Aminosilane Micropatterns on Hydroxyl-Terminated Substrates

The mPEG-silane (MW = 1000) was purchased from Laysan Bio, Inc. (Arab, AL). ...... Il-Yung Sohn , Duck-Jin Kim , Jin-Heak Jung , Ok Ja Yoon , Tien Ngu...
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Aminosilane Micropatterns on Hydroxyl-Terminated Substrates: Fabrication and Applications Hai Li,† Juan Zhang,† Xiaozhu Zhou,† Gang Lu,† Zongyou Yin,† Gongping Li,‡ Tom Wu,‡ Freddy Boey,† Subbu S. Venkatraman,† and Hua Zhang*,† †

School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore and ‡Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore Received October 15, 2009. Revised Manuscript Received November 6, 2009 The technique to pattern aminosilanes on hydroxyl-terminated substrates will open up extensive applications in many fields. There are some existing methods to pattern aminosilanes, in particular, (3-aminopropyl)triethoxysilane (APTES) on SiO2 and glass substrates through indirect routes. However, few reports focus on the direct patterning of APTES by microcontact printing (μCP), due to the volatility of “inks” which consist of APTES and organic solvents. This report shows that high-quality APTES patterns on hydroxyl-terminated substrates can be directly obtained by μCP using an APTES aqueous solution as “ink”. Gold nanoparticles (Au NPs) have been used to verify the presence and quality of APTES patterns on which they are selectively adsorbed. Thus-obtained Au NP patterns can serve as templates for the growth of ZnO nanostructures. Lectins are also successfully immobilized on the APTES patterns, with glutaraldehyde as linker. We believe that our method will serve as a general approach and find a wide range of applications in the fabrication of patterns and devices.

1. Introduction Due to their wide applications in micro- and nanofabrication,1-7 molecular electronics,8-10 array-based sensors,11,12 *To whom correspondence should be addressed. Telephone: þ6567905175. Fax: þ65-67909081. E-mail: [email protected]. Website: http://www.ntu.edu.sg/home/hzhang/. (1) Onclin, S.; Ravoo, B. J.; Reinhoudt, D. N. Angew. Chem., Int. Ed. 2005, 44, 6282–304. (2) Ulman, A. Chem. Rev. 1996, 96, 1533–1554. (3) Xia, Y.; Mrksich, M.; Kim, E.; Whitesides, G. M. J. Am. Chem. Soc. 1995, 117, 9576–9577. (4) Zhang, H.; Amro, N. A.; Disawal, S.; Elghanian, R.; Shile, R.; Fragala, J. Small 2007, 3, 81–85. (5) Zhang, H.; Elghanian, R.; Amro, N. A.; Disawal, S.; Eby, R. Nano Lett. 2004, 4, 1649–1655. (6) Zhang, H.; Jin, R.; Mirkin, C. A. Nano Lett. 2004, 4, 1493–1495. (7) Huo, F.; Zheng, Z.; Zheng, G.; Giam, L. R.; Zhang, H.; Mirkin, C. A. Science 2008, 321, 1658–1660. (8) Briseno, A. L.; Mannsfeld, S. C.; Ling, M. M.; Liu, S.; Tseng, R. J.; Reese, C.; Roberts, M. E.; Yang, Y.; Wudl, F.; Bao, Z. Nature 2006, 444, 913–7. (9) Coskun, U. C.; Mebrahtu, H.; Huang, P. B.; Huang, J.; Sebba, D.; Biasco, A.; Makarovski, A.; Lazarides, A.; LaBean, T. H.; Finkelstein, G. Appl. Phys. Lett. 2008, 93, 123101-3–. (10) Zhou, C.; Nagy, G.; Walker, A. V. J. Am. Chem. Soc. 2005, 127, 12160– 12161. (11) Basabe-Desmonts, L.; Beld, J.; Zimmerman, R. S.; Hernando, J.; Mela, P.; Parajo, M. F. G.; van Hulst, N. F.; van den Berg, A.; Reinhoudt, D. N.; CregoCalama, M. J. Am. Chem. Soc. 2004, 126, 7293–7299. (12) Basabe-Desmonts, L.; van der Baan, F.; Zimmerman, R.; Reinhoudt, D.; Crego-Calama, M. Sensors 2007, 7, 1731–1746. (13) Bae, S.-S.; Lim, D. K.; Park, J.-I.; Lee, W.-R.; Cheon, J.; Kim, S. J. Phys. Chem. B 2004, 108, 2575–2579. (14) Cau, J. C.; Cerf, A.; Thibault, C.; Genevieve, M.; Severac, C.; Peyrade, J. P.; Vieu, C. Microelectron. Eng. 2008, 85, 1143–1146. (15) Chen, C. F.; Tzeng, S. D.; Lin, M. H.; Gwo, S. Langmuir 2006, 22, 7819– 7824. (16) Khatri, O. P.; Han, J.; Ichii, T.; Murase, K.; Sugimura, H. J. Phys. Chem. C 2008, 112, 16182–16185. (17) Lin, Y.-C.; Yu, B.-Y.; Lin, W.-C.; Chen, Y.-Y.; Shyue, J.-J. Chem. Mater. 2008, 20, 6606–6610. (18) Ling, X.; Zhu, X.; Zhang, J.; Zhu, T.; Liu, M.; Tong, L.; Liu, Z. J. Phys. Chem. B 2005, 109, 2657–2665. (19) Wu, C. H.; Lee, T. M.; Sheu, J. T.; Chao, T. S. Jpn. J. Appl. Phys. 2009, 48, 04C133. (20) He, H. X.; Zhang, H.; Li, Q. G.; Zhu, T.; Li, S. F. Y.; Liu, Z. F. Langmuir 2000, 16, 3846–3851.

