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A Simple Approach to Micropatterning and Surface Modification of Poly(dimethylsiloxane) Gerardo A. Diaz-Quijada and Danial D. M. Wayner* National Institute for Nanotechnology, National Research Council of Canada, Edmonton, AB, Canada Received May 18, 2004. In Final Form: July 23, 2004 Ozone treatment is an efficient economical, alternative method for surface activation of poly(dimethylsiloxane) (PDMS). This is illustrated by the derivatization of a PDMS surface with (3-aminopropyl)triethoxysilane (APTES). The apparent surface concentration of amino groups was found to be ca. 10-8 mol/cm2 using UV/visible spectroscopy of the product from the reaction of the amino groups and fluorescamine. Potential application for micropatterning of biologically active interfaces was demonstrated by the covalent immobilization of oligonucleotides. A simple process for photolithographic patterning on PDMS surfaces has been developed.
Introduction The chemical inertness, elasticity, low cost, and biocompatibility of poly(dimethylsiloxane) (PDMS) has led to a wide range of applications.1-4 An important challenge in the development of bioanalytical applications of PDMS is the development of reliable approaches to surface modification.2 A number of methods have been reported that include the following: adsorption of polymers or biomolecules on the surface, followed by covalent immobilization of desired biomolecules on the adsorbed material,5-7 introducing reactive groups at the surface of PDMS by blending or copolymerizing PDMS with a suitable polymer or monomer, respectively,8 grafting molecules with desirable groups using a radical initiator,9,10 and oxidation of the surface by ultraviolet, X-ray, gamma ray, and infrared irradiation.4,11-17 Surface oxidation has provided the most viable approach. For example, irradiation in air with UV light at 184 and 254 nm leads to in-situ generation of ozone and * Corresponding author. Tel.: (613) 993-1212. Fax: (613) 9545242. E-mail:
[email protected]. (1) Allcock, H. R.; Lampe, F. W. Contemporary Polymer Chemistry, 2nd ed.; Prentice Hall, Inc.: NJ, 1990; pp 575-590. (2) Abbasi, F.; Mirzadeh, H.; Katbab, A.-A. Polym. Int. 2001, 50, 1279-1287. (3) Kabanov, V. Y.; Aliev, R. E.; Kudryanvtsev, V. N. Radiat. Phys. Chem. 1991, 37, 175-192. (4) Hoffman, A. S. Radiat. Phys. Chem. 1981, 18, 323-342. (5) Barker, S. L. R.; Tarlov, M. J.; Canavan, H.; Hickman, J. J.; Locascio, L. E. J. Anal. Chem. 2000, 72, 4899-4903. (6) Linder, V.; Verpoorte, E.; Thormann, W.; de Rooij, N. F.; Sigrist, H. J. Anal. Chem. 2001, 73, 4181-4189. (7) Eteshola, E.; Leckband, D. Sens. Actuators, B 2001, B72, 129133. (8) Okaniwa, M.; Ohta, Y. J. Polym. Sci., Part A: Polym. Chem. 1997, 35, 2607-2617. (9) Okaniwa, M.; Ohta, Y. Polym. Prepr. 1997, 38, 673-674. (10) Chabrecek, P.; Dietliker, K.; Lohmann, D. PCT Int. Appl.; CibaGeigy, A.-G., Switz.: WO, 1996; p 76. (11) Genzer, J.; Efimenko, K. Science 2000, 290, 2130-2133. (12) Phely-Bobin, T. S.; Muisener, R. J.; Koberstein, J. T.; Papadimitrakopoulos, F. Polym. Prepr. 2000, 41, 812-813. (13) Phely-Bobin, T. S.; Muisener, R. J.; Koberstein, J. T.; Papadimitrakopoulos, F. Adv. Mater. 2000, 12, 1257-1261. (14) Khorasani, M. T.; Mirzadeh, H.; Sammes, P. G. Radiat. Phys. Chem. 1996, 47, 881-888. (15) Hari, P. R.; Sharma, C. P. J. Biomater. Appl. 1991, 6, 170-180. (16) Khorasani, M. T.; Mirzadeh, H. Iran. J. Polym. Sci. Technol. (Persian Ed.) 1997, 10, 107-125. (17) Khorasani, M. T.; Mirzadeh, H.; Sammes, P. G. Radiat. Phys. Chem. 1999, 55, 685-689.
