Photoresponsive Surfaces with Two Independent Wavelength

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11872

Langmuir 2008, 24, 11872-11879

Photoresponsive Surfaces with Two Independent Wavelength-Selective Functional Levels Petra Stegmaier,† Jose´ Marı´a Alonso,† and Ara´nzazu del Campo*,†,‡ Max-Planck-Institut fu¨r Metallforschung, Heisenbergstrasse 3, 70569 Stuttgart, Germany, and INM - Leibniz Institut fu¨r Neue Materialien gGmbH, Campus D2 2, 66123 Saarbruecken, Germany ReceiVed NoVember 20, 2007. ReVised Manuscript ReceiVed August 19, 2008 Two photoremovable protecting groups, namely, nitroveratryloxycarbonyl (NVo) and diethylamino-coumarin-4-yl (DEACM), have been tested for wavelength-selective, independent removal. The chromophores were attached to the amine group of aminopropyltriethoxysilane and used for the modification of silica surfaces. A photolytic experiment on the photosensitive layers allowed us to identify the irradiation conditions for the selective cleavage of the chromophores. UV measurements revealed that the photolabile DEACM group can be cleaved off with UV light at 412 nm without damaging the NVo group. The NVo group could then be removed at 365 nm. Masked irradiation of substrates modified with a 1:1 molar mixture of both silanes allowed the generation of bifunctional patterns after the selective cleavage of DEACM and NVo in a sequential irradiation process. The deprotection reaction was confirmed by coupling two different fluorescent dyes to the liberated amine groups. The expected two-color pattern could be observed by fluorescence microscopy.

Introduction Miniaturized and high-throughput chemical and biological analysis systems in microarray format have moved to the forefront of the bioanalytical science area. They require only small amounts of analytes and reagents for accurate detection and allow an analysis of a variety of samples in parallel.1 The fabrication of these analytical platforms requires the development of surfacepatterning strategies that are able to create a high density of individual and isolated reactive sites on a substrate, onto which the biomolecular species will be immobilized for detection.2,3 Among them, photoreactive surface layers that can be siteselectively activated upon masked irradiation constitute an interesting patterning alternative with many possible variants. Light can be used (i) to destroy or remove molecular layers at selected positions to render a bare, inactive substrate,4,5 (ii) to graft molecular species onto irradiated regions via photogenerated radical cross-reactions occurring between a photosensitive surface layer and the biological molecule (photochemical cross-linking),6-10 or (iii) to direct the synthesis of small molecules on the surface (peptides,11,12 oligocarbamates,13 oligonucleotides,14-17 and peptoids18) through the iterative unmasking of photoreactive groups and monomer coupling cycles. Alternatively, surface layers * Corresponding author. Tel: +49 711 6893416. Fax: +49 711 6893412. E-mail: [email protected]. † Max-Planck-Institut fu¨r Metallforschung. ‡ INM - Leibniz Institut fu¨r Neue Materialien gGmbH. (1) del Campo, A.; Bruce, I. J. Biomedical Nanotechnology; Malsch N. I., Ed.; Taylor & Francis: Boca Raton, FL, 2005; pp 75-112. (2) del Campo, A.; Bruce, I. J. Top. Curr. Chem. 2005, 260, 77–111. (3) Uttamchandani, M.; Walsh, D. P.; Yao, S. Q.; Chang, Y. T. Curr. Opin. Chem. Biol. 2005, 9, 4–13. (4) Dulcey, C. S.; Georger, J. H.; Krauthamer, V.; Stenger, D. A.; Fare, T. L.; Calvert, J. M. Science 1991, 252, 551–554. (5) Tarlov, M. J.; Burgess, D. R. F.; Gillen, G. J. Am. Chem. Soc. 1993, 115, 5305–5306. (6) Monsathaporn, S.; Effenberger, F. Langmuir 2004, 20, 10375–10378. (7) Ito, Y. Biotechnol. Prog. 2006, 22, 924–932. (8) Kado, Y.; Mitsuishi, M.; Miyashita, T. AdV. Mater. 2005, 17, 1857+. (9) Kanoh, N.; Kumashiro, S.; Simizu, S.; Kondoh, Y.; Hatakeyama, S.; Tashiro, H.; Osada, H. Angew. Chem., Int. Ed. 2003, 42, 5584–5587. (10) White, M. A.; Johnson, J. A.; Koberstein, J. T.; Turro, N. J. J. Am. Chem. Soc. 2006, 128, 11356–11357. (11) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767–773. (12) Pellois, J. P.; Wang, W.; Gao, X. L. J. Comb. Chem. 2000, 2, 355–360.

