Capillary-Assembled Microchip for Universal Integration of Various

May 6, 2004 - Science and Technology, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan, and Department of Applied. Chemistry, Graduate ...
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Anal. Chem. 2004, 76, 3222-3228

Capillary-Assembled Microchip for Universal Integration of Various Chemical Functions onto a Single Microfluidic Device Hideaki Hisamoto,*,† Yuya Nakashima,† Chihiro Kitamura,† Shun-ichi Funano,† Midori Yasuoka,† Keisuke Morishima,‡ Yoshikuni Kikutani,‡ Takehiko Kitamori,‡,§ and Shigeru Terabe†

Department of Material Science, Graduate School of Science, Himeji Institute of Technology, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297 Japan, Micro Chemistry Group, Special Laboratory for Optical Science, Kanagawa Academy of Science and Technology, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan, and Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon. Microfluidic devices have been the focus of much attention in the past decade for their potential applications in micro total analysis systems or microreactors.1-5 Previously reported micro* To whom correspondence should be addressed. E-mail: hisamoto@ sci.himeji-tech.ac.jp. Fax: +81-791-58-0493. † Himeji Institute of Technology. ‡ Kanagawa Academy of Science and Technology. § The University of Tokyo. (1) Proceedings of the µTAS’2002 Symposium 2002; Baba, Y., Shoji, S., van den Berg, A., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 2002. (2) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (3) Auroux, P.-A.; Reyes, D. R.; Iossifidis, D.; Manz, A. Anal. Chem. 2002, 74, 2637-2652.

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fluidic devices allowed not only fast mixing or heat dissipation by exploiting the intrinsic physical characteristics originating from a liquid microspace but also diffusion-based mixing, separation, or multiphase flow at Y-shaped or T-shaped channel patterns by exploiting laminar flow characteristics.6-20 On the other hand, integration of chemical functions onto a microfluidic device has become a target of much current research in analytical chemistry. Whiteside’s group was the first to report laminar flow patterning of various materials inside a microchannel.6,7 Beebe’s group has reported many examples using photopolymerization techniques to get inner channel preparation of chemically actuating valves, a reaction-facilitated readout system, and so on.21 Fre´chet’s group also used photopolymerization techniques to prepare monolithic polymer structures for use in preconcentration of analyte, thermally actuating valves, or (4) Proceedings of the Third International Conference on Microreaction Technology; Ehrfeld, W. Ed.; Springer: Berlin, 1999. (5) Ehrfeld, W.; Hessel, V.; Lowe, H. Microreactors: New Technology for Modern Chemistry, Wiley-VHC: Weiheim, 2000. (6) Kenis, P. J. A.; Ismagilov, R. F.; Whitesides, G. M. Science 1999, 285, 8385. (7) Kenis, P. J. A.; Ismagilov, R. F.; Takayama, S.; Whitesides, G. M.; Li, S.; White, H. S. Acc. Chem. Res. 2000, 33, 841-847. (8) Weigl, B. H. and Yager, P. Science 1999, 283, 346-347. (9) Kamholz, A. E.; Weigl, B. H.; Finlayson, B. A.; Yager, P. Anal. Chem. 1999, 71, 5340-5347. (10) Zhao, B.; Moore, J. S.; Beebe, D. J. Science 2001, 291, 1023-1026. (11) Zhao, B.; Viernes, N. O. L.; Moore, J. S.; Beebe, D. J. J. Am. Chem. Soc. 2002, 124, 5284-5285. (12) Tokeshi, M.; Minagawa, T.; Kitamori, T. Anal. Chem. 2000, 72, 17111714. (13) Hibara, A.; Tokeshi, M.; Uchiyama, K.; Hisamoto, H.; Kitamori, T. Anal. Sci. 2001, 17, 89-93. (14) Hisamoto, H.; Horiuchi, T.; Tokeshi, M.; Hibara, A.; Kitamori, T. Anal. Chem. 2001, 73, 1382-1386. (15) Hisamoto, H.; Horiuchi, T.; Tokeshi, M.; Hibara, A.; Kitamori, T. Anal. Chem. 2001, 73, 5551-5556. (16) Surmeian, M.; Hibara, A.; Slyadnev, M. N.; Uchiyama, K.; Hisamoto, H.; Kitamori, T. Anal. Lett. 2001, 34, 1421-1429. (17) Tokeshi, M.; Minagawa, T.; Uchiyama, K.; Hibara, A.; Sato, K.; Hisamoto, H.; Kitamori, T. Anal. Chem. 2002, 74, 1565-1571. (18) Hisamoto, H.; Saito, T.; Tokeshi, M.; Hibara, A.; Kitamori, T. Chem. Commun. 2001, 2662-2663. (19) Ueno, K.; Kitagawa, F.; Kitamura, N. Lab Chip 2002, 231-234. (20) Kuban, P.; Dasgupta, P. K.; Morris, K. A. Anal. Chem. 2002, 74, 56675675. (21) Moorthy, J.; Beebe, D. J. Anal. Chem. 2003, 75, 292A-301A and references therein. 10.1021/ac035385t CCC: $27.50

