Route to Smooth Silica-Based Surfaces Decorated with Novel Self

Apr 20, 2009 - Langmuir , 2009, 25 (10), pp 5526–5535. DOI: 10.1021/la804088d ... [email protected]. Cite this:Langmuir 25, 10, 5526-553...
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Route to Smooth Silica-Based Surfaces Decorated with Novel Self-Assembled Monolayers (SAMs) Containing Glycidyl-Terminated Very Long Hydrocarbon Chains† )

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Duy Hai Dinh,‡,§ Luc Vellutini,‡ Bernard Bennetau,‡ Corinne Dejous,§ Dominique Rebiere,§  Emilie Pascal, Daniel Moynet, Colette Belin,‡ Bernard Desbat,^ Christine Labrugere,# and Jean-Paul Pillot*,‡

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‡ Universit e de Bordeaux, Institut des Sciences Mol eculaires, UMR 5255-CNRS, 351 cours de la Lib eration, 33405 Talence Cedex, France, §Universit e de Bordeaux, Laboratoire de l’Int egration du Mat eriau au Syst eme, e de ENSEIRB, UMR 5218-CNRS, 351 cours de la Lib eration, 33405 Talence Cedex, France, Universit Bordeaux, Laboratoire d’Immunologie et Parasitologie, 146 rue L eo Saignat, 33076 Bordeaux Cedex, ^ Universit e de Bordeaux, Chimie et Biologie des Membranes et des Nanoobjets, UMR 5248-CNRS, avenue des Facult es, 33402 Talence Cedex, France, and #Universit e de Bordeaux, Centre de Caract erisation des Mat eriaux Avanc es, ICMCB-CNRS, 87 avenue du Docteur A. Schweitzer, 33608 Pessac Cedex, France

Received December 11, 2008. Revised Manuscript Received March 6, 2009 Novel glycidyl-terminated organosilicon coupling agents possessing a trialkoxysilyl head group and a very long hydrocarbon chain (C22) were synthesized. Their ability to afford densely packed self-assembled monolayers (SAMs) grafted on silica-based surfaces was investigated. Transmission FT-IR spectra showed that the most regular films were obtained by using trichloracetic acid as the catalyst (10 M %). Atomic force microscopy (AFM) and optical ellipsometry were consistent with well ordered monolayers exhibiting a marked decrease of the surface roughness. Epifluorescence microscopy revealed that these SAMs possessed a better surface reactivity than monolayers obtained with the commercially available (3-glycidoxypropyl) trimethoxysilane (GPTS) upon grafting of a fluorescent probe (dansylcadaverin). Moreover, direct attachment of fluorescent antibodies (RAG-TRITC) through covalent binding led to higher mean fluorescence intensities, showing that these new SAMs possess high potential for the immobilization of biological molecules.

Introduction Over the last decades, there has been sustained interest in the development of molecular self-assembled monolayers (SAMs).1-6 Chemisorbed SAMs find interest in the tailoring of the chemical and physicochemical properties of synthetic materials, affording surfaces with a high density of functional groups. Patterned SAMs containing (sub)micron regions terminated by different chemical functionalities open up the route to a wide range of applications in areas such as nanotechnology and biological array formation. Moreover, biological applications are strongly dependent on the surface properties of SAMs, including wetting and adhesion. These properties result directly from properties of the groups at the SAM-air interface, the composition of the monolayers, and their packing. It is very important that these surfaces are very uniform and possess a very *Corresponding author. E-mail: [email protected]. † This paper was presented at the XVth International Symposium on Organosilicon Chemistry, June 1-6, 2008, Jeju, Korea. (1) Sagiv, J. J. Am. Chem. Soc. 1980, 102, 92. (2) Swalen, D.; Allara, D. L.; Andrade, D. D.; Chandross, E. A.; Garoff, D.; Israelachvili, J; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (3) Ulman, A. Chem. Rev. 1996, 96, 1533. (4) Schreiber, F. Prog. Surf. Sci. 2000, 65, 151. (5) Gooding, J. J.; Mearns, F.; Yang, W.; Liu, J. Electroanalysis 2003, 15, 81. (6) Onclin, S.; Ravoo, B. J.; Reinhoudt, S. N. Angew. Chem., Int. Ed. 2005, 44, 6282. (7) Senaratne, W.; Andruzzi, L.; Ober, C. K. Biomacromolecules 2005, 6, 2427. (8) (a) Moll, N.; Pascal, E.; Dinh, D. H.; Pillot, J.-P.; Bennetau, B.; Rebiere, D.; Moynet, D.; Mas, Y.; Mossalayi, D.; Pistre, J.; Dejous, C. Biosens. Bioelectron. 2007, 22, 2145. (b) Moll, N.; Pascal, E.; Dinh, D. H.; Lachaud, J.-L.; Vellutini, L.; Pillot, J.-P.; Rebiere, D.; Moynet, D.; Pistre, J.; Mossalayi, D.; Mas, Y.; Bennetau, B.; Dejous, C. ITBM-RBM 2008, 29, 155. (9) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103. (10) Gupta, P.; Loos, K.; Korniakov, A.; Spagnioli, C.; Cowman, M.; Ulman, A. Angew. Chem., Int. Ed. 2004, 43, 520.

