Photochemical Microcontact Printing by Thiol−Ene and Thiol−Yne

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Photochemical Microcontact Printing by Thiol-Ene and Thiol-Yne Click Chemistry Christian Wendeln,† Stefan Rinnen,‡ Christian Schulz,† Heinrich F. Arlinghaus,‡ and Bart Jan Ravoo*,† †

Organic Chemistry Institute and Center for Nanotechnology (CeNTech), Westf€ alische Wilhelms-Universit€ at M€ unster, Corrensstrasse 40, 48149 M€ unster, Germany, and ‡Physikalisches Institut, Westf€ alische Wilhelms-Universit€ at M€ unster, Wilhelm-Klemm-Strasse 10, 48149 M€ unster, Germany Received July 26, 2010. Revised Manuscript Received September 3, 2010

This article describes the microstructured immobilization of functional thiols on alkene- and alkyne-terminated selfassembled monolayers on silicon oxide substrates by photochemical microcontact printing. A photochemical thiol-ene or thiol-yne “click” reaction was locally induced in the area of contact between stamp and substrate by irradiation with UV light (365 nm). The immobilization reaction by photochemical microcontact printing was verified by contact angle measurements, X-ray photoelectron spectroscopy, atomic force microscopy, and time-of-flight secondary ion mass spectrometry. The reaction rate of photochemical microcontact printing by thiol-ene chemistry was studied using time dependent contact angle measurements. The selective binding of lectins to galactoside microarrays prepared by photochemical microcontact printing was also demonstrated. It was found that photochemical microcontact printing results in a high surface coverage of functional thiols within 30 s of printing even for dilute (mM) ink solutions.

Introduction Although the radical addition reaction of thiols to alkenes has been known since more than a century,1 it has attracted increasing interest during the last years due to the recognition of its “click chemistry” characteristics,2 which include high yields, regiospecifity, straightforward product isolation, and mild reaction conditions with readily available starting materials and reagents. Furthermore the “thiol-ene” reaction is modular, wide in scope and proceeds in the absence of solvent. The versatility of the reaction is enhanced by the fact that it does not need any catalyst, it can be initiated thermally as well as photochemically, and it tolerates a variety of functional groups. Several recent reviews3-5 summarize the impressive progress that has been achieved in chemistry and materials science by utilization of this reaction. Thiol-ene radical chemistry also found application in the area of surface chemistry. Pioneering work was done by Waldmann and co-workers,6 who immobilized alkene-functionalized biomolecules such as biotin on thiol-modified glass substrates. Local reaction could be induced by irradiation of the surface, covered with a solution of the biotin-alkene conjugate, by laser, or through a photomask. The biotin patterns showed selective binding to streptavidin, which allowed further linkage of biotinylated proteins to the surface, offering a general method for the production of protein microarrays. In addition, Waldmann and co-workers7 showed that farnesylated (and therefore: alkenefunctionalized) proteins can also directly be photoimmobilized on *Corresponding author. E-mail: [email protected].

(1) Posner, T. Ber. Dtsch. Chem. Ges. 1905, 38, 646. (2) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004. (3) Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Chem. Soc. Rev. 2010, 39, 1355. (4) Hoyle, C. E.; Bowman, C. N. Angew. Chem., Int. Ed. 2010, 49, 1540. (5) Lowe, A. B. Polym. Chem. 2010, 1, 17. (6) Jonkheijm, P.; Weinrich, D.; K€ohn, M.; Engelkamp, H.; Christianen, P. C. M.; Kuhlmann, J.; Maan, J. C.; N€usse, D.; Schroeder, H.; Wacker, R.; Breinbauer, R.; Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int. Ed. 2008, 47, 4421. (7) Weinrich, D.; Lin, P.; Jonkheijm, P.; Nguyen, U. T. T.; Schr€oder, H.; Niemeyer, C. M.; Alexandrov, K.; Goody, R.; Waldmann, H. Angew. Chem., Int. Ed. 2010, 49, 1252.

