solution (3:1 H2SO4, 96%:H2O2, 30%) at 100 °C for 30 min then hydrogen terminated by etching in ammonium fluoride. The resulting hydrogen-terminated surfaces, Si(111)-H, were placed under argon in a Schlenk tube containing previously deoxygenated neat or 10% undecylenic acid solution in toluene and irradiated in a photochemical reactor (Rayonet model RPR-200 from Southern New England Ultra Violet Company) at 300 nm for 3 h (12 lamps RPR-3000 Å, 75 W each, were used). The excess of unreacted and physisorbed reagent was removed by rinsing, at room temperature, with tetrahydrofuran and 1,1,1-trichloroethane, and the sample was dried under a stream of nitrogen. NHS-Functionalized Surface. The conversion of the acid function to succinimidyl ester was accomplished as follows: the acid-terminated surface was covered with 500 µL of an aqueous solution of NHS (0.1 M) and 500 µL of an aqueous solution of EDC (0.4 M) and allowed to react at room temperature for 1 h (pH 6.3). The resulting surface was rinsed copiously with deionized water and 1,1,1-trichloroethane and then dried under a stream of nitrogen. TEG-Terminated Surfaces. The best solvent was found to be dry DMF. Solutions were prepared using either 30 µL TEGamine per 2.0 mL DMF and 70 µL TEA or 50 µL TEGamine per 3.0 mL
Langmuir, Vol. 20, No. 26, 2004 11715 DMF and 100 µL TEA. The results were the same with both solutions. The reaction was complete after 1 h at room temperature and the TEG-terminated surface was rinsed copiously with deionized water and 1,1,1-trichloroethane and dried under a stream of nitrogen. Monolayer Formation on Photopatterned Surfaces. Photopatterned surfaces on Si(111)-H were prepared according to our previous work.31 Irradiation in ambient air of a hydrogenterminated surface through a mask (100 µm open squares separated by 50 µm lines; the mask from Adtek is chromium on quartz with a special antiscratch coating) with UV light (Hg(Ar) pen-lamp from Oriel at 254 nm, with 18 mA operating current) for 30 min led to the formation of a surface composed of 100 µm oxide squares and 50 µm Si-H lines (oxide/Si-H surface). The resulting surface was immersed in deoxygenated undecylenic acid and irradiated at 300 nm for 3 h. This step allows selective functionalization of the Si-H lines to yield a well-ordered pattern of oxide (squares) and acid-terminated (lines) (oxide/Si-C10COOH surface) surfaces. The carboxylic acid groups were activated by reaction with NHS/EDC to form an (oxide/Si-C10COONHS) surface. The NHS-terminated regions were then reacted with TEGamine to give an (oxide/Si-C10CONHTEG) surface. The unreacted NHS groups were capped with ethanolamine. Finally, the oxide from the squares was converted to a new Si-H surface by dipping the silicon shard for 1 min in 2% HF (aqueous solution). This actually led to a reduction in the line width to approximately 20 µm. The chemical modification cycle was repeated to provide the final patterned surface, Si-C10COOH/Si-C10CONHTEG, SiC10COONHS/Si-C10CONHTEG which was used for the immobilization of DNA. DNA Immobilization on Chemically Modified Silicon Surfaces. DNA on NHS-Terminated Surfaces. The single-strand DNA (E. coli probe 16S, 5′-H2N-C12H24-TT-CCT-GTT-ACCGTT-CGA-CTT-G-3′, [DNA] ) 20 µM) in PBS tethered to a primary amine group was reacted with a NHS-terminated surface. After 3 h of reaction at room temperature, the surface was rinsed with a detergent solution (2% Tween 20 in PBS) and water and then dried under a stream of nitrogen. The resultant surface was analyzed by ATR-FTIR. DNA on Patterned Surfaces. The patterned surface (Si-C10CONHS/Si-C10CONHTEG) was reacted overnight at room temperature with H2N-dT20 in PBS solution (37.4 µM) to give Si-C10CONH-dT20/Si-C10CONHTEG surface. The remaining NHS groups were capped with ethanolamine (10-1 M solution for 2 h at room temperature). The resulting surface was copiously rinsed with water and dried under a stream of nitrogen. The hybridization reaction was performed by immersing the above surface in 30.8 µM PBS solution of the complementary labeled oligonucleotide, Cy3-dA20, overnight at room temperature. The rinsing conditions after the reactions with the oligonucleotides were 3 × 5 min of shaking in 0.2% SDS solution in PBS plus 2 min of water rinsing.