oxygen atoms that oxidize PDMS12,18 to produce a shortlived highly hydrophilic, glasslike surface.19-21 A similar approach involves the generation of ozone using a corona discharge followed by UV photolysis at 254 nm.20,22,23 Probably one of the most successful approaches involves the generation of hydroxyl groups at the surface using oxygen plasma treatments,24-27 followed by reaction with trialkoxy-based silane reagents.28-31 We demonstrate herein that the surface of PDMS can be functionalized with (3-aminopropyl)triethoxysilane (APTES) after exposure to ozone without the need for photolysis. This is in contrast to results reported by Papadimitrakopoulos and co-workers, which suggest that PDMS is stable in the presence of ozone in the absence of UV light.12,13,18 The method reported herein has a number of advantages. Besides being simple and economical, it is suitable for a more homogeneous oxidation of three-dimensional structures of PDMS such as microchannels in microfluidic devices without the need to irradiate from different angles to achieve a homogeneous oxidation. In addition, this method roughens the surface without otherwise mechanically damaging the film, unlike other surface treatments such as irradiation with UV light,22 corona discharges,32 and oxygen plasma33 that all (18) Phely-Bobin, T. S.; Muisener, R. J.; Koberstein, J. T.; Papadimitrakopoulos, F. Synth. Met. 2001, 116, 439-443. (19) Ouyang, M.; Klemchuk, P. P.; Koberstein, J. T. Polym. Degrad. Stab. 2000, 70, 217-228. (20) Ouyang, M.; Yuan, C.; Muisener, R. J.; Boulares, A.; Koberstein, J. T. Chem. Mater. 2000, 12, 1591-1596. (21) Muisener, R. J.; Koberstein, J. T. Polym. Mater. Sci. Eng. 1997, 77, 653. (22) Efimenko, K.; Wallace, W. E.; Genzer, J. J. Colloid Interface Sci. 2002, 254, 306-315. (23) Genzer, J.; Efimenko, K. PCT Int. Appl.; North Carolina State University, USA: WO, 2002; p 31. (24) Chaudhury, M. K. Biosens. Bioelectron. 1995, 10, 785-788. (25) He, Q.; Liu, Z.; He, N.; Xiao, P.; Lu, Z. Proc. SPIE-Int. Soc. Opt. Eng. 2001, 4601, 406-411. (26) Donzel, C.; Geissler, M.; Bernard, A.; Wolf, H.; Michel, B.; Hilborn, J.; Delamarche, E. Adv. Mater. 2001, 13, 1164-1167. (27) Wilson, D. J.; Pond, R. C.; Williams, R. L. Interface Sci. 2000, 8, 389-399. (28) Ferguson, G. S.; Chaudhury, M. K.; Biebuyek, H. A.; Whitesides, G. M. Macromolecules 1993, 26, 5870-5875. (29) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550-575. (30) Ferguson, G. S.; Chaudhury, M. K.; Sigal, G. B.; Whitesides, G. M. Science 1991, 253, 776-778. (31) Chaudhury, M. K.; Whitesides, G. M. Science 1992, 255, 12301232.