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and templates for adsorption of nanoparticles,13-21 biomolecules22-25 and other materials,26 self-assembled monolayers (SAMs) with different chemical functionalities have attracted extensive attention in surface chemistry1,2,27 and materials science.28 Although patterned alkanethiolate SAMs on Au substrates are well developed to study a number of biological processes and they play critical roles in the formation of protein microarrays23,29,30 and geometric control of cell growth and migration,31,32 the long-term instability and biocompatibility hinder their applications.33,34 Therefore, the use of other substrates, such as SiO2, glass, hydroxyl-terminated diamond, metal oxides, and polymers, is required for SAMs.35 The SAMs of silanes formed on SiO2 have been found in a lot of applications in bio- and nanotechnology.1 Although the micropatterns of alkylsilanes on SiO2 generated by microcontact (21) Li, B.; Lu, G.; Zhou, X.; Cao, X.; Boey, F.; Zhang, H. Langmuir 2009, 25, 10455–10458. (22) Lee, K.-B.; Kim, E.-Y.; Mirkin, C. A.; Wolinsky, S. M. Nano Lett. 2004, 4, 1869–1872. (23) Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702–1705. (24) Shim, H. Y.; Lee, S. H.; Ahn, D. J.; Ahn, K.-D.; Kim, J.-M. Mater. Sci. Eng., C 2004, 24, 157–161. (25) Zhang, H.; Li, Z.; Mirkin, C. A. Adv. Mater. 2002, 14, 1472–1474. (26) (a) Zhang, H.; Grim, P. C. M.; Liu, D.; Vosch, T.; De Feyter, S.; Wiesler, U. M.; Berresheim, A. J.; Mullen, K.; Van Haesendonck, C.; Vandamme, N.; De Schryver, F. C. Langmuir 2002, 18, 1801–1810. (b) Zhang, H.; M€ullen, K.; De Feyter, S. J. Phys. Chem. C 2007, 111, 8142–8144. (27) Smith, R. K.; Lewis, P. A.; Weiss, P. S. Prog. Surf. Sci. 2004, 75, 1–68. (28) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498–1511. (29) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055–2060. (30) Tan, J. L.; Tien, J.; Chen, C. S. Langmuir 2002, 18, 519–523. (31) Brock, A.; Chang, E.; Ho, C.-C.; LeDuc, P.; Jiang, X.; Whitesides, G. M.; Ingber, D. E. Langmuir 2003, 19, 1611–1617. (32) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696–698. (33) Pulsipher, A.; Westcott, N. P.; Luo, W.; Yousaf, M. N. J. Am. Chem. Soc. 2009, 131, 7626–7632. (34) Yanker, D. M.; Maurer, J. A. Mol. BioSyst. 2008, 4, 502–504. (35) Shirahata, N.; Nakanishi, J.; Echikawa, Y.; Hozumi, A.; Masuda, Y.; Ito, S.; Sakka, Y. Adv. Funct. Mater. 2008, 18, 3049–3055.

