Integration of Multianalyte Sensing Functions on a Capillary

Dec 23, 2006 - A general and simple implementation of simultaneous multiparametric sensing in a single microchip is presented by using a capillary-ass...
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Anal. Chem. 2007, 79, 908-915

Integration of Multianalyte Sensing Functions on a Capillary-Assembled Microchip: Simultaneous Determination of Ion Concentrations and Enzymatic Activities by a “Drop-and-Sip” Technique Terence G. Henares,† Masayuki Takaishi,† Naoya Yoshida,† Shigeru Terabe,† Fumio Mizutani,† Ryuichi Sekizawa,‡ and Hideaki Hisamoto*,†

Graduate School of Material Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan, and Metaboscreen Co. Ltd., 34-1-412, Terakubo Naka-ku, Yokohama, 231-0855, Japan

A general and simple implementation of simultaneous multiparametric sensing in a single microchip is presented by using a capillary-assembled microchip (CAsCHIP) integrated with the plural different reagent-release capillaries (RRCs), acting as various biochemical sensors. A novel “drop-and-sip” technique of fluid handling is performed with a microliter droplet of a model sample solution containing proteases (trypsin, chymotrypsin, thrombin, elastase) and divalent cations (Ca2+, Zn2+, Mg2+) that passes through the microchannel with the aid of a micropipette as a vacuum pump, concurrently filling each RRC via capillary force. To avert the evaporation of the nanoliter sample volume in each capillary, PDMS oil is dropped on the outlet hole of the CAs-CHIP exploiting the capillary force that results in spontaneous sealing of all the RRCs. In addition, this high-speed sample introduction alleviates the possibility of protein adsorption and capillary cross-contamination, allowing a reliable and multianalyte determination of a sample containing many different proteases and divalent cations by using the fluorescence image analysis. Presented results suggested the possible application of this microchip in the field of drug discovery and systems biology. Over the years, the remarkable progress of micro total analysis systems or lab-on-a-chip is fueled by the demands from different fields of science.1-5 The development of an overall easy-to-use microfluidic device that can simultaneously measure diverse * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +81-791-58-0493. † University of Hyogo. ‡ Metaboscreen Co. Ltd. (1) Dittrich, P. S.; Tachikawa, K.; Manz, A. Anal. Chem. 2006, 78, 3887-3908. (2) Proceedings of mTAS 2005 Conference; Jensen, K. F., Han, J., Harrison, J. D., Voldman, J., Eds.; Transducer Research Foundation, Inc., 2005. (3) Lab-on-a-Chip: Miniaturized Systems for (Bio) Chemical Analysis and Synthesis; Oosterbroek, R. E., van de Berg, A., Eds.; Elsevier: Boston, MA,2003. (4) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, 1, 244-248. (5) Sia, S. K.; Whitesides, G. M. Electrophoresis 2003, 24, 3563-3576. (6) Weston, A. D.; Hood, L. J. Proteome Res. 2004, 3, 179-196.

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multiplexed panels of analytes on a single microchip is one of the indispensable research issues that need to be addressed. When dealing with real sample that contains a broad spectrum of analytes, it will be cumbersome to analyze a set of biomolecules from one chip and another panel of analytes from another microchip. It must be really interesting and practical to study interrelated biomolecular response, whether it is antagonistic or synergistic, on a global perspective using a single miniaturized device.6 Thus, integration of multiparametric biochemical sensors on a single microfluidic device is inevitable. Microfluidic networks prove to be a suitable platform for the simultaneous biochemical sensing by patterning lines of proteins onto the microchannel.7 This type of device has an enormous potential to be an important tool in the fertile field of systems biology,6 which investigates the relationship among biomolecular events of a certain system in response to genetic or environmental perturbation, while taking advantage of the extremely low sample/ reagent consumption inside the chip microspace. Currently, a putative chip design formed in poly(dimethylsiloxane) (PDMS) is being carried out to pattern and array different biomolecules onto the microfluidic networks, which is aimed toward simultaneous multianalyte detection.8-11 The immobilization of various types of probes such as carbohydrates,12 peptides,13 proteins,14 and antigens15 are patterned on a self-assembled monolayer (SAM) while enzymes16 and bacteria17 are entrapped inside the hydrogel (7) Delmarche, E.; Bernard, A.; Schmid, H.; Michel, B.; Biebuyck, H. Science 1997, 276, 779-781. (8) Rowe, C. A.; Scruggs, S. B.; Feldstein, M. J.; Golden, J. P.; Ligler, F. S. Anal. Chem. 1999, 71, 433-439. (9) Bernard, A.; Michel, B.; Delmarche, E. Anal. Chem. 2001, 73, 8-12. (10) Jiang, X.; Ng, J. M. K.; Stroock, A. D.; Dertinger, S. K. W.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 5294-5295. (11) Su, J.; Bringer, M. R.; Ismagilov, R. F.; Mrksich, M. J. Am. Chem. Soc. 2005, 127, 7280-7281. (12) Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am. Chem. Soc. 2003, 125, 6140-6148. (13) Wegner, G. J.; Lee, H.-J.; Corn, R. M. Anal. Chem. 2002, 74, 5161-5168. (14) Wegner, G. J.; Lee, H.-J.; Marriott, G.; Corn, R. M. Anal. Chem. 2003, 75, 4740-4746. (15) Kanda, V.; Kariuki, J. K.; Harrison, D. J.; McDermott, M. T. Anal. Chem. 2004, 76, 7257-7262. (16) Heo, J.; Crooks, R. M. Anal. Chem. 2005, 77, 6843-6851. 10.1021/ac061245i CCC: $37.00