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thiol-modified surfaces. Bertin and Schlaad8 used mercaptopropylterminated surfaces for the attachment of allyl-R-D-glucopyranoside, perfluoro-1-decene, and 1,2-polybutadiene. The immobilization was carried out homogeneously by exposure of the thiolmodified surfaces to concentrated alkene solutions (1-4 wt %) and irradiation with UV-visible light for 24 h. The polybutadiene surfaces could also be functionalized by reaction with tetra-Oacetyl-1-thio-D-glucopyranose. The immobilized carbohydrates bind to lectins. In addition, thiol-ene chemistry has recently been applied for the modification of planar polymer surfaces9 as well as polymer microspheres,10,11 the covalent layer-by-layer assembly of dithiols and dienes12 and for the fabrication of polymer-coated surfaces by free-radical polymerization.8,13-16 Similar to alkenes, also alkynes can act as a substrate for the radical addition of thiols. The most important difference between “thiol-ene” and “thiol-yne” reactions is that the alkyne bond can react with two thiols to form a double addition product with 1,2-regioselectivity. Although less investigated than the thiol-ene reaction, thiol-yne chemistry is gaining increasing relevance and was used for the preparation of multifunctional thioethers17 and highly refractive networks18 as well as for end-functionalization of poly(N-isopropylacrylamide) synthesized by RAFT.19 Furthermore, (8) Bertin, A.; Schlaad, H. Chem. Mater. 2009, 21, 5698. (9) Wickard, T. D.; Nelsen, E.; Madaan, N.; ten Brummelhuis, N.; Diehl, C.; Schlaad, H.; Davis, R. C.; Linford, M. R. Langmuir 2010, 26, 1923. (10) Diehl, C.; Schlaad, H. Chem.;Eur. J. 2009, 15, 11469. (11) Goldmann, A. S.; Walther, A.; Nebhani, L.; Joso, R.; Ernst, D.; Loos, K.; Barner-Kowollik, C.; Barner, L.; M€uller, A. H. E. Macromolecules 2009, 42, 3707. (12) Li, Y.; Wang, D.; Buriak, J. M. Langmuir 2010, 26, 1232. (13) Reddy, S. K.; Sebra, R. P.; Anseth, K. S.; Bowman, C. N. J. Polym. Sci. Part A 2006, 44, 7027. (14) Khire, S. V.; Yi, Y.; Clark, A. A.; Bowman, C. N. Adv. Mater. 2008, 20, 3308. (15) Hagenberg, E. C.; Malkoch, M.; Ling, Y.; Hawker, C. J.; Carter, K. R. Nano Lett. 2007, 7, 233. (16) Khire, V. S.; Benoit, D. S. W.; Anseth, K. S.; Bowman, C. N. J. Polym. Sci. Part A 2006, 44, 7027. (17) Chan, J. W.; Hoyle, C. E.; Lowe, A. B. J. Am. Chem. Soc. 2009, 131, 5751. (18) Chan, J. W.; Zhou, H.; Hoyle, C. E.; Lowe, A. B. Chem. Mater. 2009, 21, 1579. (19) Yu, B.; Chan, J. W.; Hoyle, C. E.; Lowe, A. B. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 3544.