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printing (μCP) have been reported, only octadecyltrichlorosilane (OTS) was studied extensively.3,34,36-38 As a long-chain alkyltrichlorosilane (RSiCl3), OTS is a typical “ink” to be patterned on oxide substrates by μCP,37 since the -SiCl3 groups can react with surface -OH groups to form the Si-O-Si bonds. Aminosilanization of hydroxyl-terminated surfaces finds numerous applications in immobilization of biomolecules,39,40 electrostatic assembly of inorganic nanoparticles,13,15,18,41 and fabrication of sensors.11,12 The patterned amino-functionalized SAMs have been used as templates for adsorption of a wide variety of materials on Si, SiO2, and glass surfaces, including gold nanoparticles (Au NPs)9,15-19 and biomolecules.24,42 Previous studies reported that aminosilanes are inconvenient “inks” for μCP due to their rather low affinity in poly(dimethylsiloxane) (PDMS) stamps and easy hydrolysis and polymerization in ambient conditions;43 only few groups reported the direct patterning of aminosilanes on SiO2 or glass substrates by μCP, in which the aminosilanes are usually dissolved in an organic solvent used as “ink”.24,43-45 Many efforts have been applied to develop indirect methods for patterning aminosilanes.9,15,17,18,36 For example, the scanningprobe-based oxidation method18 and vacuum UV (193 nm) photolithography17 were used to fabricate oxide patterns in OTS SAMs on Si and glass, respectively. E-beam lithography was directly used to generate patterns on poly(methyl methacrylate) (PMMA)-coated SiO2.9 Then the patterned substrates were immersed into (3-aminopropyl)triethoxysilane (APTES) solution to form APTES patterns. In another example, the aminosilane patterns were generated by immersing the OTSpatterned SiO2 substrate into a APTES solution.36 The electricfield-induced electrochemical reaction has also been used to convert (3-aminopropyl)trimethoxysilane (APTMS) SAMs to oxide patterns by applying a positive voltage between the conductive Si stamp and the Si substrate. The rest of the APTMS SAMs formed the aminosilane patterns.15 However, all of these aforementioned methods are indirect routes for fabrication of aminosilane patterns. Although gas-transfer lithography has been used to fabricate APTMS patterns on SiO2 directly, it needs the overnight inking of the PDMS stamp.46 Several groups have tested the efficiency of direct patterning of APTES on SiO2 and glass by using APTES dissolved in volatile organic solvent as “ink” during the μCP experiment, but poor quality APTES patterns were obtained.24,45 Due to their relatively low molecular weight, APTES molecules are volatile and easily present in the gas phase. After they are dissolved in the volatile organic solvent and used as “ink” to coat PDMS stamps for μCP experiments, a considerable amount of APTES molecules from the gas phase will be deposited on the (36) Cau, J.-C.; Cerf, A.; Thibault, C.; Genevieve, M.; Severac, C.; Peyrade, J.-P.; Vieu, C. Microelectron. Eng. 2008, 85, 1143–1146. (37) Jeon, N. L.; Finnie, K.; Branshaw, K.; Nuzzo, R. G. Langmuir 1997, 13, 3382–3391. (38) St. John, P. M.; Craighead, H. G. Appl. Phys. Lett. 1996, 68, 1022–1024. (39) Almeida, A. T.; Salvadori, M. C.; Petri, D. F. S. Langmuir 2002, 18, 6914–6920. (40) Shlyakhtenko, L. S.; Gall, A. A.; Weimer, J. J.; Hawn, D. D.; Lyubchenko, Y. L. Biophys. J. 1999, 77, 568–576. (41) Zhang, F.; Srinivasan, M. P. Langmuir 2004, 20, 2309–2314. (42) Sugimura, H.; Nakagiri, N. J. Am. Chem. Soc. 1997, 119, 9226–9229. (43) Geissler, M.; Kind, H.; Schmidt-Winkel, P.; Michel, B.; Delamarche, E. Langmuir 2003, 19, 6283–6296. (44) Fujita, M.; Mizutani, W.; Gad, M.; Shigekawa, H.; Tokumoto, H. Ultramicroscopy 2002, 91, 281–285. (45) Kim, Y. K.; Park, S. J.; Koo, J. P.; Kim, G. T.; Hong, S.; Ha, J. S. Nanotechnology 2007, 18, 6. (46) de la Rica, R.; Baldi, A.; Mendoza, E.; Paulo, A. S.; Llobera, A.; Fernandez-Sanchez, C. Small 2008, 4, 1076–1079. (47) Mewe, A. A.; Kooij, E. S.; Poelsema, B. Langmuir 2006, 22, 5584–5587.