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low roughness.7-10 One of the most versatile routes to SAMs relies on the condensation of hydroxylated surfaces with organosilicon coupling agents. When they contain a long, linear hydrocarbon chain, these precursors lead to well ordered SAMs, providing molecularly defined platforms for the control of the surface chemistry of materials. Hence, they offer new possibilities in various fields ranging from molecular electronics to biomedicine and catalysis.7,11-14 Various reports have pointed out the crucial role of the molecular structure of precursors on self-assembly, and it has been demonstrated that the chain length determined the formation of high quality SAMs.15-27 (11) Chaki, N. K.; Vijayamohanan, K. V. Biosens. Bioelectron. 2002, 17, 1. (12) Luderer, F.; Walschus, U. Top. Curr. Chem. 2005, 260, 37. (13) Singamaneni, S.; LeMieux, M. C.; Lang, H. P.; Gerber, C.; Lam, Y.; Zauscher, S.; Datskos, P. G.; Lavrik, N. V.; Jiang, H.; Naik, R. R.; Bunning, T. J.; Tsukruk, V. Adv. Mater. 2008, 20, 653. (14) Feng, T. Pure Appl. Chem. 2008, 80, 45. :: (15) Pomerantz, M.; Segmuller, A.; Netzer, L.; Sagiv, J. Thin Solid Films 1983, 105, 674. (16) Brzoska, J. B.; Shahidzadeh, N.; Rondelez, F. Nature (London) 1992, 360, 719. (17) Cave, N. G.; Kinloch, A. J. Polymer 1992, 33, 1162. (18) Parikh, A. N.; Allara, D. L.; Ben Azouz, I.; Rondelez, F. J. Phys. Chem. 1994, 98, 7577. (19) Brzoska, J. B.; Ben Azouz, I.; Rondelez, F. Langmuir 1994, 10, 4367. :: :: (20) Bierbaum, K.; Kinzler, M.; Woll, Ch.; Grunze, M.; Haner, G.; Heid, S.; Effenberger, F. Langmuir 1995, 11, 512. (21) Hoffmann, H.; Mayer, U.; Krischanitz, A. Langmuir 1995, 11, 1304. (22) Atre, S. V.; Liedberg, B.; Allara, D. L. Langmuir 1995, 11, 3882. (23) Duchet, J.; Chabert, B.; Chapel, J. P.; Gerard, J. F.; Chovelon, J. M.; Jaffrezic-Renault, N. Langmuir 1997, 13, 2271. (24) Stevens, M. J. Langmuir 1999, 15, 2773. (25) Martin, P.; Marsaudon, S.; Thomas, L.; Desbat, B.; Aime, J.-P.; Bennetau, B. Langmuir 2005, 21, 6934. (26) Navarre, S.; Choplin, F.; Bousbaa, J.; Bennetau, B.; Nony, L.; Aime, J.-P. Langmuir 2001, 17, 4844. (27) Pillot, J. P.; Birot, M.; Tran, T. T. T.; Dao, T. M.; Belin, C.; Desbat, B.; Lazare, S. Langmuir 2005, 21, 3338.

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The mechanism of monolayer formation is very dependent on experimental parameters such as water content and temperature. Thus, an island-growth mechanism predominates below a critical temperature, close to 20 C for octadecyltrichlorosilane (OTS), whereas a homogeneous deposition mechanism takes place above the critical temperature.6,15,19,20 A mixed regime may occur at intermediate temperatures, and it has been postulated that the molecules can migrate at the surface. Thus, infrared studies of the adsorption of octadecyltrichlorosilane or octadecyltrimethoxysilane on Si/SiO2 surfaces, performed both in the solid/gas interface and the solid/liquid interface, have shown that a layer of adsorbed water on the surface is necessary to hydrolyze the trichloro- or alkoxy silane head groups and form trisilanols.28-30 This thin film of water allows lateral mobility of the long alkyl chain silanes to form a homogeneous and densely packed monolayer with parallel chains covalently bonded to the surface. In addition, intermolecular cross-linking can occur between the trisilanol head groups. As a result, a robust and dense network of polysiloxane is formed on the substrate. In spite of the high flexibility of the Si-O-Si angle, it has been pointed out that extensive cross-polymerization of the polysiloxane network should be avoided to form dense, fully covered monolayers because the length of the Si-O-Si cross-link must be less than twice the Si-O bond length or 3.2 A˚ in the final molecular edifice.5,24,31 One-pot grafting of well-ordered functional SAMs is not trivial since covalent attachment via siloxane linkages may have the restriction that any functional group present on the alkyl tether must be sufficiently small.32 Moreover, the large hydrodynamic volume of a functional organosilane may prevent migration of surface-bound molecules, which self-assemble. In other words, lateral packing of the head group limits the possible monolayer structures at full coverage because the intermolecular interactions between certain functional tail groups during monolayer formation or between these functional groups and the surface may be stronger than the alkyl chain interactions, which normally drive self-assembly. At all coverages, steric constraints may limit cross-linking to small clusters, resulting in some disorder in the monolayer structure.33 To overcome these difficulties, a few attempts involving the use of very long chain molecules were developed successfully, for example, with 23-trichlorosilanyl-tricosanoic acid methyl ester and N-(21-trichlorosilanylhenicosyl)-phthalimide.15,25,34 A quite different approach, involving strong intermolecular π-π interaction, has also been described.35 Alternatively, chemical transformations at the solid-liquid interface of 2D model surfaces formed by self-assembled monolayers have been reported.36-38 However, these postgrafting methods are not exempt from severe limitations due to steric congestion inherent to SAM interface-crowding effects that are not generally encountered in the analogous bulk solution reactions. In the case of bulky functional tail groups, this difficulty may lead to poor yields of product. In this context, the functionalization of surfaces exhibiting a high density of epoxide groups is a very important goal. (28) Silberzan, P.; Leger, L.; Ausserre, D.; Benattar, J. J. Langmuir 1991, 7, 1647. (29) Angst, D. L.; Simmons, G. W. Langmuir 1991, 7, 2236. (30) Tripp, C. P.; Hair, M. L. Langmuir 1992, 8, 1961. (31) Blaudez, D.; Bonnier, M.; Desbat, B.; Rondelez, F. Langmuir 2002, 18, 9158. (32) Heid, S.; Effenberger, F. Langmuir 1996, 12, 2118. :: (33) Benters, R.; Niemeyer, C. M.; Wohrle, D. ChemBioChem. 2001, 2, 686. (34) Tillman, N.; Ulman, A.; Penner, T. L. Langmuir 1989, 5, 101. (35) Nam, H.; Granier, M.; Boury, B.; Park, S. Y. Langmuir 2006, 22, 7132. (36) Fryxell, G. E.; Rieke, P. C.; Wood, L. L.; Engelhard, M. H.; Williford, R. E.; Graff, G. L.; Campbell, A. A.; Wiacek, R. J.; Lee, L.; Halverson, A. Langmuir 1996, 12, 5064. :: (37) Sullivan, T. P.; Huck, W. T. S. Eur. J. Org. Chem. 2003, 1, 17. (38) Monsathaporn, S.; Effenberger, F. Langmuir 2004, 20, 10375.