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enzyme immobilization.22-24 Crooks’s group reported preparation of a hydrogel micropatch including living cells or enzymes by photopolymerization of hydrogels.25,26 Corn’s group used a poly(dimethylsoloxane) (PDMS) channel to prepare a patterned surface and successfully applied the technique to surface plasmon resonance sensing of biological substances.27-29 We have reported the in situ preparation of a chemically functional membrane structure in a glass microchip and successfully demonstrated membrane permeation and catalytic reaction processes.30,31 In all cases, immobilization of highly functional molecules (or polymers) to an appropriate position in the microchannel played an important role. These techniques are quite promising for integrating a single chemical function onto a microfluidic device; however, an experimental difficulty arises when different and plural chemical functions are integrated into a single microfluidic device. This is because most techniques listed above require introduction of reagent solutions into the “whole microchannel”, although the chemical modification is only carried out at a defined position of it. Therefore, in general, once a certain chemical function is patterned at part of the channel, position selectively, other reagents should be introduced over another patterned surface to integrate plural chemical functions into a single microchannel. This may lead to contamination or deterioration of the previously patterned chemical function. Furthermore, choice of the initial surface modification method on the channel surface, i.e., surface silanization or some other methods, is generally dependent on the structure or functional groups of the target molecules or polymers to be patterned, so that the initial surface modification method limits the molecules or polymers to be immobilized on channel surfaces. According to these considerations, development of a simple and universal methodology for integrating various chemical functions into a single microfluidic device is an indispensable research subject. We propose a simple and promising approach for assembling various chemical functions onto a single microfluidic device as shown in Figure 1. Our concept, called a capillaryassembled microchip (CAs-CHIP), involves embedding of chemically functionalized square capillaries into the lattice microchannel network fabricated on PDMS having the same channel dimensions as the outer dimensions of the square capillaries. Square capillaries are well known in the field of capillary electrophoresis.32 They have been utilized to improve optical detection by eliminating unfavorable reflection or refraction, which (22) Yu, C.; Mutlu, S.; Selvaganapathy, P.; Mastrangelo, C. H.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2003, 75, 1958-1961. (23) Yu, C.; Davey, M. H.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2001, 73, 5088-5096. (24) Peterson, D. S.; Rohr, T.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2002, 74 4081-4088. (25) Zhan, W.; Seong, G. H.; Crooks, R. M. Anal. Chem. 2002, 74, 4647-4652. (26) Heo, J.; Thomas, K. J.; Seong, G. H.; Crooks, R. M. Anal. Chem. 2003, 75, 22-26. (27) Lee, H. J.; Goodrich, T. T.; Corn, R. M. Anal. Chem. 2001, 73, 5525-5531. (28) Wegner, G. J.; Lee, H. J.; Corn, R. M. Anal. Chem. 2002, 74, 5161-5168. (29) Wegner, G. J.; Lee, H. J.; Marriott, G.; Corn, R. M. Anal. Chem. 2003, 75, 4740-4746. (30) Shimizu, Y.; Hisamoto, H.; Hibara, A.; Tokeshi, M.; Kitamori, T. Digest of Papers, 2001 International Microprocesses and Nanotechnology Conference, Japan Society of Applied Physics: Tokyo, Japan, 2001; pp20-21. (31) Hisamoto, H.; Shimizu, Y.; Uchiyama, K.; Tokeshi, M.; Kikutani, Y.; Hibara, A.; Kitamori, T. Anal. Chem. 2003, 75, 350-354. (32) Tsuda, T.; Sweedler, J. V.; Zare, R. N. Anal. Chem. 1990, 62, 2149-2152.

