Anal. Chem.

2007, 79, 1725-1730

Microfluidic Device for the Detection of Glucose Using a Micro Direct Methanol Fuel Cell as an Amperometric Detection Power Source
Takeshi Ito,*, Masayuki Kunimatsu, Satoru Kaneko, Seishiro Ohya, and Koji Suzuki,

Kanagawa Industrial Technology Center, Shimoimaizumi, Ebina, Kanagawa 243-0435, Japan, Faculty of Science and Technology, Keio University, Hiyoshi, Yokohama, Kanagawa 223-8522, Japan, and Japan Science and Technology Agency, CREST, Honcho, Kawaguchi, Saitama 332-0012, Japan

We designed and prepared a novel microbiosensing system consisting of a microbioreactor fabricated using photosensitive sheets intercalated between Pyrex wafers as a dam structure, together with a micro fuel cell as a power source device between the electrodes for amperometric detection. The dam structure retains enzyme (glucose oxidase, GOx)-immobilized microbeads in a microchannel. Microelectrodes are used as an integrated detector within a microchannel located downstream of the dam structure, and these are used to detect the oxidation current of hydrogen peroxide produced from a glucose sample and GOx. A micro direct methanol fuel cell (DMFC, i.d. 500 m) was fabricated on a polymeric substrate and was used to supply a potential for the electrochemical detector. In this case, two -DMFCs were stacked on one substrate to increase the voltage for the oxidation of hydrogen peroxide. A linear response curve was obtained in range from 0.1 to 10 mM glucose for the designed microbiosensing system. These results show that a microfluidic biosensing system designed with a -DMFC device is useful and has the potential to assist minuaturization and simplification of the sensing system, in addition to increasing disposability of the device.
Miniaturized chemical and biochemical sensing systems are regarded as micro total analysis systems (-TAS) or a lab-on-achip.1 Microfluidic devices for biosensing provide a new alternative with great potential for decentralized clinical analysis and personal home monitoring. Flow-through analysis systems and biosensors are commonly based on sensing selectivity for the enzymatic detection of the chemical species of interest. Recently, enzyme microreactors fabricated on a silicon wafer have been reported using micromachining technologies.2-5 Murakami et al.2 reported
* Corresponding author. Tel: +81-46236-1500. Fax: +81-43236-1525. E-mail: taito@kanagawa-iri.go.jp. Kanagawa Industrial Technology Center. Keio University. Japan Science and Technology Agency. (1) Manz, A.; Graber, N.; Widmer, M. Sens. Actuators, B 1990, 1, 244-248. (2) Murakami, Y.; Takeuchi, T.; Yoyoyama, K.; Tamiya, E.; Karube, I.; Suda, M. Anal. Chem. 1993, 65, 2731-2735. (3) Laurell, T.; Rosengren, L. Sens. Actuators, B 1994, 18-19, 614-617. (4) LHostis, E.; Michel, P. E.; Fiaccabrino, G. C.; Strike, D. J.; de Rooji, N. F.; Koudelka-Hep, M. Sens. Actuators, B 2000, 64, 156-162. 10.1021/ac0618167 CCC: $37.00 Published on Web 01/10/2007 2007 American Chemical Society

