pubs.acs.org/Langmuir © 2010 American Chemical Society
Fabrication of Honeycomb-Structured Porous Surfaces Decorated with Glycopolymers Alexandra Mu~noz-Bonilla,† Emmanuel Ibarboure,† Vanesa Bordege,‡ Marta Fernandez-Garcı´ a,‡ and Juan Rodrı´ guez-Hernandez*,†,‡ †
Laboratoire de Chimie des Polym eres Organiques (LCPO), CNRS, Universit e Bordeaux I, ENSCPB. 16, Avenue Pey Berland 33607, Pessac-Cedex, France, and ‡Instituto de Ciencia y Tecnologı´a de Polı´meros (ICTP-CSIC) C/Juan de la Cierva no. 3, 28006 Madrid, Spain Received December 3, 2009. Revised Manuscript Received January 29, 2010
We prepared breath figure patterns on functional surfaces by the surface segregation of a statistical glycopolymer, (styrene-co-2-(D-glucopyranosyl) aminocarbonyloxy ethyl acrylate (S-HEAGl). The synthesis of the statistical glycopolymer is prepared in a straightforward approach by conventional free radical copolymerization of styrene and the unprotected glycomonomer. Blends of this copolymer and high-molecular-weight polystyrene were spin coated from THF solutions, leading to the formation of surfaces with both controlled functionality and topography. AFM studies revealed that both the composition of the blend and the relative humidity play key roles in the size and distribution of the pores at the interface. Thus, the topographical features obtained on the polymer surfaces during film preparation by the breath figure methodology varied from 200 to 700 nm. Moreover, this approach leads to porous films in which the hydrophilic glycomonomer units are oriented toward the pore interface because upon soft annealing in water the holes are partially swelled. The self-organization of the glycopolymer within the pores was additionally confirmed by the reaction of carbohydrate hydroxyl groups with rhodamine isocyanate. Equally, we demonstrate the bioactivity of the anchored glycopolymers by means of the lectin binding test using concanavalin A (Con A).
Introduction The design and control of structural and chemical-physical properties of polymer surfaces are crucial for practical use because a large number of properties in polymeric materials, including adhesion, wettability, permeability, and biocompatibility, are controlled by the interface.1 Moreover, the development of methods to control surface structures in the submicrometer range is of interest for microelectronics,2 membrane technologies,3 microfluidic devices,4 and microbiology.5 Other micrometrically scaled structures are promising for applications including photonic crystals and biomedical and superhydrophobic interfaces.6 The surface engineering of a biomaterial surface has to take into account several aspects such as control of the chemical composition, surface morphology (roughness and topography), and coexistence and distribution of micro/macrodomains.7 A large variety of examples have evidenced the key role of these parameters in the surface properties, including adhesion/friction, wettability, and surface conductivity. For instance, the wettability *Corresponding author. Fax: (34) 91 564 48 53. Tel: (34) 91 258 75 05. E-mail:
[email protected]. (1) (a) Tsukruk, V. V. Prog. Polym. Sci. 1997, 22, 247–311. (b) Lafuma, A.; Quere, D. Nat. Mater. 2003, 2, 457–460. (c) Graighead, H. G. Science 2000, 290, 1532–1535. (2) Moreau, W. M. Semiconductor Lithography: Principles and Materials; Plenum: New York, 1988. (3) Kesting, R. E. Synthetic Polymer Membranes; Wiley: New York, 1985. (4) (a) Quake, S. R.; Scherer, A. Science 2000, 290, 1536–1540. (b) Thorsten, T.; Roberts, R. W.; Arnold, F. H.; Quake, S. R. Phys. Rev. Lett. 2001, 86, 4163–4166. (5) Singhi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696–698. (6) (a) Bormashenko, E.; Pogreb, R.; Stanevsky, O.; Bormashenko, Ye.; Socol, Y.; Gendelman, O. Polym. Adv. Technol. 2005, 16, 299–304. (b) Nishikawa, T.; Nishida, J.; Ookura, R.; Nishimura, Sh.-I.; Wada, Sh.; Karino, T.; Shimomura, M. Mater. Sci. Eng., C 1999, 10, 141–146. (7) (a) Bhushan, B.; Koch, K.; Jung, Y. C. Soft Matter 2008, 4, 1799–1804. (b) Tsai, I. Y.; Crosby, A. J.; Russell, T. P. Methods Cell Biol. 2007, 83, 67–87. (c) Chan, E. P.; Smith, E. J.; Hayward, R. C.; Crosby, A. J. Adv. Mater. 2008, 20, 711–716. (d) Nosonovsky, M.; Bhushan, B. Mater. Sci. Eng. R 2007, 58, 162–193.