Figure 1. General concept for fabricating CAs-CHIP by embedding square capillaries into the lattice microchannel network.

may occur in a cylindrical capillary, or to allow multiple detection in capillary array electrophoresis.33,34 In this work, we focus on the outer shape and size of a square capillary, which are very close to those of microchannels fabricated on a microfluidic device. Therefore, when a lattice microchannel having the same size as the outer size of the square capillary is fabricated on a soft material such as PDMS, embedding square capillaries into the microchannel would allow easy fabrication of the microfluidic device with a freely designed channel (capillary) network. In this case, the square shapes of the glass capillary and PDMS channel play important roles, not only for successful embedding without any solution leakage but also for easy formation of multilayer flow at the confluence point in the planar microfluidic device. Furthermore, many types of surface modification procedures for the capillary inner surface35,36 or monolithic structure fabrication methodologies in fused-silica capillaries are well known,37,38 so that integration of various chemical functions such as chemical sensing, catalytic reaction, and chromatographic separation can be expected by cutting chemically functionalized capillaries into appropriate lengths and embedding them into the lattice PDMS microchannel network. From the viewpoint of industrial produc(33) Li, L.; McGown, L. B. Electrophoresis 2000, 21, 1300-1304. (34) Lu, S. X.; Yeung, E. S. J. Chromatogr., A 1999, 853, 359-369. (35) Doherty, E. A. S.; Meagher, R. J.; Albarghouthi, M. N.; Barron, A. E. Electrophoresis 2003, 24, 34-54. (36) Guzman, N. A.; Stubbs, R. J. Electrophoresis 2001, 22, 3602-3628. (37) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3646-3649. (38) Tanaka, N.; Kobayashi, H.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Hosoya, K.; Ikegami, T. J. Chromatogr., A 2002, 965, 35-49.