10.1021/la048761t CCC: $27.50 © 2004 American Chemical Society Published on Web 10/02/2004
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lead to physical damage to the surface of PDMS producing “cracks” or other microstructures on the surface. These modified surfaces are suitable for subsequent chemical modification with trialkoxy-based silane reagents. The suitability of these surfaces for the covalent immobilization of biomolecules is demonstrated by the reversible hybridization of DNA. In addition, we report a new method for micrometer scale photolithographic patterning on PDMS surfaces that overcomes problems with lack of adhesion of resists. Experimental Section Sylgard 184 silicone elastomer kit was purchased from Dow Corning. Microscope slides (cat. # 48312-024) were from VWR Scientific, Inc. Sodium phosphate monobasic, disodium phosphate dibasic, and trisodium citrate were from BDH, Inc. Ethylenediamine tetraacetic acid (EDTA) and sodium chloride were from EM Science. Fluorescamine, 3-(aminopropyl)triethoxysilane (APTES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), Denhardt’s solution, tetramethylammonium chloride (TMACl), and sodium dodecyl sulfate (SDS) were from Aldrich. Molecular Biology (MB) grade DNA from fish sperm was from Roche Diagnostics. Single-stranded oligonucleotides were custom-synthesized by Biocorp Ltd. (Montreal, QC). Ozonolysis was performed with an Ozone generator (OZO2VTT) from O3zomax Ltd. Infrared spectra were collected on a Nicolet Magna 860 Fourier transform infrared spectrometer. UV/ visible spectroscopy was carried out with a Cary 3 spectrometer from Varian, Inc. Aluminum deposition was carried out with a Balzer BAK 600 electron beam evaporation system from Balzers Instruments. HPR-504 photoresist was purchased from OCG Materials, and the Microposit 354 developer was from Shipely. Irradiation of photoresist was accomplished with a 100 W Hg lamp, model 68806, from Oriel Instruments. Thin films (25-30 µm) of poly(dimethylsiloxane) (PDMS) were spin cast on microscope cover slips. Typically, surface functionalization of PDMS films involved exposure to ozone for 1 h followed immediately by immersion in a freshly prepared solution containing 5% APTES, 5% water, and 90% absolute ethanol for 30 min. Films were thoroughly rinsed with ethanol and water, dried under a stream of dry nitrogen, and finally heated to 60 °C for at least 2 h. Fluorescence detection of microarrays was accomplished with a Virtek chipreader from Carsen Group, Inc. Atomic force microscopy (AFM) images were acquired in air with a Nanoscope IIIa multimode microscope (Digital Instruments). The pristine PDMS sample was studied in contact mode using a silicon nitride tip (DNP, Digital Instruments), while the images of the chemically modified surfaces were obtained in tapping mode with a silicon cantilever with a resonance frequency of ∼300 kHz (TESP, Digital Instruments). All images were acquired unfiltered at a rate between 1.5 and 2 Hz. Root-meansquare (RMS) roughness was calculated on 10 × 10 µm regions after applying a second-order flattening procedure.
Results and Discussion We find that exposure of the surface of PDMS to ozone generates a significant number of hydroxyl groups as indicated by the broad band for the O-H stretch at 3400 cm-1 in the infrared spectrum of a free-standing film (Supporting Information, Figure S1) concomitant with a rapid disappearance of the absorption at 2158 cm-1 (due to the Si-H stretch in pristine PDMS from residual unreacted cross-linker). Oxidation of these Si-H groups probably involves the formation of a hydroperoxide intermediate.34,35 In addition to the loss of the Si-H peak, (32) Wang, B.; Chen, L.; Abdulali-Kanji, Z.; Horton, J. H.; Oleschuk, R. D. Langmuir 2003, 19, 9792-9798. (33) Owen, M. J.; Smith, P. J. J. Adhes. Sci. Technol. 1994, 8, 10631075. (34) Lacoste, J.; Israeli, Y. Polym. Prepr. 1993, 34, 127-128. (35) Israe¨li, Y.; Philippart, J.-L.; Cavezzan, J.; Lacoste, J.; Lemaire, J. Polym. Degrad. Stab. 1992, 36, 179-185.