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between hydrophilic and superhydrophobic behavior can be varied by choosing the appropriate chemical function at the interface.8 Equally, patterned surfaces serve as templates to create a pattern of proteins that can, in turn, be employed to study the interactions between proteins or antibodies with other biomolecules.9 The design of polymer surfaces with either suitable chemical composition or surface topography has been extensively studied.10 As a result, a variety of methodologies are available to modify the surface chemical composition: chemical and physical treatments such as plasma,11 surface grafting,12 metal coating,13 and surface segregation.14 Likewise, surface topography has been controlled by using lithographic techniques,15 imprinting methods,16 self-assembly to reach micro and nanopatterned surfaces,17 and “breath figure” formation.18 However, examples of polymer surfaces in which both parameters (i.e., surface chemistry and topography) are simultaneously controlled are somewhat scarce. Attempts have been made to manufacture ordered surfaces (8) (a) Holmes-Farley, S. R.; Reamey, R. H.; McCarthy, T. J.; Deutch, J.; Whitesides, G. M. Langmuir 1985, 1, 725–740. (b) Yarbrough, J. C.; Rolland, J. P.; DeSimone, J. M.; Callow, M. E.; Finlay, J. A.; Callow, J. A. Macromolecules 2006, 39, 2521–2528. (9) Zhang, Y.; Wang, C. Adv. Mater. 2007, 19, 913–916. (10) Mittal, K. L. Polymer Surface Modification: Relevance to Adhesion; VSP BV: The Netherlands, 1996. (11) Denes, F. S.; Manolache, S. Prog. Polym. Sci. 2004, 29, 815–885. (12) Jordan, R. Adv. Polym. Sci. 2006, 197/198 . (13) Bowden, N.; Brittain, S.; Evans, A. G.; Hutchinson, J. W.; Whitesides, G. M. Nature 1998, 393, 146–149. (14) (a) Koberstein, J. T. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 2942– 2956. (b) Senshu, K.; Yamashita, S.; Mori, H.; Ito, M.; Hirao, A.; Nakahama, S. Langmuir 1999, 15, 1754–1762. (15) Xia, Y.; Rogers, J. A.; Paul, K. E.; Whitesides, G. M. Chem. Rev. 1999, 99, 1823–1848. (16) Dusseiller, M. R.; Schlaepfer, D.; Koch, M.; Kroschewski, R.; Textor, M. Biomaterials 2005, 26, 5917–5925. (17) Matsen, M. W. Curr. Opin. Colloid Interface Sci. 1998, 3, 40–47. (18) Stenzel, M. H. Aust. J. Chem. 2002, 55, 239–243.