Published on Web 09/21/2010

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patterned bifunctional surfaces have been made by the sequential immobilization of thiols on alkyne-modified polymer brushes. To this end, a first thiol was immobilized in a pattern by local irradiation of a substrate covered with thiol (neat or highly concentrated in THF) through a photomask and unreacted areas were subsequently filled by photochemical reaction with a second thiol.20 In contrast to photochemical lithography, which is limited to a few reactions (including the thiol-ene and thiol-yne free radical reaction), microcontact chemistry is emerging as a more general method for the patterning of surfaces.21 Microcontact chemistry is also known as reactive microcontact printing (μCP). In microcontact chemistry a polydimethylsiloxane (PDMS) stamp is “inked” with molecules of interest and placed on a reactive substrate. The ink reacts exclusively in the area of contact. Using microcontact chemistry, functional microarrays can be obtained in a single, rapid printing step. Microcontact chemistry has been used for the microstructured immobilization of RGD-containing proteins22 and amine-terminated DNA23 on aldehyde-terminated self-assembled monolayers (SAMs), for the preparation of carbohydrate microarrays by azide-plus-alkyne click chemistry24,25 and Diels-Alder reactions,26 as well as for the attachment of amine-terminated biotin on pentafluorophenol active ester modified polyolefin and polyester surfaces.27 Important advantages of microcontact chemistry are that reactions proceed very fast due to the high concentration of ink and SAM molecules in the contact area of stamp and substrate and that only very small amounts of ink are necessary for surface patterning. In this respect, microcontact chemistry is superior to solution based surface reactions. Furthermore, μCP can provide high resolution patterns independent from the optical properties of the substrate, whereas the quality of the patterns made by photolithography is limited by reflection and scattering. In this article we describe the immobilization of functional thiols on alkene-terminated SAMs through thiol-ene radical addition induced by photochemical μCP. In addition, we describe the immobilization of thiols on alkyne-terminated SAMs through thiol-yne radical addition induced by photochemical μCP. During the preparation of this manuscript, a communication28 on the modification of alkene monolayers on oxide-free silicon via thiol-ene chemistry was published. In that paper, the authors report the photochemical μCP of thioglycolic acid on an alkeneterminated SAM by using a PDMS stamp inked with a highly concentrated (M) solution of thiol. In contrast, this work describes photochemical μCP of a range of functional thiols on alkene-terminated as well as alkyne-terminated SAMs on glass and silicon wafers by using oxidized PDMS stamps, which allows a drastic decrease of the concentration (mM) of the ink solution. Polar and/or high molecular weight inks do not permeate into an oxidized PDMS stamp but instead adsorb to its surface. This is a well-known phenomenon in μCP which implies that a high surface coverage of ink on the stamp can be achieved also with (20) Hensarling, R. M.; Doughty, V. A.; Chan, J. W.; Patton, D. L. J. Am. Chem. Soc. 2009, 131, 14673. (21) Ravoo, B. J. J. Mater. Chem. 2009, 19, 8902. (22) Rozkiewicz, D. I.; Kraan, Y.; Werten, M. W. T.; de Wolf, F. A.; Subramaniam, V.; Ravoo, B. J.; Reinhoudt, D. N. Chem.;Eur. J. 2006, 12, 6290. (23) Rozkiewicz, D. I.; Brugman, W.; Kerkhoven, R. M.; Ravoo, B. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 2007, 129, 11593. (24) Michel, O.; Ravoo, B. J. Langmuir 2008, 24, 12116. (25) Godula, K.; Rabuka, D.; Nam, K. T.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 4973. (26) Wendeln, C.; Heile, A.; Arlinghaus, H. F.; Ravoo, B. J. Langmuir 2010, 26, 4933. (27) Hyun, J.; Zhu, Y.; Liebmann-Vinson, A.; Beebe, T. P.; Chilkoti, A. Langmuir 2001, 17, 6358. (28) Campos, M. A.; Paulusse, J. M. J.; Zuilhof, H. Chem. Commun. 2010, 46, 5512. (29) Kaufmann, T.; Ravoo, B. J. Polym. Chem. 2010, 1, 371.

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a dilute ink solution.29 This is particularly advantageous for biological inks such as carbohydrates, proteins and nucleic acids which are typically available only in small amounts. Moreover, it is shown in this article that photochemical μCP yields dense monolayers of functional molecules within 30 s.

