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Langmuir 1998, 14, 6610-6612
pH-Sensitive Thin Hydrogel Microfabricated by Photolithography Guoping Chen,† Yukio Imanishi,† and Yoshihiro Ito*,†,‡ Graduate School of Materials Science, NAIST, Ikoma 630-0101, Japan, and PRESTO, Japan Science and Technology Corporation, Keihanna Plaza, Hikaridai 1-7, Seika-cho, Kyoto 619-0237, Japan Received May 29, 1998
Introduction Signal-responsive hydrogels have been the focus of increasing interest in various fields.1-7 They undergo abrupt changes in volume in response to external stimuli such as changes in solvent composition,8 pH,9 temperature,10 electric field,11,12 and light irradiation.13 Their sensitivity is controlled by molecular design technologies.14-16 To accelerate the rate of swelling and shrinkage, the introduction of pores into hydrogel17 or the downsizing of string or spherical hydrogels was attempted.18 However, the pH control of microgel shape on a microscopic scale has not been performed thus far. Recently, we used photolithography to pattern-immobilize bioactive molecules and stimuli-responsive polymers onto polymer films for cell culture engineering.19,20 In this study, photolithography was used to synthesize pHsensitive microgel networks. The pH response of the microgel networks was observed by optical microscopy and by atomic force microscopy (AFM) in an aqueous solution. Experimental Section Materials. Poly(acrylic acid) (MW ) 10 000) was purchased from Aldrich Chemical Co. (Milwaukee, WI). 4-Azidoaniline hydrochloride and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (water-soluble carbodiimide, WSC) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Photolithographic masks made of quartz plate deposited with chrome were purchased from Nippon Filcon Co., Ltd. (Osaka, Japan). † ‡
NAIST. PRESTO.
(1) Steinberg, I. Z.; Oplatka, A.; Katchalsky, A. Nature 1966, 210, 568. (2) Sussman, M. V.; Katchalsky, A. Science 1970, 167, 45. (3) Tanaka, T. Polymer 1979, 20, 1404. (4) Kwon, I. C.; Bae, Y. H.; Kim, S. W. Nature 1991, 354, 291. (5) Kajiwara, K.; Ross-Murphy, S. B. Nature 1992, 355, 208. (6) Peppas, N. A.; Langer, R. Science 1994, 263, 1715. (7) Hu, Z.; Zhang, X.; Li, Y. Science 1995, 269, 525. (8) Hirokawa, E.; Tanaka, T. J. Chem. Phys. 1984, 81, 6379. (9) Tanaka, T.; Fillmore, D.; Sun, S.-T.; Nishio, I.; Swislow, G.; Shah, A. Phys. Rev. Lett. 1980, 45, 1636. (10) Hoffman, A. S. J. Controlled Release 1987, 6, 297. (11) Tanaka, T.; Nishio, I.; Sun, S.-T.; Ueno-Nishio, S. Science 1982, 218, 467. (12) Osada, Y.; Okuzaki, H.; Hiro, H. Nature 1992, 355, 242. (13) Suzuki, A.; Tanaka, T. Nature 1990, 346, 345. (14) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.; Okano, T. Nature 1995, 374, 240. (15) Chen, G.; Hoffman, A. S. Nature 1995, 373, 49. (16) Yoshida, R.; Takahashi, T.; Yamaguchi, T.; Ichijo, H. Adv. Mater. 1997, 9, 175. (17) Dong, L. C.; Hoffman, A. S. J. Controlled Release 1987, 13, 21. (18) Park, M. H.; Saito, R.; Ishizu, K.; Fukutomi, T. Polym. Commun. 1988, 29, 230. (19) Ito, Y.; Chen, G.; Guan, Y.; Imanishi, Y. Langmuir 1997, 13, 2756. (20) Ito, Y.; Chen, G.; Imanishi, Y. Bioconjugate Chem. 1998, 9, 277.
Figure 1. Scheme showing azidophenyl-derivatized poly(acrylic acid) (AzPhPAA) synthesis (a) and that showing photolithographic fabrication of microgels (b). Synthesis of Azidophenyl-Derivatized Poly(acrylic acid). Azidophenyl-derivatized poly(acrylic acid) was synthesized by coupling with 4-azidoaniline, as shown in Figure 1a. Poly(acrylic acid) (40.0 mg), 4-azidoaniline hydrochloride (4.7, 18.9, 42.6, or 66.3 mg), and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (water-soluble carbodiimide, WSC, 106.5 mg) were dissolved in deionized water (20 mL), of which the pH was adjusted to 7.0 by NaOH or HCl. After stirring at 4 °C for 24 h, the solution was ultrafiltered (Millipore MoleCut II, cutoff molecular weight below 5 kDa) and the polymer was washed with distilled water until the absence of 4-azidoaniline in the filtrates was confirmed by ultraviolet absorption measurement at 280 nm. The azidophenylderivatized poly(acrylic acid) was referred to as AzPhPAA. The content of azidophenyl groups in AzPhPAA was determined spectroscopically from the absorbance of azidophenyl groups at 280 nm. Synthesis of Microgels. The photolithographic fabrication of microgels is shown in Figure 1b. AzPhPAA was dissolved in water (500 µg/mL) and the solution (100 µL) was deposited on a glass plate. After air-drying at room temperature, the AzPhPAA-deposited glass was covered with a patterned photomask having 2-µm-wide networks and irradiated with an ultraviolet lamp (Koala, 100 W) from a distance of 5 cm for 10 s. Subsequently, the glass was immersed in and rinsed with
10.1021/la9806303 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/09/1998
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Figure 3. Cross-sectional plots of the atomic force micographs (AFM) of immobilized AzPhPAA microgel network (azidophenyl group content, 14.8%) at pH 2 and 10.
