Anal. Chem. 2008, 80, 1323-1327
Second-Generation Maskless Photolithography Device for Surface Micropatterning and Microfluidic Channel Fabrication Kazuyoshi Itoga, Jun Kobayashi, Yukiko Tsuda, Masayuki Yamato, and Teruo Okano*
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan
We have previously reported on a maskless photolithography device for surface micropatterning and microfabrication by modifying a commercially available liquid crystal display projector. For the prototype, 10-µm resolution was achieved by downsizing the image on a 0.7-in. liquid crystal display panel to an area of 8 × 6 mm and projecting it on a fixed stage. Here, we report on a secondgeneration maskless photolithography device having two novel features. First, the sliding lens system with variable focal distances and exchangeable objective lenses achieves a variable resolution of 2-8 µm. Second, the synchronous control of displayed images generated by a personal computer and the movement of a XY-positioning stage allows for the fabrication of micropatterns over a larger area (over 50 × 50 mm). Here, we show examples fabricated with the two novel features. Microfabrication techniques such as photolithography have been applied to micropatterning of polymers, biomolecules, and cells.1-4 However, most microfabrication techniques require specialized equipment and the preparation of photomasks, resulting in a costly and time-consuming process. It is therefore desirable to develop simple methods for rapid prototyping of micropatterned surfaces. Whitesides et al. reported rapid prototyping of poly(dimethylsiloxane) (PDMS) microfluidic devices using masks composed of laser-printed commercial transparency sheets on which the desired channel design was printed in place of expensive chrome masks.5-7 However, it is difficult to achieve line resolutions as high as 10 µm/dot with this approach. We previously developed an all-in-one photolithography device for the rapid fabrication of micropatterned surfaces by modifying a commercially available liquid crystal display projector (LCDP).8-11 * To whom correspondence should be addressed. E-mail: tokano@abmes. twmu.ac.jp. (1) Bhatia, S. N.; Yarmush, M. L.; Toner, M. J. Biomed. Mater. Res. 1997, 34, 189-199. (2) McFarland, C. D.; Thomas, C. H.; DeFilippis, C.; Steele, J. G.; Healy, K. E. J. Biomed. Mater. Res. 2000, 49, 200-210. (3) Chen, G.; Imanishi, Y.; Ito, Y. J. Biomed. Mater. Res. 1998, 42, 38-44. (4) Matsuda, T.; Sugawara, T. J. Biomed. Mater. Res. 1995, 29, 749-756. (5) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984. (6) Ng, J. M.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Electrophoresis 2002, 23, 3461-3473. (7) McDonald, J. C.; Whitesides, G. M. Acc. Chem. Res. 2002, 35, 491-499. 10.1021/ac702208d CCC: $40.75 Published on Web 01/23/2008
© 2008 American Chemical Society
The features of the device are as follows: 1) Photomasks are not required (i.e., maskless), 2) the all-in-one system does not require an external optical system, and 3) there is no need for clean rooms. To enhance the capabilities of the prototype maskless device, we designed a second-generation device having two additional features. First, the sliding lens system with variable focal distances and exchangeable objective lenses achieves variable resolution. Second, the synchronous control of displayed images generated by a personal computer and the movement of a XYpositioning stage allows for fabrication over a larger area (over 50 × 50 mm). EXPERIMENTAL SECTION Materials. g-line positive photoresist (OFPR-800LB, 34cp), i-line negative thick photoresist (TSMR-iN1000PM, 3000cp), and developer solvent (2.38% tetramethylammonium hydroxide solution, NMD-3) were obtained from Tokyo Ohka Kogyo Co., Ltd. (Kanagawa, Japan). The LCDP (PLC-XW20A) was kindly provided by Sanyo Electric Co. (Osaka, Japan). The X-axis stage (ALS-301CM) and dual-axis controller driver (QT-CM2-35) were purchased from Chuo Precision Industrial Co. (Osaka, Japan). Two types of objective lens having long working distances (M Plan Apo 2×, NA 0.055, and M Plan Apo 5×, NA 0.14), tube lens (MT-40), and a 3× objective lens for measuring microscope MF series (ML3X, NA 0.07) were purchased from Mitutoyo Co. (Kanagawa, Japan). 