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For example, they can find many uses as templates for chemical tethering of polymer layers or platforms for robust biofunctional surfaces.39-43 Their access via organosulfur monolayers adsorbed on noble metals, which are widely used for biotechnological applications, would probably be quite difficult because epoxide groups easily react with nucleophiles. Previously reported studies on the chemical modification of Si/SiO2 surfaces by epoxy groups almost exclusively repose on the use of (3-glycidoxypropyl) trimethoxysilane (GPTS). Thus, continuous films of monolayer thickness with intergrown nodules have been described.44-46 In addition to inhomogeneous surface silylation and a low surface coverage, difficulties in the case of nonatomically flat substrates, the short tethers can adopt various orientations. Therefore, some of the epoxide groups may not be accessible to the species to immobilize. 11-Glycidoxy-1-undecyltrimethoxysilane was used in the chemical modification of silica gels, and a molecular monolayer containing a C11 hydrocarbon chain with polyethylene glycol units and glycidyl end-groups revealed efficiency for single-step immobilization of DNA on Si-H substrates.47,48 From a theoretical point of view, molecular dynamics simulations on the formation of SAMs on iron and alumina with various alkoxysilane models possessing glycidyl end-groups, comprising the C16 and C20 long chains molecules, have led the authors to conclude that the interactions between organosilane molecules increased with the chain length and always favor dense, ordered films.49,50 Moreover, the molecules adopt extended conformations with the molecular orientation placing the epoxide group toward the vacuum interface. Since this kind of derivative has never been synthesized, it was challenging to develop a route to very long chain organosilicon coupling agents containing terminal glycidyl groups and to investigate their direct grafting on hydroxylated surfaces. Thus, the ability of trialkoxy-(22-oxiranylmethoxy-docosyl)-silanes to give well ordered SAMs on glass surfaces was examined as well as their potential to attach biological molecules by covalent links.

Experimental Procedures Transmission Fourier-transform IR spectra of long-chain SAMs grafted on glass slides both faces were obtained on a Nicolet Nexus 670 spectrometer. The length of the grafted silylated coupling agent was estimated by molecular modeling (ChemOffice Ultra 2006, Cambridgesoft). Toluene for the synthesis of monolayers was dried and distilled under inert atmosphere immediately before use according to a literature procedure. Epifluorescence microscopy images were obtained with a Leica DM R video microscope at the Plate-Forme d’Imagerie Cellulaire, Institut des Neurosciences (PICIN, Universite Bordeaux). Optical ellipsometry measurements were performed with an I-elli2000 NFT ellipsometer (laser: λ = 532 nm, 50 mW). (39) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 1679. :: (40) Elender, G.; Kuhner, M.; Sackmann, E. Biosens. Bioelectron. 1996, 11, 565. (41) Mateo, C.; Fernandez-Lorente, G.; Abian, O.; Fernandez-Lafuente, R.; Guisan, J. M. Biomacromolecules 2000, 1, 739. (42) Penn, L. S.; Hunter, T. F.; Quirk, R. P.; Lee, Y. Macromolecules 2002, 35, 2859. (43) Cloarec, J.-P.; Chevolot, Y.; Laurenceau, E.; Phaner-Goutorbe, M.; Souteyrand, E. ITBM-RBM 2008, 29, 105. (44) Luzinov, I.; Julthongpiput, D.; Liebmann-Vinson, A.; Cregger, T.; Foster, M. D.; Tsukruk, V. V. Langmuir 2000, 16, 504. (45) Cloarec, J.-P.; Deligianis, N.; Martin, J.-R.; Lawrence, I.; Souteyrand, E.; Polychronakos, C.; Lawrence, M. F. Biosens. Bioelectron. 2002, 17, 405. (46) Wong, K. Y.; Krull, U. J. Anal. Bioanal. Chem. 2005, 383, 187. (47) Sudo, Y.; Akiba, M.; Sakaki, T.; Takahata, Y. J. Liquid Chromatogr. 1994, 17, 1743. :: (48) Bocking, T.; Kilian, K. A.; Gaus, K.; Gooding, J. J. Langmuir 2006, 22, 3494. (49) Hobbs, P. M.; Kinloch, A. J. J. Adhes. 1998, 66, 203. (50) Hayes, R. A.; Watson, G. W.; Willock, D. J. Mol. Simul. 2006, 32, 1095.