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industrially by introducing, for instance, hydrophobicity and/or roughness. The preparation of structured functional surfaces15,19 requires the combination of different fabrication processes. Bottomup techniques have been employed to obtain nanoscale order at surfaces (e.g., by the self-assembly of block copolymers).20 However, even with the development of recent approaches such as controlled solvent annealing or the use of crystallizable blocks, this approach provides limited in-plane ordering.21 On the contrary, microstructuration has been typically created by using topdown techniques such as lithography, writing, and printing.22 In this case, the diffraction depth focus and/or electrostatic interactions are important drawbacks resulting in expensive facilities and slow pattern writing.23 An alternative approach to overcoming the limitations of both approaches is to combine their strengths using a top-down fabrication mechanism to produce controlled surface features on the micrometer length scale and a bottom-up approach based on self-assembly to create desired structures at the nanometer level. For instance, bottom-up and top-down strategies have been combined in the so-called templated-selfassembly (TSA) approach.24 Hence, topographically patterned surfaces25 (lithographically defined grooved substrates, polydimethylsiloxane (PDMS) stamps, and hard imprint molds) and/or chemically patterned substrates26 are typical templates used to order block copolymer films on larger length scales. Also, hydrophobic surfaces in which the roughness has been artificially modified have been fabricated with hierarchical structures such as electrodeposition, nanowire arrays, colloidal systems, and photolithography.27 Breath figures formed by water condensation has been employed as physical micropatterning (top-down approach) to vary the topography of the polymer surface by creating holes with controlled dimensions. Because of the interference between the hydrophilic segments of the copolymer and the condensed water droplets, the pore surface will be mainly decorated with glycomonomer units. The surfaces prepared could be of potential interest as 3D cell culture platforms that have been otherwise obtained by using tedious multistep procedures using lithographic techniques.28 It is important to notice that glycopolymers (i.e., synthetic polymers containing carbohydrate pendant groups), because their “glyco-cluster effect,”29,30 could be interesting candidates for modifying polymer surfaces, creating bioactive (19) Xia, Y.; Rogers, J. A.; Paul, K. E.; Whitesides, G. M. Chem. Rev. 1999, 99, 1823–1848. (20) Darling, S. B. Prog. Polym. Sci. 2007, 32, 1152–1204. (21) Cheng, J. Y.; Ross, C. A.; Smith, H. I.; Thomas, E. L. Adv. Mater. 2006, 18, 2505–2521. (22) Nanotechnologie: Eine Einf€ uhrung in Die Nanostrukturtechnik, K€ohler, M., Ed.; , Wiley-VCH: New York, 2001. (23) Nie, Z.; Kumacheva, E. Nat. Mater. 2008, 7, 277–290. (24) (a) Gorzolnik, B.; Mela, P.; Moeller, M. Nanotechnology 2006, 17, 5027– 5032. (b) Seagalman, R. A.; Yokoyama, H.; Kramer, E. J. Adv. Mater. 2001, 13, 1152– 1155. (c) Cheng, J. Y.; Ross, C. A.; Thomas, E. L.; Smith, H. I.; Vancso, G. J. Adv. Mater. 2003, 15, 1599–1602. (d) Sundrani, D.; Darling, S. B.; Sibener, S. J. Nano Lett. 2004, 4, 273–276. (e) Sundrani, D.; Darling, S. B.; Sibener, S. J. Langmuir 2004, 20, 5091–5099. (25) (a) McCord M. A.; Rooks M. J. In Handbook of Microlithography, Micromachining, and Microfabrication; Rai-Choudhury, P., Ed.; SPIE Optical Engineering Press: Bellingham, WA, 1997; Vol. 1 (b) Xia, Y. N.; Whitesides, G. M. Annu. Rev. Mater. Sci. 1998, 28, 153–184. (c) Chou, S. Y.; Krauss, P. R.; Renstrom, P. J. J. Vac. Sci. Technol., B 1996, 14, 4129–4133. (d) Resnick, D.; Sreenivasan, S. V.; Wilson, C. G. Mater. Today 2005, 8, 34–42. (26) Fasolka, M. J.; Mayes, A. M. Annu. Rev. Mater. Res. 2001, 31, 323–355. (27) (a) Chong, M. A. S.; Zheng, B.; Gao, H.; Tan, L. K. Appl. Phys. Lett. 2006, 89, 233104. (b) Shirtcliffe, N. J.; McHale, G.; Newton, M. I.; Chabrol, G.; Perry, C. C. Adv. Mater. 2004, 16, 1929–1932. (28) (a) Dusseiller, M. R.; Smith, M. L.; Vogel, V.; Textor, M. Biointerphases 2006, 1, P1–P4. (b) Dusseiller, M. R.; Schlaepfer, D.; Koch, M.; Kroschewski, R.; Textor, M. Biomaterials 2005, 26, 5917–5925. (29) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321–327. (30) Miura, Y.; Ikeda, T.; Wada, N.; Sato, H.; Kobayashi, K. Green Chem. 2003, 5, 610–614.