3-Methacryloxypropyltrimethoxysilane (MPTMS; Shin-Etsu Chemical Co., Tokyo, Japan), potassium peroxodisulfate (KPS; Nacalai Tesque, Inc., Kyoto, Japan), acrylamide, (AAm; Wako Pure Chemical Industries Ltd., Osaka, Japan), Alexa488-phallodin, and ethidium homodimer-1 (Invitrogen, Carlsbad, CA) were all used as received. Device Design. The basic structure is essentially the same as that of the prototype reported previously (Figure 1a).8 The LCDP used here has three liquid crystal panels (1.78 cm in diagonal), each consisting of 1024 × 768 square pixels (13.9 µm (8) Itoga, K.; Yamato, M.; Kobayashi, J.; Kikuchi, A.; Okano, T. Biomaterials 2004, 25, 2047-2053. (9) Itoga, K.; Yamato, M.; Kobayashi, J.; Kikuchi, A.; Okano, T. J. Biomed. Mater. Res., A 2004, 69, 391-397. (10) Itoga, K.; Kobayashi, J.; Yamato, M.; Kikuchi, A.; Okano, T. Biomaterials. 2006, 27, 3005-3009. (11) Kobayashi, J.; Yamato, M.; Itoga, K.; Kikuchi, A.; Okano, T. Adv. Mater. 2004, 16, 1997-2001.
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Figure 1. Photograph of the maskless photolithography device equipped with an LCD projector. (a) Appearance of the device with an XYstage. (b) The resolution is variable by sliding function even without exchanging objective lens. The device presented here is equipped with a 2× objective lens. The resolution is 5 × 5 µm/pixel in the upper setting and 8 × 8 µm/pixel in the lower setting.
Figure 2. Confocal laser scanning micrographs of bovine endothelial cells on the PAAm micropatterns 2 days after seeding. DNA was stained with ethidium homodimer-1 (red), and cytoskeletal actin microfilaments of the ECs were stained with Alexa488-phallodin (green). (a) Unpatterned surface; (b) 20-µm-width line pattern; (c) 10-µm-width line pattern; (d) 20-µm-width square pattern; (e) 10-µm-width square pattern. Scale bar, 50 (a-c) and 20 µm (d, e).
in sides). By exchanging the objective lens, the projected image resolution can be changed. In addition, the slide function of the projector unit facilitates the variable resolution of projected images (Figure 1b). Since a higher power lens is employed in the present model, the projected area is smaller than that of the prototype. To compensate for this, the present device is equipped with a XY-stage composed of two orthogonal X-axis stages (ALS-301-CM) with a travel range of 50 mm. A personal computer generating images controls a dual-axis controller driver (QT-CM2-35) via an RS-232C interface. The software used to generate mask patterns and synchronously control the XY-stage was programmed inhouse. Functionalization of Glass Surfaces and Photoresist Coating. Glass coverslips (24 × 50 mm, 0.2-mm thickness, Matsunami Glass Ind., Osaka, Japan) were cleaned by oxygen plasma treatment and placed in a 500-mL separable flask with 3 mL of MPTMS. The coupling reaction of vaporized MPTMS with the 1324
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coverslip surfaces was performed under N2 gas at 70 °C for 16 h, as described previously.12 Coverslips were rinsed with ethanol, methanol, and distilled water and dried for 5 h at 80 °C. Silanized coverslips were mounted on a spin coater (ACT-300D, Active Co., Ltd., Saitama, Japan), covered with positive photoresist, spun at 2000 rpm for 30 s and prebaked in a heating chamber for 4 h at 80 °C. Surface Micropatterning with Polyacrylamide. The fabrication of polyacrylamide (PAAm) micropatterns was performed with a 5× objective lens to adjust the final resolution to 2.5 µm/pixel, and the two-step procedure was employed as described previously.10 Cell Culture. Bovine aortic endothelial cells (JCRB0099) were provided from the Japan Health Science Foundation and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (12) Okusa, H.; Kurihara, K.; Kunitake, T. Langmuir 1994, 10, 3577-3581.