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tion, cutting long capillaries into small pieces would be effective for getting low-cost fabrication of chemically functionalized microfluidic devices. In Figure 1, a lattice structure was employed to allow universal embedding of various capillaries; thus, the volume of the access channel becomes relatively large compared to inner volumes of the chemically functionalized square capillaries. However, reducing the access channel size can be done easily when assembly positions and lengths and kinds of capillaries are optimized for their respective purposes. To realize our concept, (1) the fabrication of a capillaryembedded microchannel (capillary) network without any solution leakage and (2) the preparation of various types of chemically functionalized square capillaries are indispensable. Here we report the first example implementing the concept, and we give a simple example application of the method for preparing a dual-chemical sensing chip. For the sensing application, ion sensing using a highly selective ionophore-incorporated ion-selective optode membrane and pH sensing using a fluoresceinimmobilized hydrophilic membrane are performed in a single microfluidic device. These immobilization techniques employ different surface modification technologies; thus, it is generally difficult to integrate both of them using typical surface modification techniques for microchips such as photopolymerization or laminar flow patterning. EXPERIMENTAL SECTION Square Capillaries and Reagents. Square capillaries having 300-µm outer widths (flat to flat) and 100- or 50-µm inner widths were purchased from Polymicro (Phoenix, AZ). The polyimide coating of these capillaries was removed by heating before use. Sylgard 184 silicone elastomer was purchased from Dow Corning (Midland, MI). Reagents of the highest grade commercially available were used for the preparation of the aqueous test electrolytes. 2-Nitrophenyl octyl ether and 3-(trimethoxysilyl)propyl methacrylate were purchased from Tokyo Chemical Industry (Tokyo, Japan). 4,5-Dibromofluorescein octadecyl ester was purchased from Fluka (Ronkonkoma, NY). Fluorescein isothiocyanate (FITC) was purchased from Sigma (St. Louis, MO). Poly(ethlene glycol) diacrylate, poly(vinyl chloride)-co-vinyl alcohol vinyl acetate (90 wt % vinyl chloride, 6 wt % vinyl alcohol, and 4 wt % vinyl acetate), and Coumarin 343 were purchased from Aldrich (Milwaukee, WI) Calcium ionophore, 10,19-bis[(octadecylcarbamoyl) methoxyacetyl]- 1,4,7,13,16-pentaoxa-10,19-diazacycloheneicosane (K23E1)39-41 was kindly donated by Professor Koji Suzuki of Keio University. Polyethyleneimine (PEI, 30% solution, average molecular weight ∼70 000) and 2,2′-azobis(2,4-dimethylvaleronitrile) were purchased from Wako Chemical (Osaka, Japan). All reagents were used without further purification. Distilled and deionized water used had resistivity values of more than 1.7 × 107 Ω cm-1 at 25 °C. Fabrication of a Lattice Microchannel on a PDMS Plate. First, a glass mold with a lattice structure was prepared by cutting (39) Suzuki, K.; Watanabe, K.; Matsumoto, Y.; Kobayashi, M.; Sato, S.; Siswanta, D.; Hisamoto, H. Anal. Chem. 1995, 67, 324-334. (40) Hisamoto, H.; Watanabe, K.; Nakagawa, E.; Shichi, Y.; Suzuki, K. Anal. Chim. Acta 1994, 299, 179-187. (41) Hisamoto, H.; Miyashita, N.; Watanabe, K.; Nakagawa, E.; Suzuki, K. Sens. Actuators, B 1995, 29, 378-385.

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a 300-µm depth with a 1-mm pitch using a dicing saw (DAD522, Disco Corp., Tokyo, Japan) possessing an edge of 300-µm width. The glass mold surface was first treated with a water-resistant spray to avoid irreversible adhesion of the cured PDMS plate, and then PDMS prepolymer was coated onto it and cured at 70 °C for 3 h. This first PDMS plate was peeled off from the glass mold surface in order to use it as a PDMS mold. This PDMS mold was treated with the water-resistant spray again, and PDMS prepolymer was coated onto it. Curing was done at the same temperature and for the same time to give the lattice microchannel network on the second PDMS plate. Embedding Square Capillaries on a PDMS Plate and Bonding of PDMS Cover. Square capillaries were cut into appropriate lengths (usually 1-3 mm) and embedded into the lattice microchannel network fabricated on the PDMS plate. Plugged capillaries were prepared by introduction of PDMS prepolymer into square capillaries (inner width, 50 µm) and cured at 70 °C for more than 5 h. These plugged capillaries were also cut and used for preparing the designed channel network. After embedding all the capillaries, a PDMS cover was bonded on top. For this, a spin-coated PDMS prepolymer on a glass slide was used as a cover plate. PDMS prepolymer was spin coated on the glass slide at 5000 rpm and then adhered to the capillaryembedded PDMS plate with clippers before curing. Bonding was carried out by curing at 70 °C for 5 h. Preparation of Chemical-Sensing Capillaries. All the capillaries used in this work were washed with 1 M sodium hydroxide solution (30 min), flushed with pure water and then acetone, and heated at 70 °C for 30 min prior to use. This washing procedure ensured the following surface modifications could be made. Ion-Sensing Capillary. The ion-sensing capillary was prepared by a two-step procedure. First, a square capillary was filled with an aliquot of membrane cocktail solution consisting of calcium ionophore (1.8 mg), lipophilic dye (3.0 mg), o-nitrophenyl octyl ether (66 mg), PVC copolymer (33 mg), and tetrahydrofuran (250 mg). Then air was introduced into the capillary by a microsyringe pump at a flow rate of 100 µL/min to push out the solution. This treatment allows membrane materials to remain at the four corners of the inner capillary. This capillary was dried at 50 °C for 12 h to evaporate tetrahydrofuran. These treatments allowed us to prepare ion-selective optode membranes at the four corners of the inner capillary. pH-Sensing Capillary. The pH-sensing capillary was prepared by a procedure similar to that reported by Zhan et al.,25 in which they entrapped a large molecule into a PEG hydrogel network. Here a FITC-labeled polymer was entrapped. The inner volume of the capillary was filled with 3-(trimethoxysilyl)propyl methacrylate-0.12 M HCl mixture (volumetric ratio, 4:1) and left for 30 min. Then the inner volume was flushed with methanol and acetone and dried in an oven at 80 °C for 1 h. FITC (10 mg) was dissolved into a mixture of water (400 µL) and PEI (100 mg), which was stirred for 30 min. This treatment covalently immobilized fluorescein to the PEI. A portion of FITC-labeled PEI solution (50 µL) was mixed with 0.05 M Tris-HCl buffer solution (pH 7, 150 µL), poly(ethlene glycol) diacrylate (200 mg), and 2,2′azobis(2,4-dimethylvaleronitrile)) (10 mg) and the resultant mixture was stirred. This mixture was introduced into the surfacemodified square capillary, which had the methacryl group, and

