© Copyright 2004 American Chemical Society
OCTOBER 12, 2004 VOLUME 20, NUMBER 21
Letters High-Resolution Technique for Fabricating Environmentally Sensitive Hydrogel Microstructures Ming Lei,† Yuandong Gu,‡ Antonio Baldi,†,| Ronald A. Siegel,‡,§ and Babak Ziaie*,†,§ Departments of Electrical and Computer Engineering, Pharmaceutics, and Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, and Centro Nacional de Microelectro´ nica (CNM-IMB), CSIC, Campus UAB, E-08193 Bellaterra, Spain Received May 24, 2004 This communication introduces a novel high-resolution technique for fabricating hydrogel microstructures using photoresist lithography and dry etching. This method alleviates the need for photoinitiators used in conventional approaches and is applicable to a broad range of hydrogels. This technique is also compatible with traditional microfabrication methods, thus allowing the integration of hydrogels with microelectronics and microelectromechanical system microstructures. Environmentally sensitive hydrogels of different shapes and sizes were patterned in a batch scale (wafer-level) with resolutions down to 2.5 µm. The patterned pH-sensitive hydrogels with micron-sized dimensions exhibit volume responses within seconds of change in pH. Deposited aluminum thin films on top of hydrogel microstructures were used to fabricate environmentally sensitive free-standing micromirrors with a vertical displacement sensitivity of 7 µm/pH at pH 5.0. These structures open the possibility of fabricating on-chip photonic-based hydrogel microsensors.
Introduction Environmentally sensitive hydrogels offer numerous opportunities in biomedical sensing and active flow control.1-5 These three-dimensional cross-linked polymer networks are capable of undergoing reversible volume * Corresponding author. Phone: (612) 625-1574. Fax: (612) 6254583. E-mail:
[email protected]. † Department of Electrical and Computer Engineering, University of Minnesota. ‡ Department of Pharmaceutics, University of Minnesota. § Department of Biomedical Engineering, University of Minnesota. | Centro Nacional de Microelectro ´ nica (CNM-IMB), CSIC. (1) Siegel, R. A.; Falamarzian, M.; Firestone, B. A.; Moxley, B. C. J. Controlled Release 1988, 8(2), 179-82. (2) Cao, X.; Lai, S.; Lee, J. L. Biomed. Microdevices 2001, 3 (2), 10917. (3) Han, I. S.; Han, M. H.; Kim, J.; Lew, S.; Lee, Y. J.; Horkay, F.; Magda, J. J. Biomacromolecules 2002, 3 (6), 1271-5. (4) Liu, R. H.; Yu, Q.; Beebe, D. J. J. Microelectromech. Syst. 2002, 11 (1), 45-53.
change in response to different stimuli, such as temperature,6 pH,7 glucose concentration,8 and so forth. Such behavior makes hydrogels attractive as components of sensors and actuators operating in aqueous media such as body fluids. However, practical applications of macrosized (millimeter range) hydrogels have been limited due to their long response time (several hours). Response times of hydrogels can be reduced to minutes or seconds by reducing their dimensions and integrating them into microelectromechanical systems (MEMS).8,9 (5) De, S. K.; Aluru, N. R.; Johnson, B.; Crone, W. C.; Beebe, D. J. J. Microelectromech. Syst. 2002, 11 (5), 544-55. (6) Hu, Z.; Zhang, X.; Li, Y. Science 1995, 218, 525-7. (7) Tanaka, T.; Fillmore, D.; Sun, S. T.; Nishio, I.; Swislow, G.; Shah, A. Phys. Rev. Lett. 1980, 45, 1936-9. (8) Baldi, A.; Gu, Y.; Loftness, P. E.; Siegel, R. A.; Ziaie, B. J. Microelectromech. Syst. 2003, 12 (5), 613-21. (9) Baldi, A.; Lei, M.; Gu, Y.; Siegel, R. A.; Ziaie, B. MEMS 2003: The 16th IEEE International Conference on Microelectromechanical Systems, Kyoto, Japan, January 19-23, 2003; IEEE: Piscataway, NJ, 2003; Vol. 12, pp 84-7.
10.1021/la048719y CCC: $27.50 © 2004 American Chemical Society Published on Web 09/17/2004
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Figure 1. Sequence of patterning steps. Scheme I, pattern transfer using photoresist mask. Scheme II, pattern transfer using resist/aluminum mask.
