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Langmuir 2008, 24, 448-457
Photopatterned Surfaces for Site-Specific and Functional Immobilization of Proteins Jose´ Marı´a Alonso,† Annett Reichel,‡ Jacob Piehler,‡ and Ara´nzazu del Campo*,† Max-Planck-Institut fu¨r Metallforschung, Heisenbergstraβe 3, 70569 Stuttgart, Germany, and Institute of Biochemistry, Johann Wolfgang Goethe-UniVersity, 60438 Frankfurt/Main, Germany ReceiVed August 31, 2007. In Final Form: October 8, 2007
Photosensitive silanes containing nitroveratryl (Nvoc)-caged amine groups and protein repellent tetraethylene glycol units were synthesized and used for modification of silica surfaces. Functional surface layers containing different densities of caged amine groups were prepared and activated by UV-irradiation of the surface. The performance of these layers for functional and site-selective immobilization of proteins was tested. For this purpose, biotin and tris-nitrilotriacetic acid (tris-NTA) were fist coupled to the activated surface, and the interaction of streptavidin and His-tagged proteins with the functionalized surfaces was monitored by real-time label-free detection. After optimizing the coupling protocols, highly selective functionalization of the deprotected amine groups was possible. Furthermore, the degree of functionalization (and therefore the amount of immobilized protein) was controlled by diluting the surface concentration of the amine-functionalized silane with a nonreactive (OMe-terminated) tetraethylene glycol silane. Immobilized proteins were highly functional on these surfaces, as demonstrated by protein-protein interaction assays with the type I interferon receptor. Protein micropatterns were successfully generated after masked irradiation and functionalization of the caged surface following the optimized coupling protocols.
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 analysis of a variety of samples in parallel.1 The fabrication of these analytical platforms requires the development of surface patterning strategies 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 which can be site-selectively 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 the bare, inactive substrate,4,5 (ii) to graft molecular species to irradiated regions via photogenerated radical cross-reactions occurring between a photosensitive surface layer and the biological molecule (photoaffinity),6-10 or (iii) to direct synthesis * To whom correspondence should be addressed. Telephone: +49 711 6893416. Fax: +49 711 6893412. E-mail:
[email protected]. † Max-Planck-Institut fu ¨ r Metallforschung. ‡ Johann Wolfgang Goethe-University. (1) del Campo, A.; Bruce, I. J. Diagnostics and High Throughput Screening. In Biomedical Nanotechnology; Malsch, I., Ed.; CRC Press: 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 (1), 4-13. (4) Dulcey, C. S.; Georger, J. H.; Krauthamer, V.; Stenger, D. A.; Fare, T. L.; Calvert, J. M. Science 1991, 252 (5005), 551-554. (5) Tarlov, M. J.; Burgess, D. R. F.; Gillen, G. J. Am. Chem. Soc. 1993, 115 (12), 5305-5306. (6) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251 (4995), 767-773. (7) Pellois, J. P.; Wang, W.; Gao, X. L. J. Comb. Chem. 2000, 2 (4), 355-360. (8) 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 (5126), 1303-1305. (9) Li, S. W.; Bowerman, D.; Marthandan, N.; Klyza, S.; Luebke, K. J.; Garner, H. R.; Kodadek, T. J. Am. Chem. Soc. 2004, 126 (13), 4088-4089.