Figure 2. Optical micrographs of a photomask having a network structure (a) and prepared microgel network (azidophenyl group content, 14.8%) at pH 2 and 10 (b). an alkaline solution (pH 10.0). The microgel was washed by distilled water until pH change of the washing solution was no longer detected. Observation by Optical Microscopy. The microgel networks were immersed in 0.1 M NaCl solution of pH 2.0 or pH 10.0 and observed by optical microscopy (Olympus Co., Tokyo, Japan). The microgel networks were first equilibrated in 0.1 M NaCl solution of pH 10.0, and then the solution was replaced with a 0.1 M NaCl solution of pH 2.0. After 1, 2, 3, 4, and 5 s, micrographs were taken. Then the microgel networks were soaked in 0.1 M NaCl solution of pH 10.0 and micrographs were taken at 1 s intervals. The void areas of the microgel network were measured from the micrographs. The swelling and deswelling kinetics was defined as the change in the relative mesh area (SG/SC) of the microgel network with time. SG represents the mesh area of swelled microgel network, and SC, that of shrunken microgel network. Observation by AFM. A Nanoscope IIIa (Digital Instrument Co.) equipped with a fluid cell containing an aqueous solution, was utilized to record the images. The microgel network was immobilized on a polystyrene plate under the same conditions as those for preparation on the glass plate. The immobilized microgel network was immersed in a 0.1 M NaCl solution of pH 10.0 or 2.0 and one side of the network mesh square was observed by AFM. The pH was adjusted by NaOH or HCl. For the
Figure 4. Time course of swelling-deswelling behavior of the microgel network. The void areas were determined using the micrographs shown in Figure 2. repulsive mode, commercial Si3N4 cantilevers (Olympus Co.) with a nominal force constant of 0.06 N/m were used.
Results and Discussion The content of azidophenyl groups in AzPhPAA was dependent on the relative concentrations of poly(acrylic acid) and 4-azidoaniline in the aqueous solution. Table 1 shows that the content of azidophenyl groups increased with increasing 4-azidoaniline concentration. However, the coupling yield decreased with increasing concentration.
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Table 1. Preparation of Azidophenyl-Derivatized Poly(acrylic acid) (AzPhPAA) 4-azidoaniline:poly(acrylic acid) in solution (molar ratio) (%)
azidophenyl in AzPhPAA (mol %)
yield (%)
5:100 20:100 45:100 70:100
3.8 7.1 14.8 20.1
76 36 32 28
The photomask used is shown in Figure 2a. AzPhPAA in the ultraviolet light-irradiated areas was inter- and intramolecularly cross-linked. AzPhPAA in other areas was not cross-linked and was removed by washing with an alkaline solution. Figure 2b shows a micrograph of the microgel networks prepared from AzPhPAA (azidophenyl content, 14.8%). The shape of the microgel was identical to that of the photomask. The AzPhPAA microgel network shrank at pH 2 and swelled at pH 10. The area of the square mesh at pH 10 was 2.5 times that at pH 2. The AzPhPAA microgel network was immobilized on a polystyrene plate and observed by AFM. The height of the immobilized microgel changed in response to pH. The height at pH 10 was 1.67 times that at pH 2, although the width was not changed. The time course of swelling and shrinkage of the microgel network was micrographically determined (Figure 4). The behavior was expressed as the change in void areas in the state of swelling (SG) and shrinkage (SC). The microgel network swelled within 4 s when the pH was raised from 2 to 10 and shrank within 1 s when the pH was lowered from 10 to 2. This pH-induced behavior was reversible, and the response was very rapid. The pH responsiveness of microgels was dependent on the content of azidophenyl groups, as shown in Figure 5. As the content of azidophenyl groups in the microgel increased, the swelling-shrinkage inflection point shifted toward higher pH and the extent of swelling decreased.
Figure 5. pH dependence of mesh area of microgel network synthesized from AzPhPAA. The content of azidophenyl groups in AzPhPAA was 3.8% (O), 7.1% (b), 14.8% (4), or 20.1% (2).
This was explained by the enhancement of cross-linking and hydrophobicity by azidophenyl group introduction. It was also reported that the hydrophobization of poly(acrylic acid) led to the shift of inflection point from low pH to high pH.21 In the present study, a microscopic pH-sensitive hydrogel network was synthesized by photolithography. The microgel network showed rapid and reversible response to pH change. Furthermore, the swelling-shrinkage inflection point and the extent of swelling were controlled by the content of azidophenyl groups. Photolithography provides a convenient and useful means for fabricating hydrogels on a microscopic scale. LA9806303 (21) Philippova, O. E.; Hourdet, D.; Audebert, R.; Khokahlov, A. R. Macromolecules 1997, 30, 8278.