Experimental Section General. Chemicals were purchased from Sigma Aldrich or from AcrosOrganics and used without further purification, unless otherwise noted. 11-Undecenyltrichlorosilane was obtained from ABCR. Silicon wafers (B-doped, 100 orientation, 20-30 Ω) were kindly donated by Siltronic AG (Burghausen, Germany). Glass substrates were prepared from IDL microscope slides (Interressengemeinschaft der Laborfachh€andler). Surfaces were cleaned with dry ethanol p.a. and Milli-Q water, which was prepared from distilled water using a PureLab UHQ deionization system (Elga). Fluorescein isothiocyanate (FITC)-labeled concanavalin A (ConA) was purchased from Sigma Aldrich. Tetramethylrhodamine isothiocyanate (TRITC)-labeled peanut agglutinin (PNA) was obtained from Vector Laboratories. Surface irradiation was carried out using a high power UV-LED (P8D236, Seoul Semiconductor, 365 nm peak wavelength, 18 nm spectrum half width, 90 mW optical power output) which was supplied by Conrad Electronics. Detailed information concerning the synthesis of galactopyranosyl-thiolconjugates (4-5), 2-(2-(2-methoxyethoxy)-ethoxy)ethanethiol (6), and methyl-11-(trichlorosilyl)undecanoate (7) is provided in the Supporting Information. SAM Preparation. Glass slides and Si wafers were cut into pieces of 1.4
2.6 cm2, cleaned by sonication in pentane, acetone and Milli-Q water, dried and then treated with a freshly prepared Piranha solution (H2O2/H2SO4 = 1/3) for 30 min. Caution: piranha is a very strong oxidant and reacts violently with many organic materials. The substrates were thoroughly washed with Milli-Q water and again dried. Monolayer formation was carried out by immersing the freshly oxidized surfaces in a stirred solution of trichlorosilane in toluene (0.1 vol %) for 40 min. 11-Undecenyltrichlorosilane was used to prepare alkene-terminated SAMs. Unreacted silanes were removed from the surface by washing with ethanol and water. Alkyne-terminated SAMs were prepared in a three-step procedure: (1) Piranha treated substrates were covered with an ester-terminated SAM by reaction with methyl-11-(trichlorosily)undecanoate (40 min, 0.1% vol.) and unreacted material was removed by washing (ethanol, water). (2) Ester groups were hydrolyzed by treatment with 2.5 M HCl at 85 °C for 2 h.30 The substrates were thoroughly washed with water and dried. (3) The acid SAMs were coupled to propargylamine using a standard protocol for inverse solid phase peptide synthesis:31 Substrates were immersed into a freshly prepared solution of propargylamine (0.2 M), diisopropylethylamine (0.2 M), and TBTU (0.16 M) in DMF (peptide synthesis grade) and stirred for 2 h at room temperature. Finally the surfaces were cleaned with ethanol and water. PDMS Stamps. PDMS stamps were prepared by casting a 10:1 (v/v) mixture of poly(dimethylsiloxane) and curing agent (Sylgard 184, Dow Corning) on a patterned silicon master. The PDMS was heated in an oven at 80 °C overnight for curing. The patterned sections were cut out with a knife and oxidized in an UV-ozonizer (PSD-UV, Novascan Technologies Inc.) for 50-60 min prior to use. Flat PDMS stamps were made analogously, using a flat silicon wafer as master. Photochemical Microcontact Printing. A total of 1-3 drops of a freshly prepared solution of thiol (30 mM) and R,Rdimethoxy-R-phenylacetophenone (Irgacure 651, 15 mM) in oxygen-free ethanol was spread on the surfaces of oxidized PDMS stamps. For the printing of 3-mercaptopropionic acid, a 50 mM (30) Al-Abadleh, H. A.; Voges, A. B.; Bertin, P. A.; Nguyen, S. T.; Geiger, F. M. J. Am. Chem. Soc. 2004, 126, 11126. (31) Johansson, A.; A˚kerblom, E.; Ersmark, K.; Lindeberg, G.; Hallberg, A. J. Comb. Chem. 2000, 2, 496.

