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Protein Micropatterning on Bifunctional Organic-Inorganic Sol-Gel Hybrid Materials Woo-Soo Kim,†,‡ Min-Gon Kim,§ Jun-Hyeong Ahn,§ Byeong-Soo Bae,† and Chan Beum Park*,† Department of Materials Science and Engineering, Korea AdVanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon, 305-701, Republic of Korea, and Bio-Nano Technology Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Oeun-dong, Yuseong-gu, Daejeon, 305-806, Republic of Korea ReceiVed January 10, 2007. In Final Form: February 25, 2007 Active protein micropatterns and microarrays made by selective localization are popular candidates for medical diagnostics, such as biosensors, bioMEMS, and basic protein studies. In this paper, we present a simple fabrication process of thick (∼20 µm) protein micropatterning using capillary force lithography with bifunctional sol-gel hybrid materials. Because bifunctional sol-gel hybrid material can have both an amine function for linking with protein and a methacryl function for photocuring, proteins such as streptavidin can be immobilized directly on thick bifunctional sol-gel hybrid micropatterns. Another advantage of the bifunctional sol-gel hybrid materials is the high selective stability of the amine group on bifunctional sol-gel hybrid patterns. Because amine function is regularly contained in each siloxane oligomers, immobilizing sites for streptavidin are widely distributed on the surface of thick hybrid micropatterns. The micropatterning processes of active proteins using efficient bifunctional sol-gel hybrid materials will be useful for the development of future bioengineered systems because they can save several processing steps and reduce costs.
Introduction The ability to control the selective immobilization of biomaterials on micro and nanoscale areas is important for the development of bioengineered systems, such as biosensor, labon-a-chip, or bio-MEMS structures.1 Selectively immobilized microarray technology enables high-throughput drug screening and rapid sensing analysis for medical diagnostics.2 Reliable, simple, and inexpensive patterning methods are essentially required for the commercialization of protein microarrayed chips in the biomedical diagnostic field.3 The design and fabrication of biologically functional surfaces are significant steps in the production of such cheap microdevices. Two existing processes for producing patterned surface modifications for bioapplications are optical photolithography4 and soft lithography.5 Photolithography is a well-established technique for the bulk manufacture of integrated circuits. However, the conventional solutions used to develop and strip the photoresist are largely incompatible with biological molecules. * Corresponding author. Phone: +82-42-869-3340. Fax: +82-42-8693310. E-mail:
[email protected]. † Korea Advanced Institute of Science and Technology. ‡ Present Address: Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA. § Korea Research Institute of Bioscience and Biotechnology. (1) (a) Macbeath, G.; Schreiber, S. L. Science 2000, 289, 1760. (b) Frey, W.; Meyer, D. E.; Chilkoti, A. AdV. Mater. 2003, 15, 248. (c) Wilson, D. S.; Nock, S. Angew. Chem., Int. Ed. 2003, 42, 494. (d) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55. (2) (a) Patel, N.; Padera, R.; Sanders, G. H. W.; Cannizzaro, S. M.; Davies, M. C.; Langer, R.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Shakesheff, K. M. FASEB J. 1998, 12, 1447. (b) Ostuni, E.; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2000, 16, 7811. (3) (a) Lee, K. B.; Yoon, K. R.; Woo, S. I.; Choi, I. S. J. Pharm. Sci. 2003, 92, 933. (b) Lee, K. B.; Lim, J. H.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 5588. (c) Lim, J. H.; Ginger, D. S.; Lee, K. B.; Heo, J.; Nam, J. M.; Mirkin, C. A. Angew. Chem., Int. Ed. 2003, 42, 2309. (4) Brack, H. P.; Padeste, C.; Slaski, M.; Alkan, S.; Solak, H. H. J. Am. Chem. Soc. 2004, 126, 1004. (5) Lopez, G. P.; Biebuyck, H. A.; Harter, A. R.; Kumar, A.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10774.
