Integration of Valving and Sensing on a Capillary-Assembled

A simple integration of both flow control valves and a reaction-based sensing ... on-chip valving systems, where the simple valving functions were int...
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Anal. Chem. 2005, 77, 2266-2271

Integration of Valving and Sensing on a Capillary-Assembled Microchip Hideaki Hisamoto,* Shun-ichi Funano, and Shigeru Terabe

Graduate School of Material Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297 Japan

A simple integration of both flow control valves and a reaction-based sensing function on a single microchip was performed by using capillary-assembled microchip (CAsCHIP: Hisamoto, H.; Nakashima, Y.; Kitamura, C.; Funano, S.-i.; Yasuoka, M.; Morishima, K.; Kikutani, Y.; Kitamori, T.; Terabe, S. Anal. Chem. 2004, 76, 32223228.). In contrast to the previously reported on-chip valving systems, where the simple valving functions were integrated, our system can integrate not only valving function but also many other chemical functions to perform a complex chemical operation on a single microchip. Here, an enzymatic reaction-based readout system is employed as an example. A square capillary immobilizing N-isopropylacrylamide polymer monolith (referred to as “valving capillary”) is used as a thermoresponsive “valving part” and the immobilizing enzyme-modified glycidyl methacrylate polymer monolith (referred to as “sensing capillary”) is used as a “sensing part” of the CAsCHIP. These capillaries are embedded into a lattice microchannel network fabricated on poly(dimethylsiloxane), which has the same channel dimensions as the outer dimensions of the square capillaries. After bonding, a small Peltier device (2 mm × 2 mm) for temperature control is placed on the embedded valving capillaries to control fluid flow. Using this for heating or cooling, fast operation times of 1.4 and 3.2 s for opening and closing valves, respectively, are successfully achieved. Finally, two valving capillaries are independently controlled to trap sample solution within a bypass channel, where the enzyme-immobilized capillary is embedded, and then enzymatic reaction-based sensing of chemical species is performed as an example. The fundamental characteristics of the valve-integrated microchip are fully investigated, and an application to the analysis of an enzyme substrate by using two independent valving capillaries and a sensing capillary is demonstrated. Recently, micro total analysis systems (µ-TAS) have progressed extensively, and many fundamental operations such as mixing, separation, reaction, sensing, and valving have been integrated on a microchip.1-4 Among these, valving is one of the most important parts for the development of complex chemical * To whom correspondence should be addressed. E-mail: hisamoto@ sci.u-hyogo.ac.jp. Fax: +81-791-58-0493. (1) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636.