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network exploiting this aforementioned format. For SAM, an amine-terminated surface of an alkanethiol monolayer on a gold substrate requires surface activation using a discrete reagent that has the specific biomolecular affinity to fully attach the probe on the surface. In contrast, hydrogel-based biosensing is governed by the pore size of the hydrogel; i.e., it is designed to prevent biomaterial leaching while allowing the small molecules to move in and out of the hydrogel. Moreover, the proposed parallel microfluidic format that uses the microbead technology as a solid phase to support immune complex formation would also be a potential design toward multiple analyte sensing.18,19 Additionally, multi-ion detection is implemented in a different planar format using various sensor technologies such as ion-selective electrodes and ion-sensitive field-effect transistors,20 light-addressable potentiometric sensor,21 and neutral ionophore-based ion pair extraction in an intermittent multiphase flow.22 In contrast to the planar layout, a capillary microfluidic format has also been tapped for its multiple analyte sensing capacity.23-25 These microfluidic designs offer an elegant solution for the concurrent and precise measurement of similar analytes on a single microchip. However, the above-mentioned microfluidic layouts may fail to enhance assay heterogeneity due to various issues like surface modification, chemical manipulation of solidphase support, and difficulty in introducing different biochemical functions in a confined space of an already fabricated microchip. To circumvent these predicaments, our group has proposed a simple microfluidic format that incorporates plural chemical principles on a single microchip, and it is called capillaryassembled microchip (CAs-CHIP)26 (see Figure 1). In our system, specific chemical modification is done on each square glass capillary and not on the PDMS microchannel. The functionalized capillaries are then embedded on the native PDMS plate for the final fabrication of the CAs-CHIP. A variety of applications have already been demonstrated using CAs-CHIP that includes valving with enzyme sensing,27 multiple ion sensing,28 and capillary electrophoresis integrated with pretreatment.29 However, simultaneous sensing of different chemical species such as electrolyte concentrations and enzymatic activities has not been realized yet. In this paper, we propose a facile approach to analyze the different chemical species present in a microliter sample solution (17) Tani, H.; Maehana, K.; Kamidate, T. Anal. Chem. 2004, 76, 6693-6697. (18) Sato, K.; Tokeshi, M.; Odake, T.; Kimura, H,; Ooi, T.; Nakao, M.; Kitamori, T. Anal. Chem. 2000, 72, 1144-1147. (19) Herrmann, M.; Veres, T.; Tabrizian, M. Lab Chip 2006, 6, 555-560. (20) Igarashi, I.; Ito, T.; Taguchi, T.; Tabata, O.; Inagaki, H. Sens. Actuators, B 1990, 1, 8-11. (21) Yoshinobu, T.; Iwasaki, H.; Ui, Y.; Furuichi, K.; Ermolenko, Yu.; Mourzina, Yu.; Wagner, T.; Na¨ther, N.; Scho ¨ning, M. J. Methods 2005, 37, 94-102. (22) Hisamoto, H.; Horiuchi, T.; Uchiyama, K.; Tokeshi, M.; Hibara, A.; Kitamori, T. Anal. Chem. 2001, 73, 5551-5556. (23) Balakirev, M. Y.; Porte, S.; Vernaz-Gris, M.; Berger, M.; Arie´, J-P.; Fouque, B.; Chatelain, F. Anal. Chem. 2005, 77, 5474-5479. (24) Petrou, P. S.; Kakabakos, S. E.; Christofidis, I.; Argitis, P.; Misiakos, K. Biosens. Bioelectron. 2002, 17, 261-268. (25) Ligler, F. S.; Breimer, M.; Golden, J. P.; Nivens, D. A.; Dodson, J. P.; Green, T. M.; Haders, D. P.; Sadik, O. A. Anal. Chem. 2002, 74, 713-719. (26) 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. (27) Hisamoto, H.; Funano, S.-i.; Terabe, S. Anal. Chem. 2005, 77, 2266-2271. (28) Hisamoto, H.; Yasuoka, M.; Terabe, S. Anal. Chim. Acta., 2006, 556, 164170. (29) Hisamoto, H.; Takeda, S.; Terabe, S. Anal. Bioanal. Chem. 2006, 386, 733738.