For example, proteins easily malfunction in the harsh environments of photolithography. It is difficult to preserve their character during processes due to their complex and fragile structures. As a result, the processes for protein microarray formation must be gentle enough to preserve the protein functionality. Soft lithography, including imprint lithography,6 microcontact printing,7 inkjet printing process,8 and capillary force lithography (CFL),9 are emerging lithographies for bioapplications. In particular, CFL uses capillary force as a tool for the patterning of polymeric materials and fulfills patterning without harsh solvents or chemicals. When a liquid wets a capillary tube and if the wetting results in the reduction of free energy, the wetting leads to a capillary rise in the liquid.10 Selectively immobilized substrate materials for proteins may have carboxylic acid11 or aldehyde-terminated12 chemistry to connect with different kinds of proteins. Proteins can be attached to aldehydes through free amines, forming imine bonds.13 Because photoactive materials typically used for patterning do not contain these kinds of bioactive chemistry, there have been many studies for conferring these biofunctionalities to patterned surfaces. Molecular assembly patterning by lift-off (MAPL) was introduced recently.14 The MAPL technique converts a pre-structured resist (6) (a) Hoff, J. D.; Cheng, L. J.; Meyhofer, E.; Guo, L. J.; Hunt, A. J. Nano Lett. 2004, 4, 853. (b) Kim, W. S.; Choi, D. G.; Bae, B. S. Nanotechnology 2006, 17, 3319. (7) Shirahata, N.; Yonezawa, T.; Minura, Y.; Kobayashi, K.; oumoto, K. Langmuir 2003, 19, 9107. (8) Kimura, J.; Kawana, Y.; Kuriyana, T. Biosensors 1988, 4, 41. (9) (a) Suh, K. Y.; Kim, Y. S.; Lee, H. H. AdV. Mater. 2001, 13, 1386. (b) Suh, K. Y.; Seong, J.; Khademhossenini, A.; Laibinis, P. E.; Langer, R. Biomaterials 2004, 25, 557. (10) Kim, E.; Xia, Y.; Whitesides, G. M. Nature 1995, 376, 581. (11) Kenseth, J. R.; Harnisch, J. A.; Jones, V. W.; Porter, M. D. Langmuir 2001, 17, 4105. (12) Wadu-Mesthrige, K.; Xu, S.; Amro, N. A.; Liu, G. Y. Langmuir 1999, 15, 8580. (13) Christman, K. L.; Requa, M. V.; Enriquez-Rios, V. D.; Ward, S. C.; Bradley, K. A.; Turner, K. L., Maynard, H. D. Langmuir 2006, 22, 7444. (14) (a) Falconnet, D.; Koenig, D.; Assi, A.; Textor, M. AdV. Funct. Mater. 2004, 14, 749. (b) Falconnet, D.; Pasqui, D.; Park, S.; Eckert, R.; Schift, H.; Gobrecht, J.; Barbucci, R.; Textor, M. Nano Lett. 2004, 4, 853.
10.1021/la070074p CCC: $37.00 © 2007 American Chemical Society Published on Web 03/23/2007
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Langmuir, Vol. 23, No. 9, 2007 4733
Figure 1. (a) Schematic diagram of major components and synthesized oligomer units of the biohybrimer. (b) Schematic diagram of the fabrication process for the biohybrimer micropatterns by capillary force lithography and SEM images of biohybrimer replica structures with ∼20 µm line widths, respectively.
film into a pattern of biointeractive chemistry in a non-interactive background using the spontaneous, monomolecular assembly of biofunctional moieties.14 An attractive feature of MAPL lies in its ability to vary and control the surface density of bioactive materials in the biointeractive regions of the pattern. However, this process needs photoresist patterning and the self-assembly of biofunctional materials onto the pre-patterned surface. Unfortunately, these several steps have drawbacks. The cost is high and they require long, serial processes in sample preparation. In this work, we present a simple fabrication process for a thick, protein micropatterning using capillary force lithography with bifunctional sol-gel hybrid materials. Because bifunctional sol-gel hybrid material has both an amine function for linking with streptavidin and a methacryl function for photocuring, a protein can be attached directly to the thick bifunctional sol-gel hybrid micropatterns. This thick, hybrid pattern has advantages not only with the organic flexibility of the polymeric surface but also with the inorganic mechanical stability from the inorganic glassy nature of the hybrid material. Amino-functional materials
that attach biomolecules to surfaces are versatile because they react with many proteins and peptides under mild conditions.15 There is no need for any special chemical etching step of patterning because the capillary force lithography allows rapid prototyping for high volume production of fully patterned substrates. Other advantages of the hybrid bioactive pattern are thermal stability over 300 °C and easy pattern size control by changing the pattern size of the PDMS mold. Experimental Section Fabrication of a Bifunctional Sol-Gel Hybrid Material. The bioactive hybrid material was newly designed and synthesized to induce selective immobilization of proteins and to be easily UVcurable. Silicon alkoxide precursors for the bifunctional sol-gel hybrid material (biohybrimer) were (3-aminopropyl)trimethoxysilane(APTMS, Aldrich) for amino-functionality and 3-(trimethoxysilyl) propyl methacrylate (MPTMS, Aldrich) as a polymerizable (15) Lemieux, G. A.; Bertozzi, C. R. Trends Biotechnol. 1998, 16, 506.