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systems,5-25 because flow control in microfluidic channels becomes more complicated with system integration. On the other hand, application fields of µ-TAS research are widely expanded by the integration of chemical functions by position-selective immobilization of highly functional molecules, biomolecules, or polymers inside the microchannel.15-21,26-31 Thus, one important direction of future µ-TAS research is the integration of complex (2) Auroux, P.-A.; Reyes, D. R.; Iossifidis, D.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (3) Vilkner, T.; Janasek, D.; Manz, A. Anal. Chem. 2004, 76, 3373-3386. (4) Lab-on-a-Chip: Miniaturized Systems for (Bio) Chemical Analysis and Synthesis; Oosterbroek, R. E., van den Berg, A. Eds.; Elsevier: Enschede, 2003. (5) Go, J. S.; Shoji, S. Sen. Actuators, A 2004, 114, 438-444. (6) Hosokawa, K.; Maeda, R. J. Micromech. Microeng. 2000, 10, 415-420. (7) Unger, M. A.; Chou, H.-P.; Thorsen, T.; Scherer, A.; Quake, S. R. Science 2000, 288, 113-116. (8) Lagally, E. T.; Medintz, I.; Mathies, R. A. Anal. Chem. 2001, 73, 565-570. (9) Ismagilov, R. F.; Rosmarin, D.; Kenis, P. J. A.; Chiu, D. T.; Zhang, W.; Stone, H. A.; Whitesides, G. M. Anal. Chem. 2001, 73, 4682-4687. (10) Selvaganapathy, P.; Carlen, E. T.; Mastrangelo, C. H. Sens. Actuators, A 2003, 104, 275-282. (11) Klintberg, L.; Svedberg, M.; Nikolajeff, F.; Thornell, G. Sens. Actuators, A 2003, 103, 307-316. (12) Liu, R. H.; Bonanno, J.; Yang, J.; Lenigk, R.; Grodzinski, P. Sens. Actuators, B 2004, 98, 328-336 (13) Pal, R.; Yang, M.; Johnson, B. N.; Burke, D. T.; Burns, M. A. Anal. Chem. 2004, 76, 3740-3748. (14) Hartshorne, H.; Backhouse, C. J.; Lee, W. E. Sens. Actuators, B 2004, 99, 592-600. (15) Beebe, D. J.; Moore, J. S.; Bauer, J. M.; Yu, Q.; Liu, R. H.; Devadoss, C.; Jo, B.-H. Nature 2000, 404, 588-590. (16) Eddington, D. T.; Beebe, D. J. Adv. Drug Delivery Rev. 2004, 56, 199210. (17) Harmon, M. E.; Tang, M.; Frank, C. W. Polymer 2003, 4547-4556. (18) Yu, C.; Mutlu, S.; Selvaganapathy, P.; Mastrangelo, C. H.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2003, 75, 1958-1961. (19) Luo, Q.; Mutlu, S.; Gianchandani, Y. B.; Svec, F.; Fre´chet, J. M. J. Electrophoresis 2003, 24, 3694-3702. (20) Hasselbrink, E. F., Jr.; Shepodd, T. J.; Rehm, J. E. Anal. Chem. 2002, 74, 4913-4918. (21) Reichmuth, D. S.; Shepodd, T. J.; Kirby, B. J. Anal. Chem. 2004, 76, 50635068. (22) Feng, Y.; Zhou, Z.; Ye, X.; Xiong, J. Sens. Actuators, A 2003, 108, 138143. (23) Juncker, D.; Schmid, H.; Drechsler, U.; Wolf, H.; Wolf, M.; Michel, B.; de Rooij, N.; Delamarche, E. Anal. Chem. 2002, 74, 6139-6144. (24) Hosokawa, K.; Fujii, T.; Endo, I. Anal. Chem. 1999, 71, 4781-4785. (25) Yamada, M.; Seki, M. Anal. Chem. 2004, 76, 895-899. (26) Kenis, P. J. A.; Ismagilov, R. F.; Whitesides, G. M. Science 1999, 285, 8385. (27) Kenis, P. J. A.; Ismagilov, R. F.; Takayama, S.; Whitesides, G. M.; Li, S.; White, H. S. Acc. Chem. Res. 2000, 33, 841-847. (28) Peterson, D. S.; Rohr, T.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2002, 74, 4081-4088. (29) Zhan, W.; Seong, G. H.; Crooks, R. M. Anal. Chem. 2002, 74, 4647-4652. (30) Lee, H. J.; Goodrich, T. T.; Corn, R. M. Anal. Chem. 2001, 73, 55255531. 10.1021/ac0484528 CCC: $30.25

© 2005 American Chemical Society Published on Web 02/11/2005

chemical operation involving position-selective, immobilized functional chemistry using on-chip valving systems. Many types of on-chip valving systems such as pneumatic valves,5-9 phase-change valves,10-13 ferrofluid-based valves,14 stimuliresponsive polymer valves,15-19 movable piston-type valves,20,21 and hydrophobic passive valves22-25 have been reported. Each is quite promising for its own described purpose. However, from the viewpoint of the number of integrated (immobilized) chemical functions in a single microfluidic device, most of the works listed above are limited in that they integrate a valving function only or that together with simple mixing-type reactions or capillary electrophoresis-based separations. Thus far, not many chemical functions have been integrated, because of the difficulties imposed by current fabrication methods of the on-chip valve. Generally, reported fabrication procedures of on-chip valves are complicated because they entail step-by-step micromachining. Therefore, when another chemical function is integrated into the microchannel after the fabrication of valves, the entire fabrication procedure should be reconsidered again to achieve total system integration. Among the reported valve fabrication methods, a photopolymerization technique for in situ preparation is well known for its simplicity in integrating a valving function as compared to those of micromachining techniques. However, this method requires the introduction of reagent solution into the entire system of microchannels, although the chemical modification is carried out only at a defined position in the microchannel. Thus, once a valving function is patterned at a part of the channel, position-selectively, other reagents should be introduced over the previously patterned valve surface to integrate multiple chemical functions into a single microchip. In this case, experimental difficulty arises due to pressure problems when reagent solutions are introduced over the existing valve structure. Moreover, this procedure can be sometimes risky because some reagents, which usually include a radical initiator or other highly reactive organic species, have a detrimental effect on the previously patterned valving position. Therefore, integration of other chemical functions such as enzymatic reaction, chemical sensing, filtering, and so on, which are usually carried out by the introduction of functional molecule solution containing hazardous reagents in the microchannel, is technically difficult once the valving function is set in place. Moorthy et al. recently reported a total ELISA analysis system involving check valves.32 The concept and the developed system are very elegant; however, the fabrication procedure seems complicated because it requires multiple photopolymerization procedures and surface protection process for integrating chemical functions in the same microchip platform. On the other hand, we have recently developed a new method for integrating multiple chemical functions onto a single microchip, called a capillary-assembled microchip (CAs-CHIP).33 In this case, various types of chemically functionalized square capillaries are easily assembled onto a single microchip by embedding them onto a lattice microchannel network. Thus, even if the valving function is already set in place, many other chemical functions can be (31) Hisamoto, H.; Shimizu, Y.; Uchiyama, K.; Tokeshi, M.; Kikutani, Y.; Hibara, A.; Kitamori, T. Anal. Chem. 2003, 75, 350-354. (32) Moorthy, J.; Mensing, G. A.; Kim, D.; Mohanty, S.; Eddington, D. T.; Tepp, W. H.; Johnson, E. A.; Beebe, D. J. Electrophoresis 2004, 25, 1705-1713. (33) Hisamoto, H.; Nakashima, Y.; Kitamura, C.; Funano, S.-i.; Yasuoka, M.; Morishima, K.; Kikutani, Y.; Kitamori, T.; Terabe, S. Anal. Chem. 2004, 76, 3222-3228.