that glucose oxidase (GOx) was immobilized in a V-shaped microchannel surface that was 1 m long, 100 m wide, and 70 m deep. In fact, a very long microchannel was required for the reactor. A major problem in immobilization of an enzyme on the surface of a microchannel is the small total surface area, while a standard microbead offers a relatively large surface area. For this study, in order to increase the enzyme-immobilized surface area, a microfluidic device was fabricated within a dam structure to retain the enzyme-immobilized microbeads in the microchannel. Microbeads immolibized with enzymes,6 antibodies,7,8 and other biological materials9 were introduced into the microchannels. To construct a dam structure, Sato et al. used fast atom beam fabrication on a quartz wafer,7 and Uchiyama et al. used a process involving the inductively coupled plasma etching of Si and anodic bonding with a Pyrex wafer.8 These reports were focused on the immunoassay method used to detect biological materials, and the fabrication techniques were costly, due to their high precision. Seong et al. fabricated microbead-based microfluidic systems using poly(dimethylsiloxane).6,9 However, this method has not been adapted for standardized mass products and batch processing. Low-cost and disposable microfluidic devices are desirable in the analytical field, especially for medical applications, in order to prevent the risk of secondary infection. Based on these requirements, we propose the fabrication of a dam structure using photosensitive sheets similar to a dry film photoresist, which is widely used as a sacrificial layer for the patterning of wiring on a printed-wiring assembly. This technology has been recently introduced for fabrication of microfluidic devices.10,11 Compared with a liquid-type photoresist, a photosensitive sheet has advantages, such as good adhesion to other substrates including Si, glass, and polymers, uniform distribution, no requirement for liquid handling, short processing time, and uniform thickness.
(5) Xie, B.; Danielsson, B.; Norberg, P.; Winquist, F.; Lundstrom, I. Sens. Actuators, B 1992, 6, 127-130. (6) Seong, G. H.; Heo, J.; Crooks, R. M. Anal. Chem. 2003, 75, 3161-3167. (7) Sato, K.; Tokeshi, M.; Odake, T.; Kimura, H.; Ooi, T.; Nakao, M;, Kitamori, T. Anal. Chem. 2000, 72, 1144-1147. (8) Uchiyama, K.; Yang, M.; Sawazaki, T.; Shimizu, H.; Ito, S. Sens. Actuators, B 2004, 103, 200-205. (9) Seong, G. H.; Zhan, W.; Crooks, R. M. Anal. Chem. 2002, 74, 3372-3377. (10) Vulto, P.; Glade, N.; Altomare, L.; Bablet, J.; Tin, L. D.; Medoro, G.; Chartier, I.; Manaresi, N.; Tartagni, M.; Guerrieri, R. Lab Chip 2005, 5, 158-162. (11) Tsai, Y. C.; Jen, H. P.; Lin, K. W.; Hsieh, Y. Z.; J. Chromatogr., A 2006, 1111, 267-271.

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The micro fuel cell is an attractive power source for mobile devices such as cellular phones, laptop computers, and various microsystems, including -TAS. Many studies on the miniaturization of fuel cells have been reported using micromachining technologies.12-15 However, these micro fuel cells have a maximum power density of less than 1 mW/cm2 at room temperature, despite costly fabrication difficulties, and some require the pumping of fuel. Direct methanol fuel cells (DMFCs) are attractive as portable power sources, because only a methanol solution is required as the fuel for power generation. We propose a novel manufacturing method for a micro-DMFC (-DMFC), fabricated with microholes on a polymeric substrate.16 The proposed -DMFC does not require precision microfabrication techniques and is cost-effective, due to the use of a polymeric substrate. We demonstrate in this study that stacked -DMFCs are able to supply sufficient potential to drive an electrochemical (EC) detector. The EC detector was composed of a thin Pt layer working electrode (WE) and a thin Ag layer reference electrode (RE) that also functioned as the counter electrode (CE). The electrodes were located downstream of the dam structure for the detection of hydrogen peroxide, which was generated by the enzyme reaction of glucose oxidase immobilized with micro glass beads. In this paper, we describe the integration of a micro fuel cell and a microbead-based microfluidic device, and its subsequent application to glucose detection. The high performance for glucose detection and inexpensive fabrication techniques demonstrate the potential use of this integrated microfluidic device and -DMFC as a disposable microbiosensing system. EXPERIMENTAL SECTION Fabrication of the Microfluidic cdevice. Three photosensitive sheets were used to fabricate the dam structure. A schematic of the device is shown in Figure 1. The upper Pyrex wafer has an inlet port for the microbeads and solutions and an outlet port for waste. The bottom Pyrex wafer has an EC detector composed of a thin Pt layer for the WE (0.5 mm long) and a thin Ag layer for the RE/CE (2 mm long). These electrodes were separated from each other by a distance of 0.5 mm. Pyrex wafers (0.5 mm thick) were purchased from Mitorika Glass Co., Ltd. (Ibaraki, Japan). A schematic of the fabrication procedure is presented in Figure 2. The thin Ag layer (200 nm) was deposited on the bottom wafer using a heating deposition apparatus (Ulvac Kiko Inc., VPC-410A) after a thin Cr layer (20 nm) was deposited as a buffer layer. A positive-type photoresist (Tokyo Ohka Kogyo Co., Ltd., OFPR800) was coated on the wafer using a spin coater (Mikasa, 1HDX2), and the electrode was patterned using a mask aligner (Mikasa, MA-20). A prebake process was carried out at 85 C for 30 min. After etching of the thin Ag and Cr layers, the photoresist was removed by dipping in acetone. The etching reagents for the thin Cr and Ag layers were prepared from a mixture of the following materials: Cr etchant, Ce(SO4)22(NH4)2SO4H2O:HClO4:
(12) Min, K. B.; Tanaka, S.; Esashi, M. Electrochemistry 2002, 70, 924-927. (13) Hayase, M.; Kawase, T.; Hatsuzawa, T. Electrochem. Solid-State Lett. 2004, 7, A231-A234. (14) Kelly, S. C.; Deluga, G. A.; Smyrl, W. H. Electrochem. Solid-State Lett. 2000, 3, 407-409. (15) Motokawa, S.; Mohamedi, M.; Momma, T.; Shoji, S.; Osaka, T. Electrochem. Commun. 2004, 6, 562-565. (16) Ito, T.; Kimura, K.; Kunimatsu, M. Electrochem. Commun. 2006, 8, 973976.