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In most experiments, the monolayers were grafted on glass :: microscope slides (slides for biology SuperFrost6 Menzel-Glazer & Co.) cut into pieces of 2.5  1.8 cm2 before use (rms e 1 nm). To overcome the perturbation of the ellipsometric measurement by the back-side reflection beam of the microscopic thin glass slide, 3 mm thick flat glass slides were used (float glass, Saint-Gobain). Biological Materials. A buffer solution (PBS, pH 7.2, Sigma-Aldrich), was used in all experiments. SBBB (StartingBlock Blocking Buffer) saturating agent was provided by Pierce. Fluorescent RAG-TRITC antibodies were purchased from Sigma-Aldrich. Activation of Glass Supports. The glass slides were washed by acetone and treated by ultrasound in chloroform (10 min at least). Then, they were exposed to UV-ozone (homemade apparatus, λ = 254 nm) for 30 min on each side and immediately introduced into the reactor flask. This treatment was checked by XPS as to whether it was very efficient to strongly lower the residual carbon percentage. However, traces of this element due to air contamination during handling of the samples were still present. Besides the peaks of the main constituting elements (O (1s) at 532.43 eV and Si (2p) at 103.35 eV), traces of the following elements were found: Na (1s) (1070.8 eV), Ca (2p) (347.84 eV), and C (1s) (285.25 eV). :: Contact angles were obtained on a Kruss (Drop shape analysis system DSA 10 Mk2) at 20 C in static mode. The results correspond to the mean of at least 3 measurements. Atomic force microscopy (AFM) images were obtained on a ThermoMicroscope Autoprobe CP Research (Park Scientific Instrument) instrument. Scanning was done at constant temperature (25 C) in air on 5  5 μm2 surface areas (tapping mode, tip curvature radius 6 nm). X-ray Photoelectron Spectroscopy (XPS). Analyses were conducted using a 220i-XL VG-ESCALAB system. The spectrophotometer was calibrated and operated in the 10-8 Pa range. The X-ray radiation was a nonmonochromatic Mg KR (100 W, 1253.6 eV) source, collection of photoelectrons being perpendicular to the samples surface. An electron-gun charge neutralization was used to compensate for the nonconductive samples. Surveys in the 0-1000 eV binding energies range were used to determine the chemical elements present at the surface (spectrum not shown). In addition to the very weak residual carbon signal, traces of Na and Ca were detected (very likely present in the glass slides). The high-resolution spectra were provided through a pass energy of 40 eV. High resolution spectra were fitted using the AVANTAGE software provided by ThermoFischer Scientific. Synthesis of Monolayers. The GPTS monolayer was synthesized according to the procedure previously reported in the literature.63 The long-chain glycidyl-ended SAM was grafted as follows: a solution of compound 3 (12.5 mg) freshly synthesized in dried toluene (100 mL) was first prepared to obtain the desired concentration (i.e., C = 2.5  10-4 M 3 L-1). The catalyst (TCA, 0.4 mg, 10 M %) was then added. This mixture was introduced into the silanization flask and stirred at 20 C under inert atmosphere over 20-24 h. The samples were then washed by toluene, ethanol, and sonicated in ethanol during 5 min, then dried under inert gas stream, and kept in the dark. Reaction with 2-Amino Ethanol. The reaction was performed in water under exactly the same conditions for both moloayers at pH 7. The slides were dipped into a solution of 2-aminoethanol (C = 100 mM 3 L-1) and kept at 25 C during 20 h. Then, they were successively rinsed with pure water and alcohol before drying under inert gas flow.

Immobilization of RAG-TRITC Antibodies onto Monolayers. The glass slides functionalized by GPTS and long-chain SAM monolayers were dipped into the solution of RAG-TRITC antibodies (20 μg/mL) during 120 min at 37 C, before being rinsed abundantly with a buffer solution (PBS). Beforehand, the negative control was made by saturating the free sites with the

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blocking agent (SBBB) to avoid the immobilization of antibodies on the surface and measure the fluorescence background. Epifluorescence microscopy experiments were conducted in all cases in less than 2 h after the antibody deposition.

Results and Discussion Synthesis of Triethoxy- and Trimethoxy-(22-oxiranylmethoxy-docosyl)-silanes 3 and 4. One of the most popular routes to access to long-chain functional compounds consists of the acylation of an enamine with an acyl chloride.51-53 This technique has previously been used to access to the 22-tricosen1-yl skeleton, but difficulties arise, such as some degree of migration of the terminal double bond during the reduction of the intermediate keto-ester.34 The cross-coupling of organic halides via organometallic derivatives in the presence of an organolithium cuprate has emerged as a very efficient technique (e.g., Corey-House synthesis).15,54 Joining together two C(sp3) centers often requires stoichiometric amounts of copper, but purification of the products may be difficult.55 In this work, the synthesis of the 21-docosen-1-yl skeleton was achieved by using a Grignard reagent and copper iodide.56 Thus, a method that proved to be more practical than the previously reported one using methylcopper in stoichiometric amount was developed (Figure 1). Experimental details and spectroscopic data are given in the Supporting Information. Synthesis and Characterization of the SAM Monolayer. To conveniently carry out IR and AFM studies, glass microscope slides were used in the present study. Both faces were silylated by immersing the slides in solutions of the coupling agents 3 and 4 in toluene under various experimental conditions. IR spectroscopy is a convenient method to monitor monolayer deposition and to probe van der Waals interactions between alkylene chains. Thus, specific information about the conformation of the alkylene chain can be obtained.57,58 In particular, the positions of the asymmetric and symmetric CH2 stretching modes provide qualitative measurements of conformational orders changes. Indeed, crystalline-like packing of the alkyl chains is expected to lead to νas(CH2) and νs(CH2) bands at approximately 2918 and 2850 cm-1, respectively. In contrast, the frequencies of methylene vibration bands increase with chain disorder. Preliminary attempts performed without any catalyst, as frequently reported in the literature with GPTS, showed that the grafting of these molecules was very slow. Thus, the CH2 IR bands in the 2800-3000 cm-1 region were very weak after 24 h of reaction time, more particularly with 3 (Figure 2). To optimize the synthesis of densely packed monolayers, the effect of various experimental parameters was examined (Figure 3). First, the addition of a weak acid to the solution, e.g., acetic acid (pKa = 4.76) led to a significant grafting enhancement in the case of the trimethoxysilyl derivative 4 (Figure 3a). Thus, the corresponding film displayed strong IR bands centered at 2925 and 2854 cm-1 after 24 h. In spite of this marked reactivity increase, these high frequency values led us to infer that a monolayer containing disordered hydrocarbon :: (51) Hunig, S.; Salzwedel, M. Chem. Ber. 1966, 99, 823. (52) Veale, G.; Girling, I. R.; Peterson, I. R. Thin Solid Films 1985, 127, 293. (53) Barraud, A.; Rosilio, C.; Ruaudel-Teixier, A. J. Colloid Interface Sci. 1977, 62, 509. (54) Bergbreiter, D. E.; Whitesides, G. M. J. Org. Chem. 1975, 40, 779. (55) Effenberger, F.; Heid, S. Synthesis 1995, 1126. (56) Derguini-Boumechal, F.; Linstrumelle, G. Tetrahedron Lett. 1976, 36, 3225. (57) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558. (58) Parikh, A. N.; Allara, D. L. J. Chem. Phys. 1992, 96, 927.