integrated with relative ease. This concept is quite promising for realizing the complex operations involving valving function and other chemical functions in the microdevice format. Moreover, this concept can involve many techniques reported in the literature. For example, photolithographically prepared polymer valves15,16,18-21 can also be prepared inside square capillaries and then assembled on a PDMS plate. This point is one of the advantages of using CAs-CHIP.33 To this end, we focused on work reported by Fre´chet’s group.18,19 They performed immobilization of a thermoresponsive polymer inside a microchannel for use in the fluid control valve of the microfluidic device. Fundamental characteristics of valving function were fully studied. This immobilization technique is deemed suitable as well for setting the valving function to a square capillary, as the valving component of CAs-CHIP. We prepared a valving capillary using the above-mentioned technique and a small Peltier device for rapid and convenient flow control. Finally, two valving capillaries are independently controlled to trap the sample solution into a bypass channel, where the sensing capillary is embedded. The feasibility of performing a complex chemical operation inside a microchannel using this configuration is demonstrated by enzymatic reaction-based sensing of chemical species under stopped-flow condition. EXPERIMENTAL SECTION Square Capillaries and Reagents. Square capillaries having 300-µm outer widths (flat-to-flat) and 100- or 50-µm inner width 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. N-Isopropylacrylamide (pNIPAAm), N,N′-methylenebisacrylamide, glycidyl methacrylate, ethylene glycol dimethacrylate, ethylenediamine, glutaraldehyde, sodium borohydride, 2,2′azobis(isobutyronitrile), and 3-(trimethoxysilyl)propyl methacrylate were purchased from Tokyo Chemical Industry (Tokyo, Japan). β-Galactosidase from Escherichia coli and fluorescein di(β-Dgalactopyranoside) were purchased from Sigma (St. Louis, MO). 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. Preparation of Functional 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 that surface modifications could be made. Valving Capillary. A valving capillary was prepared by a procedure similar to that reported by Yu et al.,18 in which they immobilized a thermoresponsive polymer by in situ polymerization of a monomer inside the microchannel. The inside 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 liquid in the capillary was flushed with methanol and acetone and dried in an oven at 80 °C for 1 h. pNIPAAm (191.9 mg), N,N′-methylenebisacrylamide (10.3 mg), and 2,2′-azobis(isobutyronitrile) (2.4 mg) were dissolved into a mixture of water (1 mL) and methanol (1 mL), which was stirred and purged with nitrogen for 10 min. This mixture was introduced into the surface-modified square capillary, Analytical Chemistry, Vol. 77, No. 7, April 1, 2005