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nonpatterned region. This greatly degrades the pattern quality.47 Meanwhile, the organic solvent can swell PDMS stamps, and the oligomers from PDMS dissolving in the solvent bring another form of contamination.48 Therefore, the PDMS stamps should be cleaned before being coated with “inks” in organic solvent. However, the cleaning of PDMS stamps is time-consuming and tedious.49 It has been reported that the hydrolysis and condensation of APTES in aqueous solution are autocatalyzed and the hydrolysis completes in a few minutes.50 However, in the dilute APTES aqueous solution, the concentration of reactive silanol groups is still more than 50% even after 80 h of condensation.50 Since PDMS and its nonpolymerized oligomers are insoluble in water,48 it could be a promising method to use PDMS stamps coated with APTES aqueous solution to fabricate high-quality APTES patterns. Although APTES aqueous solution has been used as “ink” in reactive μCP by using a wet agarose stamp, it is difficult to fabricate patterns with the small feature size (e.g., less than 20 μm), because of the lateral diffusion of “inks” induced by the wet stamp surface.51 Herein, we report a facile method for fast and easy fabrication of high-quality APTES patterns on hydroxyl-terminated substrates directly by μCP. In our method, only APTES aqueous solution and common PDMS stamps are employed. APTES patterns with feature size ranging from several tens of micrometers to sub-micrometers are fabricated on mica, glass coverslips, SiO2, sapphire, and quartz. Au NPs were selectively adsorbed on APTES patterns without any notable nonspecific adsorption in the nonpatterned area. The crossed patterns of Au NPs and graphene oxide (GO) were also fabricated on SiO2 substrates. The generated APTES patterns were used as templates for immobilization of proteins, and growth of ZnO nanostructures in solution and by the chemical vapor deposition (CVD) method. We believe that this direct patterning method will find wide applications in biology and devices.

2. Experimental Section 2.1. Materials. (3-Aminopropyl)triethoxysilane (APTES), zinc acetate dehydrate, ammonia (25%), glutaraldehyde solution (50%), hydrogen peroxide (30%), concentrated sulfuric acid (98%), FITC-lectin (L4895), phosphate buffered saline (PBS) solution, and Tween-20 were purchased from Sigma-Aldrich Pte Ltd. (Singapore). The mPEG-silane (MW = 1000) was purchased from Laysan Bio, Inc. (Arab, AL). Silicone elastomer and silicone elastomer curing agent (Sylgard 184 silicone elastomer kit) were purchased from Dow Corning Corporation (Midland, MI). Silicon oxide wafers were purchased from Bonda Technology Pte Ltd. (Singapore). Muscovite mica was purchased from Sichuan Meifeng Co. (Ya’an, Sichuan, China). ST-cut quartz and sapphire were purchased from Roditi International Corporation Ltd. (London, England). Nature graphite (SP-1) was purchased from Bay Carbon (Bay City, MI) and used for synthesizing GO. All chemicals (analytical grade reagent) were used as received without further purification. Milli-Q water (Milli-Q System, Millipore, Billerica, MA) was used in all experiments. 2.2. Preparation of PDMS Stamps. PDMS stamps were fabricated by pouring a mixture of Sylgard 184 elastomer and curing agent (w/w = 10:1) over a master and heating at 70 °C for 12 h after degassing. (48) Lee, J. N.; Park, C.; Whitesides, G. M. Anal. Chem. 2003, 75, 6544–6554. (49) Thibault, C.; Severac, C.; Mingotaud, A.-F.; Vieu, C.; Mauzac, M. Langmuir 2007, 23, 10706–10714. (50) Beari, F.; Brand, M.; Jenkner, P.; Lehnert, R.; Metternich, H. J.; Monkiewicz, J.; Siesler, H. W. J. Organomet. Chem. 2001, 625, 208–216. (51) Campbell, C. J.; Smoukov, S. K.; Bishop, K. J. M.; Grzybowski, B. A. Langmuir 2005, 21, 2637–2640.

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2.3. Substrate Cleaning and Microcontact Printing of (3-Aminopropyl)triethoxysilane (APTES). After the SiO2 substrates, quartz, and glass coverslips were sonicated in a mixture of acetone and water (v/v = 1:1) for 15 min, they were immersed in piranha solution (mixture of 98% H2SO4 and 30% H2O2 with v/v = 7:3) for 1 h at 120 °C (Caution: strongly corrosive!). They were thoroughly washed with Milli-Q water (18.2 MΩ cm) and dried with nitrogen gas. Mica was freshly cleaved prior to use. Sapphire disks were sonicated in a soap solution for 15 min. After being rinsed with water, the cleaned substrates were treated with an oxygen plasma cleaner (PDC-32G-2, Harrick Plasma, Ithaca, NY) with a power of 10.8 W for 5 min at a pressure of 120 mTorr. A total of 100 μL of 1% APTES aqueous solution was dropped on a PDMS stamp. After inking for 2 min, the stamp was dried with nitrogen gas. It was brought into contact with the substrates for ca. 15 s before it was withdrawn from their surface.