Figure 3. Fabrication of PDMS microchannels using the LCDP-modified device with the XY positioning stage. (a) Schematic representation of fabrication of PDMS microchannels. The synchronous control of displayed images generated by a PC and the movement of an XY-positioning stage achieves fabrication of the micropattern over a larger area. Three-dimensional topography of (b) photoresist master, (c) resulting PDMS microchannel, and (d) cross-sectional profile of PDMS microchannel was observed by scanning electron microscopy (SEM). (e) Macroscopic view of “Nazca Lines” PDMS microchannels fabricated with the LCDP device. PDMS microchannels with large area and complex shapes were fabricated by using the XY-stage. Scale bar. 200 (b, c) and 50 µm (d).
fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin. Cells were recovered from tissue culture poly-
(styrene) dishes by treatment with 0.05% trypsin-0.05% ethylenediaminetetraacetic acid (EDTA) in PBS and were routinely Analytical Chemistry, Vol. 80, No. 4, February 15, 2008
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passaged at 1/4 confluency. Endothelial cells (1.0 × 105 cells/ cm2) were seeded onto treated glass coverslips. After the actin filaments of the cells were stained with Alexa488-phallodin and the DNA of the cells was stained with ethidium homodimer-1, the cells were observed under a confocal fluorescence laserscanning microscope. Cells on the nonmodified surface and the stripe patterns were monitored under a LSM510 confocal microscope (Carl Zeiss, Hallbergmass, Germany), and the cells on the square patterns were monitored under a TCS-PS confocal microscope (Leica Microsystems, Wetzlar, Germany). Fabrication of PDMS Microchannel with XY-Positioning Stage. For the fabrication of PDMS microchannels, a 3× objective lens was employed to adjust the resolution to 5 µm/pixel. The procedure to fabricate PDMS microchannels is outlined in Figure 3a. First, surface patterning with a thick negative-type photoresist on the silicon wafer was performed by irradiation of visible light through patterned images on LCDP. silicon wafers (50 mm in diameter, 280 µm in thickness; Mitsubishi Materials Co., Tokyo, Japan) were mounted on a spin coater (ACT-300D, Active Co.), covered with a thick negative-type photoresist, spun at 800 rpm for 40 s and prebaked in a heating chamber for 30 min at 120 °C. Then, the photoresist-coated silicon wafer was placed in the LCDP apparatus and visible-light irradiation from the LCDP was performed for 60 s at each shot. The exposed photoresist was postexposure baked in a heating chamber at 120 °C for 30 min. The nonexposed area of the photoresist on the silicon wafer was stripped in developer solvent for 25 min. Surfaces were washed with distilled water and air-dried. Then, a mixture of PDMS base and curing agent (Sylgard 184, Dow Corning, Midland, MI) was applied onto the photoresist master and cured for 1 h at 80 °C. The resultant PDMS patterned substrate was cut and peeled from the master mold and then trimmed to provide the final patterned piece. Three-dimensional surface profiles were obtained with a scanning electron microscope (VE-9800, Keyence Co., Osaka, Japan). RESULTS AND DISCUSSION In the prototype maskless photolithography system reported previously,8 emitted light from the lamp was not decomposed into red, green, and blue but directly subjected to only one LCDP in the optical path by removing dichroic mirrors. This is in contrast to the original projector in which the light was decomposed into three colors prior to passing through three separate LCDPs and then resynthesized with dichroic prisms prior to entering projection lenses. Therefore, the prototype device emitted white color light onto the reaction stage. However, since g-line (436 nm) and i-line (365 nm) photoresists were expected to be used, the optical path before the projection lens was not modified in the present model. In the case that reduction of light amount emitted onto LCD is needed, a neutral density filter was inserted to protect the LCD panel units. Besides the projection lens, the objective lens was placed outside the LCDP to focus on the reaction stage. In the present device, the resolution could be changed by adjusting the sliding lens system to vary focal distance or exchangeable objective lens (Figure 1). Variable resolution ranged from 5.0 to 8.0, 3.0 to 5.0, and 2.0 to 3.0 µm/pixel using 2×, 3×, and 5× objective lenses, respectively. In order to confirm this 1326