containing the reactive functionalities protected with a photocleavable19-21 group (cage) can be used for the site-specific coupling of complementary functionalities after activation by light deprotection.11,22-29 The latter is a particularly flexible approach because many photoremovable groups are known that can be combined with many different reactive species. Moreover, it has been recently demonstrated that different protecting groups may be independently cleaved using light of different wavelengths.30,31 This approach has been successfully used in solidphase peptide synthesis32 or in generating complex chemical patterns with more than a single functionality.24,25 The application of this strategy to biosensors would allow the generation of (13) Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.; Fodor, S. P. A.; Adams, C. L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. G. Science 1993, 261, 1303–1305. (14) McGall, G.; Labadie, J.; Brock, P.; Wallraff, G.; Nguyen, T.; Hinsberg, W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13555–13560. (15) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022–5026. (16) Gao, X. L.; Yu, P. L.; LeProust, E.; Sonigo, L.; Pellois, J. P.; Zhang, H. J. Am. Chem. Soc. 1998, 120, 12698–12699. (17) Singh-Gasson, S.; Green, R. D.; Yue, Y. J.; Nelson, C.; Blattner, F.; Sussman, M. R.; Cerrina, F. Nat. Biotechnol. 1999, 17, 974–978. (18) Li, S. W.; Bowerman, D.; Marthandan, N.; Klyza, S.; Luebke, K. J.; Garner, H. R.; Kodadek, T. J. Am. Chem. Soc. 2004, 126, 4088–4089. (19) Pillai, V. N. R. Synthesis 1980, 1–26. (20) Bochet, C. G. J. Chem. Soc., Perkin Trans. 1 2002, 125–142. (21) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900–4921. (22) Vossmeyer, T.; Jia, S.; DeIonno, E.; Diehl, M. R.; Kim, S. H.; Peng, X.; Alivisatos, A. P.; Heath, J. R. J. Appl. Phys. 1998, 84, 3664–3670. (23) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117, 12050–12057. (24) del Campo, A.; Boos, D.; Spiess, H. W.; Jonas, U. Angew. Chem., Int. Ed. 2005, 44, 4707–4712. (25) Jonas, U.; del Campo, A.; Kruger, C.; Glasser, G.; Boos, D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5034–5039. (26) Yan, F. N.; Chen, L. H.; Tang, Q. L.; Rong, W. Bioconjugate Chem. 2004, 15, 1030–1036. (27) Ryan, D.; Parviz, B. A.; Linder, V.; Semetey, V.; Sia, S. K.; Su, J.; Mrksich, M.; Whitesides, G. M. Langmuir 2004, 20, 9080–9088. (28) Dendane, N.; JHoang, A.; Guillard, L.; Defrancq, E.; Vinet, F.; Dumy, P. Bioconjugate Chem. 2007, 18, 671–676. (29) Critchley, K.; Jeyadevan, J. P.; Fukushima, H.; Ishida, M.; Shimoda, T.; Bushby, R. J.; Evans, S. D. Langmuir 2005, 21, 4554–4561. (30) Bochet, C. G. Tetrahedron Lett. 2000, 41, 6341–6346. (31) Bochet, C. G. Synlett 2004, 2268–2274. (32) Bochet, C. G. Angew. Chem., Int. Ed. 2001, 40, 2071–2073.

10.1021/la802052u CCC: $40.75  2008 American Chemical Society Published on Web 09/26/2008

PhotoresponsiVe Surfaces with Functional LeVels

surfaces with wavelength-triggerable ability to capture more than one biomolecular species with spatiotemporal resolution. Until now, only two pairs of photoremovable groups have been selectively removed at two different wavelengths. Nitroveratryl esters (NVo) have been selectively cleaved at 350 nm in 87% yield in the presence of the pivaloylpropanediol (Piv) group, with virtually no absorbance at this wavelength, and can be subsequently cleaved at 300 nm.33 The wavelength-selective cleavage works only when NVo is removed before Piv because NVo also shows appreciable photosensitivity at 300 nm. A more interesting combination was demonstrated with 3,5-dimethoxybenzoin esters (Bnz), which were effectively cleaved at wavelengths below 300 nm, whereas nitroveratryl esters (NVo) were cleaved at wavelengths of up to 420 nm.24,32 These two protecting groups are orthogonal and can be individually addressed in any chronological sequence. (Bnz shows virtually no absorbance at λ >380 nm, and NVo photolysis proceeds with a much lower quantum yield (Φ ≈ 0.023) than Bnz photolysis (Φ ≈ 0.64) at λ < 300 nm). Both the Piv and Bnz groups need irradiation wavelengths below 320 nm for wavelength-selective cleavage. This fact limits their application to biological issues because many biomolecules show appreciable absorption at λ < 320 nm and would interfere with the photocleavage process. Therefore, the identification of additional cages that are cleavable at longer wavelengths remains a challenging task. Among the reported candidates, the members of the coumarin-4-ylmethyl family with electron-donating substituents at C6 or C7 positions show promising properties because they display high photolytic cross-sections at long wavelegths.34 In particular, (7-diethylaminocoumarin-4-yl)methyl (DEACM) has a strong absorption maximum at 390-400 nm (ε ) 20 000 M-1 cm-1), which extends up to 470 nm.35-42 For comparison, the NVo absorption maximum is located at 356 nm (ε ≈ 4500 M-1 cm-1) and tails up to 420 nm.43 The photolysis quantum yield of DEACM approaches 0.3, which is the highest among the reported coumarin cages. In this work, we will test the wavelength-selective photolysis of DEACM and NVo derivatives. These two groups will be used to cage the amine group of aminopropyltriethoxysilane. Resulting (33) Kessler, M.; Glatthar, R.; Giese, B.; Bochet, C. G. Org. Lett. 2003, 5, 1179–1181. (34) Trenor, S. R.; Shultz, A. R.; Love, B. J.; Long, T. E. Chem. ReV. 2004, 104, 3059–3077. (35) Eckardt, T.; Hagen, V.; Schade, B.; Schmidt, R.; Schweitzer, C.; Bendig, J. J. Org. Chem. 2002, 67, 703–710. (36) Furuta, T.; Wang, S. S. H.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1193–1200. (37) Geissler, D.; Kresse, W.; Wiesner, B.; Bendig, J.; Kettenmann, H.; Hagen, V. ChemBioChem 2003, 4, 162–170. (38) Hagen, V.; Bendig, J.; Frings, T.; Eckhardt, T.; Helm, S.; Reuter, D.; Kaupp, B. Angew. Chem., Int. Ed. 2001, 40, 1045–1048. (39) Hagen, V.; Frings, S.; Wiesner, B.; Helm, S. U.; Kaupp, B.; Bendig, J. ChemBioChem 2003, 4, 434–442. (40) Schonleber, R. O.; Bendig, J.; Hagen, V.; Giese, B. Bioorg. Med. Chem. 2002, 10, 97–101. (41) Shembekar, V. R.; Chen, Y.; Carpenter, B. K.; Hess, G. P. Biochemistry 2005, 44, 7107–7114. (42) Suzuki, A. Z.; Watanabe, T.; Kawamoto, M.; Nishiyama, K.; Yamashita, H.; Ishii, M.; Iwamura, M.; Furuta, T. Org. Lett. 2003, 5, 4867–4870. (43) Goeldner, M., Givens, R., Eds. Dynamic Studies in Biology: Phototriggers, Photoswitches and Caged Biomolecules; Wiley-VCH: Weinheim, Germany, 2005.