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Figure 1. Synthetic route to triethoxy- and trimethoxy-(22-oxiranylmethoxy-docosyl)-silanes 3 and 4. Reagents and conditions: (a) Mg

turnings (1.1 equiv.), THF, 55 C; (b) CuI (5 mol %), THF, -78-0 C; (c) CH3Li (1 equiv.), THF, -78 C; (d) -78 C, 3 h; (e) H3O+; (f) NaH, THF; (g) Cl-CH2-CH(O)CH2; (h) (RO)3SiH (excess), toluene, Karstedt cat., 60 C, 4 h (R = Et), 16 h (R = Me).

chains was mainly formed. However, only a very weak change was observed under the same conditions with 3. In this case, low intensity IR bands at 2926 and 2856 cm-1 clearly showed a largely incomplete, highly disordered monolayer (spectrum not shown). Interestingly, the use of a stronger acid (trichloracetic acid, TCA), (pKa = 0.66) at a concentration of 10 mol % led to dramatic changes in the IR bands, more particularly with compound 4 (Figure 3a). Thus, the νas(CH2) and νs(CH2) IR bands rapidly displayed very strong intensities and their frequencies shifted to low frequencies at 2920 cm-1 and 2851 cm-1, respectively. These values were close to the ones usually obtained with well-ordered monolayers, including ω-tricosenoic acid-derived monolayers,34,53 showing a very strong catalytic effect of TCA on the grafting process. However, in the case of 3, this effect was appreciably less, whereas only IR bands corresponding to poorly ordered monolayers were observed (spectrum not shown). Consequently, all subsequent studies were carried out on compound 4 containing a trimethoxysilyl group using TCA as the catalyst. Increasing the TCA concentration (up to 100 mol %) rapidly led to significant broadening of the IR bands as well as frequency shifts to values of about 2925 and 2855 cm-1 (IR spectrum not given). Thus, above a TCA concentration threshold of 10 M %, chain ordering decrease was observed, probably due to the formation of multilayers or aggregates. Conversely, it turned out that low concentrations of TCA (5 mol % or less) had only a poor catalytic effect. Therefore, the best results were obtained with 4, for a TCA concentration of 10 mol %. Then, experiments at different temperatures were performed (Figure 3b, reaction time: 24 h). Lowering the temperature down to 15 C led to a dramatic grafting rate decrease (spectrum not given). Thus, the corresponding IR spectrum exhibited very weak νas(CH2) and νs(CH2) IR bands. Conversely, at 20 C, these bands were significantly stronger and their frequencies corresponded to monolayers with a high degree of ordering (i.e., 2920 and 2851 cm-1). At 25 C, their intensities appreciably decreased, and their frequencies increased, leading us to assume that more disordered monolayers were formed. Therefore, the best temperature for the actual SAM synthesis was 20 C. These results as a whole revealed that the glycidyl-terminated C22 chains exhibited efficient self-assembly. Considering that the ideal grafting temperature and TCA concentration were close to 20 C and 10 M %, respectively, the effect of various coupling agent concentrations was examined (Figure 3c). For a concentration C = 1.0  10-4 M, only weak Langmuir 2009, 25(10), 5526–5535

Figure 2. Effect of the nature of hydrolyzable alkoxysilane head groups on νas(CH2) and νs(CH2) IR absorption bands (substrates, glass microscope slides; coupling agent concentration, 5  10-4 M; temperature, 20 C; reaction time, 24 h).

intensity IR bands were obtained after 24 h, with frequency values corresponding to disordered chains. For C = 2.5  10-4 M, a dramatic increase in the intensities was observed, whereas νas(CH2) and νs(CH2) frequencies decreased to 2920 (or less in a few experiments) and 2851-2850 cm-1, respectively, showing that monolayers with a high degree of ordering were formed, as in the case of the SAM with the same chain length and terminal CH3-O-CO- groups reported by Pomerantz et al. on glass.15 However, these values did not exactly match those generally reported for the OTS monolayer, typically 2918-2917 cm-1 for the νas(CH2) band, which has no functional end-group. They are :: consistent with the work of Woll et al. showing that the grafting of trimethoxysilyl head groups is slightly less efficient than that of trichlorosilyl groups.20 In contrast to the direct grafting of monolayers with terminal amino groups described by these authors, no interaction of the glycidyl terminal groups and the hydroxyl groups of the substrate, which might lead to significant disorder in the deposited films, took place. For C = 5.0  10-4 M, a slight additional intensity increase was observed after 24 h, but no concomitant down shift frequency, leading us to assume that optimal chain ordering was reached at a concentration of 2.5  10-4 M. To check the consistency of these results, SAMs prepared in the same runs on 3 mm thick glass slides instead of microscope slides were studied by optical ellipsometry (Figure 4). DOI: 10.1021/la804088d

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Figure 3. Effect of various experimental conditions on νas(CH2) and νs(CH2) IR absorption bands for compound 4 (substrates, glass microscope slides): (a) nature of the catalyst; (b) temperature; (c) coupling agent concentration; (d) duration.