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exposure to ozone generates carbonyl species as indicated by growth of peaks at 1725 and 1718 cm-1 followed by the appearance of a band at 1699 cm-1 (Supporting Information, Figure S2). The latter two absorptions have been observed during the photooxidation of PDMS and are believed to be associated with the formation of carboxylic acids.36 Overall, changes in the infrared absorption spectra are very similar to those reported for the oxidation of PDMS during ozone photolysis.37 It is also noteworthy that cleavage of the cross-links appears to be taking place at very long exposures of ozone because thin PDMS films dissolve in ethanol after exposure to ozone for 10 h. The generation of hydroxyl groups after exposure to ozone enables the subsequent functionalization of the PDMS surface with alkoxysilanes. For instance, 3-(aminopropyl)triethoxysilane (APTES) reacts efficiently with the oxidized surface. The presence of primary amino groups was confirmed by reacting the surface with fluorescamine to yield a highly fluorescent product.38,39 Fluorescamine itself and its hydrolysis products do not fluoresce. A control experiment indicated that ozone is required for functionalization. The actual concentration of the amino groups, which are chemically accessible, on the surface film was determined in situ using UV/visible spectroscopy. Fluorescamine is a colorless compound that reacts selectively with primary amines to yield a yellow product. A solution of fluorescamine (1.8 mg) and APTES (1 uL) in acetone (200 uL) was prepared. The surface concentration of accessible primary amino groups was determined in the following way. A calibration curve was constructed by dispensing different amounts of a stock solution containing fluorescamine and APTES into wells formed by a PDMS O-ring (0.7 thick, 6 and 9 mm inner and outer diameters) on the surface of microscope slides. The purpose of the O-ring was simply to constrain the area that contains the yellow product on the glass slide and to ensure that the entire volume was absorbing in the spectroscopic measurement. The solvent was evaporated under vacuum, and the absorbance of the films was measured by absorption spectrophotometry. A linear correlation was observed when plotting the amount of dispensed stock solution and the absorbance at 395 nm (Supporting Information, Figure S3). The density of amino groups on the APTES-modified PDMS surface was determined first by immersing a PDMS film (17 × 7 × 0.7 mm) in acetone (250 uL) containing fluorescamine (1.5 mg) to form the chromophore within the film. The film was dried under vacuum, and the absorbance was measured by spectrophotometry providing a linear calibration curve. The absorbance of the film was within the linear range of the calibration curve, leading to a primary amino group concentration of 4 × 10-8 mol/ cm2 (average of three samples). This surface concentration appears to be approximately 1 order of magnitude higher than that expected for a monolayer of APTES on a flat nonporous surface and implies the surface is surprisingly porous. The expected concentration was simply estimated from geometric considerations for the limiting case of a close-packed monolayer of 3-aminopropylsilanetriol molecules cross-linked through siloxane bonds on a flat surface using PCMODE (88.0), which employs an extended MM2 force field. (36) Israe¨li, Y.; Lacoste, J.; Cavezzan, J.; Lemaire, J. Polym. Degrad. Stab. 1993, 42, 267-279. (37) Xiao, D.; Zhang, H.; Wirth, M. Langmuir 2002, 18, 9971-9976. (38) Udenfriend, S.; Stein, S.; Bo¨hlen, P.; Dairman, W. Science 1972, 178, 871-872. (39) Bo¨hlen, P.; Stein, S.; Udenfriend, S. Arch. Biochem. Biophys. 1974, 163, 390-399.