Results and Discussion The carboxylic acid functional group is particularly useful because of its versatile chemical versatility and wetting properties. The carboxylic acid-terminated monolayer was obtained through the photochemical hydrosilylation of undecylenic acid with a hydrogen-terminated Si(111) surface (Scheme 1). Scheme 1
Figure 1a shows the IR spectrum of a hydrogenterminated Si(111)-H surface freshly prepared which consists of a narrow absorption centered at 2084 cm-1. After irradiation (UV, 300 nm) of the hydrogen-terminated surface in a deoxygenated neat or toluene solution of undecylenic acid (10%) for 3 h, new peaks associated with C-H stretching in the region of 2800-2980 cm-1, a strong
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Figure 1. ATR-FTIR of freshly prepared Si(111)-H before (a) and after (b) functionalization with undecylenic acid, (c) surface (b) reacted with NHS/EDC for 1 h at room temperature and (d) surface (c) after reaction with TEGamine. The background used is the spectrum of a clean ATR Si(111) crystal for (a) and the spectrum of a Si(111)-H surface for (b) and (c). The IR spectra are baseline-corrected.
peak at 1715 cm-1 due to carbonyl (CdO) stretching and the stretching of the OH group at 3100 cm-1 appeared (Figure 1b). It is difficult to assign unambiguously the absorption at 1715 cm-1 to a free acid because a similar absorption has been observed for the carbonyl function of silyl esters obtained by electrochemical oxidation of hydrogen-terminated porous silicon (PSi-H) surfaces with formic acid.42,43 However, the absence of the carboncarbon (CdC) double bond stretching at 1640 cm-1, the presence of an OH stretching, and the disappearance of the silicon-hydrogen vibration44 in the IR spectrum are consistent with at least a significant fraction of the reaction proceeding by hydrosilylation of the CdC double bond and leaving the acid terminal function intact. Moreover, we found that the thermal reaction of a porous Si-H surface with undecylenic acid led to the formation of an organic monolayer covalently attached to the PSi surface through Si-C bonds.45,46 This process was not expected since the exposed siliconhydrogen sites on the surface are acidic (Lewis acid) and the oxophilic nature of silicon should thermodynamically favor reaction of the Si-H bonds with the hydroxyl group of the acid. It is well established that the thermal reaction of alcohols,47 Grignard,48 and alkyllithium49 reagents with (42) Lee, E. J.; Ha, J. S.; Sailor, M. J. J. Am. Chem. Soc. 1995, 117, 8295. (43) Lee, E. J.; Bitner, T. W.; Ha, J. S.; Shane, M. J.; Sailor, M. J. J. Am. Chem. Soc. 1996, 118, 5375. (44) In all experiments, the ATR-FTIR measurements suggest the complete disappearance of the Si(111)-H vibrational mode. This is inconsistent with the estimated 50% coverage of the surface proposed by Chidsey (ref 18). In fact, this absorption can be observed on alkylmodified Si(111) using high-resolution electron energy loss spectroscopy (to be published) and appears to have been heterogeneously broadened to the extent that it cannot be observed by IR absorption. (45) Boukherroub, R.; Wojtyk, J. T. C.; Wayner, D. D. M.; Lockwood, D. J. J. Electrochem. Soc. 2002, 149, H59. (46) Boukherroub. R.; Petit. A.; Loupy. A.; Chazalviel. J-N.; Ozanam. F. J. Phys. Chem. B 2003, 107, 13459-13462. (47) Kim, N. Y.; Laibinis, P. E. J. Am. Chem. Soc. 1997, 119, 2297. (48) Kim, N. Y.; Laibinis, P. E. J. Am. Chem. Soc. 1998, 120, 4516. (49) Song, J. H.; Sailor, M. J. J. Am. Chem. Soc. 1998, 120, 2376.