of small molecules on the surface (peptides,6,7 oligocarbamates,8 oligonucleotides,14-17 and peptoids9) through iterative unmasking of photoreactive groups and monomer coupling cycles. Alternatively, surface layers containing the reactive functionalities protected with a photocleavable19-21 group can be used for sitespecific coupling of complementary functionalities after lightdeprotection.6,10-17 The latter is a particularly flexible approach, since a good number of photoremovable groups are known that can be combined with many different reactive species. Compared to the immobilization of oligonucleotides or peptides, functional immobilization of proteins is much more demanding: surface layers are required for minimizing nonspecific interactions of the protein with the surface, which lead to protein denaturation and loss of function of the immobilized protein. Furthermore, functional groups for site-specific tethering of the protein to the surface have to be incorporated. In the case of proteins, surfaces containing oligoethylene glycol (OEG) units within the surface layer are well-known to effectively reduce (10) 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 (7), 3664-3670. (11) Sundberg, S. A.; Barrett, R. W.; Pirrung, M.; Lu, A. L.; Kiangsoontra, B.; Holmes, C. P. J. Am. Chem. Soc. 1995, 117 (49), 12050-12057. (12) del Campo, A.; Boos, D.; Spiess, H. W.; Jonas, U. Angew. Chem., Int. Ed. 2005, 44 (30), 4707-4712. (13) Jonas, U.; del Campo, A.; Kruger, C.; Glasser, G.; Boos, D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (8), 5034-5039. (14) Yan, F. N.; Chen, L. H.; Tang, Q. L.; Rong, W. Bioconjugate Chem. 2004, 15 (5), 1030-1036. (15) Ryan, D.; Parviz, B. A.; Linder, V.; Semetey, V.; Sia, S. K.; Su, J.; Mrksich, M.; Whitesides, G. M. Langmuir 2004, 20 (21), 9080-9088. (16) Dendane, N.; Hoang, A.; Guillard, L.; Defrancq, E.; Vinet, F.; Dumy, P. Bioconjugate Chem. 2007, 18 (3), 671-676. (17) Critchley, K.; Jeyadevan, J. P.; Fukushima, H.; Ishida, M.; Shimoda, T.; Bushby, R. J.; Evans, S. D. Langmuir 2005, 21 (10), 4554-4561. (18) Oklejas, V.; Harris, J. M. Langmuir 2003, 19 (14), 5794-5801. (19) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M. Langmuir 1992, 8 (5), 1330-1341. (20) Tamada, K.; Hara, M.; Sasabe, H.; Knoll, W. Langmuir 1997, 13 (6), 1558-1566. (21) Fadeev, A. Y.; McCarthy, T. J. Langmuir 1999, 15 (21), 7238-7243.
10.1021/la702696b CCC: $40.75 © 2008 American Chemical Society Published on Web 12/20/2007
Photopatterned Surfaces for Protein Immobilization
nonspecific interactions.30,31 Accordingly, different strategies have been followed to incorporate OEG units to a functional surface: direct coupling of surface active functionality through OEG linkers, coadsorption from a solution of OEG and functional molecules (in either parallel18-20 or sequential21 surface reactions), or the use of branched surface coupling agents containing protein repellent and protein attractive arms.36-40 From these three possibilities, competitive chemisorption by treating the surface with a mixture of reagents allows controlled dilution of surface functionality to adjust the surface properties precisely. Here, we have synthesized novel photosensitive silanes designed for patterned and functional protein immobilization, namely, a tetraethylene glycol (TEG) triethoxysilane with a terminal amino group protected by the nitroveratryl (Nvoc) group, and a methoxy-terminated