Figure 1. Fabrication of CAs-CHIP for heterogeneous multiplexed analyte sensing.

droplet on a CAs-CHIP using the novel chemically modified capillaries, called reagent-release capillaries (RRCs), and “dropand-sip” fluid-handling technique with a micropipette as a negative pressure pump. RRC implements a straightforward method of reagent immobilization in which molecular probes, whether it is for enzyme activity or ion sensing, are noncovalently immobilized inside the square glass capillary in the same manner. Here, the selective enzyme activity sensing in the presence of proteases and inhibitor is demonstrated using the RRCs-based CAs-CHIP. Also, to display the utility of the device for simultaneous multicomponent sensing, we have chosen panels of proteases (trypsin, chymotrypsin, thrombin, elastase) and divalent cations (Mg2+, Zn2+, Ca2+) as a model sample, because of the importance of analyzing the dependence of the protease’s activity on divalent cations.30,31 This simple-to-use microfluidic device may be considered as one of the first generation of heterogeneous multiparametric sensing microchips that can be used for a wide range of bioanalytical applications. EXPERIMENTAL SECTION Square Capillaries and Reagents. Square capillaries having 300-µm outer widths (flat to flat) and 100-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 kit and PDMS oils SH 200 with specified viscosities (50, 200, 500, and 1000 cSt) were purchased from Dow Corning (Midland, MI). Reagents of the highest grade commercially available were used for the preparation of the aqueous test electrolytes. Molecular probes such as Fluo-4, Mag-Fluo-4, and FluoZn-3 for sensing Ca2+, Mg2+, and Zn2+, respectively, were purchased from Invitrogen (Eugene, OR). Peptidyl-4-methylcoumaryl-7-amide (peptidyl-MCA) substrates such as Bz-Arg-MCA and Boc-Gln-Ala-Arg-MCA for trypsin; Boc-Val-Pro-Arg-MCA and Boc(30) Sousa, M. C.; McKay, D. B. Acta Crystallogr. 2001, D57, 1950-1954. (31) Hamilton, J. M. U.; Simpson, D. J.; Hyman, S. C.; Ndimba, B. K.; Slabas, A. R. Biochem. J. 2003, 370, 57-67.

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Figure 2. Basic operational procedure using the multianalyte sensing CAs-CHIP by the novel “drop-and-sip” technique of fluid handling.