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materials. As is well known, carbohydrates are involved in various biological functions in living systems and participate in a variety of mutual recognition processes including immunological protection, virus infection, and recognition in the nervous system, among others.31 These recognition processes are thought to proceed by specific carbohydrate-protein interaction. Hence, progress in glycoscience and glycotechnology32 has emerged as important materials in the basic exploration of proteinsaccharide interactions29,33 and cell adhesion. In this sense, the modification of polymer surfaces with glycopolymers having both controlled topography and chemistry may allow us to obtain further information on the cell-material interaction processes. Herein, we propose a straightforward methodology to obtain regularly 3D patterned polymer surfaces from blends of polystyrene and a statistical glycopolymer (styrene-co-2-(D-glucopyranosyl)aminocarbonyloxyethyl acrylate, S-HEAGl) using the breath figure methodology as a dynamic templating method. Whereas several examples have been reported in which the pores formed by using this methodology have been decorated with different functional groups34 or nanoparticles,35 the preparation of breath figures with a glycopolymer is rather unusual. Nishikawa et al.36 in 1999 described the preparation of polyion complexes at the surface with anionic polysaccharides. Very recently, Ting et al.37 reported the patterning of proteins onto galactosylated porous films. This article presents regular film formation by using six-arm star polystyrene that by itself presents honeycomb structure with an ordered hexagonal array. Moreover, the preparation of the copolymer was carried out in multiple polymerization and protection/deprotection steps. In the present article, the methodology employed leads to glycopolymers in a single step without additional protective chemistry. In addition, the parameters (humidity, temperature, polymer concentration, etc.) that significantly modify the surface topography in the breath figure mechanism have been widely investigated. Finally, we demonstrate the capability of the surfaces to interact selectively with both small molecules and proteins within the cavities where the swelling behavior of the glycopolymers contained within the holes appears to be crucial for the aggregation of the protein-carbohydrate.
Experimental Section Materials. Styrene (St) (Aldrich, 99%) was distilled under reduced pressure. 2,20 -Azoisobutyronitrile (AIBN) (Aldrich, 98%) was purified by successive crystallizations from methanol. The synthesis of 2-{[(D-glucosamin-2-N-yl) carbonyl]oxy}ethyl acrylate (HEAGl) will be described elsewhere.38 All of the solvents (31) (a) Tsuchida, A.; Kobayashi, K.; Matsubara, N.; Muramatsu, T.; Suzuki, T.; Suzuki, Y. Glycobiology 2008, 18, 779–788. (b) Tsuchida, A.; Kobayashi, K.; Matsubara, N.; Muramatsu, T.; Suzuki, T.; Suzuki, Y. Glycoconjugate J. 1998, 15, 1047–1054. (32) (a) Okada, M. Prog. Polym. Sci. 2001, 26, 67–104. (b) Varma, A. J.; Kennedy, J. F.; Galgalia, P. Carbohydr. Polym. 2004, 56, 429–445. (33) (a) Bovin, N. V.; Gabius, H. J. Chem. Soc. Rev. 1995, 24, 413–421. (b) Tagawa, K.; Sendai, N.; Ohno, K.; Kawaguchi, T.; Kitano, H.; Matsunaga, T. Bioconjugate Chem. 1999, 10, 354–360. (c) Ohno, K.; Fukuda, T.; Kitano, H. Macromol. Chem. Phys. 1998, 199, 2193–2197. (34) (a) Stenzel, M. H.; Barner-Kowollik, C.; Davis, T. P. J. Polym. Sci. Part A: Polym. Chem. 2006, 44, 2363–2375. (b) Barner-Kowollik, C.; Dalton, H.; Davis, T. P.; Stenzel, M. H. Angew. Chem., Int. Ed. 2003, 42, 3664–3668. (c) Nygard, A.; Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H. Aust. J. Chem. 2005, 58, 595–599. (d) Stenzel, M. H.; Davis, T. P. Aust. J. Chem. 2003, 56, 1035–1038. (35) B€oker, A.; Lin, K .; Chiapperini, K.; Horowitz, R.; Thompson, M.; Carreon, V.; Xu, T.; Abetz, C.; Skaff, H.; Dinsmore, A. D.; Emrick, T.; Rusell, T. P. Nat. Mater. 2004, 3, 302–306. (36) Nishikawa, T.; Nishida, J.; Ookura, R.; Nishimura, S.-I.; Wada, S.; Karino, T.; Shimomura, M. Mater. Sci. Eng., C 1999, 8-9, 495–500. (37) Ting, S. R. S.; Min, E. H.; Escale, P.; Save, M.; Billon, L.; Stenzel, M. H. Macromolecules 2009, 42, 9422–9434. (38) Bordege, V.; Leon, O.; Mu~noz-Bonilla, A.; Cuervo-Rodrı´ guez, R.; Sanchez-Chaves, M.; Fernandez-Garcia, M. Submitted to Macromolecules.