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which had the methacryl group. This capillary was heated at 60 °C for 17 h to complete monolithic polymer formation. 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 performance of the valve was evaluated using an HPLC pump and a pNIPAAm-immobilized capillary (valve size: 100 µm × 100 µm × 10 mm). In this case, flow rates were varied from 1 to 5 µL/min, and back pressures in close and open modes were measured. Sensing Capillary. An enzyme-immobilized capillary was prepared using the procedure reported by Petro et al.,34 with slight modification: they prepared a glycidyl methacrylate polymer monolith followed by trypsin immobilization, while here, βgalactosidase was immobilized. The inner wall of the capillary was treated with 3-(trimethoxysilyl)propyl methacrylate-0.12 M HCl mixture in the same manner as the valving capillary. Glycidyl methacrylate (180 mg), ethylene glycol dimethacrylate (120 mg), 2,2′-azobis(isobutyronitrile) (3 mg), dodecanol (70 mg), and cyclohexanol (630 mg) were mixed and purged with nitrogen for 10 min. This mixture was introduced into the surface-modified square capillary and heated at 60 °C for 2 h to complete glycidyl methacrylate polymer monolith formation. The resulting capillary was fully washed by introducing tetrahydrofuran at 1 µL/min for 12 h to remove unreacted chemical species. Then, the ethylenediamine was introduced into the monolith capillary and allowed to react at 80 °C for 2 h. In the next step, 2.5% glutaraldehyde solution was introduced into the capillary at 5 µL/min for 1 h to form a Schiff base on the monolith surface, followed by washing with pure water at 5 µL/min for 30 min. β-Galactosidase solution (0.06 mg/mL, 0.1 M phosphate buffer pH 7.0) was introduced at 2.5 µL/min for 3 h for immobilization of the enzyme on the surface by Schiff base formation. After washing with pure water at 5 µL/ min for 30 min, 0.1% sodium borohydride solution was introduced at 5 µL/min for 5 min to reduce the Schiff base. Finally, washing with buffer at 5 µL/min for 30 min to remove unreacted sodium borohydride completed the enzyme immobilization on the monolith polymer surface. The prepared capillaries were cut into ∼2mm lengths using a ceramic cutter and embedded into the PDMS plate having lattice channel network. Construction of Final Channel System on Chip. Figure 1 shows a typical procedure for fabricating a capillary-assembled microchip (CAs-CHIP) with valving function. Chemically functionalized square capillaries were cut into appropriate lengths and embedded into the lattice microchannel network fabricated on the poly(dimethylsiloxane) (PDMS) plate. A fabrication procedure of a lattice microchannel on a PDMS plate has already been reported elsewhere.33 Briefly, a glass mold with a lattice structure was prepared by cutting a 300-µm depth with a 1-mm pitch using a dicing saw possessing an edge of 300-µm width. Then, a conventional PDMS molding process using a glass mold was carried out to prepare the PDMS mold. The second molding process using this PDMS mold reproduced the lattice microchannel network on the second 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 (34) Petro, M.; Svec, F.; Fre´chet, J. M. J. Biotechnol. Bioeng. 1996, 49, 355363.

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Figure 1. Typical procedure for fabricating a CAs-CHIP with valving function by embedding square capillaries into the lattice microchannel network, followed by bonding a thin PDMS layer on a glass plate and attaching a small Peltier device on a glass plate side. The plugged capillaries indicated as gray areas are actually square capillaries possessing 50-µm square-sized conduits, but these conduits were blocked with PDMS. In this figure, these conduits were not shown in order to avoid complexity.

channel network. After embedding all the capillaries, a PDMS cover was bonded on top. For this, a spin-coated PDMS prepolymer on a cover glass slide (∼147 µm thick) allowing relatively fast heat transfer was used as a cover plate. Because the valving function was controlled by a small Peltier device attached on the glass plate. PDMS prepolymer was spin coated on the glass slide at 5000 rpm and then adhered to the capillary-embedded PDMS plate before curing. Bonding was carried out by curing at 40 °C for 12 h. Flow Control of the Valve-Integrated Chip by a Small Peltier Device. A commercially available small Peltier device (2 mm × 2 mm × 1 mm, model CTN20201012, Citizen Watch Co., Ltd., Tokyo, Japan.) was used for actuating the thermoresponsive polymer located inside the microchannel network. Since ∼2-mmlong valving capillaries were used in our experiments, the Peltier device size of 2 mm × 2 mm covering valving capillary size was chosen. The device was attached on the glass plate side of the chip, just above the embedded position of the valving capillary (see figures). To ensure the attachment between the Peltier device and the glass surface, a small portion of thermal conduction glue was used. Temperature of the Peltier device was controlled by a Peltier controller (DPC-100, Daitron Technology Co., Ltd., Osaka, Japan.), and the actual temperature of the device itself was measured by a small thermocouple embedded inside. Optical Detection and Data Processing. Optical and fluorescence images of the microchannel were obtained using an optical/fluorescence inverted microscope (Eclipse TS100-F, Nikon, Tokyo, Japan). 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 filter block (B-2A, Nikon, Tokyo, Japan).