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Figure 1. AFM images (10 × 10 µm2) of PDMS as a function of chemical modification. (A) Pristine PDMS (RMS ) 1.2 nm); (B) after exposure to O3 for 1 h (RMS ) 1.4 nm); and (C) after modification with APTES (RMS ) 0.7 nm). The RMS roughness indicates that surface oxidation under the conditions employed in this work does not lead to an appreciable increase in surface roughness. Moreover, chemical modification with APTES leads to surfaces with a decreased surface roughness. Chart 1
Analysis of the PDMS surfaces by AFM does not show any significant increase in roughness (Figure 1). This is in contrast to other surface treatments such as irradiation with UV light,22 corona discharges,32 and oxygen plasma33 that all lead to physical damage to the surface of PDMS producing “cracks” or other microstructures on the surface. The possibility that the higher apparent surface concentration may be due to permeation of APTES into the polymeric matrix from swelling in ethanol was considered. Indeed, it has been reported that PDMS increases its mass by 5.5% upon immersion for 24 h in ethanol.40 However, in-situ measurement of amino groups indicates that APTES within the polymeric matrix is still available for reaction, at least with relatively small molecules. Further spectroscopic evidence for the presence of the amino groups on the PDMS surface was obtained by acylation with an activated carboxylic acid. This condensation reaction is particularly useful because it may be used for immobilizing biomolecules on the surface of PDMS. The amino groups on the surface were reacted with acetic acid in the presence of EDC and NHS. The characteristic CdO and N-H stretches of the acetamide derivative were observed at 1655 and 3290 cm-1, respectively (Supporting Information, Figures S4 and S5). These are consistent with infrared bands observed in N-methyl acetamide (1657.9 and 3297.9 cm-1).41 Immobilization of DNA was illustrated with a singlestranded oligonucleotide composed of 20 Thymidine (ss(dT)20) units modified at the 5′ end with a carboxylic acid group (Chart 1). We estimated that 50% of the amino groups are accessible for this reaction. This was estimated by first reacting the APTES-modified surface with ss(dT20) in the presence of EDC/NHS followed by reaction (40) Duineveld, P. C.; Lilja, M.; Johansson, T.; Inganas, O. Langmuir 2002, 18, 9554-9559. (41) Pouchert, C. J. The Aldrich Library of FT-IR Spectra; 1st ed.; The Aldrich Chemical Co., Inc.: Milwaukee, WI, 1985; Vol. 1, p 754.
Scheme 1. Photolithographic Procedure for Selective Surface Chemical Modification of PDMS
with fluorescamine as described above. UV-visible absorption and fluorescence spectroscopy of the resulting film showed that only about one-half of the available reactive sites remained accessible. A microarray with this oligonucleotide (ss(dT)20) was constructed using a simple photolithographic process. One of the problems in carrying out photolithographic processing of PDMS is that photoresists do not adhere well to the polymer. We found that this problem may be circumvented by first evaporating a thin film of aluminum under vacuum. To illustrate the concept, aluminum metal (30 nm) was deposited on a thin film (25-30 µm) of PDMS. Photoresist (HPR 504) was spin coated and irradiated for 30 s through a photomask consisting of 100 µm squares with a 135 µm pitch. The irradiated photoresist was selectively removed with Microposit 354 developer for 13 s. The exposed regions of aluminum metal were then etched with a 1.6 M aqueous solution of H3PO4 (Scheme 1 and Supporting Information, Figure S6). The patterned PDMS film was oxidized and derivatized with APTES in the same manner as before (Scheme 2). Covalent immobilization of ss(dT)20 having a carboxylic acid linker was carried out with 2 mL of a 6.4 µM solution of the oligonucleotides in 0.01 M phosphate buffered saline (PBS) in the presence of EDC (79 mg) and NHS (13 mg) for 2 h. To minimize nonspecific binding, the microarray was subjected to a prehybridization and a hybridization procedure which employs fragmented DNA from fish sperm as a blocking agent. This protocol is described in the literature in great detail (protocol 8).42 Briefly, the sample was incubated with 30 uL of the prehybridization solution for at least 12 h at 37 °C. Hybridization was
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Scheme 2. Procedure for the Chemical Immobilization of OligoDNA on the Surface of PDMS
Figure 2. Fluorescent image of a PDMS surface patterned with 100 µm squares containing hybridized oligoDNA. The complementary strand was modified with Cy3 at the 5′ end. The fluorescent squares are not evident after denaturation but reappear after rehybridization. Chart 2