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Article used in the course of experiments and characterization (dimethyl formamide (DMF) and dimethyl sulfoxide-d) were employed without further purification. Rhodamine B isothiocyanate (Aldrich) and lectin-fluorescein isothiocyanate conjugate from Canavalia ensiformis (ConA-FTIC) (Sigma) were used as received. Copolymerization Reaction. Free-radical copolymerization of styrene and HEAGl with a molar feed composition in styrene of 0.80 was carried out in a pyrex ampule sealed in an argon atmosphere at 70 °C at a global concentration of 1 mol/L DMF solution with 3 10-2 mol/L AIBN as the initiator concentration. The reaction was conducted up to total conversion. The resulting copolymer, S8Gl2, was purified by dialysis and further liophilization. Characterization. The statistical glycopolymer was characterized by 1H NMR with a Bruker Advanced 400 MHz spectrometer at room temperature. A probe of deuterated DMSO was introduced into a copolymer solution of dimethylformamide (DMF). Average molecular weights and dispersities were determined by size exclusion chromatography (SEC) using a Jasco system equipped with two PL gel 5 μm (300 7.5 mm2) mixed-C columns and a PL gel 5 μm (50 7.5 mm2) guard column, a Jasco 1530 differential refractive index detector, and a Jasco 875 UV detector. N,N-Dimethylformamide (HPLC grade) was used as an eluent containing 0.1 M LiBr with a flow rate of 0.8 mL/min at 60 °C. Calibration was obtained from narrowly distributed polystyrene standards. Film Preparation. Mixtures having 10-50 wt % copolymer S8Gl2 and 90-50% high-molecular-weight homopolystyrene (Mn = 250 000 g/mol) were dissolved in THF (solution concentration 30 mg/mL). The polymer solutions were filtered with a 0.1 μm Millipore membrane and spin coated (4000 rpm for 60 s) onto silicon wafers purchased from SC (Siegert Consulting e.K., Germany). The silicon wafers were cleaned prior to use in piranha solution (3:1 v/v of H2SO4/H2O2) and rinsed several times with ethanol. For the preparation of samples under controlled relative humidity, beakers containing water and saturated aqueous solutions of sodium bromide were placed inside the spin-coating chamber to obtain values of the relative humidity of between ∼40 and ∼57%, respectively. The degree of humidity and the temperature were measured by means of a hygrothermograph. Annealing. Sample annealing was carried out to study the variations of the surface chemical composition as a function of the environment. Upon analyzing (by AFM imaging) the films obtained after spin coating, the samples were exposed either to air at 90 °C for 3 days or placed in a tightly closed stainless steel vessel saturated with water vapor. The annealing in a humid environment was carried out for 36 h at 90 °C. After each treatment, the samples were dried under vacuum at room temperature. Rhodamine-Isocyanate Immobilization. The films previously annealed to water vapor were immersed in an aqueous rhodamine isocyanate solution (0.25 mg/mL) for 2 h at room temperature. After being washed with deionized water, the film was dried at room temperature. Lectin Interaction. The film was immersed into a phosphatebuffered saline solution (PBS, 7.4) at room temperature containing 0.2 mg/mL fluorescein-conjugated concanavalin A (Con A-FITC). After 6 h, the film was sequentially washed with PBS buffer and distilled water. Methods. Atomic Force Microscopy. AFM images were recorded in air at room temperature with a Nanoscope IIIa microscope operating in tapping mode. The probes were commercially available silicon wafers with a spring constant of 42 N/ m, a resonance frequency of 285 kHz, and a typical radius of curvature in the 10-12 nm range. Both the topography and the phase-signal images were recorded with a resolution of 512 512 data points. Fluorescence Microscopy and Image Processing. Images were acquired with a Zeiss Axiovert 40 CFL inverted microscope 8554 DOI: 10.1021/la904565d
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Figure 1. Synthesis approach to preparing S-co-HEAGl statistical glycopolymers by conventional free radical polymerization. equipped with a 12 V, 35 W halogen lamp (for the phase-contrast images) and an HBO 50 W/AC mercury lamp (for the fluorescence images). The objectives used were a 5/0.12 A-Plan, a 10/ 0.25 A-Plan, a 20/0.50 EC Plan-NEOFLUAR, and a 40/0.50 LD A-Plan (Zeiss). Images were acquired by using a Canon A640 CCD camera. Scanning Electron Microscopy. SEM experiments for crosssectional material analysis were carried out at room temperature in a XL30 ESEM Philips microscope working at 25 kV. Samples were in situ cryofractured prior to observations of the film cross section. The samples were coated with gold-palladium (80:20) with a sputter coater (Polaron SC7640) working at 800 V and 5 mA.