4734 Langmuir, Vol. 23, No. 9, 2007 organo-alkoxy silane. APTMS and MPTMS were mixed at molar ratios of 1:5, 1:3, 1:1, 3:1, and 5:1, respectively. These ratios were chosen to balance the two functionalities to make an optimal biohybrimer. In addition, control of the quantities of APTMS and MPTMS could tune the photoactivity and immobilization site of the fabricated biohybrimer. APTMS and MPTMS were first hydrolyzed with 0.75 equiv of 0.01 M hydrochloric acid (HCl) in an N2 atmosphere. After that, the hydrolyzed APTMS solution was added to the pre-hydrolyzed MPTS solution and stirred for 1 h to advance hydrolysis and condensation with propylene glycol methyl ether acetate (PGMEA, Aldrich, purity 99%) as an unreactive solvent. The mixed solution was reacted with additional water for 10 h to complete the hydrolysis and condensation reactions. The total amount of water contained 1.5 equiv of total alkoxides in the solution. After the reaction to synthesize the biohybrimer solution, any residual products, such as alcohols, were removed at 50 °C with an evaporator. The biohybrimer was optically transparent due to the highly condensed methacrylic-oligosiloxane structure and highly aminofunctionalized due to the large quantity of amino-groups. The viscosity of the hybrimer could be controlled from 20 to 220 mPa.s by altering the content of PGMEA solvent and through thermal heating.16 Capillary Force Lithography of Bifunctional Biohybrimer. In the CFL presented here, the essential feature of imprinted lithography, i.e., molding a liquid biohybrimer, was combined with the major process of microcontact printing, i.e., using a poly (dimethylsiloxane) (PDMS) elastomeric mold.17 As an advantage over microcontact printing, a stringent thick micropattern for selective immobilization of proteins can be acquired with the CFL method. We used PDMS (Sylgard 184, Dow Corning, Midland) as an elastomeric mold and fabricated a PDMS master that has a planar surface with protruding micropatterns by casting PDMS against a complimentary relief structure prepared by photolithography or electron beam lithography. Silicon wafer (100) was used as the substrate. After conformal contact of the PDMS mold to the bare silicon substrate, the biohybrimer was injected from the one side edge of the mold for capillary injection along pattern lines. For enhancing capillary force injection of the biohybrimer into pattern lines, the PDMS mold, while in contact with the substrate, was pressed in the vacuum chamber with vacuum pressure of 1000 mbar and a compression time of 60 s. Because the biohybrimer has photocurable moiety, i.e., methacryl group containing 1.0 wt % of photoinitiator (Irgacure369, Ciba Geigy), UV light (80 mJ/cm2, 365 nm wavelength) cross-linked biohybrimer oligomers only within 20-30 s. Finally, the negative replica of the thick biohybrimer micropattern was obtained after peeling off the PDMS mold. Characterization of Biohybrimer. The synthesized transparent biohybrimer was passed through a 0.45 µm filter to remove impurities and bubbles. Then, the synthesis result of the structural evolutions in the biohybrimer was examined through Fourier transform-infrared (FT-IR) spectroscopy (JASCO, FT-IR 460plus) and direct 29Si NMR spectroscopy. Direct 29Si NMR spectra were recorded using a Bruker FT 500 MHz instrument from a sample consisting of 30% volume of the biohybrimer resin in chloroform-d. Chromium (III) acetylacetonate was added at a concentration of 30 mg/L as a relaxation agent for silicon. Pulse delays were 30 s, the sample temperature was 300K, and TMS was used as a reference. Immobilization of Streptavidin onto the Biohybrimer Micropattern. To confirm the biohybrimer pattern on silicon wafer, streptavidin-biotin affinity binding was applied to the formation of the biohybrimer pattern. The amine-functionalized, biohybrimer patterned surface was washed and ultrasonicated with ethanol. Next, the biohybrimer pattern was reacted with 1 mg/mL NHS-biotin (EZLink, Pierce, USA) in a pH 7.4 phosphate-buffered saline (PBS) buffer to form the biotin surface. NHS-biotin was dissolved in dimethyl sulfoxide to achieve a 10 mg/mL concentration and diluted to a 1 mg/mL concentration with a pH 7.4 PBS buffer. Then, the (16) Kim, W. S.; Kim, K. S.; Kim, Y. C.; Bae, B. S. Thin Solid Fims 2005, 476, 181. (17) Park, J. P.; Lee, K. B.; Lee, S. J.; Park, T. J.; Kim, M. G.; Chung, B. H.; Lee, Z. W.; Choi, I. S.; Lee, S. Y. Biotechnol. Bioeng. 2005, 92, 160.