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photosensitive silanes 1 and 2 will be reacted with silica surfaces, and the wavelength-selective response at the surface will be characterized. We will apply this system to generate bifunctional patterns with two independent functional levels by two-step irradiation of the photosensitive layers using masks of different geometries and subsequent labeling with fluorescent labels of different colors.

Experimental Section Synthesis. Chemicals and solvents were purchased from Fluka Chemie AG (Taufkirchen, Germany), Fisher Scientific UK Ltd. (Loughborough, Leics. GB), Merck KGaA (Darmstadt, Germany), Sigma-Aldrich Chemie GmbH (Steinheim, Germany), and ABCR (Karlsruhe, Germany). Analytical thin-layer chromatography was performed on TLC plates (Alugram Sil G/ UV254) from MachereyNagel (Du¨ren, Germany). Preparative column chromatography and flash column chromatography were carried out using silica gel (60 Å pore size, 63-200 µm particle size) from Merck KGaA (Darmstadt, Germany). Solution 1H and 13C spectra were recorded on either a Bruker Ultra Shield 250 MHz or a Bruker Spectrospin 300 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). UV spectra were recorded on a Varian Cary 4000 UV-vis spectrometer (Varian Inc. Palo Alto, CA). 3-Triethoxsilylpropyl-N-(4,5-dimethoxy-2-nitrobenzyloxycarbonyl)amine (1). To a solution of 3-(isocyanopropyl)triethoxysilane (2.09 mL, 8 mmol) and 4,5-dimethoxy-2-nitrobenzylalcohol (1.74 g, 8 mmol) in dry THF, dibutyltin dilaurate (40 µL) was carefully added dropwise. The mixture was stirred at room temperature for 24 h. After the organic solvent was removed under vacuum, the residue was purified by chromatography performed with passivated silica gel24 using ethyl acetate/hexane 1/1 as the eluent to afford 1 (2.36 g, 64%) as a pale-yellow solid. 1H NMR in d-THF: δ 0.62-0.70 (m, 2H), 1.25 (t, J ) 7.0 Hz, 9H,), 1.58-1.72 (m, 2H), 3.13-3.23 (m, 2H), 3.86 (q, J ) 7.0 Hz, 6H); 3.96 (s, 3H), 4.00 (s, 3H), 5.48 (s, 2H), 7.24 (s, 1H), 7.79 (s, 1H). 7-(Diethylamino)-4-(hydroxymethyl)-2H-chromen-2-one (3). 41 Selenium(IV) dioxide (3.32 g, 30 mmol) was added to a solution of 7-diethylamino-4-methylcoumarin (4.64 g, 20 mmol) in xylene (120 mL, mixture of isomers). The reaction mixture was heated under reflux with vigorous stirring. After 24 h, the brown mixture was allowed to cool to room temperature, filtered, and concentrated under reduced pressure. The dark-brown residual oil was dissolved in ethanol (130 mL). Sodium borohydride (0.38 g, 10 mmol) was added, and the solution was stirred for 3.5 h at room temperature. Hydrochloric acid (1 N, 20 mL) was carefully added. After dilution with water, the red solution was extracted three times with dichloromethane. The combined organic extracts were washed with water and brine (saturated sodium hydrogen carbonate solution) and dried over anhydrous sodium sulfate. Removal of the solvent under reduced pressure gave a dark-brown oil (7.39 g). Column chromatography using dichloromethane/acetone 5/1 as the eluent afforded desired alcohol 3 (2.19 g, 8.9 mmol, 29.5% yield). 1H NMR (CDCl3): δ 7.32 (d, J ) 9.0 Hz, 1H), 6.55-6.58 (m, 1H), 6.51-6.52 (m, 1H), 6.25 (t, J ≈ 1.2 Hz, 1H), 4.84 (s br, 2H), 3.41 (q, J ) 7.1 Hz, 4H), 1.55 (s br, 1H), 1.20 (t, J ) 7.1 Hz, 6H). (7-(Diethylamino)-2-oxochroman-4-yl)methyl allylcarbamate (4). 36 3 (0.3 g, 1.1 mmol) was dissolved in dry dichloromethane (30 mL). 4-Dimethylaminopryridine (DMAP, 0.28 g, 2.3 mmol) and 4-nitrophenylchloroformate (0.26 g, 1.3 mmol) were added simultaneously. The dark-brown-yellow solution was stirred at room