The thicknesses of monolayers were calculated by using a refractive index value of 1.47 (values in the range of 1.45 to 1.50 have previously been reported for SAMs possessing close structures).3,34,59,60 For C = 1.0  10-4 M, an average thickness value of only 1.7 nm was found, very far from the calculated one for the corresponding coupling agent molecule in its extended conformation (Figure 4d). This result led us to infer that at this coupling agent concentration the monolayer was incomplete. It is corroborated by the corresponding ellipsometry mapping image, which displayed a heterogeneous, grainy surface (Figure 4a). For C = 2.5  10-4 M, the mean thickness value reached 3.2 nm, very close to the calculated one (3.3 nm), while the image showed a surface with a very good homogeneity (Figure 4b). For C = 5.0  10-4 M, the calculated mean thickness strongly increased to 4.3 nm, a value much higher than the expected one, whereas aggregates or multilayers begun to appear (Figure 4c). These images were consistent with the corresponding IR spectra (Figure 3c). They led to the conclusion that the best coupling agent concentration was close to 2.5  10-4 M. The effect of reaction time was investigated by IR spectroscopy for TCA and coupling agent concentrations of 10 M % and 2.5  10-4 M, respectively, at the temperature of 20 C (59) Wassermann, S. R.; Whitesides, G. M.; Tidswell, I. M.; Ocko, B. M.; Pershan, P. S.; Axe, D. J. J. Am. Chem. Soc. 1989, 11, 5852. (60) Tillman, N.; Ulman, A.; Schildkraut, J. S.; Penner, T. L. Langmuir 1988, 110, 6136.

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Figure 4. Ellipsometry mapping images of long chain SAM surfaces (450 μm  600 μm; glass slide thickness, 3 mm) obtained with 4 at different concentrations: (a) C = 1.0  10-4 M; (b) C = 2.5  10-4 M; (c) C = 5.0  10-4 M; (d) molecule in its extended conformation (temperature, 20 C; reaction time, 24 h).

(Figure 3d). For reaction times increasing up to 20-24 h, the stretching frequencies progressively decreased and reached values that could be expected for well-ordered monolayers (2920 and 2851 cm-1 ,respectively) while the band intensities increased. Unlike the intensities, which continued to increase, along with band broadening, no further frequency decrease was observed for longer reaction times, e.g., 43 h. Langmuir 2009, 25(10), 5526–5535

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In parallel, ellipsometry mapping images of samples corresponding to different reaction times were obtained. The surfaces corresponding to times in the range of 12-24 h looked homogeneous (Figure 5). However, the experimental thickness values obtained for the 12 and 16 h samples only correspond to incomplete monolayers (0.6 and 1.0 nm, respectively). The monolayer thickness markedly increased for longer reaction times, e.g., 3.0 nm after 20 h and 3.2 nm after 24 h, a value close to the expected one. After 43 h, a grainy aspect emerged, while the monolayer thicknesses markedly increased above the calculated value (4.0 nm instead of 3.3 nm for the calculated value). These results led us to infer that aggregates were formed extensively and corroborate that optimal SAM ordering could be obtained for reaction times in the range of 20-24 h at 20 C. The calculated length of the chain (including the head group silicon atom) was of 3.4 nm. Optical ellipsometry measurements using a refractive index value close to those previously reported in the literature (η = 1.47) lead to a slightly lower estimated length of the chain (3.2 nm including the silicon atom head group). This difference led us to assume that the monolayer was oriented with a lowered tilt angle of 15-20. AFM studies in the tapping mode were performed with the same samples (3 mm thick glass slides) (Figure 6). They were consistent with ellipsometry results and IR spectra on glass microscope slides. In spite of its relatively low mean square roughness values (rms e 1.0 nm), the starting glass slide exhibited numerous surface flaws (Figure 6a). At t = 16 h (Figure 6b), its surface became appreciably more homogeneous, and flaws disappeared. In parallel, rms values decreased to 0.45-0.5 nm. For longer reaction times (t = 20 and 24 h) (Figure 6c and d), the surface looked quite smooth, consistent with rms values in the same range in both cases. Few small grains could be seen on the surfaces. However, a more grainy aspect was observed after 43 h (Figure 6e), in agreement with ellipsometry results, along with an appreciable rms increase (0.7 nm). Because attempts to scratch out the SAM monolayer with the tip remained unsuccessful, it turned out to be impossible to confirm by AFM the thickness values found by ellipsometry. Thus, no exploitable hole allowing measurement of monolayer thickness could be observed, as inferred from the corresponding rms profile (Figure 6g). Nevertheless, the lower and upper edges of the corresponding 1  1 μm2 pressed flat zone showed a few white grains that have been dragged along by the AFM tip, probably corresponding to the aggregates that begin to form for times longer than 20 h. It is noteworthy that the mean air-layer interface rms value, which was approximately 1.0 nm for the bare substrate, significantly decreased to values in the range of 0.45 to 0.50 nm for an optimized SAM, showing appreciable surface smoothing. This phenomenon could be explained upon considering that only the surface Si-OH functions located on the peaks of the glass slides condensed with the hydrolyzed alkoxysilane groups of the incoming molecules at the liquid-solid interface, whereas those located in the valleys were poorly or not at all accessible. In the case of the long-chain SAM, it is likely that part of the alkoxysilane groups did not bind to the surface, accounting for the fact that the self-assembly driving force should be mainly found in the strong van der Waals interactions between the very long alkyl chains methylene groups, yielding densely packed molecules and surface flattening. Accordingly, 2D cross-linking of hydrolyzable alkoxysilane head groups, leading to a polysiloxane network at the glass surface is not necessarily the Langmuir 2009, 25(10), 5526–5535

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Figure 5. Ellipsometry mapping images of long chain SAM surfaces (450 μm  600 μm; glass slide thickness, 3 mm) for different grafting times (temperature, 20 C; concentration of 4, 2.5  10-4 M).