hydrogen-terminated porous Si surfaces induces cleavage of the silicon-silicon back-bonds. Moreover, short-chain alcohols such as methanol are known to etch silicon-based semiconductors. There is evidence that the oxophilic nature of the silicon surface will result in direct reactions with OH groups. We found that decanol reacts with the Si(111)-H surface to yield an organic monolayer covalently attached to the surface through Si-O-C bonds.30 The surface morphology and its chemical composition are comparable to the silicon surface obtained using a thermal reaction of Si(111)-H with decanal.30 This implies that the thermal reaction of long-chain alcohols with Si(111)-H occurs with the loss of dihydrogen. The relative reactivity of the SiH surface with the alkenyl end of the undecylenic acid is consistent with a free radical, rather than a nucleophilic mechanism. Chemical activation of the acid function with NHS also supported the presence of a significant fraction of carboxylic acid functional groups on the surface(Scheme 2). Scheme 2
This is a characteristic esterification reaction widely used to prepare NHS-activated esters. Figure 1c displays a spectrum of an acid-terminated surface that has been chemically activated with aqueous NHS/EDC for 1 h at room temperature. It shows a complete disappearance of the peaks due to the acid (3100 and 1715 cm-1) and appearance of new peaks (1815, 1787, and 1744 cm-1) assigned to the succinimidyl ester (stretching modes of the carbonyl at 1815 and 1787 cm-1 are characteristic of the succinimidyl moiety). Again, these results are consistent with a hydrosilylation reaction taking place mainly at the carbon-carbon double bond since there does not appear to be any residual absorption at 1715 cm-1 that would be expected if more than 10% of the monolayer was in the form of the silyl ester.
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The availability of the NHS groups on the surface for further reaction was demonstrated by the aminolysis with TEGamine, which is known to inhibit nonspecific adsorption (Scheme 3). Scheme 3
The choice of TEGamine was based on previous work published in the literature that established the efficiency of short oligoethylene glycol to prevent the nonspecific adsorption of biomolecules on solid substrates.50-53 Figure 1d illustrates the IR spectrum of the ester-activated surface after reaction with TEGamine and shows the disappearance of the NHS ester and the appearance of peaks at 1650 and 1550 cm-1 assigned to the carbonyl function and the CNH vibration (includes both N-H bending and C-N stretching) of the amide. Peaks at 3300 and 3100 cm-1 were observed as well and attributed to the NH stretch and an overtone of the 1550 cm-1 peak, respectively. We have used X-ray photoelectron spectroscopy to analyze the chemical composition of the surfaces before and after chemical modifications to evaluate the nature of the chemical bonding associated with the transformations that occurred on the surface. Figure 2a displays an XPS survey of the hydrogen-terminated Si(111) surface showing signals due to silicon 2p and 2s at 99 and 151 eV, respectively. The spectrum did not show any other peaks due to surface contamination (e.g., carbon and oxygen peaks due to contamination during sample handling and transferring). After irradiation of the Si(111)-H surface at 300 nm for 3 h in neat or in a toluene solution of undecylenic acid, new signals due to C 1s (285 eV) and O 1s (532 eV) were observed, consistent with the incorporation of an organic monolayer on the surface (Figure 2b). An additional peak due to N 1s (absent in the acidterminated surface XPS survey) and a relative increase of the C 1s and O 1s signals were obtained when the acidterminated surface was converted to an activated ester by chemical treatment with NHS/EDC (Figure 2c). Figure 3 shows the high-resolution spectrum (silicon peak) of freshly prepared Si(111)-H. There are no apparent peaks at higher binding energies that would be indicative of adventitious oxidation of the surface. Similar spectra were obtained after chemical functionalization of the surface with undecylenic acid and after conversion of the acid function to an NHS-activated ester. Again, the absence of peaks at higher binding energies in the highresolution spectrum of the silicon surface modified with undecylenic acid is consistent with a process occurring without any apparent oxidation of the surface. The activation process (NHS/EDC) did not induce any measurable oxidation of the surface, implying a good passivation of the surface by the acid monolayer. However, it is important to note that the shift of the Si 2p might not be the most sensitive measure of oxidation54 (vide infra). The carbon 1s signal of the Si(111)-undecanoic acid surface shows three different peaks. The main peak (50) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605. (51) Papra, A.; Gadegaard, N.; Larsen, N. B. Langmuir 2001, 17, 1457. (52) Zhu, X.-Y.; Jun, Y.; Staarup, D. R.; Major, R. C.; Danielson, S.; Boiadjiev, V.; Gladfelter, W. L.; Bunker, B. C.; Guo, A. Langmuir 2001, 17, 7798. (53) Benesch, J.; Svedhem, S.; Svensson, S. C. T.; Valiokas, R.; Liedberg, B.; Tengvall, P. J. Biomater. Sci., Polym. Ed. 2001, 12, 581. (54) Fabre, B.; Lopinski, G. P.; Wayner, D. D. M. J. Phys. Chem. B 2003, 107, 14326.