11874 Langmuir, Vol. 24, No. 20, 2008 temperature. A small sample was taken for analysis after 17.5 h. The formation of the carbamate intermediate could be confirmed by 1H NMR spectroscopy, with the observed shift of the signals attributed to H14 from 4.83/4.84 ppm in alcohol 2 to 5.22 ppm in the carbamate. 4-Dimethylaminopyridine (DMAP, 0.29 g, 2.4 mmol) and allylamine (0.1 g, 1.75 mmol) were then added to the stirring solution. After 23.5 h at room temperature, the reaction mixture was quenched with citric acid (15% w/V, 15 mL) and diluted with dichloromethane. After phase separation, the aqueous phase was extracted with dichloromethane. The combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained dark-orange oil (0.69 g) was subjected to column chromatography using ethyl acetate/petroleum ether 3/1 as the eluent. Fractions containing compounds with an Rf of 0.9 (ethyl acetate/ petroleum ether 3/1) were combined and concentrated. Desired product 4 was allowed to crystallize from the obtained orange oil. The mixture was then taken up in ethylacetate/petroleum ether 3/1 and filtered. The residual yellow solid (0.08 g, 0.2 mmol, 22% yield) was determined to be desired product 4. 1H NMR (CDCl3): δ 1.17-1.22 (t, J ) 7.1 Hz, 1H), 3.40 (q, 4H), 3.86 (tt, J ) 5.7 Hz, J ≈ 1.4 Hz, 2H), 5.04 (s, 1H), 5.14-5.26 (m, 4H), 5.80-5.93 (m, 1H), 6.12 (s br, 1H), 6.50-6.51 (m, 1H), 6.54-6.58 (m, 1H), 7.29 (d, J ) 8.7 Hz, 1H). 13C NMR (CDCl3): δ 162.1, 156.4, 155.5, 150.8, 150.4, 134.2, 124.5, 116.6, 108.8, 106.4, 98.0, 62.0, 44.9, 43.8, 12.6. (7-(Diethylamino)-2-oxo-2H-chromen-4-yl)methyl-3-(triethoxysilyl)propylcarbamate (2). (Method A) 4 (0.07 g, 0.21 mmol) and triethoxysilane (1.8 g, 10.8 mmol) were placed into a previously HMDS-passivated dry, round-bottomed flask and heated under an argon atmosphere to about 80 °C. After the addition of isopropanolic H2PtCl6 solution (50 µL, 0.027 mg/µL), the mixture was allowed to react for 4 h at 80 °C and then to cool. Excess triethoxysilane was removed in vacuum, and the solid residue was taken up in dichloromethane and filtered through Celite500. The filtrate was concentrated under reduced pressure and determined to be 2 in a 45% mixture with the reduced compound. 1H NMR (CDCl3): δ 7.30 (d, J ) 8.9 Hz, 1H), 6.58 (dd, J ) 8.9 Hz, J ) 2.5 Hz, 1H), 6.51 (d, J ) 2.5 Hz, 1H), 6.12 (s br, 1H), 5.22 (s br, 2H), 4.91 (s br, 1H), 3.91-3.79 (q, J ) 7.2 Hz, 6H), 3.41 (q, J ) 7.2 Hz, 4H), 3.27-3.17 (m, 2H), 1.72-1.62 (m, 2H), 1.26-1.18 (m, 15H), 0.63-0.68 (m, 1H). 13C NMR (CDCl3): δ 98.1, 106.4, 108.9, 124.6, 124.6, 150.5, 150.7, 155.7, 156.4, 162.1, 61.8, 58.7, 45.0, 43.1, 12.6, 7.9. (7-(Diethylamino)-2-oxo-2H-chromen-4-yl)methyl-3-(triethoxysilyl)propylcarbamate (2). (Method B) 3-Isocyanato propyltriethoxysilane (0.53 g, 2 mmol) and 3 (0.5 g, 2 mmol) were dissolved in dry THF (20 mL). After the addition of 20 µL of dibutyltin dilaureate, the mixture was allowed to react at room temperature for 3 h. The solvent was removed under reduced pressure, and the product was purified by column chromatography. 2 was obtained purified in a yield of 72%. Passivation of Glassware. Glass reaction vessels used for silanization were previously passivated either by leaving them in a desiccator overnight under vacuum and in the presence of HMDS or by wetting the glass surface with pure HMDS. Posterior washing with THF and water is needed for the complete removal of excess of HMDS. Substrate Cleaning. Quartz slides (Suprasil 1) with a thickness of 1 mm were purchased from Heraeus Quarzglas (Hanau, Germany). Silicon wafers in (100) orientations were provided by Crystec (Berlin, Germany). For the experiments, slides of 1 × 2.5 cm2 were cut and modified as indicated below. Quartz slides and silicon wafers were cleaned by soaking them in Piranha solution (H2SO4(conc)/ H2O2(30%) 5/1) overnight and subsequent rising with Milli-Q water and drying in vacuum at 90 °C for 1 h. Surface Modification. A 1% w/v solution of 1 in 17 mL of THF was stirred for 1 h after the addition of 17 µL of 1 mN NaOH(aq). The solution was then filtered through a 0.2 µm-pore-size PTFE filter, and clean substrates (either quartz or silicon) were immersed in it. After 18 h of reaction time, the substrates were successively rinsed with THF and Milli-Q water to remove physisorbed molecules. The chemisorbed silane molecules were fixed to the silica surface