Figure 1. Schematic figure of a microfluidic device integrated with a dam structure and an electrochemical detector. (A) Top view of the device. The diameter of the inlet and outlet ports is 1 mm. The WE and RE/CE are 0.5 mm and 2 mm long, respectively, and they are separated by a distance of 0.5 mm. (B) Closeup view of the dam structure. The distance from the center of the inlet port to that of the dam structure was 7.5 mm. The WE was located 0.2 mm downstream of the dam structure. (C) Cross section view at the dam structure. The microchannels upstream and downstream of the dam structure are 75 and 25 m deep, respectively.

H2O ) 17:5:100 (weight ratio); and the Ag etchant, NH4OH:H2O2: H2O ) 1:1:20 (volume ratio). Ammonia water, hydrogen peroxide, ammonium cerium(IV) nitrite, perchloric acid, and acetone were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). A thin Pt layer (200 nm) was deposited on the bottom wafer using an electron beam deposition apparatus (Ulvac Co., Ltd., DRP-40E) after a thin Ti layer (50 nm) was deposited as a buffer layer. The thin Pt layer was patterned using a liftoff technique with a negative-type photoresist (Zeon Cor., ZPN-1150). After the wafer was dipped in acetone to remove the resist layer, it was laminated to a photosensitive sheet (Hitachi Chemical Co., Ltd., ME-1025 (25 m thick)) using standard industrial lamination equipment (GBC Japan, GBC 2500) at 110 C with a feed rate of 1.05 cm/s. The microchannel downstream of the dam structure was patterned using the mask aligner, and dip development (developer, 1 wt % Na2CO3) was carried out for 5 min followed by rinsing with ultrapure water. Photosensitive sheets of ME-1025 and ME-1050 (50 m thick) were laminated in sequence to increase the channel depth (75 m thick). The microchannel upstream of the dam structure was patterned and developed in the same manner. Both microchannels had a width of 300 m. After the upper and bottom wafers were aligned and fixed in position by lamination, the wafers were adhered together at 160 C for 1 h.

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Figure 2. Schematic procedure for fabrication of the microfluidic device. After the upper wafer was drilled with through-holes, the photosensitive sheet was laminated and patterned. Ag/Cr and Pt/Ti thin layers were deposited on the bottom wafer for the electrochemical detector, the photosensitive sheet was then laminated and patterned. They were then adhered to each other by a lamination and curing process.

Fabrication of -DMFCs. Two -DMFCs were stacked on one wafer to increase the voltage. The fabrication process has been described elsewhere.16 An engineering thermoplastic, polysulfone (PSF), substrate was chosen because of its high resistance to chemicals and heat. Two -DMFCs were placed in microholes (i.d. 500 m) on an array and connected by a thin Au layer. A proton exchange membrane (PEM) was made by dehydrating a Nafion dispersion solution (Wako Pure Chemical Industries, Ltd., DE2020) injected into the microholes. Pt/Ru black (HiSPEC 6000) for the anode and Pt black (HiSPEC 1000) for the cathode were purchased from Johnson Matthey Fuel Cells (London, UK). The