2.4. Selective Adsorption of Gold Nanoparticles (Au NPs) and Graphene Oxide (GO) on APTES Patterns.

Au NPs (13 nm) were synthesized based on the Frens method.52 Briefly, after 20 mL of 1.0 mM HAuCl4 in a 50 mL glass bottle was heated to boiling with stirring, 2 mL of a 1% solution of trisodium citrate dihydrate, Na3C6H5O7 3 2H2O, was added. The gold sol gradually formed as the citrate reduced Au(III). A total of 20 μL of thus-synthesized Au NP solution was dropped on the APTESpatterned substrates for 2 min, followed by rinsing with Milli-Q water, and then drying with N2. GO was synthesized from natural graphite (SP-1) by using a modified Hummers’ method.53,54 The resulting graphite oxide was dispersed in water with a certain concentration and subsequently sonicated to give GO. The APTES-patterned substrates were immersed in a GO aqueous solution for 1 h, resulting in single layer GO patterns.54,55 Optical images were captured with a Nikon microscope (Eclipse LV100, Nikon Instruments Inc., NY).

2.5. Growth of ZnO Nanostructures in Solution and by Chemical Vapor Deposition (CVD) Method. In the solution

method, ZnO nanostructures were grown as follows.56 After 55 mg of zinc acetate dehydrate was dissolved in 50 mL of Milli-Q water under sonication for 30 min, 0.5 mL of ammonia was added into the solution. Subsequently, the Au NP-patterned SiO2 was loaded into the above solution and then maintained in an oven at 75 °C for 1 h. The resulting solid product was washed with ethanol and finally dried at 50 °C in air. In the CVD method, ZnO nanostructures were grown by thermally vaporizing a mixed source of ZnO and graphite powder in a tube furnace.57 Before heating, the tube was evacuated down to 10-3 mbar. Argon mixed with 0.5% oxygen was used as the carrying gas with a controlled flow rate of 50 sccm. The pressure inside the tube was maintained at 50 mbar during the growth. Typically, the temperature of the furnace was raised to 950 °C with a ramping rate of 50 °C/min and cooled down to room temperature after growth of ZnO for a few minutes. Details of the growth process were described elsewhere.57

2.6. Protein Immobilization on APTES Patterned Glass Coverslips and the Fluorescent Images. After the APTESpatterned glass coverslip was immersed into 2% mPEG-1000 silane toluene solution for 2 days at room temperature, it was washed with toluene and ethanol to remove any physisorbed materials and then dried with N2. The patterned substrate was immersed into a 0.5% glutaraldehyde solution for 1 h and (52) Frens, G. Nature Phys. Sci. 1973, 241, 20–22. (53) Hummers, W. S.; Offeman, R. E. J. Am. Chem. Soc. 1958, 80, 1339. (54) Zhou, X.; Huang, X.; Qi, X.; Wu, S.; Xue, C.; Boey, F. Y. C.; Yan, Q.; Chen, P.; Zhang, H. J. Phys. Chem. C 2009, 113, 10842–10846. (55) Wang, Z.; Zhou, X.; Zhang, J.; Boey, F.; Zhang, H. J. Phys. Chem. C 2009, 113, 14071–14075. (56) Xu, H.; Wang, H.; Zhang, Y.; He, W.; Zhu, M.; Wang, B.; Yan, H. Ceram. Int. 2004, 30, 93–97. (57) Wang, X.; Li, G.; Chen, T.; Yang, M.; Zhang, Z.; Wu, T.; Chen, H. Nano Lett. 2008, 8, 2643–2647.

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Article subsequently rinsed with Milli-Q water. After 20 μL of 0.1 mg/ mL FITC-lectin was added on the substrate and incubated at 37 °C in a dark humid box for 1 h, the sample was washed with PBST buffer (0.1% Tween-20 in PBS buffer), PBS buffer, and water, and then dried with N2. The immobilized fluorescent lectins were imaged with a Leica TCS SP5 confocal system (Leica, Wetzlar, Germany).