Stegmaier et al. by baking for 1 h at 90-95 °C. The experimental procedure used for surface modification with 2 was analogous, but a 0.2% w/v solution of the silane in THF was used and the maximum absorbance was obtained at 70 h. In the case of the 1/1 molar ratio mixture of silanes 1 and 2, a 0.1% w/v solution of the silanes in THF was used, and the silanation time was 45 h. After baking and before further application, all substrates were sonicated in THF for 3 min, washed with Milli-Q-water, and dried with in an N2 stream. Characterization of the Surface Layers. The surface modification process was followed by recording UV spectroscopy (Varian Cary 4000 UV-vis spectrometer, Varian Inc., Palo Alto, CA) on quartz substrates at different reaction times and by measuring ellipsometry layer thickness (ELX-02C ellipsometer, DRE-Dr. Riss Ellipsometerbau GmbH, Ratzeburg, Germany) on modified silicon wafers under equivalent reaction conditions. Static water contact angles (OCA 30 contact angle system with SCA 202 software, DataPhysics Instruments GmbH, Filderstadt, Germany) were used to determine changes in the hydrophilicity after surface reaction. For the determination of the ellipsometric thickness and contact angle, substrates were previously dried under vacuum at 90 °C for 30 min. In the ellipsometric measurements, the refractive index was assumed to be 1.4571 in the deposited layers in a three-phase silicon substrate/silica + organic layer/air model. Surface loading, ∆Γ, in terms of molecules mm-2, was estimated from the ellipsometric thickness assuming a refractive index of 1.4571 for the silane layer, a typical density F of 1.0 g cm-3 for an organic layer with a refractive index n of 1.43, and an increment dn/dF of 0.24 g cm-3.44,45 Irradiation (Photodeprotection) of the Substrates. Irradiation experiments with monochromatic light were carried out using a Polychrome V system (TILL Photonics GmbH, Gra¨felfing, Germany) at the following wavelengths and intensities: 345 nm (0.78 mW cm-2) and 412 nm (1.6 mW cm-2). After irradiation, substrates were washed with THF and rinsed with Milli-Q water before monitoring the deprotection reaction by UV-vis spectroscopy. For laterally structured deprotection, quartz slides with 1 × 2.5 cm2 chrome patterned fields containing micrometric stripes (50µm-wide chrome lines, spaced by 10 µm, provided by ML&C, Jena, Germany) were placed on top of the substrate during irradiation. Fluorescence Labeling. The generation of free surface amine groups after irradiation was verified by fluorescence staining of patterned substrates using Alexa Fluor dyes (Alexa Fluor 647 and Alexa Fluor 488 carboxylic acid succinimidyl esters from Invitrogen, Eugene, OR). A quartz substrate was masked irradiated at 412 nm for 20 min and then washed with THF and Milli-Q water. A solution (150 µL) of Alexa Fluor 647 succinimidyl ester in dry DMSO (0.14 mg/mL) was deposited on one side of the substrate and left at RT for 12 h, shielded from light. The droplet was then removed with a pipet, and unbounded dye was removed by washing with DMSO, THF (sonication for 3 min), and Milli-Q water. After drying in an N2 stream, the substrate was again masked irradiated for 20 min at 345 nm (quartz mask bearing the same pattern rotated around 90° with respect to the first mask). The photolysis products were washed off, and the dried substrate was covered with 150 µL of a solution of Alexa Fluor 647 succinimidyl ester in dry DMSO (0.1 mg/mL) and incubated at RT for 8.5 h. Unbound dye was again washed off, and the dried substrate was analyzed with a fluorescence microscope (Axio Imager Z1 with Axio Vision Rel. 4.6 software, Carl Zeiss GmbH, Go¨ttingen, Germany) using the corresponding filters.

Results and Discussion Synthesis. Silane 1 was synthesized in one step with good yield by the reaction of 3-(isocyanopropyl)triethoxysilane and 4,5-dimethoxy-2-nitrobenzylalcohol (Figure 1). The synthesis of 2 was performed by two methods (A and B). Both paths start (44) Piehler, J.; Brecht, A.; Hehl, K.; Gauglitz, G. Colloids Surf., B 1999, 13, 325–336. (45) Piehler, J.; Brecht, A.; Valiokas, R.; Liedberg, B.; Gauglitz, G. Biosens. Bioelectron. 2000, 15, 473–481.