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Figure 1. (A) Schematic illustration of photochemical μCP by thiol-ene chemistry: An oxidized PDMS stamp inked with a thiol is placed on an alkene-terminated SAM and irradiated with UV light (365 nm). Immobilization of the thiol occurs exclusively in the area of contact. (B) Light microscopy of water condensation experiments on surfaces patterned by printing N-acetyl-L-cysteine (1), D,L-dithiothreitol (2), 3-mercaptopropionic acid (3), tetraacetylgalactoside-thiol conjugate (4), and galactoside-thiol conjugate (5).

thiol solution (30 mM Irgacure 651) was used. After an incubation time of 1 min, the stamps were dried in a stream of argon and immediately placed on alkene- and alkyne-functionalized substrates. The PDMS stamps were irradiated for 5-600 s with a 365 nm high power UV-LED, which was placed approximately 2 cm above the interface of stamp and substrate. After irradiation, the PDMS was removed and the surfaces were washed with ethanol, sonicated in ethanol (1 min), and dried. Please note: For successful μCP, PDMS stamps must be oxidized so that dilute thiol solutions in ethanol can be used for inking. Oxidation results in a high density coating of polar thiols on the PDMS surface. Contact Angle Measurements. Water contact angles were measured on glass substrates by means of the sessile drop method on a DSA 100 goniometer (Kr€ uss GmbH Wissenschaftliche Laborger€ate). Three measurements were performed for every sample. For μCP reactivity studies three samples were prepared independently for every reaction time and three measurements were carried out on every sample. The evaluation of the measurements was performed using the software Drop Shape Analysis. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectra were recorded with the Kratos Axis Ultra system (Kratos Analytical) and monochromatized Al KR radiation (1486.6 eV) as the excitation source. The obtained spectra were evaluated by using the software Kratos Vision 2 and were referenced by setting the C(1s)-peak of the saturated hydrocarbons to 284.5 eV. All measurements were carried out on glass substrates. Time-of-Flight Secondary Ion Mass Spectrometry. SIMS experiments were performed using a type IV compatible TOF-SIMS instrument equipped with a liquid metal ion gun (IONTOF GmbH). As primary ions Bi3þ with an energy of 25 keV were used. The samples were imaged with a primary ion dose density of up to 5
1012 ions/cm2. All measurements were carried out on silicon substrates. Atomic Force Microscopy. AFM mesurements were carried out using a Veeco Nanoscope 3 (Digital Instruments) in the 15968 DOI: 10.1021/la102966j

tapping mode, equipped with a BS-Tap300 AFM tip (Budget Sensors). Fluorescence Microscopy. Fluorescence microscopy images were made by using an Olympus inverted research microscope CKX41 equipped with a mercury burner U-RFL-T as light source and a DX 20 L-FW camera (Kappa opto-electronics GmbH) for image acquisition. The camera was controlled by the program Kappa CameraControl (version 2.7.5.7032). All investigations were carried out on glass substrates. Lectin-Carbohydrate Interactions. Patterned galactose terminated surfaces were covered with a solution of R,R-dimethoxy-Rphenylacetophenone in neat 2-(2-(2-methoxy-ethoxy)ethoxy)ethanethiol (3 wt %) and irradiated for 10 min at 365 nm. The substrates were washed with ethanol and dried. The carbohydrate chips were exposed to a solution of fluorescence labeled lectin (FITC labeled Con A and TRITC labeled PNA, 10 μg/mL) in HEPESbuffer (20 mM HEPES, pH 7.5, 0.15 M NaCl, 1.0 mM CaCl2). In the case of ConA, MnCl2 was added to a concentration of 1 mM. After 30 min of incubation, the surfaces were washed with the same buffer without lectin, carefully dried with a tissue, and analyzed.