air was pumped in by the microsyringe pump. This capillary was heated at 50 °C for 2 h to complete fluorescein-labeled polymerentrapped hydrophilic membrane formation at the four corners of inner capillary. The resulting capillary was fully washed by introducing buffer solution (0.05 M Tris-HCl, pH 7) at 1 µL/min for 12 h to remove unreacted chemical species. The capillary was cut into ∼2-mm lengths, using a ceramic cutter, and embedded into the PDMS plate having lattice channel network. Optical Detection and Data Processing. Optical images of the microchannel and chemically modified membranes in square capillaries were obtained using an optical/fluorescence inverted microscope (Eclipse TS100-F, Nikon, Tokyo, Japan). Depending on the experiment, 4×, 10×, and 20× objective lenses were used to obtain data. Photographs were captured using a 3CCD color camera (HV-D28S, Hitachi Kokusai Electric Inc., Tokyo, Japan) installed at the front port of the microscope. Fluorescent images were collected using a mercury lamp as a light source and a blue or green filter block (B-2A or G-2A, Nikon).

Figure 2. Typical cross sections of 300- and 100-µm channel (capillary) before and after bonding spin-coated PDMS prepolymer plate.

RESULTS AND DISCUSSION Preparation of the Designed Microchannel Network by Embedding Bare Square Capillaries and Plugged Capillaries into the Lattice Microchannel Network on a PDMS Plate. In the first experiment, we tried to prepare a designed microchannel (capillary) network on a lattice PDMS plate. Commercially available square capillaries have a square cross section; however, their outer corner edges are not actually at right angles, so that there are slight spaces of a few micrometers between the capillary and the PDMS channel wall when capillaries are embedded. The bottom shape of the PDMS channel can easily be transformed by embedding a hard square glass capillary, so there is little space between the capillary bottom and the PDMS channel when slight pressure is applied. However, there is a slight space between the top edges of the square capillary and the PDMS cover plate. Therefore, bonding of a PDMS cover plate plays a crucial role for fabricating the designed microchannel (capillary) network without any solution leakage. Usually, oxygen plasma-treated PDMS plate or a chemically hydrophilized PDMS plate prepared using an oxidant is used for PDMS bonding. However, all preliminary experiments using these techniques failed, and solutions introduced into the microchannel leaked out, due to the existence of a slight space between the square capillaries and the PDMS plate (data not shown). Therefore, we took a different approach that involves bonding between the capillary-embedded PDMS plate and spin-coated PDMS prepolymer. In this case, the spin-coated thin PDMS prepolymer penetrates into the slight space between the top edges of square capillaries and the PDMS plate by wetting force and completely fills all these spaces. Therefore, curing treatment with slight pressure after this procedure is expected to block both voids existing at the top and bottom edges of square capillary, which leads to fabrication of the microchannel network with no solution leakage. Figure 2 shows a typical cross section of the 300-µm channel and 100-µm channel (embedded capillary) before and after the cover-bonding procedure. Since PDMS prepolymer wets the top edges of the 300-µm PDMS channel, the cross section of the 300µm channel formed is not a completely square shape (see left photograph of “After bonding”); however, the slight space existing