Results and Discussion The amphiphilic statistical glycopolymer (S8Gl2) copolymer (Mn = 42 900 g/mol; PD = 1.73) used throughout this study was prepared by conventional free radical copolymerization of styrene and HEAGl at a global concentration of 1 mol/L using 3 10-2 mol/L AIBN as an initiator in DMF solution at 70 °C (Figure 1). Among the main advantages of this methodology is worth mentioning that no additional protection/deprotection steps are needed to obtain the desired glycopolymer.39 The composition of the copolymer can be varied by modifying the initial feed. Nevertheless, herein we focused on a copolymer in which a relative high proportion of styrene (80% styrene and 20% HEAGl) units is employed for two main reasons. First, by increasing the styrene content, the compatibility between the glycopolymer and the homopolymer matrix is enhanced. Second, as we will describe below, in a subsequent step the prepared films will be annealed in water. The amount of hydrophilic glycomonomer has to be small in order to form stable films that do not dissolve in aqueous media and could resist the annealing conditions. Films of the blends containing between 10 and 100% glycopolymer and 90 to 0% linear homopolystyrene matrix were prepared by spin coating from THF solutions (30 mg/mL) in an atmosphere with controlled humidity (44% RH). As has been reported, during solvent evaporation the temperature of the solvent-air interface decreases and water vapor starts to condense. As solvent evaporation continues, the condensed water droplets grow until, finally, upon complete evaporation of both solvent and water the resulting surface contains randomly distributed holes reflecting the positions where the water droplets condensed. The conditions under which the films were prepared influence the size and distribution of the holes obtained. Hence, the solvent, blend composition, concentration of solution, and humidity are important external parameters that have to be controlled.40 In Figure 2 are illustrated the AFM images of films (39) 1H NMR and FTIR spectra and an SEC chromatogram of the copolymer are provided in the Supporting Information. (40) Bunz, U. H. F. Adv. Mater. 2006, 18, 973–989.
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Figure 2. AFM height and phase images (10 μm 10 μm) of films obtained by spin coating in a 44% humidity atmosphere by varying the composition of the blend (w/w, S8Gl2/PS): (a) 10/90, (b) 20/80, and (c) 50/50. The surface morphology (size and density of the holes) depends on the amount of glycopolymer included in the mixture.
Figure 3. Illustrative AFM height and section images of the holes obtained by spin coating in a 44% humidity atmosphere by varying the composition of the blend (w/w, S8Gl2/PS): (a) 10/90, (b) 20/80, and (c) 50/50. The average diameter increases from ∼220 to ∼700 nm by increasing the amount of copolymer in the mixture.
obtained from different blend compositions in which the amount of glycopolymer was varied between 10 and 50%. As depicted in Figure 2, by maintaining the humidity and glycopolymer concentration constant (30 mg/mL and 44% RH), the average diameter of the holes increases with the amount of copolymer introduced into the feed. Amphiphilic copolymers are hygroscopic and may interact with water during the spin-coating process.41 Thus, increasing the amount of hydrophilic polymer (41) Wong, K. H.; Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H. Polymer 2007, 48, 4950–4965.