Results and Discussion μCP by thiol-ene chemistry was carried out on glass and silicon substrates, which were functionalized by an 11-undecenyltrichlorosilane SAM. In a first step, oxidized PDMS stamps were covered with 1-3 drops of ethanol solutions of the respective thiol plus R,R-dimethoxy-R-phenylacetophenone and dried. Next, the stamps were placed on the substrate and irradiated for 10 min at 365 nm to induce the reaction selectively in the area of contact (Figure 1A). Effective wetting of the stamp with thiol ink is required in order to achieve high quality patterns with high reproducibility. This could generally be ensured by using oxidized PDMS stamps and ink solutions of 30 mM thiol and 15 mM Langmuir 2010, 26(20), 15966–15971

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Figure 2. Static water contact angles versus printing time of galactoside-thiol conjugate (5) on an undecenyl-terminated SAM on glass.

Irgacure 651 in ethanol. N-Acetyl-L-cystein (1), D,L-dithiothreitol (2), and 3-mercaptopropionic acid (3) were immobilized homogeneously on the surface by printing with a flat stamp under irradiation at 365 nm for 10 min. In each case the water contact angle decreased from 111/88 (adv/rec) to less than 60/20 (see the Supporting Information for exact values for each thiol), proving the successful immobilization of the hydrophilic molecules on the surface. Moreover, if printing was carried out by using a patterned stamp (10 μm lines spaced by 5 μm), selective reaction in the area of contact could easily be verified by condensing water on the surface. In each case the water droplets aligned according to the dimensions of the stamp (Figure 1B). In another experiment, tetraacetylgalactoside-thiol conjugate (4) and its deprotected analogue (5) were immobilized by printing homogenously with a flat stamp on the alkene-terminated monolayer. Water contact angles of 68°/24° (protected carbohydrate) and 46° /14° (unprotected carbohydrate) were observed, which is in good agreement with the polarity of the molecules as well as previous reports.24,26 Printing with a patterned stamp and subsequent water condensation again resulted in aligned water droplets (Figure 1B), illustrating the efficient patterning of the alkene surface by photochemical μCP. An important advantage of surface reactions induced by μCP is that reactions by μCP usually proceed much faster in comparison to reactions from (dilute) solution.21 The immobilization rate for photochemical mCP of galactoside-thiol conjugate (5) was estimated by contact angle measurements (Figure 2). The exposure time was varied from 5 to 80 s. It was found that even after only a short irradiation time of 5 s the contact angle decreases from 103 ( 3° to 71 ( 10°. After a printing time of 20 s, a value of 49 ( 5° was observed. Printing for 40 s resulted in a complete surface coverage with a water contact angle of 43 ( 3, and longer printing times did not lead to a further significant decrease. We conclude that thiols can be immobilized in high density within 30 s by photochemical μCP. The successful immobilization of the carbohydrate conjugates was furthermore investigated by X-ray photoelectron spectroscopy (XPS). μCP was carried out with a flat stamp in order to cover the entire surface with carbohydrates. In comparison to the undecenylterminated SAM (I), the C(1s)-signal of the tetraacetylgalactoside (4) modified surface (II) is significantly broader, which is in agreement with the diverse chemical surroundings of the carbon atoms (Figure 3A). The small shoulder at 289 eV in the tetraacetylgalactoside spectra can be attributed to the ester groups. In the case of the galactoside-thiol conjugate (5) modified surface, the shoulder is not present (III). Also the N(1s)-region (Figure 3B) and the S(2s) region (Figure 3C) of the latter sample were investigated (see the Supporting Information). The observation of these heteroatoms in the XPS spectrum indicates the successful immobilization of the carbohydrate-thiol conjugate by photochemical μCP. Furthermore, the surface modification was studied by atomic force microscopy (AFM). An undecenyl-terminated substrate was patterned Langmuir 2010, 26(20), 15966–15971