Asp(OBzl)-Pro-Arg-MCA for thrombin; Glt-Ala-Ala-Phe-MCA and Suc-Ala-Ala-Pro-Phe-MCA for chymotrypsin; Suc-Ala-Ala-Ala-MCA and Suc(OMe)-Ala-Ala-Pro-Val-MCA for elastase, and leupeptin as inhibitor for trypsin and thrombin were all acquired from Peptide Institute, Inc. (Osaka, Japan). The following enzymes trypsin, thrombin, chymotrypsin, and elastase were obtained from Sigma (St. Louis, MO). Poly (ethylene glycol) (PEG; Mn ) 20 000) was 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. Preparation of Reagent-Release Capillaries. All the capillaries employed in this work were washed with water and then methanol and dried at 70 °C for 1 h prior to use. This washing procedure ensured a homogeneous distribution of solution cocktail inside the capillary. Enzyme Activity-Sensing Capillary. The cocktail solution was prepared by dissolving 10 mg of various peptidyl-MCA substrates into 50 µL of dimethyl sulfoxide (DMSO) and diluting with 150 µL of 3 mM of PEG in DMSO. A micropipette was used to thoroughly mix the cocktail solution. The “reverse-pumping” method was used to homogeneously immobilize the cocktail inside the square capillary. Using a syringe pump, the PEG-peptidylMCA cocktail was filled in the ∼20-cm-long square capillary at 20 µL/min flow rate, and air was introduced by a suction flow rate of 30 µL/min. The capillary was dried at 40 °C for 12 h. The bulk solution of the 3 mM PEG in DMSO showed slow gelation at room temperature (25 °C) and exhibited transition temperature from gel to solution phase between 47 and 50 °C. Thus, it was presumed that the state of the cocktail inside the square capillary was in gel form, which resulted in a physisorption on the four corners of the inner capillary. These capillaries were stored in a desiccator cabinet with 500-nm UV cut and found to be stable at least for 6 months at room temperature, which may lead to a very practical preparation of an enzyme activity sensor. Ion-Sensing Capillary. The preparation of the cocktail solution is similar to that of the enzyme activity-sensing capillary. PEGion-fluorescent probe cocktail was prepared by dissolving 500 µg of the molecular probe in 50 µL of methanol and further diluted using 10 mM PEG in methanol to achieve a final concentration of 910

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0.3 µM Fluo-4, 1.5 mM Mag-Fluo-4, 1.2 mM FluoZn-3, and 1 µM fluorescein for Ca2+, Mg2+, Zn2+, and pH sensing, respectively. The same immobilization technique as that of the peptidyl-MCA cocktail was carried out into the 100-µm square capillary. These capillaries were dried at 50 °C for 2 h to complete the noncovalent immobilization on the four corners of the inner capillary. The RRC was cut into ∼2.3-mm lengths using a ceramic cutter and used for the fabrication of CAs-CHIP. Fabrication of CAs-CHIP. The fabrication procedure of the lattice microchannel on a PDMS plate has already been reported elsewhere.26 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 with a glass mold was carried out to prepare a PDMS mold. The second molding process by this PDMS mold gave the lattice microchannel network on the second PDMS plate. Plugged capillaries were prepared by introduction of PDMS prepolymer into the square capillaries and cured at 70 °C for more than 5 h. These plugged capillaries were also cut and used for preparing the designed channel network. The square capillaries’ outer dimension and PDMS microchannel’s dimension is the same, 300 µm. This facilitates an easy integration of all the sensing and plugged capillaries into the PDMS lattice. After embedding all the capillaries, a PDMS cover was bonded on top. A spin-coated PDMS prepolymer on an acrylic plate (∼2 mm thick) bearing the 0.8-mm holes (see Figure 1) was used as a cover plate. It was spin coated at 7000 rpm and then adhered to the capillary-embedded PDMS plate before curing for 2 h at 70 °C. Simultaneous Multiparametric Sensing Using CAs-CHIP. The simple-to-use nature, in terms of operational procedure, even highlighted the ease of CAs-CHIP’s heterogeneous multiplexed measurement potential (see Figure 2). Using the drop-and-sip technique of fluid handling, a single drop (a few microliters) of sample solution containing mixtures of proteases and divalent cations was put on the inlet hole. After covering the two air vent holes, a negative pressure was applied on the outlet hole using a micropipette. A long sample plug advanced into the PDMS microchannel while concurrently introducing sample solution into each RRC via capillary force. The air vent hole covers were

Figure 3. (a) General concept of the reagent-release capillary. (b) Reagent immobilized inside the 100-µm-i.d. (flat to flat) glass square capillary and the chemical principle involved during sensing. The P1, P2, P3, and P4 of the peptidyl-MCA are the N-terminal side of the amino acid.