Several hydrogel-based microfluidic structures have been fabricated through the infusion of pre-gel solution in microchannels, followed by in situ photopolymerization.10 Hydrogel patterning on various surfaces has also been attempted in fabricating biochemical microsensors.11 The conventional approach has been to photopolymerize a mixture of monomers and a photoinitiator through a photomask.12 For example, Revzin and co-workers spincoated a viscous precursor solution, a mixture of poly(ethylene glycol) macromer and a photoinitiator, and then patterned the resulting thin film using UV lithography.13 The patterned hydrogel immobilized a pH-sensitive dye and demonstrated a pH response. More recently, micromolding has been proposed as a means for selectively depositing hydrogel on various surfaces.14 In this communication, a new patterning approach is introduced that involves traditional photolithography and is applicable to a wide variety of hydrogels. This method alleviates the need for a photoinitiator through a combination of photoresist lithography and dry etching in oxygen plasma, hence simplifying the chemistry. Using this technique, we were able to pattern hydrogel features with 2.5 µm resolution on silicon and glass substrates. We have also fabricated free-standing environmentally (10) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; Yu, Q.; Liu, R. H.; Devadoss, C.; Jo, B. H. Nature 2000, 404, 588-90. (11) Hilt, J. Z.; Gupta, A. K.; Bashir, R.; Peppas, N. A. J. Biomed. Microdevices 2003, 5, 177-84. (12) Hoffmann, J.; Plotner, M.; Kuckling, D.; Fischer, W. Sens. Actuators 1999, 77, 139-44. (13) Revzin, A.; Russell, R. J.; Yadavalli, V. K.; Koh, W. G.; Deister, C.; Hile, D. D.; Mellott, M. B.; Pishko, M. V. Langmuir 2001, 17, 5440-7. (14) Tang, M. D.; Golden, A. P.; Tien, J. J. Am. Chem. Soc. 2003, 125, 12988-9.
Figure 2. A variety of hydrogel shapes patterned on a glass substrate. All black bars are 40 µm long.
sensitive micromirrors on top of hydrogel columns with potential applications to chemo-optical microsensors. Materials and Methods Three hydrogel compositions were used in this study. Recipes for pre-gel solutions are listed below.
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Figure 3. SEM images of (a) 5 µm wide lines, (b) 5 µm squares of MPBA-co-AAm hydrogel and (c) 2.5 µm wide lines, and (d) 2.5 µm squares of NIPA-co-mAA which became circles due to the overetch. The bars are 10 µm long.
Figure 4. Optical microscopy images of hydrogel in (a) the dry state and (b) pH 3.0, (c) pH 5.0, and (d) pH 7.4 buffer solutions. The bars are 40 µm long. (1) Poly(N-isopropylacrylamide-co-methacrylic acid), (NIPAco-mAA, temperature- and pH-sensitive): 100 mg of N-isopropylacrylamide (NIPA, Polysciences), 8.3 µL of methacrylic acid (MAA, Sigma-Aldrich), 1.3 µL of ethylene glycol dimethacrylate (EGDMA, Polysciences, cross-linker), 1.1 mg of ammonium persulfate (APS, Polysciences, initiator), and 4.4 µL of N,N,N′,N′tetramethylethylenediamine (TEMED, Sigma-Aldrich, accelerator), all dissolved in 230 µL of deionized water. (2) Poly(methylacrylamidophenylboronic acid-co-acrylamide), (MPBA-co-AAm, glucose- and pH-sensitive): 80 mg of acrylamide (AAm, Sigma-Aldrich), 52 mg of methylacrylamidophenylboronic acid (MPBA), 0.5 mg of methylenebisacrylamide (Bis, Polysciences, cross-linker), 5 µL of TEMED, and 0.5 mg of APS, all dissolved in 0.7 mL of deionized water. MPBA was synthesized in the lab from 3-aminophenylboronic acid hemisulfate (SigmaAldrich) and methacrylic acid.15
(3) Poly(methacrylic acid-co-acrylamide) (mAA-co-AAm, pH sensitive): 251.2 mg of acrylamide, 25.0 µL of methacrylic acid, 1.3 µL of EGDMA, 13.2 µL of TEMED, and 2.2 mg of APS, all dissolved in 0.7 mL of deionized water. The patterning process is illustrated in Figure 1. A pre-gel solution is clamped between a silanized glass wafer and a treated silicon wafer, separated by 17-µm-thick photoresist spacers (Figure 1a). To improve adhesion between the silicon substrate and the hydrogel, a thin layer of thermal oxide is grown on the silicon wafer, which is then soaked in a 10 vol % solution (in acetone) of the organosilane coupling agent, γ-methacryloxypropyl trimethoxysilane (γ-MPS, Sigma-Aldrich).11 To prevent (15) Gu, Y. Swelling Properties of Phenylboronic Acid-Containing Hydrogels and their Application in Microfluidic Drug Delivery Devices. Ph.D. Thesis, University of Minnesota, Minneapolis, MN, 2003.