Figure 2. XPS survey of (a) the hydrogen-terminated Si(111)-H surface (b) after derivatization with undecylenic acid and (c) after conversion of the acid to the NHS ester.
Figure 3. High-resolution XPS spectra of Si 2p of the Si(111)-H surface.
centered at 285 eV is characteristic of the alkyl chain, while the two other peaks at 286 and 290 eV correspond to C-O and to the carbon bearing the carbonyl function, respectively (Figure 4a). The NHS-activated surface has four peaks, at 285, 286, 286.6, and 290 eV, attributed to the chemically different carbons of the alkyl chain, C-O, C(N)O and C(O)O, respectively (Figure 4b). Figure 5a is a high-resolution XP spectrum of the O 1s region of the acid-terminated Si(111) surface.The oxygen signal exhibits three peaks at 531.7, 533.0, and 534.4 eV. The peak at 531.7 eV represents 10% of the total area for O 1s, and it may be due to the Si-O bonds (either from adventitious oxidation or otherwise undetectable Si-OC-
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Figure 6. High-resolution N 1s XP spectrum of the NHSactivated ester surface. Table 1. Atomic Concentrations (%) and Relative Atomic Concentrations for H-, Acid- and Ester-Terminated Surfaces from XPS Data sample
Figure 4. High-resolution C 1s XP spectra of (a) the carboxylic acid-terminated surface and (b) after conversion of the acid to the NHS ester.
Si 2p
C 1s
O 1s
N 1s
C/Oa
N/Oa
Si(111)-H 91.90 6.00 1.40 0.60 Si(111)-C10H20- 55.90 35.30 8.30 0.30 4.25 (5.5) COOH Si(111)-C10H20- 49.70 36.10 11.00 3.10 3.28 (3.75) 0.28 (0.25) COONHS a The expected values (according to the chemical structure) are in parentheses.
Figure 5. High-resolution O 1s XP spectra of (a) acidterminated surface and (b) after conversion of the acid to the NHS ester.
(O)R). The other two peaks are consistent with the elemental composition of the organic molecule. Chemical transformation of the acid function to the NHS ester results in three O 1s peaks at 531.8, 533, and 535.6 eV (Figure 5b). The peak at 535.6 eV is shifted by about 1.2 eV with respect to the corresponding peak (534.4 eV) in the acid spectrum due to the formation of a C-O-N bond in the NHS ester. The more electronegative element, N,
will shift the peak toward higher binding energy compared with the C-O bond in the COOH group. Figure 6 indicates clearly the occurrence of the N 1s signal after the formation of the NHS ester. The high-resolution spectrum for N 1s exhibits two peaks at 400 and 402.6 eV, corresponding to N-C and N-O, respectively, consistent with the formation of the NHS ester. Analysis of the atomic concentrations of C, O, and N in the monolayers (Table 1) provides values in good agreement with those expected. Sensitivity factors of 0.733 (O 1s), 0.314 (C 1s), 0.368 (Si 2p), and 0.499 (N 1s) were used in these calculations to convert peak areas into concentrations. Slightly higher concentrations of oxyen than expected (including for the acid-modified surface) suggest that there is some oxidation of the Si surface in competition with Si-C formation. Immobilization of DNA on NHS-Modified Surfaces. In the above section, we demonstrated that simple amines (e.g., TEG amine) can be easily immobilized by reaction with the NHS-terminated surfaces. In principle, the reactions should be amenable to any accessible primary amine, including those of biologically active molecules such as proteins and DNA. We have demonstrated the utility of this approach by the immobilization of single-strand DNA (E. coli probe 16S, [DNA] ) 20 µM in PBS) tethered to a primary amine linker in the 5′-position. After 3 h of reaction at RT, characteristic peaks of the amide function were observed (1650 and 1550 cm-1) along with the NHS ester peaks at 1815, 1787, and 1744 cm-1 in the IR spectrum, suggesting that not all NHS groups are accessible, presumably because of steric hindrance of the DNA oligomers. The resulting surface was then exposed to a capping agent (ethanolamine) to deactivate the remaining reactive NHS groups on the surface (Figure 7). In addition to the amide absorption, characteristic peaks for CdO at
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Figure 7. ATR-FTIR spectrum of single-strand DNA immobilized on the activated ester surface.