electrode precursors were agitated with a Nafion dispersion solution using a magnetic stirrer. The Pt/Ru catalyst was coated using a pipetter, followed by drying at room temperature for 30 min. After the Pt catalyst was coated in the same way as the anode catalyst, the excess was wiped off and cleaned with a plastic plate. Finally, the substrate was pressed (0.2 MPa) at 135 C for 5 min to solidify the catalysts. For each cell, 5 mg/cm2 of both Pt/Ru and Pt were used. The thin Au layer (200 nm thick) was used only as an electronic conductor and was fabricated using the heating deposition apparatus. The Au wire (>99.99%) was obtained from Tanaka Kikinzoku Kogyo (Tokyo, Japan). The electric conductor was patterned using a polyethylene terephthalate (PET) film mask. We inserted electrically conductive paste (Dohtent NH070A, Nohon Handa Co., Ltd.) into the microholes, then the heating procedure was carried out at 110 C for 30 min, in order to connect the anode and cathode sides of the substrate. Immobilization of Glucose Oxidase. Glucose oxidase (Roche, 300 units/mg) was immobilized with micro glass beads (i.d. 50 m, Union) using a glutaraldehyde cross-linking technique. After the beads (100 mg) were fed into a microtube and dipped in 1 mL of glutaraldehyde solution (8 wt %, TAAB) for 45 min, the beads were rinsed 5 times with 1 mL of ion-exchanged water. A 100 units/mL enzyme solution was prepared as GOx was dissolved into phosphate buffer solution (1/15 M, pH 7.0, Wako Pure Chemical Industries, Ltd.). The microbeads were immobilized with GOx by mixing with 200 L of GOx solution at 4 C for 24 h. The GOx solution was skimmed off after immobilization, and the microtube was filled with 200 L of phosphate buffer solution. System Setup and Electrochemical Detection. A schematic of the glucose detection system using -DMFCs is presented in Figure 3. After the immobilized microbeads were introduced from the inlet port and filled the microchannel upstream of the dam structure, a fused-silica capillary (o.d. 360 m, i.d. 100 m) was attached to the inlet port. A microsyringe (1 mL, Hamilton Co.) was connected to the capillary, and then phosphate buffers with specific concentrations of D-glucose (Wako Pure Chemical Industries, Ltd.) were injected using a microsyringe pump (CMA Co.,

Figure 3. Schematic figure and photographs of the glucose detection system using a -DMFC. Five -DMFCs were fabricated on one PSF substrate and three microfluidic devices were prepared on a Pyrex substrate. The -DMFC cathode was connected to the WE, and the anode was connected to a nonresistant ammeter.

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Ltd., CMA-102) at a flow rate of 5 L/min. Two EC detection modes were used. One used the -DMFC for supply of a potential between the WE and RE/CE (DMFC mode). The cathode of the -DMFC was connected to the WE, and the anode was connected to a potentiostat (ALS Co., Ltd., ALS 832) that was used as a nonresistant ammeter. The other mode used only the potentiostat in order to supply the potential and to detect the current for comparison with each detection system (normal mode). Chronoamperometry was used to measure the oxidation current of hydrogen peroxide. The WE potential was stepped from -0.01 to 0.0 V and from 0 to 0.7 V versus the RE in the DMFC mode and normal mode, respectively. The current was adopted at 10 s after the potential was stepped. RESULTS AND DISCUSSION Fabrication of the Microfluidic Device with a Dam Structure Using a Photosensitive Sheet. A Pyrex wafer laminated with the photosensitive sheet was cut into rectangular pieces using a dicing saw (Disco, DAD-321), in order to verify the adherence of the sheet. No samples revealed delamination after the 160 C postbake, indicating that the photosensitive sheet has good adhesion to the Pyrex glass wafer. To obtain a thick sheet, the lamination procedure was repeated after removal of the covered PET layer. Wafers were able to be tightly adhered by a postbake at 160 C for 1 h after the sheet was intercalated between the substrates by lamination. These procedures provide a convenient way of fabricating the microfluidic device. Fusion bonding,17 anodic bonding,18 and HF bonding19 have usually been employed to seal the microchannel. These techniques are limited in use to steps such as a thin-film electrode. A bonding technique using SU-8 has been reported;20 however, this technique has several disadvantages compared to use of a photosensitive sheet, such as a prebake being required, double bonding requiring additional preparative steps, and the high-viscosity photoresisit is difficult to handle. Since the heating procedures for bonding wafers can cause damage to biological materials after coating them within the microchannel, most of reported studies2-4,7,8,20 treated the biological materials after the bonding procedure. Immobilized microbeads were also introduced after the bonding procedures used for our system. Figure 4 shows the microbeads that remained in the dam structure. The displacement between the upper and bottom wafers was 15 m along the lateral direction. The mask aligner used in this study did not have control for the pressure and heat during bonding. Use of a flip chip bonding machine may result in an improved adhesion process. For the preparation of a dam structure,6-9 the fabrication technique used in this study has advantages over other fabrication techniques; the processing time is short, materials and process are inexpensive, all of which is adapted for a roll to roll process, standardized mass production, batch processing, 3-D configuration, and integration with thin layer electrodes for EC detection. These characteristics enable disposability of the device. In the proposed device, the microbeads could be removed for recycling
(17) Harrison, D. J.; Manz, A.; Fan, Z.; Ludi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (18) Shoji, S.; Esashi, M.; Matsuo, T. Sens. Actuators 1988, 14, 101-107. (19) Nakanishi, H.; Nishimoto, T.; Nakamura, R.; Yotsumoto, A.; Yoshida, T.; Shoji, S. Sens. Actuators, A 2000, 79, 237-244. (20) Heuschkel, M. O.; Guerin, L.; Buisson, B.; Bertrand, D.; Renaud, P. Sens. Actuators, B 1998, 48, 356-361.