at the capillary-embedded channel is completely filled with PDMS (see right photograph of “After bonding”). Figure 3 shows crossing points where various patterns of capillary-embedded PDMS channel networks are prepared. As expected, in all cases, slight spaces between the square capillaries and PDMS plate are completely filled with PDMS prepolymer. Figure 3 also shows the fluorescent images observed by introducing fluorescent solution (Coumarin 343) into the microchannel network. No solution leakage is seen. Wetting of PDMS prepolymer is significant at the channel crossing point where several capillaries (three or four) face each other. To achieve successful bonding, bonding temperature and thickness of PDMS prepolymer layer should be considered. Concerning the bonding temperature, We have tried several temperatures between 60 and 80 °C; a big difference was not observed. However, the thickness of the PDMS prepolymer layer determined by spinning speed strongly affected successful bonding. In our case, spinning speed of 5000 rpm and spinning time of 10 s were used for preparing spin-coated PDMS prepolymer on the glass slide. Reducing this speed to 4000 rpm (10 s) resulted in blocking of the capillary hole when bonding was performed, due to the excess volume of PDMS prepolymer on the glass slide. On the other hand, increasing the speed above 6000 rpm resulted in lowered probability for successful bonding. In the case of using a spinning speed of 5000 rpm, PDMS prepolymer layer thickness calculated by the weight of PDMS prepolymer and surface area of glass plate before curing was ∼18 µm. However, thicknesses under conditions of 4000 and 6000 rpm were about 22 and 15 µm, respectively. From Figure 2 (see “Before bonding”), the distance between the outer edge of the capillary and top edge of the PDMS channel is ∼20 µm. Since the spincoated PDMS prepolymer wets top edges of square capillary, the actual thickness after bonding would be changed. However, the calculated thicknesses shown above are very close, so that there must be a critical thickness for successful bonding. We chose 5000 rpm for further experiments. Combining these connections allowed us to fabricate many types of microfluidic networks on the lattice PDMS plate. Figure 4 shows typical fluorescence micrographs of a Tjunction type microchannel, 300 µm downstream from the Tjunction, and inside the connected 100-µm capillary, when fluoAnalytical Chemistry, Vol. 76, No. 11, June 1, 2004

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Figure 3. Microscope images of typical channel structures at crossing points. (a) Through channel. Two channels are blocked by plugged capillaries to form a straight channel. (b) T-junction. Two open capillaries face each other (left and right). The top side of PDMS channel is blocked by a plugged capillary. (c) Straight connection between 300-µm PDMS channel and 100-µm glass channel (capillary). Left and right channels are blocked by plugged capillaries.

rescent solution and aqueous buffer solution were mixed at the T-junction with different flow rates. As expected, typical laminar flow property of diffusional mixing is observed at each position, indicating that various types of reported chemical processes, such as diffusional separation or mixing, should be possible by using prepared capillary-embedded microfluidic devices. Preparation of Chemical-Sensing Square Capillaries and Dual-Sensing Microchip. The general concept of employing a surface-modified capillary for use in optical chemical sensors has been reported by Weigl and Wolfbeis.42 Since the inner cross section of the square capillary used in this work is square, the cross section of the chemically functionalized layer is different from that reported for a normal cylindrical capillary.42 Usually, the chemically functionalized layer is prepared by introducing reagent solutions into the capillary followed by aspirating out the reagent solution or introducing an air flow to blow out the solution so that the reagent solution remains evenly on the inner surface of the capillary.42 In this case, layer thickness is homogeneous on the inner wall of the capillaries. However, in the case of square capillaries, a larger portion of the reagent solution remains at the four corners of the inner capillary, and the layer thickness at these corners depends on the viscosity of the reagent solution.43 (42) Weigl, B. H.; Wolfbeis, O. S. Anal. Chem. 1994, 66, 3323-3327.