Figure 8. Fluorescence confocal microscope image of: (a) patterned silicon surface bearing hybridized oligonucleotides (dT20 and Cy3-dA20) on the squares (b) after de-hybridization and (c) after three rehybridization cycles.
1700 cm-1 (shoulder of 1740 cm-1 peak) and for CdN at 1608 cm-1 associated with the DNA are evident (Figure 7). Other DNA peaks below 1500 cm-1, such as the phosphate diester bands, cannot be observed because of strong absorptions from bulk silicon (the ATR crystals have a 1 mm thickness, so the absorption from silicon is strong). Immobilization of DNA on Photopatterned Surfaces. Photopatterning provides an approach for creating reactive domains on the surface with micrometer resolution31 and allow site-directed immobilization of biomolecules. The photopatterning technique, which is described elsewhere, takes advantage of the selective photooxidation of the silicon surface when irradiated through a mask with UV light in air. In this case, a mask composed of 100 µm squares with a 150 µm pitch was used. After irradiation, the surface, with a pattern consisting of oxide squares with hydrogen-terminated lines, was reacted with undecylenic acid, activated with NHS, and then reacted with TEGamine to produce a surface in which the lines were resistant to nonspecific adsorption or other chemical reactions. Removal of the oxide from the squares was achieved by immersing the surface in dilute HF aqueous solution followed by reaction of the newly formed hydrogen-
terminated silicon with undecylenic acid. The squares were then activated with NHS. The resulting substrate (SiC10COONHS/Si-C10CONHTEG) was then reacted with H2N-dT20 (overnight at room temperature with 37.4 µM H2N-dT20 solution in PBS) followed by a final capping with ethanolamine of the remaining NHS groups. The hybridization reaction of the immobilized DNA target was performed with its complementary oligonucleotide bearing a Cy3 fluorescent label, Cy3-dA20. After an overnight reaction at room temperature with 30.8 µM Cy3-dA20 solution in PBS, the surface was imaged using confocal fluorescence microscopy (Figure 8a). The fluorescence signal was observed in the expected regions (squares), implying efficient inhibition of the nonspecific adsorption. The following control experiment was carried out to determine the extent of nonspecific binding of Cy3-dA20. The derivatized Si-C10CONH-dT20/Si-C10CONHTEG surface was exposed overnight to the PBS solution of the Cy3-dA20 and Cy5-dC20. Any nonspecifically bound DNA would be demonstrated by the presence of emission from Cy5. We found that, while there was some nonspecifically bound DNA, the amount was close to the detection limit of the instrument and, as such, was difficult to quantify. Given that there is significant room to optimize further
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the hybridization conditions, we are satisfied that these surfaces exhibit satisfactory binding specificity. The denaturation and the reversibility of these surfaces were explored as well. Denaturation was accomplished by heating the surface bearing the double-strand DNA at 65 °C for 1 h in 0.2% SDS (in PBS) + 0.1 M NaCl mixture (followed by 3 × 5 min shaking in 0.2% SDS solution in PBS and 2 min of water rinsing). Imaging of the resulting surface showed that the pattern was no longer observed (Figure 8b). Subsequent hybridization was performed by re-immersing the resulting surface in the Cy3-dA20 solution overnight at room temperature (Figure 8c). The hybridization/denaturation cycle was repeated three times without further changes to the fluorescence image. Conclusion In conclusion, we have shown that the reaction of hydrogen-terminated silicon surfaces with undecylenic acid takes place under photochemical conditions primarily, perhaps exclusively, at the carbon-carbon double bond to provide a carboxylic acid terminated surface. The acid-
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terminated surface can be easily converted to an activated ester by a simple chemical route (NHS/EDC). Simple amines and single-strand DNA tethered to a primary amine are covalently immobilized to the surface by amide bond formation at room temperature in a relatively fast process. Site-directed immobilization of DNA on Si(111)modified patterned surfaces with limited nonspecific adsorption was achieved by suitable surface chemistry manipulation. In addition, this surface chemistry provides excellent stability under hydbridization/denaturation conditions. This reasonably straightforward method provides a potential platform for immobilization of complex structures on silicon surfaces for applications in biosensing and the fabrication of new hybrid materials and devices. Acknowledgment. We thank G. I. Sproule and Simona Moisa (IMS) for the XPS measurements and Gerardo Diaz-Quijada and Robert Donkers for technical assistance with control experiments. LA047886V