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adhesion of the hydrogel to glass, the glass wafer is hydrophobized by treatment with a 5 vol % solution of dichlorodimethylsilane (DCMS) (Sigma-Aldrich) in toluene. After 30 min of polymerization at room temperature, the wafers are separated, with the hydrogel remaining on the silicon surface. The hydrogel is then dehydrated in acetone, shrinking vertically. A layer of photoresist (STR 1045, Shipley Co., L.L.C.) is spun onto the hydrogel, soft-baked at 70 °C for 10 min, and exposed to the UV source through a photomask having patterns of different dimensions ranging from 2.5 to 40 µm (Figure 1b). Development of the photoresist is carried out using Microposit 351 developer (Shipley Co., L.L.C.). The development time is tuned to slightly underdevelop the photoresist, leaving a 1-2-µm-thick layer on the exposed areas (Figure 1c). In this way, swelling of the hydrogel upon contact with the water-based developer is avoided. Such swelling could damage or detach the photoresist mask. The photoresist and hydrogel are subsequently reactive ion etched with oxygen16-19 (100 sccm, 100 mTorr, 100 W; STS 320 PC, Surface Technology Systems) to remove the remaining photoresist and hydrogel (Figure 1d). The photoresist step height required between the exposed and unexposed areas depends on the differential etch rate between the hydrogel and photoresist. (For an equal etch rate, the step height should be more than the thickness of the dried hydrogel.) In this way, the pattern on the photoresist is transferred to the underlying hydrogel. Finally, the remaining photoresist is washed off with acetone, and the hydrogel is rehydrated (Figure 1e). A simple variation of the process enables fabrication of freestanding hydrogels with reflective tops. These structures can be used as environmentally sensitive micromirrors for chemo-optical sensing. In this process, a thin metal (aluminum in this case) layer is evaporated on top of the dehydrated hydrogel prior to applying the photoresist (Figure 1f). Subsequently, the resist is patterned and the metal layer and hydrogel are dry etched using reactive ion etching (RIE) (8 sccm Cl2, 30 sccm BCl3, 30 mTorr pressure, and 25 W power for aluminum and the same recipe as above for the hydrogel) (Figure 1g,h). Photoresist removal and hydrogel rehydration are then performed as described previously (Figure 1i). Since the metal provides a protective layer for the dehydrated hydrogel, the photoresist can be fully developed.
Results and Discussion The thickness of the patterned hydrogel on the substrate in its dry state was measured using a Dektak 3030 surface profilometer. Dimensions of free swollen hydrogels in solution were measured using an optical microscope (Nikon Optiphot, Japan) with a digimatic indicator (Mitutoyo, Japan). Optical microscopy images of patterned NIPA-co-mAA hydrogel of different shapes and sizes are shown in Figure 2. Due to the low contrast of swollen hydrogels in water, these micrographs were taken in the dehydrated state. Figure 3 shows scanning electron microscopy (SEM) images of patterned MPBA-co-AAm and NIPA-co-mAA hydrogel lines and squares. The edges of the features are slightly overetched, forming round corners. The isotropic nature of hydrogel etching in oxygen RIE limits the achievable minimum feature size. Resolution also depends on the dried hydrogel’s thickness. We were able to achieve a resolution of 2.5 µm for a 1.3-µmthick dried hydrogel, Figure 3c,d. Figure 4 shows cross-sectional optical micrographs of mAA-co-AAm hydrogel lines. Dried hydrogel lines (3.5 µm thick and 10 µm wide) absorbed significant amounts (16) Hartney, M. A.; Hess, D. W.; Soane, D. S. J. Vac. Sci. Technol. 1989, B7 (1), 1-13. (17) Pilz, W.; Janes, J.; Muller, K. P.; Pelka, J. Advanced Techniques for Integrated Circuit Processing; Proceedings of SPIE, Vol. 1392; SPIE: Bellingham, WA, 1990; pp 84-94. (18) Graham, S. W.; Steinbruchel, C. Mater. Res. Soc. Symp. Proc. 1993, 282, 617-22. (19) Soane, D. S.; Martynenko, Z. Polymers in Microelectronics, Fundamentals and Applications; Elsevier: New York, 1989.