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Figure 4. Design of the T-junction type microchannel and typical fluorescence micrographs obtained 300-µm downstream from the T-junction, and inside the connected 100-µm capillary, when a fluorescent solution and an aqueous buffer solution are mixed at the T-junction with different flow rates.

In the first experiment, we prepared an ion-sensing capillary by attaching an ion-selective optode membrane to the inner surface. The ion-selective optode membrane is well known as a way to selectively detect a specific ion, and many applications have been reported.44-53 The general principle of ion determination is based on the ion pair extraction mechanism. Briefly, when the primary ion is extracted into the organic membrane phase by selective complexation between the ionophore and the primary ion, a proton of the lipophilic fluorescent pH indicator dye is (43) Kolb, W. B.; Cerro, R. L. Chem. Eng. Sci. 1991, 46, 2181-2195. (44) Hisamoto, H.; Satoh, S.; Satoh, K.; Tsubuku, M.; Siswanta, D.; Shichi, Y.; Koike, Y.; Suzuki, K. Anal. Chim. Acta 1999, 396, 131-141. (45) Hisamoto, H.; Kim, K.-H.; Manabe, Y.; Sasaki, K.; Minamitani, H.; Suzuki, K. Anal. Chim. Acta 1997, 342, 31-39. (46) Kurihara, K.; Ohtsu, M.; Yoshida, T.; Abe, T.; Hisamoto, H.; Suzuki. K. Anal. Chem. 1999, 71, 3558-3566. (47) Hisamoto, H.; Tani, M.; Mori, S.; Yamada, T.; Ishigaki, T.; Tohma, H.; Suzuki, K. Anal. Chem. 1999, 71, 259-264. (48) Tsagkatakis, I.; Peper, S.; Retter, R.; Bell, M.; Bakker, E. Anal. Chem. 2001, 73, 6083-6087. (49) Johnson, R. D.; Badr, I. H. A.; Barrett, G.; Lai, S.; Lu, Y.; Madou, M. J.; Bachas, L. G. Anal. Chem. 2001, 73, 3940-3946. (50) Kurihara, K.; Nakamura, K.; Hirayama, E.; Suzuki, K. Anal. Chem. 2002, 74, 6323-6333. (51) Fujii, E.; Koike, T.; Nakamura, K.; Sasaki, S.-i.; Kurihara, K.; Citterio, D.; Iwasaki, Y.; Niwa, O.; Suzuki, K. Anal. Chem. 2002, 74, 6106-6110. (52) Suzuki, K.; Hirayama, E.; Sugiyama, T.; Yasuda, K.; Okabe, H.; Citterio, D. Anal. Chem. 2002, 74, 5766-5773. (53) Hirayama, E.; Sugiyama, T.; Hisamoto, H.; Suzuki, K. Anal. Chem. 2000, 72, 465-474.

Figure 5. Top and cross sectional fluorescence images of PVC membrane-attached square capillary.

Figure 7. Top and cross sectional fluorescence images of hydrogelattached square capillary.