Figure 4. Photograph of a microchannel filled with immobilized microbeads. The diameter of the microbeads was 50 m.

by adding negative pressure from the inlet port or by ultrasonication for 30 s. This feature enables the exchange of microbeads immobilized with other enzymes suitable for other purposes. Characteristics of -DMFCs. The design of the -DMFCs was devised in order to realize a simple fabrication process without precision microfabrication. Lee et al. introduced two patterns of stacking: banded and flip-flop.21 The banded type is suitable for power generation in microdevices, because fuel supply is carried out on the same surface when many cells are integrated in one substrate. A desktop drill was used to fabricate the through-holes, and vacuum deposition was used to coat the electrical wiring with a film mask. Preparation of the PEM within the microhole was a simple task requiring the hole to be filled with a Nafion dispersion solution and then dried. The process of coating the catalyst is the same as that used for larger fuel cells. These processes can reduce the cost and time of fabrication. When large-scale production is required, injection molding technology can be used to manufacture substrates with through-holes at one time. For the detection of hydrogen peroxide, a potential of 0.7 V was applied between the WE and RE/CE in order to oxidize the hydrogen peroxide. To obtain this potential, two -DMFCs were stacked on one wafer. A 5 wt % methanol solution was used as the fuel in this study. Figure 5A shows the fuel cell polarization curves. One -DMFC connected to two 500-m-diameter cells inseries achieved an open circuit voltage (OCV) of 750 mV and a maximum power output of 5.8 W, which is equivalent to 375 mV and 1.5 mW/cm2 per single cell. Comparison of these results with other microelectromechanical systems-based micro fuel cells12-15 revealed that the cell used in this study had better performance, despite the use of a passive-type DMFC of micrometer order with a small cell area. Figure 5B shows the time dependence of the OCV for different amounts of fuel. The fuel was supplied 30 s after monitoring of the OCV was initiated. The OCV quickly decreased when using a 1 L of fuel supply compared to the 3 and 5 L fuel supplies. The OCV response for supply of both 3 and 5 L of fuel has two plateaus from 150 to 400 s at 750 mV, and from 630 to 800 s at 700 mV. As a result, sufficient power
(21) Lee, S. J.; Cahng-Chien, A.; Cha, S. W.; OHayre, R.; Park, Y. I.; Saito, Y.; Prinz, F. B. J. Power Sources 2002, 112, 410-418.

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Figure 6. Current-concentration curve for glucose at a flow rate of 5 L/min. The WE potential was set at 0.7 V vs RE/CE. Each data point is the average of four measurements of a single flow-through experiment. The current at 10 s after the potential stepped was adopted. Open and closed circles indicate measurement conditions under normal mode and DMFC mode, respectively.]