Figure 2. Simple experiments of the valving operation on a CAsCHIP. (a) A microscope image shows an actual microchannel fabricated. The photo was obtained without a small Peltier device to avoid complexity. Fluorescence images b-e correspond to the data points of the response profile shown in (f). In (f), gray and black bars correspond to the durations for temperature application (for details, see text.).

RESULTS AND DISCUSSION The mechanism of the valving capillary is based on the temperature-dependent shrinkage or swelling of the thermoresponsive polymer, pNIPAAm, immobilized in a square capillary. When the polymer is heated above 32 °C, it shrinks to allow solution flow, and when that is cooled below that temperature, it swells to block the solution flow.18 First, we evaluated the back pressure of the prepared valving capillary in open and close modes. As expected, back pressure in open mode increased linearly with increasing flow rate and reached to 0.3 MPa at 5 µL/min. On the other hand, measured back pressure in close mode reached to 1.2 MPa and the solution leaked from the joint between the capillary and pump. In the previous report, 18 the thermoresponsive polymer valve could tolerate pressure of at least 1.38 MPa for the close mode (valve size: 100 µm × 200 µm × 2 mm), and back pressure for open mode was 0.3 MPa (50 µL/min). The difference in the performance may be attributed to the difference of the valve size and polymerization method. However, our capillary worked similarly to the reported valve. Figure 2 shows the experimental results of the valving operation on a CAs-CHIP. In this case, a fluorescent solution

flowing through the valving capillary and a water flow met at the confluence point to form a two-layer flow. Fluorescence images shown in Figure 2b-e correspond to the data points of the response profile shown in Figure 2f, which was obtained by recording the fluorescence intensity at the outlet of the valving capillary (Data collection area, see Figure 2b). Since the pNIPAAm shrinks and swells at temperatures above and below 32 °C, the Peltier device surface temperature was set at 52 and 13 °C for opening and closing the valve, respectively. As expected, fluid flow was successfully controlled by switching the temperature of the Peltier device. These open/close cycles were reversible, and more than 70 cycles of valving operation were confirmed with no significant deterioration. In our case, heat transfer occurs through a cover glass plate (thickness, 147 µm), a PDMS layer (thickness, below 10 µm), and a glass capillary (thickness, 100 µm). Therefore, the response time of valving operation was relatively slow as compared with that reported in the literature.18 However, by using a Peltier device, a fast temperature rise and drop can be easily controlled by the applied current. Thus, we investigated the initial applied temperature dependence of 95% response time required for the valving operation, in which the 95% response time was determined by the time course of the fluorescence intensity at the outlet of the valving capillary. In this case, starting temperature of the Peltier device for opening the valve was set at 25 °C (corresponding to the closed mode) and that for closing the valve was set at 52 °C (corresponding to the opened mode), and just after steady-state signals were obtained, temperatures were reset at 25 and 52 °C for closing and opening the valve, respectively. As expected, a large temperature difference gave a faster response time. At the maximum applied temperature difference we tested, the fastest operation times of 1.4 and 3.2 s for opening and closing the valve were successfully achieved at the temperatures of 120 and 3.4 °C, respectively. Since the CAs-CHIP enables universal integration of various chemical functions, demonstration of a complex chemical operation is possible by using valving capillaries and other functionalized capillaries. Here, we used two valving capillaries and an enzyme reaction-based sensing capillary to demonstrate flow switching to trap sample solution and subsequent enzymatic reaction to readout a specific chemical species contained in sample solution as an example of complex chemical operation inside a microchannel. Since a normal enzymatic reaction takes a couple of minutes, stopping the solution by using an inner-channel valve is indispensable. First, we tried to demonstrate simple trapping of a sample liquid into a bypass channel by using two valving capillaries and fluorescent solution. The microchannel design is shown in Figure 3, and the operation procedure is as follows. First, valves A and B are kept open to introduce a fluorescent liquid into a bypass channel. When liquid introduction is finished, valve B is closed to stop the liquid flow inside the bypass channel, which is then confirmed by introducing pure water through valve A. In this case, temperatures of valves A and B were kept constant at 52 and 13 °C, respectively. As can be seen in the fluorescence image, the fluorescent liquid in a bypass channel did not move when water was flowing through valve A, and no significant leaking of liquid through valve B was observed during the experiment. By using the channel design shown in Figure 3, the distance between two Analytical Chemistry, Vol. 77, No. 7, April 1, 2005

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Figure 3. Sample trapping operation using two valving capillaries and a bypass channel structure.