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Figure 2. (a) Direct 29Si NMR spectrum of biohybrimer condensed with APTMS and MPTMS. (b) FT-IR spectra of the structural evolutions of the biohybrimer before and after UV curing. surface was blocked with 1 mg/mL BSA in pH 7.4 PBST. To form a biotin surface, 10 µg/mL Cy5-labeled streptavidin (Sigma-Aldrich, Inc., USA) in pH 7.4 PBS was reacted on the biohybrimer pattern for 30 min. The fluorescence image was obtained with a GenePix professional 4200A fluorescence image scanner (Axon Instrumentsm, Union, CA) and photospectrometer (fluorescence microscope, Nikon, Japan, and CCD camera, Andor technology, USA). PBS containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4 was adjusted to pH 7.4 with HCl and filtered (0.22 µm Millipore filters). A PBST buffer was prepared with 0.05% Tween 20 in a pH 7.4 PBS buffer.
Results and Discussion A series of biohybrimer was prepared by variation of the relative content of two silanes. The ratios of the two were chosen by balancing two functionalities for the optimal biohybrimer, i.e., a relatively high portion of amino-group can bind with more protein on the surface and a relatively high portion of methacrylic group can cross-link for a much denser structural pattern. Increase of the content of APTMS in the mixture may give considerable rise in the amounts of immobilized protein, but it was difficult to synthesize a biohybrimer with a high molecular portion of APTMS at over 60 mol % in the mixture because of the easy gelation character of APTMS. From 60 to 100 mol % of APTMS in the mixture, biohybrimer became a gel within 24 h after synthesis. But the biohybrimer containing less than 60 mol % APTMS showed a stable solution condition. After all, the molar ratio 1:1 of APTMS and MPTMS was chosen for the compromising ratio of biohybrimer patterning. Figure 1a schematically illustrates major components of the biohybrimer and the
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Langmuir, Vol. 23, No. 9, 2007 4735
Figure 3. (a) A red fluorescent micrograph of site-specific streptavidin patterning that was selectively adsorbed on the 20 µm lines of the biohybrimer surface. The image was obtained by the capillary force lithography of the biohybrimer followed by immobilization of Cy5streptavidin. The graph shows the estimated fluorescence intensities. (b) A green fluorescent micrograph of site-specific streptavidin patterning on the array of 5 µm lines of the biohybrimer surface and the estimated fluorescent intensities.
synthesized different sized oligomers. These oligomer units can be cross-linked with a sufficient UV dosage during CFL patterning. A schematic of the CFL for micropatterning of the biohybrimer is shown in Figure 1b. In our experiment, a PDMS mold was placed directly onto the bare silicon substrate and the biohybrimer resin was injected into the one side edge of the PDMS mold, forming a conformal contact with the surface. By applying vacuum pressure, the biohybrimer, in contact with the protruding PDMS features, receded spontaneously and moved into the void space of the PDMS mold by means of capillary action. Figure 1b shows SEM images of the negative replica of the PDMS mold, which has 40 µm size line widths. The replicated pattern shape shows the volume shrinkage due to evaporation of the PGMEA solvent. No residual biohybrimer remained on the substrate surface between pattern lines, and the substrate was exposed as depicted in Figure 1b. The formation of biohybrimer oligomers was further studied by 29Si NMR and FT-IR. 29Si NMR was used to confirm the degree of condensation and the formulated structure of the siloxane bond in biohybrimer reacted with APTMS and MPTMS in a 1:1 ratio. Figure 2a shows 29Si NMR spectra of the biohybrimer’s siloxane structures. The notations of Si atoms in NMR
Table 1. Chemical Shifts of Silicon in the Liquid 29Si NMR Spectroscopy According to Its Bond Statesa species Tn
chemical shifts
T0
-40 ppm -48 to -52 ppm -55 to -61 ppm -65 to -71 ppm
T1 T2 T3
a Tn)RSi(OMe) 3-n-m(OH)m(OSi)n,whereR)-CH2CH2CH2O(CO)C(CH3)CH2, -CH2CH2CH2NH2.
spectroscopy are as follows: Tn represents Si from APTMS and MPTS, respectively. The superscript, n, denotes the number of siloxane bonds of the Si atom. The chemical shifts of Si in 29Si NMR spectroscopy according to its bond states are shown in Table 1.18 As shown in Figure 2a, there were no T0 peaks, which suggests that the hydrolysis and condensation reactions of two different precursor molecules proceeded almost to completion and that the biohybrimer had been successfully synthesized. T2 and T3 peaks show a much higher degree of condensation than the T1 peaks. The T2 peak is the main peak, and T3 peaks are (18) Soppera, O.; Croutxe-barghorn, C. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 716.