2.7. Atomic Force Microscopy (AFM) Characterization. A commercial AFM instrument (Dimension 3100 with Nanoscope IIIa controller, Veeco Instruments Inc., CA) equipped with a scanner (90  90 μm2) was used to image the samples in tapping mode in air. Silicon cantilevers with a normal resonance frequency of 330 kHz and spring constant of 42 N m-1 (PP-NCH, Nanosensors, NanoWorld AG, Switzerland) were used. All images were captured with a scan rate at 1-2 Hz and with 512  512 pixel resolution.

2.8. Scanning Electron Microscopy (SEM) Characterization. SEM was performed using a JEOL JSM-6700 fieldemission scanning electron microanalyzer at accelerating voltages of 5 and 10 kV.

3. Results and Discussion 3.1. Microcontact Printing of APTES on SiO2, Mica, Quartz, Sapphire, and Glass Coverslips. Scheme 1 shows the process of our experiments. In order to get the optimal concentration of APTES aqueous solution used for μCP to generate high-quality APTES patterns on the hydroxyl-terminated substrates, a series of APTES solutions with different concentrations were used as “inks” to coat PDMS stamps, which then transferred APTES onto substrates. Figure 1A-E shows the generated APTES micropatterns on SiO2 with APTES solution concentrations of 0.2%, 0.5%, 1%, 2%, and 5%, respectively. The APTES patterns become clear (Figure 1A-E), and their average height increases from ∼0.1 to 0.5 nm with the increase of “ink” concentration (Figure 1F). It means that the APTES patterns generated by μCP in our experiment are submonolayers, since the height of a fully covered APTES monolayer is 0.7 nm, based on the theoretical model (bottom inset, Figure 1 D) and ellipsometry measurement.58 Meanwhile, some condensed polymerized APTES particles with a height of ∼2.5 nm (top inset in Figure 1D) can be clearly observed in the APTES patterns due to the rapid hydrolysis and condensation of APTES in aqueous solution.50 The amount of APTES particles increases with the “ink” concentration (Figure 1). Although the generated APTES patterns are not fully covered monolayers, they can still be used as templates to adsorb nano- and biomaterials (see the following experimental results). When the concentration of APTES solution is greater than 1%, some small visible APTES aggregations can be observed on the APTES-coated PDMS stamp surface after it is dried with N2, which will encumber the fabrication of homogeneous APTES patterns in a large area. When the “ink” concentration is greater than 2%, this visible aggregation cannot be completely removed even under a very strong N2 flow. We also found that APTES patterns formed with an “ink” concentration of 0.2% or 0.5% induced the low density adsorption of Au NPs (Figure S1 in the Supporting Information), while dense Au NPs patterns were observed when 1% “ink” was patterned on mica by μCP (Figure 2). Furthermore, after Au NPs are adsorbed on the APTES patterns generated with 0.2% “ink”, some Au NPs can even be pushed away from the patterned area during tapping mode AFM imaging. It means that in this case the interaction (58) Vandenberg, E. T.; Bertilsson, L.; Liedberg, B.; Uvdal, K.; Erlandsson, R.; Elwing, H.; Lundstrom, I. J. Colloid Interface Sci. 1991, 147, 103–118.

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Figure 1. (A-E) AFM images of APTES patterns generated by μCP on SiO2 with different “ink” concentrations: (A) 0.2%, (B) 0.5%, (C) 1%, (D) 2%, and (E) 5%. Z scale: 10 nm. Inset in (D): height profile of APTES patterns (top); three-dimensional structure and theoretical length of an APTES molecule (bottom).58 (F) Height analysis of APTES patterns in (A-E). Scheme 1. Schematic Illustration of Direct Fabrication of APTES Patterns by μCP and Further Applicationsa

a (1-4) μCP of APTES patterns on a substrate. (5) Selective adsorption of Au NPs on APTES patterns. (6) Growth of ZnO nanostructures on Au NP patterns. (7) Reaction of APTES patterns with glutaraldehyde. (8) Immobilization of proteins on glutaraldehyde.

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Figure 2. AFM images of (A, B) 1.5 μm width APTES lines

generated by μCP on mica, (C, D) selective adsorption of Au NPs on the APTES patterns in (A, B), and (E) selective adsorption of Au NPs on a smaller APTES patterns (400 nm) on mica. Z scale: (A, B) 10 nm and (C-E) 50 nm.