accomplished for 12-16 h at 37 °C with 30 uL of the hybridization solution containing 1.6 uM of its complementary strand labeled at the 5′ end with Cy3 to enable fluorescent detection (Chart 2). It should be noted that dextran sulfate was excluded from this procedure because it is known to cause high background. Subsequent washings with saline sodium citrate (6× SSC) buffer (pH 7.0) and 3 M tetramethylammonium chloride washing solution were performed as described in the literature, except for treatment of the sample at 54 °C with the quaternary ammonium salt. Imaging of the microarray with a complementary strand containing only one chromophore exhibited highly intense features (Figure 2) that were approximately 3 times more intense as compared to typical DNA arrays on glass containing approximately 15 Cy3 labels per strand (i.e., about 50 times more intense on a per dye basis), which is consistent with our suggestion of a chemically accessible porous film. To evaluate the extent of nonspecific adsorption of the Cy3-labeled DNA, the following control was carried out. A PDMS film was spin coated on a microscope cover slip (18 × 18 mm, No. 1 1/2, VWR). Surface modification of PDMS with amino groups was carried out in the same manner as described above. The amino-modified film was masked with a thick PDMS film that possessed three orifices with a diameter of 3 mm. Covalent immobilization of oligonucleotides in 0.01 M PBS (100 uL, 6 uM) was achieved in the presence of EDC (31 µmol) and NHS (13 µmol) for 7 h. Two of the exposed regions of the aminomodified PDMS film were modified with (dT)20-COOH, while the third region was derivatized with (dG)20-COOH. (42) Sambrook, J.; Russell, D. W. Molecular Cloning. A laboratory Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: New York, 2001; Vol. 2, pp 10.35-10.37.
Subsequently, the film was unmasked and washed in a 0.1% solution of SDS in PBS for 5 min. Prehybridization and hybridization were carried out with a mixture of (dA)20Cy3 and (dC)20-Cy5 each at a concentration of 1.6 µM. Confocal fluorescence imaging showed that hybridization occurred exclusively in the expected regions and that nonspecific binding was undetectable (see Supporting Information, Figure S7). Dehybridization of the duplex DNA was typically accomplished by immersing the microarray in 10 mL of 10% SDS for 20 min at 70 °C. This was confirmed by the loss of fluorescence signal after this treatment. Hybridization-rehybridization was carried out three times with about 50% loss of fluorescent intensity. We did not attempt to optimize the dehybridization conditions and used a method that is known to be very harsh. Conclusions Ozone, in the absence of UV irradiation, is an effective reagent for the chemical modification of PDMS. This method is less harsh than other methods reported so far to generate reactive hydroxyl groups on the surface of PDMS. It is not only simple and economical, but also it lends itself for homogeneous modification of threedimensional structures. The oxidized surface can be further derivatized with alkyltrialkoxysilane reagents as demonstrated with APTES. The surface concentration of amino groups from APTES that are accessible for further reactions was estimated to be 4 × 10-8 mol/cm2, about an order of magnitude higher than was expected, suggesting that the surface film is porous. The utility of this method for the covalent immobilization of biomolecules was illustrated with the fabrication of a two-dimensional microarray of a single-strand 20mer oligonucleotide containing only thymine base units. Hybridization was observed using the fluorescently labeled complementary strand with adenine base units. A new method for micropatterning PDMS using a photolithographic procedure that overcomes resist adhesion problems is also presented. This high surface concentration may prove advantageous for applications in microarray and microfluidic technologies. For example, it will be possible to print smaller features on microarrays using these surfaces without sacrificing signal (total brightness) because the same amount of total DNA can be immobilized in an element about one tenth the diameter as compared to
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glass. Work using this method to chemically modify microfluidic channels is currently underway. Acknowledgment. We would like to thank the Natural Sciences and Engineering Research Council of Canada for its financial support and Shane Boisclair (Institute for National Measurement Standards, NRC) for depositing aluminum metal films on the PDMS.
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Supporting Information Available: Infrared spectra of PDMS as a function of ozonolysis time. UV/visible calibration curve for quantification of amino groups. Infrared spectra of acetamide-modified surfaces of PDMS. Micropatterned PDMS film. Competitive hybridization experiment. This material is available free of charge via the Internet at http://pubs.acs.org. LA048761T