Peltier devices was very close (∼100 µm at the closest point); therefore, an overlap of thermal diffusion in a microchip could cause some concern. However, our results revealed that it was not a problem when temperatures of two Peltier devices were independently controlled. In the second fluorescence image shown in Figure 3, the fluorescence solution in valving capillary B did not move, which also supported that leakage through the valve B did not occur at all, although the slight diffusion of fluorescent molecule at the confluence point of the main and bypass channels was seen in the fluorescence image. The present results indicated that the liquid flow inside the bypass channel was completely stopped by using valve B; thus, a stopped-flow condition was maintained in a bypass channel, although water was kept flowing in main channel. This type of flow control can be applicable to a chemical process involving sample trapping and subsequent readout of chemical species by a certain reaction that requires the stopped-flow condition. Based on these results, the microchannel embedding an enzymatic reaction-based sensing capillary in a bypass channel was designed and constructed as shown in Figure 4a. In this case, β-galactosidase and fluorescein di(β-D-galactopyranoside) were used as immobilized enzyme and fluorescent substrate, respectively. When the enzymatic reaction takes place at the sensing capillary, fluorescein molecules are released from the nonfluorescent substrate. Therefore, detecting the fluorescence intensity at the sensing capillary will allow monitoring of the substrate concentration in the main flow channel. However, if the solution flow in the bypass channel is not stopped, residence time of the substrate solution in the sensing capillary is too short to produce enough of the enzymatic reaction product (fluorescein in this case), and the little that is produced just flows away. Therefore, this system is suitable for confirming the usefulness of the valving function described above. Figure 4b shows illustrations of liquid flow inside microchannel and fluorescence images when two valves were operated. After introducing the fluorescent substrate solution (0.3 mM substrate in 0.1 M phosphate buffer, pH 7) into the bypass channel (sensing capillary), an enzymatic 2270 Analytical Chemistry, Vol. 77, No. 7, April 1, 2005

Figure 4. Valving and sensing operation of CAs-CHIP by using two valving capillaries and bypass channel structure where sensing capillary was embedded. (a) Design of the channel. (b) Illustrations of liquid flow inside microchannel and fluorescence images when two valves were operated. (c) Response profile of sensing capillary. Two data points correspond to the fluorescence images shown in (b).

reaction took place. When valve B was opened, the liquid flow in the bypass channel was not stopped. Thus, the enzymatic reaction itself did not proceed well, due to the short residence time of the substrate, and only a negligible amount of product, which flowed away to the waste. Therefore, no fluorescence increase was observed in the fluorescence image (b-1). On the contrary, when valve B was closed, the fluorescence intensity at the sensing capillary position dramatically increased, indicating that the enzymatic reaction proceeded well (see fluorescence image b-2). Figure 4c shows the fluorescence intensity profile of the data collection area shown in Figure 4b-1, where an enzyme-immobilized capillary was embedded in the bypass channel. When the substrate solution flow was stopped by valve B, the enzymatic reaction gradually proceeded and then reached a maximum at the reaction time of 120 s. The control experiment for the nonspecific fluorogenesis of the substrate was also carried out using the monolithic polymer capillary without immobilized enzyme. In this case, fluorescence increase was not observed for more than 10 min even when the solution flow was stopped by valve B. These results indicated that the model experiment of complex chemical operations was successful. This type of chemical operation is generally difficult without an on-chip valve.

By using this system, monitoring the chemical composition in a flowing sample solution without stopping the main flow is possible. Here, an enzymatic reaction-based sensing system was demonstrated. Since other types of sensing capillaries, such as ion sensing and pH sensing, have already been developed,33 multiple chemical sensing is also possible using other chemical sensing capillaries. Thus, the present system can be considered as an incremental step in the development of a multiple analyte sensing CAs-CHIP. CONCLUSIONS Using CAs-CHIP, we have demonstrated a simple integration of both flow control valves and reaction-based sensing system on a single microchip. In contrast to previously reported on-chip valving systems where only the valving functions were integrated, our system can readily incorporate other chemical functions as

well. This work opens the way for the development of systems enabling performance of complex chemical operations involving fluid control valves and other chemical functions on a single microchip format. ACKNOWLEDGMENT This work was partially supported by Grants for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Kawanishi Memorial Shinmeiwa Education Foundation, Japan.

Received for review October 20, 2004. Accepted January 7, 2005. AC0484528

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