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relatively small, which indicates the three-dimensional siloxane networks occur in a comparably smaller quantity. Moreover, the inorganic condensation degree in the biohybrimer could be calculated by integrating the individual signals from 29Si NMR spectra of the biohybrimers, according to the different chemical shift of the hydrolysis and condensation species of the silane components (eq 1):19
degree of condensation (%) ) 0 × T0 + 1 × T 1 + 2 × T 2 + 3 × T 3 3
3×
∑T
(1)
n
n)0
where T is the Si-species with three hydrolyzable silane compounds and n in Tn is the number of siloxane bonds on the Si-atom. According to the calculation, the degree of condensation of the biohybrimer was about 64%. This condensation value may be higher if the poly condensation reaction time was lengthened. Figure 2b shows the infrared spectra of the biohybrimer samples before and after UV curing. It was observed that uncured and cured biohybrimer had no absorption at 3342-3420 cm-1, a band that corresponds to OH stretching of SiOH, CH3OH, and H2O. This also means that the almost hydrolyzed species were poly condensed well to biohybrimer oligomers. The spectra of each film show signals at 1580 cm-1 that correspond to N-H scissoring vibration and signals at 1486 cm-1 that should correspond to the symmetric NH+3 deformation mode.20 According to literature,21 CdC stretching vibrational mode resulting from the methacrylic groups appears at 1640 cm-1. In the sample spectra before curing, this peak was observed, but there was no CdC stretching vibrational mode in that of the sample after UV curing. This shows the complete cross-linking reaction between the oligomers of biohybrimer during UV curing has been fulfilled for highly compacted organic-inorganic hybrid materials. In this work, streptavidin was used as a model protein. For the immobilization of streptavidin onto a biohybrimer micropattern by CFL, the NHS-biotin was first bonded with the amino(19) Sepeur, S.; Kunze, N.; Werner, B.; Schmidt, H. Thin Solid Films 1999, 351, 216. (20) Shmizu, I.; Okabayashi, H.; Taga, K.; Nishio, E. Vib. Spectrosc. 1997, 14, 113. (21) Kim, W. S.; Houbertz, R.; Lee, T. H.; Bae, B. S. J. Polym. Sci., Part B: Polym. Phys. 2004, 42, 1979.
function of biohybrimer pattern. Later, red fluorescent streptavidin was reacted on the NHS-biotin connected biohybrimer pattern. In this manner, a well-defined pattern of 20 µm stripe repeats was fabricated on the thick biohybrimer on the silicon substrate. As shown in Figure 3a,b, the selective binding of the Cy5-labeled streptavidin to two different types of biohybrimer patterns could be visualized as a red or a green fluorescent micrograph. The estimated fluorescence intensities for each case are also illustrated in the Figure 3. To examine whether the binding of Cy5-labeled streptavidin was specific to the NHS-biotin connected biohybrimer pattern, a bare silicon substrate was prepared and used in the same manner for generating the streptavidin protein micropatterns. According to our observation, no fluorescence was observed, which suggests that streptavidin was exactly bonded only onto the biohybrimer patterns. In order to confirm whether streptavidin retained its binding ability after several days, the fluorescence intensities of bound streptavidin were investigated after 15 days. Most streptavidin (∼98%) remained bound compared to the first measured intensity of fluorescence, which means that the selective immobilization of streptavidin onto the stable biohybrimer pattern could be maintained for 15 days or more.
Summary and Conclusion We report here a simple protein micropatterning process with bifunctional sol-gel hybrid materials by capillary force lithography. This process did not require any chemical etching step, high-temperature step, or solvent evaporation step for patterning protein. By introducing thermally and chemically stable bifunctional sol-gel hybrid materials as the patterned media, spatially well-defined, thick images of selective protein adsorption were observed in a wide area. Because of the amino-function of wellcondensed bifunctional hybrid material, this methodology can be easily extended to the patterning of other peptides and proteins. In addition, sub-micron scale protein patterning may be possible with a sub-micron scale mold. As demonstrated by the stable immobilization of streptavidin in a micropattern, the concept of patterning bifunctional sol-gel hybrid material appears to be very flexible for establishing platforms for biomolecular immobilization. Acknowledgment. This work was supported by the Brain Korea 21 project (BK21) and the Sol-Gel Innovation Project (SOLIP) of the Ministry of Commerce, Industry and Energy of Korea. LA070074P