Figure 3. (A) C(1s) high resolution XPS spectra of the bare undecenyl-terminated SAM (black, I) and of tetraacetylgalactoside- (blue, II) and galactoside- (red, III) modified glass substrates prepared by photochemical μCP. (B) N(1s) high resolution XPS signal of the substrate functionalized with galactoside-thiol-conjugate (5). (C) S(2s) high resolution XPS signal of the same surface. (D) AFM phase image of an undecenyl-terminated SAM on a silicon wafer patterned by photochemical μCP of tetraacetylgalactoside-thiol-conjugate (4) (5 μm lines spaced by 3 μm).

by μCP of the tetraacetylgalactoside-thiol-conjugate (4). Carbohydrate coated stripes of 5 μm, separated by 3 μm interspaces were found in the AFM phase image (Figure 3D), which is in accordance with the dimensions of the oxidized PDMS stamp. An important characteristic of microcontact chemistry is the resolution that can be achieved with a particular immobilization reaction. Diffusion of ink into noncontact areas is generally a limiting factor in μCP. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful method for the analysis of patterned surfaces since as it is very sensitive with a detection limit in the ppm range.32 Tetraacetylgalactoside-thiol conjugate (4) was printed on the undecenyl-terminated SAM in 5 μm thick lines that are spaced by 15 μm and the obtained sample was exposed to a Bi3þ primary ion beam. Various characteristic fragments such as CH3-, CN-, HS-, C2HO-, CNO-, C2H3O-, CHO2-, and C2H3O2could be identified in the negative ion mode, which proves the successful immobilization of the carbohydrate by photochemical μCP. Figure 4 shows the analysis of an area of 100
100 μm2 in the imaging mode. Each anionic fragment indicates that μCP occurs with high edge resolution, as the pattern is exactly in accordance with the dimension of the used stamp and the edges of the lines are sharp. Also the analysis of a smaller area (50
50 μm2) in the negative as well as in the positive ion mode demonstrated the excellent resolution of the pattern (see the Supporting Information). Photochemical μCP by thiol-ene chemistry was also applied for the construction of a simple carbohydrate microarray. Galactoside-thiol conjugate (5) was printed as described above on an alkene-terminated SAM on glass (5 μm lines spaced by 15 μm). Unreacted alkene groups were reacted with 2-(2-(2-methoxyethoxy)ethoxy)ethanethiol, i.e., a triethylene glycol derivative. Exposure of (32) Arlinghaus, H. F. Appl. Surf. Sci. 2008, 255, 1058.

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Figure 4. ToF-SIMS analysis (negative ion mode) of an undecenyl-terminated SAM on a silicon wafer patterned by photochemical μCP of tetraacetylgalactoside-thiol conjugate (4) (5 μm lines spaced by 15 μm).

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Figure 6. (A) Synthesis of alkyne-terminated SAM. Ester-terminated SAMs were hydrolyzed by aqueous HCl (2.5 M, 2 h, 85 °C) (I) and coupled to propargylamine under conditions for inverse solid phase peptide synthesis (0.2 M DIEA, 0,2 M propargylamine and 0.16 M TBTU in DMF, 2 h, room temperature) (II). (B) Light microscopy image of water droplets condensed on carbohydrate patterns prepared by photochemical μCP of galactoside-thiol conjugate (5) on an alkyne-terminated SAM on glass (5 μm lines spaced by 20 μm). (C) Fluorescence microscopy image of TRITCPNA bound to a β-D-galactoside microarray on glass prepared by photochemical μCP (5 μm lines spaced by 20 μm).