Figure 5. Characteristics of the stacked -DMFCs. A 5 wt % methanol solution was used as the fuel. (A) Current-voltage characteristics of two -DMFCs connected in series. (B) Time-dependent characteristics of the OCV. Fuel volumes of 1, 3, and 5 L were injected at 30 s after monitoring of the OCV began. The fine line, bold line, and bold-dashed line represent fuel volumes of 1, 3, and 5 L, respectively.

could be generated using only 3 L of fuel supplied to each cell, and the second plateau was used to apply the potential to the EC detector. Glucose Detection. Throughout the column, glucose reacts with dissolved oxygen in solution, and the reaction is catalyzed by GOx on the surface of the microbeads; this reaction produces gluconolactone and hydrogen peroxide. Quantitative analyses were carried out using both DMFC and normal modes, and the results are shown in Figure 6. Each data point given is the average of four duplicate measurements of a single flow-through experiment. The relationship between the current and glucose concentration was linear in the range from 0.1 to 10 mM, with correlation coefficients of 0.9993 for normal mode and 0.9994 for DMFC mode. The maximum oxidation current measured was 1.3 10-7 A for a 10 mM glucose solution. This current probably has no effect on the -DMFC, because such a current loading did not decrease the voltage as evaluated from the current-voltage curve in Figure 5A. Since the oxidation currents for all glucose concentrations were slightly lower in DMFC mode than that in normal mode, the IR drop in the measurement system may have increased, and then the actual potential between the two electrodes would decrease. However, this characteristic has sufficient potential for the detection of glucose in human blood. Sugar diabetes is defined as a fasting blood sugar level of 126 mg/dL or more, which is estimated to be 7 mM. A fasting blood sugar level lower than 110 mg/dL (6.1 mM) is usually considered to be normal. The results for the biosensing system indicate that

it is suitable for the analysis of blood sugar levels. We can parallel stack the same enzyme column to detect many samples at once and in series to eliminate substances interfering with electrochemical detection, such as L-ascorbic acid during vivo monitoring. In order to increase the immobilized surface area of the microfluidic device, Murakami et al.2 fabricated a long microchannel extending to 1 m, and Hayashi et al.22 made a 3-D microstructure consisting of a photoresist. When compared with these devices, our proposed device was fabricated simply and has the possibility to increase the immobilized surface area and to decrease the microchannel length to less than 1 cm, because the immobilized microbeads are introduced into the microchannel. The volume and surface of the microchannel upstream of the dam structure, including the inlet port, was calculated as 0.6 L and 9.3 mm2, respectively. Assuming the microbeads to be cubic (all sides are 50 m), the number of microbeads introduced into the microchannel was 4870, which means the total surface area of the microbeads is 38 mm2; four times greater than that of the native microchannel surface. This characteristic shows the superiority of the proposed device over a microfluidic device whose microchannel surface is directly immobilized with the enzyme, because the microchannel length can be decreased. The selection of a smaller porous enzyme carrier and extension of the column length may make it possible to detect lower concentrations of substances. CONCLUSION We fabricated a microfluidic device integrated with a dam structure consisting of photosensitive sheets to retain enzyme immobilized microbeads, with an EC detector used for oxidizing hydrogen peroxide generated from the enzyme reaction with GOx. Stacked -DMFCs (i.d. 500 m) supplied the potential between the electrodes on the device using a few microliters of methanol solution as the fuel. Convenient and inexpensive fabrication techniques were used to produce the microfluidic device and
(22) Hayashi, K.; Iwasaki, Y.; Kurita, R.; Sunagawa, K.; Niwa, O.; Tate, A. J. Electroanal. Chem. 2005, 579, 215-222.

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-DMFCs. The resulting biosensing system had high performance for glucose detection and potential supply, because the relationship between the oxidation current and the glucose concentration was linear over 2 orders of magnitude in both normal and DMFC modes. The currents measured for the same glucose concentration in both the normal and DMFC modes were approximately the same. These results demonstrate that the proposed microbiosensing system is useful and has the potential to provide a miniaturized sensing system that is both simple and disposable.

ACKNOWLEDGMENT We thank Mr. Kawaguchi and Miss Miyoshi (Hitachi Chemical Co., Ltd.) for supplying the photosensitive sheet, Dr. Niwa and Dr. Kurita (National Institute of Advanced Industrial Science and Technology) for their helpful advice, and the Japan Science and Technology Agency for the opportunity to conduct this research. Received for review September 27, 2006. Accepted December 4, 2006.
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