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Figure 1. Synthesis paths for obtaining silanes 1 and 2.

with the preparation of 7-diethylamino-4-hydroxymethylcoumarin (3) by the oxidation of commercially available 7-(diethylamino)4-methylcoumarin to the corresponding coumarin-4-carbaldehyde,46 followed by reduction to 3.40,41,47 The reaction is a onepot process and does not require the isolation of the intermediate. 3 could be purified with a yield of 44%. In method A, the hydroxyl functionality of 3 was activated with 4-nitrophenyl chloroformate to allow subsequent coupling of allylamine to obtain 4.36 The isolation of 4 from the nitrophenol byproduct could not be achieved by chromatography, and the product was purified by crystallization from ethylacetate/petroleum ether 3/1 with a yield of 22%. The hydrosilylation of 4 yielded a mixture of desired product 2 and the saturated byproduct after 2 h of reaction time. (The molar ratio of 2 in the mixture was 0.45 according to the integration of the NMR signals.) It is important to note that the basic character of the tertiary aromatic amine in 4 did not neutralize the catalyst (a Lewis acid). Separating 2 from the saturated byproduct by column chromatography did not work. For this reason, method B was tried, where the hydroxyl group of 3 was reacted directly with 3-(isocyanopropyl)triethoxysilane to give product 2 in one step in good yield. Preparation and Properties of the Surface Layers. Monocomponent layers of silanes 1 and 2 were obtained by silanization in solution. In the case of silane 2, both the pure compound and the mixture of 2 and the saturated product were used for silanization. The saturated product is not expected to react with the silica surface; therefore, it does not interfere with the silanization process. In fact, no difference could be detected between surface layers obtained with the pure compound or with the mixture. The course and extent of the surface reaction was followed by ellipsometry and UV/vis spectroscopy. Representative UV spectra of quartz substrates modified with the silanes are shown (46) Ito, K.; Maruyama, J. Chem. Pharm. Bull. 1983, 31, 3014–3023. (47) Schade, B.; Hagen, V.; Schmidt, R.; Herbrich, R.; Krause, E.; Eckardt, T.; Bendig, J. J. Org. Chem. 1999, 64, 9109–9117.

Figure 2. UV/vis spectra of quartz substrates modified by silanization from solution with silanes 1 and 2 and an equimolar mixture of both.

in Figure 2 and were found to be in good agreement with the spectra of the precursor silanes in solution (Figure 1A Supporting Information). The surface density of the chromophore, Γ (molecs cm-2), can be estimated from the UV absorbance using Γ ) 1/ [A ε -1N ] × 10-3, where A is the absorbance of the surface 2 λ λ A λ layer at a given wavelength, ελ is the molar extinction coefficient of the chromophore in solution (M-1 cm-1) at λ, and NA is Avogadro’s number.48-51 The factor of 1/2 refers to the fact that the quartz slides are modified on both sides. We measured molar extinction coefficients of 2634 M-1 cm-1 at 345 nm and 4607 (48) Bramblett, A. L.; Boeckl, M. S.; Hauch, K. D.; Ratner, B. D.; Sasaki, T.; Rogers, J. W. Surf. Interface Anal. 2002, 33, 506–515. (49) Caruso, F.; Kurth, D. G.; Volkmer, D.; Koop, M. J.; Muller, A. Langmuir 1998, 14, 3462–3465. (50) Li, D. Q.; Swanson, B. I.; Robinson, J. M.; Hoffbauer, M. A. J. Am. Chem. Soc. 1993, 115, 6975–6980. (51) Tang, T. J.; Qu, J. Q.; Mullen, K.; Webber, S. E. Langmuir 2006, 22, 26–28.

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Stegmaier et al. Table 1. Properties of the Surface Layersa estimated surface density (molecs cm-2)

silane

layer thickness (nm)

water contact angle (deg)

UV

ellipsometry

water contact angle after irradiation (deg)

1 2 1+2

0.54 ( 0.05 1.5 ( 0.1 1.4 ( 0.1

65 ( 5 65 ( 5 68 ( 1

3.5 × 1014 1.1 × 1015

9.7 × 1013 2.6 × 1014

59 ( 2 60 ( 5 63 ( 3

a The ellipsometric layer thickness and water contact angle were measured on substrates obtained under optimized silanization conditions. The molar surface loading and surface coverage were calculated from the experimental layer thickness.

Figure 3. Kinetics of the silanization process as followed by UV spectroscopy. Measurements were performed on quartz substrates during silanization from an equimolar mixture of silanes 1 and 2. On the left, the raw UV data are shown. On the right, the calculated increase in the UV signal at 377 nm with the silanization time is represented.

Figure 4. Photocleavage reaction of silanes 1 and 2 upon light exposure.

M-1 cm-1 at 385 nm for silanes 1 and 2 in dichloromethane solution. Using these values, average surface densities of 3.5 × 1014 and 1.1 × 1015 molecules cm-2 were obtained for layers of silanes 1 and 2, respectively (Figure 2 and Table 1). Note that this calculation assumes that the molar extinction coefficients of the chromophores in solution and at the surface are the same. This is true only if anchored chromophores at the surface are randomly oriented and if chromophore-chromophore or chromophore-surface interactions are disregarded.48 The ellipsometric thicknesses of the silane layers were 0.55 and 1.5 nm for silanes 1 and 2, respectively (Table 1). This value is smaller than the molecular length of the silanes (1.6 and 1.9 nm, respectively). The estimated surface density from the ellipsometric measurements gives values of 9.7 × 1013 and 2.6 × 1014 molecules cm-2 for layers of silanes 1 and 2, respectively (Table 1).44,45 These surface densities are lower than those obtained from the UV measurements. This could be due to a nonrandom orientation of the chromophores within the surface layer (which would lead to different values for the extinction coefficient in solution and at the surface) or to differences in the extent of the silanization reaction on the quartz and silicon substrates. (Note that UV spectra were taken from quartz substrates and ellipsometry was performed on silicon wafers. Both substrates were treated equally prior to, during, and after the surface reaction and measurements.)