Figure 5. (A) Fluorescence microscopy image of TRITC labeled peanut agglutinin bound to a β-D-galactoside microarray on glass prepared by photochemical μCP (5 μm lines spaced by 15 μm). (B) Fluorescence microscopy image of β-D-galactoside microarray exposed to FITC labeled concanavalin A.

the surface to tetramethylrhodamine isothiocyanate labeled peanut agglutinin (TRITC-PNA) and subsequent washing with buffer led to the selective attachment of the lectin to the carbohydrate micropattern (Figure 5A). This observation indicates that the galactoside is immobilized at a high surface density by photochemical μCP, since only in that case a stable, multivalent binding of the lectin to the substrate can occur. In a similar experiment, the galactoside microarray was exposed to fluorescein isothiocyanate labeled concanavalin A (FITC-ConA) and no significant affinity was observed, which is consistent with the selectivity of the lectins (Figure 5B).33 In addition to thiol-ene chemistry, also the thiol-yne reaction can be used for the immobilization of thiols on surfaces. In order to investigate the suitability of this reaction for μCP, alkyne-terminated SAMs on silicon and glass were prepared. To this end, esterterminated SAMs were hydrolyzed by aqueous HCl (I) and the generated carboxylic acid groups were coupled to propargylamine under conditions typical for inverse solid phase peptide synthesis (II) (33) Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637.

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Figure 7. ToF-SIMS analysis (negative ion mode) of an alkyneterminated SAM on a silicon wafer patterned by photochemical μCP of tetraacetylgalactoside-thiol conjugate (4) (5 μm lines spaced by 25 μm).

(Figure 6A; see the Supporting Information for detailed procedures). Alkyne SAMs could successfully patterned by μCP of galactoside-thiol conjugate (5), using the photochemical μCP procedure described above for the μCP by thiol-ene chemistry (10 min exposure at 365 nm). The immobilization was verified by water condensation experiments (Figure 6B) and by the selective binding of PNA to the carbohydrate structures (Figure 6C). Both experiments demonstrate that the patterning proceeds efficiently and with high resolution, since the patterns are in accordance with the microstructure of the stamp (5 μm lines spaced by 20 μm). Also in this case, a more detailed analysis of the resolution was made by Langmuir 2010, 26(20), 15966–15971

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ToF-SIMS. Investigation of a surface patterned by photochemical μCP of tetraacetylgalactoside-thiol conjugate (4) (5 μm lines spaced by 25 μm) showed the formation of the anions CH3-, HS-, C2HO-, CHO2-, and C2H3O2- within the carbohydrate covered areas (Figure 7). The characteristic C2H3O2- anion clearly proves that also in this case the carbohydrate was printed with high resolution, showing that also thiol-yne chemistry allows the preparation of high quality patterns via photochemical μCP. In comparison to alkene-terminated surfaces, in principle a double surface density of thiol can be achieved by μCP on an alkyne-terminated surface, since two thiols can react with one alkyne. However, we do not observe a significant difference in contact angle, lectin binding, or ToF-SIMS. We attribute these findings to the fact that the grafting density of thiols is limited not by the surface reactive groups but by the size of the molecules grafted onto the surface by μCP. Since carbohydrates are considerably bulkier than alkenes or alkynes, the maximum grafting density of carbohydrates is identical.

Conclusion The thiol-ene as well as the thiol-yne radical addition reaction was successfully applied for surface modifications by photochemical

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μCP. A range of functional thiols could be immobilized by this technique and the edge resolution of the microscale patterns was found to be better than 100 nm. We did not observe significant differences in the carbohydrate surface density obtained by thiolene and thiol-yne reactions, respectively. Thiol-ene reactions by photochemical μCP proceed within about 30 s and require only small amounts of thiol. Photochemical μCP is therefore a valuable alternative to photochemical lithography. In particular, we expect that photochemical μCP will be useful for the preparation of carbohydrate, protein, and DNA microarrays. Acknowledgment. Silicon wafers were kindly donated by Siltronic AG. The Deutsche Forschungsgemeinschaft (DFG) is acknowledged for financial support of this work (Grant Ra 1732/2-1). Supporting Information Available: Synthesis and analysis of carbohydrate-thiol conjugates, 2-(2-(2-methoxyethoxy)ethoxy)ethanethiol and methyl-11-(trichlorosilyl)undecanoate, contact angles, XPS, and ToF-SIMS data. This material is available free of charge via the Internet at http://pubs.acs.org.

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