between Au NPs and the APTES patterns is very weak due to the low density of amino groups in the patterned substrates. Therefore, 1% APTES aqueous solution was selected as “ink” for our further experiments. Besides SiO2, other substrates with hydroxyl groups on the surface can also be patterned with APTES via the reaction of silanol with -OH groups. By using 1% APTES aqueous solution as “ink”, high-quality APTES patterns with 400 nm linewidth can be fabricated on mica, glass coverslips, quartz, and sapphire (Figure S2 in the Supporting Information). After the patterned substrates were immersed into Au NP solution (step 5 in Scheme 1), the citrate-capped Au NPs were selectively adsorbed onto the APTES patterns via electrostatic interaction.9,15-21 If APTES dissolved in ethanol or methanol was used as “ink”, the APTES molecules were present in the volatile gas phase due to their relatively low molecular weight, resulting their migration to the nonpatterned areas, which would also adsorb Au NPs.59 Obviously, our method shows great advantages, which can generate high-quality Au NP patterns without any notable nonspecific adsorption in the nonpatterned areas (Figure 2C-E and Supporting Information Figures S1 and S2B, D, G, J). (59) Mewe, A. A.; Kooij, E. S.; Poelsema, B. Langmuir 2006, 22, 5584–5587.

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3.2. Selective Adsorption of Au NPs on APTES Patterns on SiO2 and Sapphire, Used for Growth of ZnO Nanostructure Patterns. The ability to control the periodic semiconductor nanostructure arrays at a large scale is critical for integration of them in potential nanophotonic and nanoelectronic devices.60 Many groups are devoted to develop methods to achieve it, which includes nanosphere lithography61,62 and μCP.63 The latter one has better reproducibility and position controllability. ZnO is a valuable electronic and photonic material among semiconductor materials. It is widely used for making acoustic wave filters, photonic crystals, UV photodetectors, field-effect transistors, various diodes, optical modulator wave guides, and gas sensors.60,61 It has been reported that Au NPs can act as catalyst for the growth of ZnO nanostructures.61,62,64 By using the Au NPs adsorbed on APTES patterns as catalyst, the ZnO nanostructure patterns were successfully grown not only in solution based on the hydrothermal method but also in the gas phase by the CVD method (step 6 in Scheme 1, Figure 3). The locations of the grown ZnO nanomaterial arrays are strictly confined by the Au NP patterns. It shows that our approach provides a facile and well reproduced strategy to grow nanomaterials with large-scale periodicity, which is promising in many potential applications in sensors and devices. 3.3. Immobilization of Lectin Protein on APTES Patterns. After preparation of APTES patterns, the surface reactive amino groups are crucial to many applications including the construction of biomedical devices. In this work, the reactivity of the amino groups is demonstrated by decorating the APTES patterns with lectins. It has been reported that microcontactprinted lectin patterns can retain their interaction ability with cell surface carbohydrates, and regulate cell morphology and attachment frequency in microfabricated devices without invoking cell apoptosis.65 However, the hydrophobic PDMS stamp surface is conducive to enhanced protein aggregation and uneven spreading of the water-soluble polar biomolecules, thus making the microcontact-printed lectin patterns quite heterogeneous.65 Here, we show that much more homogeneous lectin patterns can be fabricated through the immobilization of lectins on the APTESpatterned glass coverslips by using glutaraldehyde as linker (steps 7 and 8 in Scheme 1). As shown in Figure 4, the uniform fluorescence signal from the FITC-lectin patterns was achieved, indicating the reactivity of amino groups in APTES patterns on substrates.24,51 In our method, the immobilization of lectins was conducted in solution, and thus, the loss of lectin bioactivity during direct patterning by μCP in air could be avoided.65 The generated lectin micropatterns by our method might keep their bioactivity and have potential applications in the cell interaction study. 3.4. Stepwise Selective Adsorption of Au NPs and Graphene Oxide and on the Cross-Patterned APTES on SiO2. Graphene is a two-dimensional material composed of only one atomic layer of carbon atoms. It is receiving increased attraction because of its high mobility, high saturation velocity for electrons and holes, stable crystal structure, and ultrathin layer thickness.66,67 Graphene oxide (GO), obtained after oxidation of (60) Fan, H. J.; Werner, P.; Zacharias, M. Small 2006, 2, 700–717. (61) Liu, D. F.; Xiang, Y. J.; Wu, X. C.; Zhang, Z. X.; Liu, L. F.; Song, L.; Zhao, X. W.; Luo, S. D.; Ma, W. J.; Shen, J.; Zhou, W. Y.; Wang, G.; Wang, C. Y.; Xie, S. S. Nano Lett. 2006, 6, 2375–2378. (62) Wang, X.; Summers, C. J.; Wang, Z. L. Nano Lett. 2004, 4, 423–426. (63) Hochbaum, A. I.; Fan, R.; He, R.; Yang, P. Nano Lett. 2005, 5, 457–460. (64) Ito, D.; Jespersen, M. L.; Hutchison, J. E. ACS Nano 2008, 2, 2001–2006. (65) Das, T.; Mallick, S. K.; Paul, D.; Bhutia, S. K.; Bhattacharyya, T. K.; Maiti, T. K. J. Colloid Interface Sci. 2007, 314, 71–9. (66) Geim, A. K.; Novoselov, K. S. Nat. Mater. 2007, 6, 183–191. (67) Novoselov, K. S.; Jiang, D.; Schedin, F.; Booth, T. J.; Khotkevich, V. V.; Morozov, S. V.; Geim, A. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10451–10453.