Figure 5. (a) SEM image of a fractured micromirror on dry hydrogel. The bar length is 5 µm. (b) Optical micrographs of chemically sensitive micromirrors in PBS solution of pH 3.0. The bar length is 40 µm.
of water in phosphate-buffered saline (PBS) solution of pH 3.0, swelling to a height of 24 µm. The hydrogel lines are swollen further to a thickness of 32 µm in pH 5.0 and 36.6 µm at pH 7.4, demonstrating pH sensitivity. Adhesion between hydrogel and substrate confines its swelling, resulting in bulging sidewalls of hydrogel lines.20 Due to the small dimensions of the hydrogel lines, large volume transitions occur within seconds, compared to an equilibrium time of 15 min for a cylindrical gel with diameter 380 µm. In micromirror fabrication experiments, a layer of 600 nm of aluminum is evaporated and patterned on the dehydrated hydrogel in order to make the top reflective. Figure 5a is a cross-sectional SEM image of a fractured aluminum micromirror on dry hydrogel after the photoresist is removed. An undercut below the metal layer can be observed. Figure 5b shows top-view optical micrographs of chemically sensitive micromirrors in PBS solution. Micromirrors deposited on mAA-co-AAm hydrogel were equilibrated in PBS solutions at different pH values for 10 min, and their height was measured by adjusting the focus of an optical microscope to the planes of the top of the mirrors and the silicon substrate. A sensitivity of 7 µm/pH unit at pH 5.0 has been established by optically (20) Harmon, M. E.; Kuckling, D.; Pareek, P.; Frank, C. W. Langmuir 2003, 19, 10947-56.
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occurs from the dry to the swollen state at pH 3.0. Freeswelling hydrogels’ diameters increase from 380 to 900 µm (2.4-fold), while the patterned hydrogels’ heights increase from 3.6 to 24.5 µm (6.8-fold). The difference in dimension change is attributed to the strong adhesion between the patterned hydrogel and the silicon substrate. Conclusions
Figure 6. Equilibrium swelling response of patterned and free mAA-co-AAm hydrogel at various pH values.
tracking the micromirror surface. The mirrored hydrogel microstructures can serve as optical sensors working in liquids, responding to different stimuli depending on hydrogel properties. To establish the effect of constraint due to bonding of the hydrogels to the silicon surface, free swelling experiments with the MAA-AAm hydrogels have also been carried out. Cylindrical hydrogels were formed by polymerization inside glass capillaries (550 µm inner diameter). After equilibration in PBS buffer solutions with different pH values for 1 h, the diameters of gel cylinders were measured using a microscope. The relationships between pH and dimension changes, scaled to the equilibrium size of the hydrogel at pH 3.0, are shown in Figure 6. The mAA-co-AAm hydrogel shows a typical volume change between pH 3.5 and pH 6.0. The patterned hydrogels show a stronger change along a single dimension than the corresponding free-swelling hydrogels, although the volume change is greater for the free-swelling hydrogels. This is expected since attachment to the base represents an essentially biaxial confinement, and the resulting elastic stress is relieved by expansion in the unconstrained direction.21,22 A more significant change
We have developed a dry pattern transfer technique applicable to a broad range of hydrogels. This is a twostep process based on photoresist lithography and dry etching that can achieve minimum features as small as 2.5 µm. At these dimensions, the swelling response time can be a few seconds. The process is compatible with traditional photolithography techniques and can easily integrate hydrogels with microelectronic and MEMS microstructures. Metallic top hydrogel structures can also be patterned using this technique, creating multiple opportunities for optically based chemical sensors. Our experiments demonstrated a larger dimensional change for patterned hydrogels that are anchored to a substrate as compared to free swelling gel cylinders. Acknowledgment. The authors thank the staff of the Nanofabrication Center of the University of Minnesota for their assistance. Partial funding for this project was provided by the National Institutes of Health (EB-003125), the Spanish Ministry of Education, Culture and Sports through a fellowship for A. Baldi, a Samuel Melendy Fellowship to Y. Gu, and a seed grant from the Biomedical Engineering Institute of the University of Minnesota. LA048719Y (21) Li, C.; Hu, Z.; Li, Y. Phys. Rev. E 1993, 48, 603-6. (22) Toomey, R.; Freidank, D.; Ru¨he, J. Macromolecules 2004, 37, 882-7.