predominant pathway, whereas Si-OH functions may remain on the coupling agent head group near the silicon oxide surface. This inference is consistent with previous reports indicating that all molecules are not individually linked to the surface (Figure 7).28,61,62 The obtained long-chain SAM was characterized by wettability measurements, reproducibly exhibiting contact angles of 72 ( 2 with water. These values were not far from those of SAMs containing methyl ester end-groups, e.g., tricosanoic methylester SAM (69 ( 2) but remained far from those corresponding to highly hydrophobic methyl-terminated monolayers, above 110.15,34 They were in contrast to the relatively high hydrophilic character of the GPTS monolayer, which reproducibly gave low values in our hands (49 ( 2) on glass slides supports, in good agreement with the ones previously reported in the literature (55 ( 10).63 These results showed that the glycidyl-terminated long chain SAM possessed a relatively high hydrophobic character, whereas the GPTS monolayer exhibited a poor barrier effect to water. They were consistent with the high amphiphilic properties of the molecules after hydrolysis of the alkoxysilane head groups during the grafting step, as demonstrated by their good self-assembly on the hydroxylated surface. As they exhibit weak absorption bands, the surface oxirane groups cannot be conveniently characterized by IR spectroscopy under the experimental conditions used in this work. Hence, it appeared useful to ascertain the presence of reactive surface epoxide groups via a chemical modification route. The reactivity of the surface-bound epoxy groups with nucleophiles was investigated by using 2-aminoethanol and followed by XPS as well as wettability measurements. After treatment with 2-aminoethanol in toluene at room temperature (20 h), the contact angles of the SAM monolayer with water drops decreased from 72 ( 2 to 65 ( 2. These values showed that the surface became more hydrophilic due to the presence of hydroxyl groups. Conversely, in the case of the GPTS monolayer, which already possessed a marked hydrophilic character, only a small change (61) Allara, D. L.; Parikh, A. N.; Rondelez, F. Langmuir 1995, 11, 2357. (62) Tripp, C. P.; Hair, M. L. Langmuir 1995, 11, 1215.

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Figure 6. AFM images (topography) of the long chain glycidyl-terminated SAM for different grafting times (glass slide thickness, 3 mm): (a) t = 0; (b) t = 16 h; (c) t = 20 h; (d) t = 24 h; (e) t = 43 h; (f) attempts at scratching the monolayer with the AFM tip and corresponding roughness profile [t = 24 h, contact mode, erosion zone (x axis scans), 1  1 μm2].

Figure 7. Schematic view of a smooth SAM resulting from the grafting of trimethoxy-(22-oxiranylmethoxy-docosyl)-silane 4 on rough glass slides.

was found under the same treatment (i.e. 47 ( 2 instead of 49 ( 2). The surfaces were also examined by XPS (Figure 8). As previously described by Tsukruk et al.,44 the C (1s) spectrum of the starting GPTS monolayer exhibited two signals around 285.0 and 286.5 eV, corresponding to C-C and C-O carbons, respectively (Figure 8a). It is worth noting that these authors have reported that the C-C and C-O signals gave approximately the same intensities for a complete GPTS monolayer adsorbed on highly polished single-crystal silicon wafers, ascribing this feature as an indication of the presence of close epoxy 5532

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cycles on the film surface. A slight difference was observed in the present XPS spectrum as the C-O signal at 286.5 eV was slightly smaller than the C-C one. This might be due to the fact that GPTS cannot lead to high quality monolayers when relatively rough glass slides are used instead of atomically flat supports. Signals with the same binding energies were observed for the C (1s) spectrum of the long chain SAM (Figure 8b). As expected, a very strong enhancement of C-C carbons signals and concomitant decrease of C-O signals were observed, in good agreement with the SAM structure containing long methylene chains.48 Langmuir 2009, 25(10), 5526–5535

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Figure 8. (a) C (1s) XPS spectra of the GPTS monolayer ; (b) C (1s) XPS spectra of the long chain SAM; (c) N (1s) XPS spectra of the GPTS monolayer after a reaction with 2-aminoethanol; (d) N (1s) XPS spectra of the long-chain SAM monolayer after a reaction with 2aminoethanol.

Since XPS inevitably detects in these experiments the upper atoms of the support, i.e., oxygen and silicon with and unknown penetration depth, no attempt was made to precisely determine the Si/O/C ratios. Then, monolayers treated with 2-aminoethanol were examined by XPS. Survey scans showed that no nitrogen trace was present in the initial glass slide as well as in the monolayers themselves. In the case of the GPTS monolayer, a new broad signal (binding energy around 400 eV) was attributed to N (1s) (Figure 8c). Curve fitting revealed the presence of two main components at 399.8 and 401.9 eV leading us to infer that nitrogen possessed at least two different environments. These binding energy values might be assigned to secondary and tertiary amine groups (around 399.8 eV), which are known to be very close, and quaternary ammoniums (401.9 eV).64 In addition, the found N/C atomic ratio (0.08) was appreciably lower than the calculated one (0.125). In the case of the longchain SAM, the N (1s) spectrum was noisier due to a weaker signal/noise ratio resulting from its low atomic percentage. However, the experimental atomic value was not very far from (63) Tsukruk, V. V.; Luzinov, I.; Julthongpiput, D. Langmuir 1999, 15, 3029. (64) Jansen, R. J. J.; van Bekkum, H. Carbon 1995, 33, 1021.