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Figure 3. Growth of ZnO nanostructures on Au NPs adsorbed on APTES patterns on SiO2 and sapphire. (A) Optical image of selectively adsorbed Au NPs on APTES patterns on SiO2. SEM images of ZnO nanostructures grown on SiO2 in solution (B) and on sapphire by CVD (C).

Figure 4. (A) Fluorescent confocal image of FITC-lectins immobilized on APTES-patterned glass coverslip by using glutaraldehyde as linker. (B) Magnified image of (A).

Figure 5. Stepwise selective adsorption of Au NPs and GO on cross-printed APTES patterns on SiO2. (A) Optical microscope image. Vertical lines represent Au NP patterns and horizontal lines represent GO patterns, as indicated by the white and red arrows, respectively. (B) AFM image. Z scale: 40 nm.

graphite,54 is one kind of graphene derivate. Printing graphene or GO sheets on the desired area might have applications in graphene-integrated circuits and devices.68,69 Since GO is negatively charged, as shown in our previous results, GO can be adsorbed on APTES-modified SiO2,54 ITO, and glassy carbon (68) Liang, X.; Fu, Z.; Chou, S. Y. Nano Lett. 2007, 7, 3840–3844. (69) Allen, M. J.; Tung, V. C.; Gomez, L.; Xu, Z.; Chen, L. M.; Nelson, K. S.; Zhou, C.; Kaner, R. B.; Yang, Y. Adv. Mater. 2009, 21, 2098–2102.

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electrode.55 After the APTES micropatterns were vertically fabricated on SiO2 by our method, the SiO2 substrate was immersed into Au NP solution, and Au NPs were adsorbed on the vertical APTES patterns.21 Then the horizontal APTES patterns were generated on the same SiO2 substrate, followed by adsorption of single-layer GO,21,54,55 which is confirmed by AFM (Figure S3 in the Supporting Information) and is consistent with our previous result.54 Thus, crossed Au NP and GO patterns were obtained on the same SiO2 substrate (Figure 5). Langmuir 2010, 26(8), 5603–5609

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The optical image shows that the vertical and horizontal lines, as indicated by the white and red arrows, respectively, correspond to the Au NP and GO patterns, respectively (Figure 5A). Obviously, the density of Au NPs is inhomogeneous in the crosspatterned area (Figure 5B), which can be explained as follows. After Au NP patterns formed on the vertical APTES microlines, some Au NPs could be removed after they contacted the APTES-coated PDMS stamp during the second-time fabrication of horizontal APTES patterns, used for adsorption of GO sheets. Therefore, some GO sheets can also be adsorbed in the crosspatterned area (Figure 5B).

4. Conclusion In summary, a facile method used for directly generating submicrometer APTES patterns by microcontact printing on hydroxyl-terminated substrates, such as SiO2, quartz, glass coverslips, sapphire, and mica, is presented. The high-quality homogeneous APTES patterns are successfully used as templates for selective adsorption of Au nanoparticles and graphene oxide,

Langmuir 2010, 26(8), 5603–5609

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immobilization of lectins, and growth of ZnO nanostructures. We believe this simple method will have applications in patterning and device fabrications. Acknowledgment. This work was supported by a Start-Up Grant from NTU, AcRF Tier 1 (RG 20/07) from MOE, CRP (NRF-CRP2-2007-01) from NRF, an A*STAR SERC Grant (No. 092 101 0064) from A*STAR, and the Centre for Biomimetic Sensor Science at NTU in Singapore. Supporting Information Available: APTES patterns generated with the “ink” concentration of 0.2% and 0.5% on SiO2 by μCP, which were used for adsorption of Au NPs with a low density; μCP of APTES patterns on mica, sapphire, glass coverslip, and quartz, and the selective adsorption of Au NPs on these patterns. Topographic image and height profile of single-layer GO adsorbed on an APTES pattern on SiO2. This material is available free of charge via the Internet at http://pubs.acs.org.

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