TEG triethoxysilane. The properties of monocomponent and mixed surface layers obtained from these silanes were characterized in detail. The performance of these surfaces for a site-specific and functional immobilization of proteins was explored by real-time label-free detection. We demonstrate the capability of these surfaces to generate laterally resolved functional protein patterns. Experimental Section Materials. All reagents were, unless otherwise noted, used as purchased. Tetrahydrofuran (THF) and toluene were freshly distilled from sodium benzophenone. All reactions were performed under an atmosphere of dry nitrogen. Analytical thin-layer chromatography (TLC) was performed with silica gel 60 F254 plates. Visualization was accomplished by UV light and KMnO4 solution. Silicon wafers (100 orientation) were provided by Crystec (Berlin, Germany). Quartz substrates (Suprasil) were purchased from Heraeus Quarzglas (Hanau, Germany) and Quarzschmelze Ilmenau (Langewiesen, Germany). Transducer slides for reflectance interference spectroscopy (10 nm Ta2O5 and 325 nm silica on a glass substrate) were obtained from Analytik Jena GmbH, Germany. OtBu-protected tris-nitrilotriacetic acid (tris-NTA)-functionalized with a carboxyl group (9) and BTtris-NTA were synthesized as published previously.22-25 The proteins MBP-H10, ifnar2-H10, and IFNR2 were expressed in E. coli and purified as published previously.24,26 1H NMR (250 MHz) and 13C NMR (65 MHz) spectra were recorded in CDCl3 using chloroform as an internal reference with (22) Lata, S.; Gavutis, M.; Tampe, R.; Piehler, J. J. Am. Chem. Soc. 2006, 128 (7), 2365-2372. (23) Lata, S.; Reichel, A.; Brock, R.; Tampe, R.; Piehler, J. J. Am. Chem. Soc. 2005, 127 (29), 10205-10215. (24) Lata, S.; Piehler, J. Anal. Chem. 2005, 77 (4), 1096-1105. (25) Reichel, A.; Schaible, D.; Al Furoukh, N.; Cohen, M.; Schreiber, G.; Piehler, J. Anal. Chem. 2007, 79 (22), 8590-8600. (26) Piehler, J.; Schreiber, G. J. Mol. Biol. 1999, 289 (1), 57-67. (27) Delamarche, E.; Donzel, C.; Kamounah, F. S.; Wolf, H.; Geissler, M.; Stutz, R.; Schmidt-Winkel, P.; Michel, B.; Mathieu, H. J.; Schaumburg, K. Langmuir 2003, 19, 8749-8758. (28) Jeong, K. S.; Park, E. J. J. Org. Chem. 2004, 69, 2618-2621. (29) Effenberger, F.; Heid, S. Synthesis 1995, 1126-1130. (30) Brecht, A.; Gauglitz, G. Biosens. Bioelectron. 1995, 10 (9-10), 923-36. (31) Hanel, C.; Gauglitz, G. Anal. Bioanal. Chem. 2002, 372 (1), 91-100. (32) Schmitt, H. M.; Brecht, A.; Piehler, J.; Gauglitz, G. Biosens. Bioelectron. 1997, 12 (8), 809-816. (33) Bunker, B. C.; Carpick, R. W.; Assink, R. A.; Thomas, M. L.; Hankins, M. G.; Voigt, J. A.; Sipola, D.; de Boer, M. P.; Gulley, G. L. Langmuir 2000, 16 (20), 7742-7751. (34) Kallury, K. M. R.; Macdonald, P. M.; Thompson, M. Langmuir 1994, 10 (2), 492-499. (35) Bierbaum, K.; Kinzler, M.; Woll, C.; Grunze, M.; Hahner, G.; Heid, S.; Effenberger, F. Langmuir 1995, 11 (2), 512-518. (36) Britcher, L. G.; Kehoe, D. C.; Matisons, J. G.; Smart, R. S. C.; Swincer, A. G. Langmuir 1993, 9 (7), 1609-1613. (37) Fadeev, A. Y.; McCarthy, T. J. Langmuir 2000, 16 (18), 7268-7274. (38) Piehler, J.; Brecht, A.; Hehl, K.; Gauglitz, G. Colloids Surf., B 1999, 13 (6), 325-336. (39) Piehler, J.; Brecht, A.; Valiokas, R.; Liedberg, B.; Gauglitz, G. Biosens. Bioelectron. 2000, 15 (9-10), 473-481. (40) Maisch, S.; Buckel, F.; Effenberger, F. J. Am. Chem. Soc. 2005, 127, 17315-17322.