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the calculated one (calculated N/C ratio, 0.037; found, 0.03), consistent with a strong nitrogen content decrease compared to that of the former monolayer. Again, a fit curve of the nitrogen signal led to the assumption of the presence of at least two different chemical environments for this element (Figure 8d). To account for the experimental nitrogen ratios, which were lower than the calculated ones, it should be considered that a single 2-aminoethanol molecule causes the formation of various nucleophiles, which may attack glycidyl groups (Figure 9). Thus, besides the formation of the expected terminal amino alcohol functions (path b), one could assume that the intermediate anionic oxigen species (path a) might further react with a neighboring epoxide group (path c), as depicted for the deactivation of epoxide-derivatized surfaces with tertiary amines.42 The electronic doublet of a secondary amine group might also be involved (paths d and e). Epifluorescence microscopy. Additional evidence of the presence of reactive, surface epoxide groups was provided via the use of a fluorescent probe. Thus, dansylcadaverine (λex = 335 nm, λem = 508 nm) was grafted onto the surface via nucleophilic attack of the epoxide groups by the amine function and ringopening condensation (Figure 10). The same treatment was carried out with a GPTS monolayer. DOI: 10.1021/la804088d

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Figure 9. Proposed reaction scheme for the attack of glycidyl-terminated surfaces by 2-aminoethanol.

Figure 10. Fluorescence signal values obtained for GPTS monolayers and glycidyl-terminated long-chain SAMs after reaction with dansylcadaverin. (a) Dansylcadaverin (fluorescent probe); (b) epifluorescence microscopy image (long-chain SAM labeled by the fluorescent probe); (c) diagram of mean fluorescence intensities (each point corresponds to the mean value of three replicates; the errors bars represent ( SD); (d) epifluorescence mapping for monolayers grafted with dansylcadaverin.

The fluorescence image for the glycidyl-terminated longchain SAM after reaction with dansylcadaverin is shown in Figure 10b. Many luminous dots are homogenously distributed on the whole surface, and scattered bright spots are also observed. For both surfaces, epifluorescence microscopy mapping images displayed homogeneous fluorescence intensities (Figure 10d), showing that the fluorophore was homogeneously distributed on the whole surface. As expected, negative controls consisting of the direct treatment of the silica surface by the fluorescent probe only led to a much weaker fluorescence signal that could be assimilated to a background, corroborating that the fluorescent probe did not graft onto the silica surface. The signal intensity was significantly higher for the long-chain SAM 5534

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labeled with the fluorophore, leading us to assume that this last monolayer exhibited a higher density of accessible surface epoxide groups. The potential of this new SAM in the trapping of biological molecules was explored by using the same microscopy technique. Thus, both monolayers were directly treated by fluorescent RAG-TRITC antibodies under exactly the same conditions (Figure 11). Negative controls consisting of the treatment of the monolayers by a blocking agent (SBBB) prior to the addition of the fluorescent antibodies were carried out. Thus, direct treatment of the glycidyl-terminated long chain SAM by RAG-TRITC antibodies resulted in a strong fluorescence signal (Figure 11d), i.e., at least 6-fold higher than the Langmuir 2009, 25(10), 5526–5535

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(e.g., lysine residues) because of their labeling with the chromophores. In addition, the GPTS monolayer is known not to be optimal, whereas the epoxide groups of the long-chain SAM are more accessible due to better ordering of the molecules. Thus, the long-chain glycidyl-ended SAM was much more efficient in binding these fluorescent antibodies than the GPTS monolayer, looking very promising for the direct immobilization of antibodies without recourse to an organic cross-linker to increase the surface reactivity.67

Conclusions Figure 11. Mean fluorescence intensity diagram of monolayers with attached RAG-TRITC antibodies (each point corresponds to the mean value of three replicates; the errors bars represent ( SD). (a) GPTS monolayer/SBBB/RAG-TRITC (negative control); (b) GPTS monolayer/RAG-TRITC; (c) long-chain SAM/SBBB/ RAG-TRITC (negative control); (d) long-chain SAM/RAGTRITC.

corresponding negative control (background, Figure 11c). Conversely, the GPTS monolayer only gave a very weak signal (Figure 11b), hardly higher than the corresponding negative control (Figure 11a). The very poor fluorescence response in this last case showed that for the same substrate, namely, glass microscope slides, there is a marked behavior difference with respect to RAG-TRITC antibodies attachment. In these experiments, one must keep in mind that RAG-TRITC antibodies were directly attached onto the monolayers surfaces through covalent links. It is known that bioadhesion strongly depends, among several parameters, on the kind of protein and the surface energy of materials.65,66 Thus, it might be inferred from the wettability values that the antibody used in this study might have a higher affinity to the long-chain SAM than the GPTS monolayer (contact angles with water drop, 72 vs 49). Also, they should possess a lower number of hydrophilic groups (65) Sigal, G. B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 3464. (66) Agashe, M.; Raut, V.; Stuart, S. J.; Latour, R. A. Langmuir 2005, 21, 1103.

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A route to a novel organosilicon coupling agent containing a very long hydrocarbon chain (C22) and a glycidyl end-group is described. This derivative exhibits a good capacity to directly yield homogeneous SAMs possessing a high degree of ordering by using trichloracetic acid (10 mol %) as a catalyst. The reactivity of these SAMs was investigated through reaction with nucleophiles, i.e., grafting of a fluorescent probe followed by epifluorescence microcopy or addition of 2-amino ethanol followed by XPS analysis, giving evidence that the glycidyl groups were not affected by the presence of the weak carboxylic acid during synthesis. This approach leads to a new platform with considerable potential for the development of smooth silica-based surfaces in view of the covalent grafting of biological molecules. Acknowledgment. This work was supported by CRA (Conseil Regional d’Aquitaine) and ANR (Agence Nationale de la Recherche). We thank C. Poujol and P. Legros (PICIN imaging Center, Neurosciences Institute of the University of Bordeaux) for help with epifluorescence microscopy. Supporting Information Available: Synthetic methods for the preparation of organosilicon coupling agents, 1H NMR spectra, and XPS survey spectrum. This material is available free of charge via the Internet at http://pubs.acs.org. (67) Kusnezow, W.; Jacob, A.; Walijew, A.; Diehl, F.; Hoheisel, J. D. Proteomics 2003, 3, 254.

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