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in the mixture enhances water uptake and, as a consequence, the diameter of the holes formed. Blends with amounts of S8GL2 above 50% lead to rather disordered surface structures as a consequence of the coalescence of the water droplets. Also, films prepared in very moist atmospheres above 50% RH led to a similar effect. However, by adjusting the preparation conditions, it is then possible to prepare porous films in which, by the self-assembly of the water droplets, a hexagonal array, also known as a honeycomb structure, can be obtained. Hence, within this range of blend compositions the average size of the holes can be controlled. Mixtures with 10% glycopolymer DOI: 10.1021/la904565d
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Figure 4. (i) AFM images of the films: 50/50 (w/w) blend of S8Gl2/PS prepared by spin coating from THF solutions (30 mg/mL). (ii) SEM cross-sectional image of a 50/50 (w/w) blend of S8Gl2/PS.
Figure 5. Tapping-mode height AFM images (5 μm 5 μm) of (i) a film obtained by spin coating 10/90 S8Gl2/PS (w/w), (ii) the same film after annealing in water vapor at 80 °C for 3 days, and (iii) the same film upon drying at 60 °C overnight. (iv) Film obtained by spin coating of a mixture 50/50 S8Gl2/PS (w/w), (v) the same film after annealing in water vapor at 80 °C for 3 days, and (vi) the same film upon drying at 60 °C overnight.
holes with average diameters of ca. 215-230 nm are obtained, whereas for blends with 20 and 50% the average sizes are 450-470 and 650-700 nm (Figure 3). The pore depth increases accordingly, and in those blends with 10% glycopolymer, a depth of 15 nm could be measured by AFM; 16-20 nm for 20% glycopolymer and 25-30 nm for 50% glycopolymer could also be measured. A cross-sectional view of the films obtained by SEM (Figure 4ii) indicates the formation of a single layer of holes with an average depth of 200-300 nm. Thus, the AFM values appeared to underestimate the real depth values. It is important to know at this stage that, as depicted in Figure 4, porous microstructured surfaces with rather low polydispersity pores ordered over a length of several micrometers can be obtained by using this approach. Spin coating offers the possibility to obtain both long-range order and low roughness. The interaction between the polar glycomonomer units and water droplets condensed during spin coating modifies the distribution in terms of chemical composition at the interface. As a consequence, the glycopolymer in contact with water rearranges around the water droplet41 and after drying is principally localized in this area. Hence, the surface composition is different between the holes and the rest of the interface. 8556 DOI: 10.1021/la904565d
As a consequence and because of the hygroscopic nature of the carbohydrate moieties, annealing in humid air allows preferential swelling in this area. This behavior is evidenced by AFM in films obtained directly after spin coating and annealed in humid air. The AFM images obtained for films of the 10/90 and 50/50 S8Gl2/PS (w/w) blends are depicted in Figure 4. Annealing in humid water vapor provoked reversible changes in the micropatterned surface. Hence, the holes are transformed into islandlike structures after 3 days of annealing. Similarly, drying the surface under mild conditions (60 °C, overnight) removes the water and recovers the original topography. More interestingly, the pore size can be controlled by the conditions employed for the preparation of the films and the swelling of the glycopolymer-provoked topographical modifications that are directly related to the pore diameter. Figure 5 shows the AFM images and cross-sectional views of the structures formed at the interface upon annealing in humid air. For the holes obtained from blends having 10% glycopolymer with an average diameter of 200 nm, the hole is completely swollen and forms a regular hill. In those films obtained from blends with 50% glycopolymer with a higher average diameter of about 650700 nm, only the external part of the pore can be swollen. As a Langmuir 2010, 26(11), 8552–8558
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Figure 6. AFM images (2 μm 2 μm) of (a) a 10/90 S8Gl2/PS (w/w) film and (b) a 50/50 S8Gl2/PS (w/w) film after annealing in water vapor at 80 °C for 3 days. The sections of the annealed surfaces below confirmed the swelling of the pore.