Langmuir, Vol. 24, No. 2, 2008 449 a Bruker Ultra Shield 250 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). Chemical shifts (δ) are given in ppm; multiplicities are indicated by s (singlet), d (doublet), dd (doubledoublet), ddt (double-double-triplet), q (quadruplet), or m (multiplet). Coupling constants (J) are reported in Hertz. Mass spectra (MS) were obtained at 70 eV by chemical ionization (CI) in a Clarus 500 GCMS spectrometer (Perkin-Elmer, Waltham, MA). Data are reported in the form m/z (intensity relative to base ) 100). Synthesis. Tetraethylene Glycol Monoallyl Ether (1).27 Tetraethylene glycol (10 g, 51 mmol), an equimolar amount of allyl chloride (3.9 g, 51 mmol), and tetrabutyl ammonium hydrogen sulfate (1.1 g, 3 mmol) were dissolved in dichloromethane. A 50% aqueous solution of NaOH (40 mL, 0.5 mol) was added slowly under vigorous stirring. The reaction was allowed to proceed for other 20 h at room temperature. The organic layer was separated, and the aqueous layer was washed three times with dichloromethane. The organic fractions were collected, dried over sodium sulfate, and filtered. After evaporation of the solvent, the residue was purified by column chromatography on silica, using ethyl acetate/ethanol 9/1 as eluent to give compound 1 (4.1 g, 31%) as a colorless oil. Disubstituted product was also obtained in 14% yield. 1H NMR (CDCl3) δ 3.483.61 (m, 17H), 3.91 (dd, 3JHH ) 5.7 Hz, 2JHHgem ) 1.3 Hz, 2H), 5.06 (dd, 3JHHcis ) 10.4 Hz, 2JHHgem ) 1.6 Hz, 1H), 5.16 (dd, 3JHHtrans ) 18.0 Hz, 2JHHgem ) 1.6 Hz, 1H), 5.80 (ddt, 3JHHtrans ) 17.1 Hz, 3JHHcis ) 10.4 Hz, 3JHH ) 5.6 Hz, 1H) ppm. 13C NMR (CDCl3) δ 61.1, 69.1, 70.0, 70. 3, 71.8, 72.4, 116.6, 134.6 ppm. MS (CI) m/z 234 (M+ + 1, 100). Tetraethylene Glycol Monoallyl Ether Bromide (2).28 To a solution of 1 (5 g, 21 mmol) in dry THF (85 mL), CBr4 (10.5 g, 31.5 mmol) and PPh3 (8.3 g, 31.5 mmol) were added slowly at 0 °C. The mixture was stirred at room temperature for 16 h and filtered. THF was then removed under vacuum, and the residue was purified by silica column chromatography eluting ethyl acetate/hexane 1.5/1 to yield compound 2 (5.3 g, 85%) as a pale yellow oil. 1H NMR (CDCl3) δ 3.46 (t, 3JHH ) 6.4 Hz, 2H), 3.57-3.70 (m, 12H), 3.80 (t, 3JHH ) 6.3 Hz, 2H), 4.01 (d, 3JHH ) 5.7 Hz, 2H), 5.17 (dd, 3JHHcis ) 10.4 Hz, 2JHHgem ) 1.7 Hz, 1H), 5.26 (dd, 3JHHtrans ) 17.2 Hz, 2JHHgem ) 1.7 Hz, 1H), 5.91 (ddt, 3JHHtrans ) 17.2 Hz, 3JHHcis ) 10.3 Hz, 3JHH ) 5.6 Hz, 1H) ppm. 13C NMR (CDCl3) δ 30.2, 69.4, 70.5, 70.6, 71.2, 72.2, 117.1, 134.8 ppm. MS (CI) m/z 299 (M+ + 1, 2), 151 (M+ - C7H13O3, 25). Tetraethylene Glycol Monoallyl Ether Phthalimide (3).28 A dimethylformamide (DMF) solution (50 mL) containing compound 2 (2 g, 6.7 mmol), phthalimide (2 g, 13.4 mmol), and potassium carbonate (0.94 g, 6.7 mmol) was heated at 85 °C for 18 h. The mixture was filtered, and the solvent was removed under vacuum. The oily residue was subjected to column chromatography on silica, eluting with ethyl acetate/hexane 1/1 to afford compound 3 (1.7 g, 69%) as a colorless oil. 1H NMR (CDCl3) δ 3.55-3.68 (m, 12H), 3.71 (t, 3JHH ) 5.7 Hz, 2H), 3.87 (t, 3JHH ) 6.4 Hz, 2H), 3.99 (d, 3J 3 2 HH ) 5.7 Hz, 2H), 5.14 (dd, JHHcis ) 10.3 Hz, JHHgem ) 1.7 Hz, 3 2 1H), 5.24 (dd, JHHtrans ) 