A static water contact angle of 65° was measured on surfaces modified with the silanes, independently of their chemical structure. Mixed layers were prepared by competitive chemisorption from an equimolar mixture of both silanes. The kinetics of the surface reaction was followed by UV spectroscopy. Figure 3 shows representative spectra recorded after increasing silanization times. The UV absorption and therefore the amount of silane attached to the surface increase with increasing silanization time and reach a saturation level after about 45 h. At this point, longer reaction times or the addition of more catalyst does not cause any further increase in absorption. The molar ratio of the silanes at the surface can be determined by deconvoluting the UV spectrum of the mixed layers as the sum of the spectra of the two individual silanes. This analysis revealed a 3/7 molar ratio of silanes 1/2 on the surface, which is different from the 1/1 molar ratio in solution used for the modification process. This ratio does not seem to change with the silanization time. According to these results and the surface density values obtained in the monocomponent layers, it seems that silane 2 has a higher affinity for the surface. This may be a consequence of the faster hydrolysis/condensation kinetics of (52) Halik, M.; Klauk, H.; Zschieschang, U.; Schmid, G.; Dehm, c.; Schu¨tz, M.; Maisch, S.; Effenberger, F.; Brunnbauer, M.; Stellacci, F. Nature 2004, 431, 963–966. (53) Maisch, S.; Buckel, F.; Effenberger, F. J. Am. Chem. Soc. 2005, 127, 17315–17322.

PhotoresponsiVe Surfaces with Functional LeVels

Figure 5. Conversion (%) for the photocleavage of silane 2 at the surface upon irradiation at 345 and 412 nm. Data were calculated from the absorbance values at λmax ) 385 nm.

silane 2 under the reaction conditions or stronger π interactions between the terminal DEACM moieties, which promote a denser packing of silane 2 within the layer.52,53 The contact angle of the mixed layers was found to be 68°, similar to that of the monocomponent layers, and the obtained layer thickness was 1.4 ( 0.1 nm (Table 1). Photoactivation of the Surface by Irradiation: WavelengthSelective Deprotection. The photolytic activity of the surface layers was tested by performing irradiation experiments at 345 and 412 nm. Light exposure is expected to cleave the chromophores from the surface and release free amine groups (Figure 4). The photolytic reaction at the surface can be followed by the decay of the UV absorbance of the substrates after washing. From these data, the conversion (%) was calculated as follows. The ratio of the absorbance at λmax after irradiation to the amount of initial absorbance multiplied by 100 gave the percent of chromophore remaining after exposure for a given time. From this, the percent conversion caused by each exposure was found by subtraction. Note that this calculation assumes that the absorbance measured at the substrate is due to only the remaining chromophore at the surface (no side reactions). The irradiation of substrates modified with silane 2 at 345 and 412 nm induces a clear decrease in the UV absorbance (Figures 2A and 3A in Supporting Information). Figure 5 represents the conversion of the photolytic reaction after increasing exposure times. Higher conversions were reached when irradiating at 412 nm than at 345 nm at a given exposure time. This is not surprising and can be attributed to the higher extinction coefficient of DEACM at this wavelength and the higher intensity of the irradiation source at this wavelength (1.6 mW cm-2 at 412 nm and 0.78 mW cm-2 at 345 nm, respectively). The irradiation of substrates modified with silane 1 at 345 nm induces a clear drop in the UV absorption after 30 s of irradiation (Figure 4A in Supporting Information). These results are in agreement with published data on similar substrates.24,54 The absorbance drops to 70% after irradiation for 40 min, but it does not decrease further at longer irradiation times. We have already demonstrated that this is due to imine formation between the benzaldehyde photofragment and the primary amino group at the surface.24 This side reaction reattaches the chromophore to (54) Alonso, J. M.; Reichel, A.; Piehler, J.; del Campo, A. Langmuir 2008, 24, 448–457.

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Figure 6. Conversion (%) for the photocleavage of silane 1 at the surface upon irradiation at 345 and 412 nm. Data were calculated from the absorbance values at λmax ) 345 nm.

Figure 7. Decrease in the surface density of chromophores after irradiation with increasing irradiation doses.

the surface, causing residual absorbance and decreasing the final density of amine groups. The irradiation of substrates modified with silane 1 at 412 nm for 40 min did not change the UV spectrum of the substrate significantly, as expected from the very low extinction coefficient of silane 1 at this wavelength (Figure 5A in Supporting Information). The photolytic reaction occurs with a much higher conversion at 345 nm than at 412 nm, as can be appreciated in Figure 6. (Note that the residual absorbance may interfere with the calculation of the conversion.) This result corroborates our hypothesis of selective cleavage of the DEACM group at 412 nm in the presence of NVo groups. Figure 5 shows that the irradiation of substrates modified with silane 2 at 345 nm caused a significant decrease in the absorbance values, even at short irradiation times. These results indicate that the NVo group cannot be cleaved selectively in the presence of DEACM groups at 345 nm and therefore DEACM and NVo are not orthogonal under our irradiation conditions. Figure 7 represents the cleavage of DEACM and NVo chromophores from substrates with comparable initial surface densities after irradiation at 345 and 412 nm with increasing irradiation dose. This representation allows us to compare the photosensitivity (defined by the product of the extinction coefficient and the quantum yield) of the two chromophores at the different wavelengths. Irradiation at 345 nm seems to cleave

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Stegmaier et al.