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feature, surface micropatterning with noncell adhesive PAAm was performed with the device for single-cell resolution (Figure 2). Micropatterns were designed with 10- and 20-µm-wide lines (Figure 2b, c), and 10- and 20-µm-square islands surrounded with PAAm (Figure 2d, e). While cells seeded on the nonpatterned silanized coverslips attached and spread freely (Figure 2a), cell adhesion and spreading were confined to the non-PAAm-grafted domains (Figure 2b-e). This resolution was higher than that for the prototype.8 Larger patterns could be fabricated by the synchronous control of mask pattern image generation with a PC and movement of the XY-positioning stage. Since the XY-stage has a travel range of 50 mm, when the size of the projected area is w × h mm, the maximal size of a pattern that can be fabricated is (50 + w) × (50 + h) mm. The values of w and h range from 2 to 8 mm depending on the objected lens used. This feature seems sufficient for the fabrication of typical biochips.13,14 In order to confirm this feature, PDMS microfluidic channels covering a large area were prepared using molds of thick negative-type photoresist and imaged using a scanning electron microscope (Figure 3). Although this photoresist was optimized for the i-line (365 nm), it also reacts with visible-light. It took 60 s to expose the photoresist for each shot (Figure 3a). Panels b and c in Figure 3 show a mask pattern design that has three branch channels (100 µm wide) that converge into a single straight channel (300 µm wide) and could be accurately transferred from the photoresist mold to PDMS (Figure 3b, c). Figure 3d shows an example of a channel with a depth of 40 µm. The depth could be controlled within a range of 1-100 µm by changing the viscosity of the photoresist and the spin coating speed. This is the first report on the fabrication of microfluidic channels utilizing both a maskless photolithography device and a photoresist. With this combination, more complex and finer designs of microfluidic channels were easily achieved without the need for the tricks employed with the prototype using one-step photopolymerization from monomer solution without photoresists.11 For example, for the fabrication of microchannels with complex shapes like Nazca Lines, the previous method needed to add dummy lines in the mask image to keep the distances between two adjacent lines nearly identical in order to equalize the amount of exposure. However, even in the absence of such dummy lines, large microchannels with complicated shapes were easily fabricated with the present method (Figure 3e), since mass transfer of monomer solution was completely prohibited by micropatterned solid photoresists. CONCLUSIONS We have developed a second-generation maskless photolithography device with two new features. Using the new device, micropatterned surfaces and microchannels with complex designs were fabricated rapidly and inexpensively. Using this device and the methods outlined here, it is possible to achieve rapid prototyping of biochips and microplatforms with resolutions of single-cell size. (13) Zheng, B.; Tice, J. D.; Roach, L. S.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2004, 43, 2508-2511. (14) Balagadde´, F. K.; You, L.; Hansen, C. L.; Arnold, F. H.; Quake, S. R. Science 2005, 309, 137-140.
ACKNOWLEDGMENT We appreciate the useful comments and technical criticism from Dr. B.C. Isenberg (Boston University, Boston, MA). This study was partially supported by Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology “Innovation Center for Fusion of Advanced Technologies to Realize Regenerative
Medicines” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
Received for review November 30, 2007.
October
26,
2007.
Accepted
AC702208D
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