17.2 Hz, JHHgem ) 1.7 Hz, 1H), 5.88 (ddt, 3J 3 3 13 HHtrans ) 17.2 Hz, JHHcis ) 10.3 Hz, JHH ) 5.6 Hz, 1H) ppm. C NMR (CDCl3) δ 37.0, 67.5, 69.1, 69.8, 70.2, 71.7, 116.3, 122.8, 131.7, 133.7, 134.7 ppm. MS (CI) m/z 174 (M+ - C10H7NO3, 100), 218 (M+ - C8H3NO2, 5). Tetraethylene Glycol Monoallyl Ether Amine (4).28 To a solution of compound 3 (2 g, 5.5 mmol) in ethanol, hydrazine monohydrate (0.56 g, 11 mmol) was added. The mixture was heated at reflux for 6 h and allowed to warm to room temperature. The white precipitate was removed by filtration, and the solvent was removed under vacuum. The residue was then diluted with water and extracted three times with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated under vacuum to give compound 4 (1.0 g, 79%) as a colorless oil. 1H NMR (CDCl3) δ 1.71 (s, 2H), 2.85 (t, 3JHH ) 5.2 Hz, 2H), 3.50 (t, 3JHH ) 5.2 Hz, 2H), 3.59-3.68 (m, 12H), 4.01 (d, 3JHH ) 5.7 Hz, 2H), 5.17 (dd, 3JHHcis ) 10.4 Hz, 2JHHgem ) 1.4 Hz, 1H), 5.26 (dd, 3JHHtrans ) 17.2 Hz, 2JHHgem ) 1.6 Hz, 1H), 5.90 (ddt, 3JHHtrans ) 17.2 Hz, 3J 3 13 HHcis ) 10.4 Hz, JHH ) 5.6 Hz, 1H) ppm. C NMR (CDCl3) δ
450 Langmuir, Vol. 24, No. 2, 2008 40.1, 69.3, 69.6, 70.0, 70.1, 70.4, 72.6, 117.0, 134.6 ppm. MS (CI) m/z 234 (M+ + 1, 100). N-NVoc Tetraethylene Glycol Monoallyl Ether Carbamate (5).12 Compound 4 (1 g, 4.3 mmol), sodium hydrogencarbonate (0.84 g, 10.8 mmol), and water were placed into a dry round-bottom flask and cooled in an ice bath. Nvoc-Cl (1.2 g, 4.3 mmol) was dissolved in dioxane (40 mL) and added dropwise under vigorous stirring. The mixture was allowed to stir at room temperature overnight and then was extracted three times with dichloromethane. The organic layers were dried over sodium sulfate and evaporated under vacuum. The solid residue was purified by silica column chromatography eluting ethyl acetate/ethanol 98/2 to afford compound 5 (1.6 g, 79%) as a pale yellow oil. 1H NMR (CDCl3) δ 3.41 (q, 3JHH ) 5.2 Hz, 2H), 3.58 (t, 3JHH ) 5.1 Hz), 3.63-3.71 (m, 12H), 3.95 (s, 3H), 3.98 (s, 3H), 4.00-4.03 (m, 2H), 5.16 (dd, 3JHHcis ) 10.3 Hz, 2JHHgem ) 1.4 Hz, 1H), 5.25 (dd, 3JHHtrans ) 17.2 Hz, 2JHHgem ) 1.6 Hz, 1H), 5.51 (s, 3H), 5.81-5.97 (m, 1H), 7.02 (s, 1H), 7.70 (s, 1H) ppm. 13C NMR (CDCl3) δ 40.9, 56.3, 63.3, 69.3, 69.9, 70.2, 70.5, 72.1, 108.1, 110.0, 117.0, 128.4, 134.7, 139.7, 148.0, 153.5, 155.9 ppm. MS (CI) m/z 196 (M+ - C12H22NO6, 100), 203 (M+ - C11H13N2O6, 10). N-NVoc Tetraethylene Glycol Monoallyl Ether Carbamate (3Triethoxysilyl)propyl Ether (6).29 Compound 5 (1 g, 2.1 mmol) and triethoxysilane (3.4 g, 21 mmol) were placed in a previously passivated dry round-bottom flask and heated under Ar atmosphere to about 85 °C. At this temperature, both reactants mix homogeneously. A solution of H2PtCl6‚H2O in i-PrOH (0.10 mL, 65 mM) was then dropped, and the mixture was stirred for 5 h at 85 °C and then allowed to cool down. An excess of triethoxysilane was removed in vacuum, and the residue was purified by chromatography performed with passivated silica gel (see “Purification of the Derivatized Triethoxysilanes by Passivated Silica Gel Chromatography” in the Experimental section) using ethyl acetate/ethanol 97/3 as eluent to afford compound 6 (0.70 g, 52%) as a pale orange oil. The saturated byproduct (