Figure 7. Top-view images οf films obtained (i) with an optical microscope and (ii) using a fluorescence microscope. The fluorescence detected in the inner part of the pore showed the functionalization of the inside of the pore with rhodamine isocyanate by reaction with the hydroxyl groups of the glycomonomer units. (iii) Images of the surface obtained upon chemical recognitition between the glycopolymer and fluorescein-conjugated concanavalin A within the pores. Langmuir 2010, 26(11), 8552–8558
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consequence, the hole functionalized with the glycopolymer can be employed as a template to guide the patterning of biomolecules.9 It is worth mentioning that the system described above significantly simplifies the fabrication of 3D templates in comparison with other micropatterning techniques, such as photoresist lithography or soft lithography, that require the employment of expensive techniques.42 The employment of the surfaces as templates requires the availability of recognition sites to which the biomolecules could be attached. Whereas the presence of glycopolymer and therefore of saccharide units within the pore surface has been proven by AFM upon imaging the surfaces obtained after annealing in water vapor, the availability of the hydroxyl groups of the saccharide units is evidenced by reaction with rhodamine isothiocyanate (Rho-FITC). The images obtained with a fluorescence microscope for the blends with 50% S8Gl2 are depicted in Figure 6. The ring-shaped fluorescent pattern indicated the attachment of rhodamine to the pore surface by the covalent reaction between the isothiocyanate and hydroxyl moieties. The interior of the pore is out of focus and does not exhibit fluorescence. Similar behavior has been described by other groups, which elaborated on breath figures decorated with other functionalities.35,9 These images clearly indicate the capability of the hydroxyl groups to accomplish chemical reactions with other functional groups. Beyond the above-proven accessibility of hydroxyl groups, it is interesting to analyze the ability of these glycopolymer-modified surfaces to recognize lectins specifically. In this sense, concanavalin A is a protein that specifically interacts with glucosyl residues. Therefore, films were immersed into PBS solution at room temperature containing 0.2 mg/mL fluorescein-conjugated concanavalin A (Con A-FITC). After 6 h, the film was sequentially washed with PBS buffer and distilled water. Figure 6 also shows the fluorescence microscopy of the interaction between the S8Gl2 glycopolymer with Con A-FITC. As can be observed, such an interaction is produced in the external part of the pore, (42) (a) He, W.; Halberstadt, C. R.; Gonsalves, K. E. Biomaterials 2004, 25, 2055–2063. (b) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363–2376. (c) Nicolau, D. V.; Taguchi, T.; Taniguchi, H.; Yoshikawa, S. Langmuir 1999, 15, 3845–3851. (d) Tan, J. L.; Tien, J.; Chen, C. S. Langmuir 2002, 18, 519–523. (e) Wang, C.; Zhang, Y. Adv. Mater. 2005, 17, 150–153.
8558 DOI: 10.1021/la904565d
Mu~ noz-Bonilla et al.
confirming that the hydrophilic part of the glycopolymer is mainly exposed to the surface.
Conclusions In this article, we described the elaboration of micropatterned surfaces by using breath figure methodology. By controlling the parameters involved in film preparation such as the composition of the blend, humidity, and temperature, we were able to prepare a surface with a defined pore size and distributions. Also, by swelling the polar carbohydrate moieties, we showed the selective enrichment of glycopolymer in the inner part of the pore. Depending on the size of the pores created, the pores are either transformed in hills or only partially swollen. Finally, the potential of these structures to serve as templates for the attachment of bioactive molecules is clearly evidenced. First, the hydroxyl groups of the saccharide are able to react, imparting specific functionality as demonstrated by the reaction with the isothiocyanate functional group of rhodamine. Second, the specific interaction with Con A lectin clearly verified the carbohydrate groups’ accessibility to the surface, again indicating the transformation from a hydrophobic to a hydrophilic surface. Acknowledgment. The authors thank the financial support given by the Centre National de la Recherche Scientifique, the Agence National de la Recherche (Jeunes Chercheurs program ANR-07-JCJC-0148), and the Spanish National Research Council (PI 200860I037, CCG08-CSIC/MAT-3643, MAT2009-12251, MAT2007-60983). Note Added after ASAP Publication. This article was published ASAP on February 12, 2010. An Acknowledgment section has been added to the manuscript. The correct version was published on February 18, 2010. Supporting Information Available: Characterization of copolymer 2-{[(D-glucosamin-2-N-yl) carbonyl]oxy}ethyl acrylate (HEAGl) via FTIR, 1H NMR, and SEC. This material is available free of charge via the Internet at http://pubs.acs.org.
Langmuir 2010, 26(11), 8552–8558