Figure 6. Fluorescence response for calcium ion (for details, see text).

released into the aqueous phase, so that the reaction leads to an increase of fluorescence intensity of the membrane.40,54-58 Figure 5 shows top and cross sectional views of the PVC membrane-modified square capillary. In our experiment, PVC copolymer containing the hydroxyl group rather than the normal PVC was used in order to achieve good adhesion of the membrane to the capillary inner surface.46 After injecting an air flow into the capillary containing the PVC membrane cocktail, most of the PVC membrane cocktail remained at the four corners of the capillary. In this case, maximum membrane thickness along the diagonal line direction is ∼7-8 µm. Fluorescence images of the capillary when introducing a buffer solution and 0.1 M calcium ion solution in buffer are shown in Figure 6. Fluorescence from the membrane increases due to the ion pair extraction reaction that took place between the organic membrane phase and the aqueous phase. Thus, ion determination in a sample solution can be carried out by the fluorescence intensity change. Figure 7 shows fluorescence images for the pH-sensing capillary. In contrast to the simple adhesion technology employed for the ion-sensing membrane, the pH-sensing PEG membrane is covalently bonded to the four corners of the square capillary. Since the prepared membrane is hydrophilic, aqueous sample solution penetrates into the membrane phase and the fluorescence intensity of the membrane changes upon pH change due to the protonation-deprotonation equilibrium of fluorescein molecule (54) Bakker, E.; Buhlmann, P.; Pretsch, E. Chem. Rev. 1997, 97, 3083-3132. (55) Buhlmann, P.; Pretsch, E.; Bakker, E. Chem. Rev. 1998, 98, 1593-1687. (56) Suzuki, K.; Ohzora, H.; Tohda, K.; Miyazaki, K.; Watanabe, K.; Inoue, H.; Shirai, T. Anal. Chim. Acta 1990, 237, 155-164. (57) Watanabe, K.; Nakagawa, E.; Yamada, H.; Hisamoto, H.; Suzuki, K. Anal. Chem. 1993, 65, 2704-2710. (58) Seiler, K.; Simon, W. Anal. Chim. Acta 1992, 266, 73-87.

Figure 8. Dual-sensing chip and calibration curves. (a) Design and photograph of the dual-sensing chip. (b) Calibration curve for pH. (c) Calibration curve for calcium ion.

immobilized in the membrane. Estimated membrane thickness of the hydrophilic membrane-modified capillary is ∼3-4 µm. Determination of pH is also made in the same manner as for the ion-sensing capillary. Figure 8 shows a photograph of a dual-sensing chip and calibration curves for pH and calcium ion concentration. Two types of chemical-sensing capillaries were cut into ∼2-mm lengths each and embedded into the PDMS channel network. To obtain fluorescence intensity data for the calibration curve, sample solutions containing different concentrations of calcium ion (constant pH, 7.0) and different pH buffer (calcium ion-free) were introduced from the side inlet capillary, and fluorescence intensity of either capillary was recorded. As expected, each capillary responds selectively for its respective analyte; detectable calcium ion concentration range is 10-5-1 M and pH range is 4-8. Analytical Chemistry, Vol. 76, No. 11, June 1, 2004

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These results suggest that two different chemical-sensing capillaries were successfully assembled, and selective detection of calcium ion concentration or pH was successfully performed by detecting fluorescence intensities of either membrane integrated into a CAs-CHIP. Thus, development of a multiple-analytesensing CAs-CHIP is expected by using many kinds of different sensing capillaries. CONCLUSIONS We have proposed a novel concept for assembling various chemical functions onto a single microfluidic device, called a CAsCHIP. By employing spin-coated PDMS prepolymer, we fabricated a capillary-embedded microchannel (capillary) network with no solution leakage. Typical two-layer flow for diffusional mixing could be established with CAs-CHIP in the same manner as for other reported microchips. Two different types of chemical-sensing layers were prepared inside the square capillaries, and these modified capillaries were embedded into a lattice PDMS channel

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network to fabricate a parallel dual-sensing microchip. Many types of surface modification methods inside capillaries are well known, so that the proposed method has great potential to fabricate different microfluidic devices having various chemical functions for analytical or synthetic applications. Application studies are currently underway in our laboratories. ACKNOWLEDGMENT We gratefully acknowledge Prof. Koji Suzuki, Department of Applied Chemistry, Keio University, for supplying the highly calcium-selective neutral ionophore (K23E1). This work was partially supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and Japan Science and Technology Agency. Received for review November 21, 2003. Accepted March 25, 2004. AC035385T