Figure 8. Fluorescence pattern expected after two-step masked irradiation of mixed layers of silanes 1 and 2 and subsequent fluorescent staining as follows: (i) deprotection of silane 2 at 412 nm by masked irradiation through a vertical stripe pattern, (ii) coupling Alexa Fluor 647 to free amine groups, (iii) deprotection of silanes 1 and 2 at 345 nm through a horizontal stripe pattern, and (iv) coupling Alexa Fluor 488 to free amine groups.

Figure 9. Fluorescence microscope images of substrates modified with mixed layers of silanes 1 and 2 after processing as indicated in Figure 7: (a) fluorescence pattern of Alexa Fluor 647, (b) fluorescence pattern of Alexa Fluor 488, (c) overlap of images in panels a and b, and (d) fluorescence pattern of DEACM.

more NVo molecules from the surface than DEACM molecules. This result is surprising because both compounds have similar extinction coefficients at this wavelength (according to their UV spectra in solution, see Figure 1A Supporting Information), and reported quantum yields of the photolysis of NVo in solution are notably lower than those of DEACM.43 However, quantum yields in solution are usually measured after irradiation experiments performed at λmax, which is not 345 nm for DEACM. Quantum yields may vary depending on the irradiation wavelength as a consequence of different photolytic mechanisms. In addition, quantum yields at the surface and in solution may also be different. More experiments are required to clarify these questions. The photolytic reaction also reduces the water contact angle of the surface by 5°, indicating the liberation of the more polar amine group.

Generating Bifunctional Patterned Surfaces after TwoStep Irradiation at Different Wavelengths. The capability of mixed layers of silanes 1 and 2 t generate chemical patterns with two different functional levels was explored by irradiating substrates through a mask in a two-step process at the selected wavelengths. Upon masked irradiation, a pattern of activated (irradiated and deprotected) and nonactivated areas reflecting the shape of the mask is generated. The resulting chemical contrast between exposed and nonirradiated regions can be used to direct the assembly process of specific targets onto activated areas, such as fluorescent dyes. If the different dyes are site-selectively assembled onto the desired active areas of a multifunctional surface, then the underlying chemical pattern will be reflected in the form of different colors under the fluorescence microscope (Figure 8).

PhotoresponsiVe Surfaces with Functional LeVels

Initially, the mixed surface was irradiated at 412 nm through a quartz mask consisting of vertical, 50-µm-thick chrome stripes separated by 10-µm-thick spaces. Surface regions with free amine groups were generated as a consequence of the cleavage of DEACM from the exposed (quartz) areas. These groups were then reacted with Alexa Fluor 647 succinimidyl ester, and the resulting pattern was visualized by fluorescence microscopy. Bright fluorescent stripes with a width of 10 µm on a nonfluorescent background could be clearly observed and proved the selective binding of the dye to the free amine groups after deprotection (Figure 9a). A second irradiation step at 345 nm through a quartz mask with horizontal 50 µm chrome stripes separated by 10 µm cleaved the DEACM and NVo groups at the exposed areas. These were reacted with Alexa Fluor 488 succinimidyl ester. A pattern with fluorescent, 10 µm horizontal stripes could be observed (Figure 9b). Figure 9c shows the two color pattern after the superposition of both fluorescent images. The fluorescence contrast at the horizontal pattern is lower than at the vertical pattern. These results were attributed to two factors: (a) the lower extinction coefficient of Alexa Fluor 488 and (b) background fluorescence as a consequence of the presence of DEACM (fluorescence maximum at 491 nm) in the nonirradiated regions. The fluorescence spectrum of DEACM overlaps with the emission spectra of Alexa Fluor 488 and makes monitoring difficult.39 This was confirmed by recording the fluorescence images of DEACM with an appropriate filter (Figure 9d).

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Conclusions We have tested the possibility of the selective cleavage of NVo and DEACM chromophores using light of different wavelengths. For this purpose, UV spectral regions where the two chromophores show marked differences in their extinction coefficients were identified. NVo absorption is almost negligible at wavelengths above 400 nm, whereas λmax of DEACM is located at around 400 nm. Irradiation experiments performed at 412 nm demonstrated the selective removal of DEACM in the presence of NVo, which could then be cleaved at 345 nm. The inverse irradiation sequence leads to the removal of both chromophores after the first exposure at 345 nm because both groups show similar extinction coefficients at this wavelength. As a consequence, the photocleavage reaction is not orthogonal. The selected wavelengths are suitable for application of the wavelengthselective deprotection strategy to biological issues. Acknowledgment. We thank Dr. G. Rodrı´guez Munˇiz for her support in the synthesis of the DEACM-derived silane. Supporting Information Available: UV spectra of silanes in solution and results of irradiation experiments. This material is available free of charge via the Internet at http://pubs.acs.org. LA802052U