removed, and then the RRC were spontaneously sealed using PDMS oil SH 200 dropped on the outlet hole wherein the air was displaced out to the air vent and inlet holes. After a 30-min incubation32 at room temperature, the fluorescence images were acquired and measured. Optical Detection and Data Processing. Optical and fluorescence images of the CAs-CHIP system were obtained using an optical/fluorescence inverted microscope (Keyence MultiViewer System VB-S20). Photographs of the multianalyte sensing were captured using a 3CCD color camera (VB-7010, Keyence) installed at the front port of the microscope. Fluorescent images were collected using a 120-W mercury lamp as a light source and a filter pair (enzyme activity sensing, excitation filter at 387/28 nm, emission filter at 430 nm; ion sensing, excitation filter at 470/ 40 nm, emission filter at 510 nm) (VB-L11, Keyence). On the other hand, characterization of the RRC was performed using a different optical/fluorescence inverted microscope (Eclipse TS100-F Nikon, Tokyo, Japan) through 3CCD color camera (HV-D28S, Hitachi Kokusai Electric Inc., Tokyo, Japan) with 50-W mercury lamp light source and a filter block (FITC and order-made UV filter (Excitation filter, 405/20 nm; dichroic mirror, 425 nm long pass; absorption filter, 460/50); Nikon, Tokyo, Japan). Fluorescence (32) Yamaguchi, N.; Okui, A; Yamada, T.; Nakazato, H.; Mitsui, S. J. Biol. Chem. 2002, 277, 6806-6812.

images were converted to numerical response using Scion Image software to obtain fluorescence response. Using the line scan mode of the software, the fluorescence signal of each RRC was compared. It should be noted that all fluorescence readings of the sample were subtracted from that obtained from the buffer reading, which does not contain the enzymes or primary ions. RESULTS AND DISCUSSION Reagent-Release Capillary. The basic concept of RRC is shown in Figure 3a. The RRC contains PEG-substrate cocktail that is noncovalently immobilized on the four corners of the square capillary (100-µm internal width). The presence of PEG in the mixture functions as a scaffold for the substrate and at the same time not interfering in the sensing process. The sample solution is introduced into the RRC via capillary force, and upon contact, reagent is released leading to nanoliter solution-based fluorescence detection. Typical examples of the RRCs are demonstrated in Figure 3b. The peptidyl-4-methyl-coumaryl-7-amide (peptidylMCA) and metal ion-fluorescent probes are used to demonstrate its selectivity to specific proteases and primary ions, respectively. The chemical principle relies on the inherent ability of the protease to selectively cleave specific peptide bonds of peptidyl-MCA liberating a fluorescent molecule, 7-amino-4-methylcoumarin (AMC), while the ion-fluorescent probe forms a complex with a specific metal ion that significantly increases its fluorescence relative to Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

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its uncomplexed form. By using the RRCs, various chemical species can be detected by simply replacing the molecular probe inside the glass square capillary while taking advantage of the ease of sensor preparation. Characterization of RRC. To develop a reliable RRC for a precise and accurate analyte sensing, two basic factors must be considered: (1) the distribution of the molecular probes inside the square capillary must be uniform, and (2) the response with specific analytes at given concentration must be selective and sensitive. The homogeneity of cocktail distribution was investigated by immobilizing the PEG-fluorescein (1 µM fluorescein) cocktail inside a 21-cm-long square capillary using the push-pull method of capillary filling. Using this method, we vary the suction flow rate by 1, 10, 30, and 50 µL/min keeping the infusion flow rate at 20 µL/min, whereas the viscosity and temperature of immobilization solution were held constant. The long capillary was cut into 21 pieces (10 mm long), and the fluorescence intensities obtained by introducing phosphate buffer (pH 7) were compared. The corresponding average fluorescence response from the aforementioned flow rates were 150, 141, 148, and 140 arbitrary units (au), respectively, whereas the RSDs (n ) 21) were 4.9, 5.6, 3.0, and 7.9%, respectively. Although there is no trend observed relative to change in suction flow rates, the homogeneity of reagent immobilized within the capillary among the RRCs did not vary significantly, i.e., RSD is between 3 and 8%. These data implied that a precise analytical measurement using RRC could be achieved. On the other hand, the enzyme activity-sensing capillary showed a typical enzyme profile with respect to change in enzyme concentration like thrombin activity-sensing capillary as illustrated in the inset of Figure 4a. The initial slope of the reaction profile was calculated and used to plot against the thrombin concentration. The linear response signified that the immobilized peptidylMCA was in excess, which is the basic requirement for an enzyme activity-sensing capillary. This could lead to enzyme activity measurement from an unknown sample. Additionally, the linear response of peptidyl-MCA immobilized capillaries to changes in protease concentration is still observed after 6 months of storage in a desiccator cabinet at room temperature (data not shown). Apparently, the practical implication of this type of RRC preparation is that it will allow a reagent immobilization on a very long square capillary and alleviate the process of sensor preparation by simply cutting the RRC into small pieces (millimeter scale). It is also noteworthy to mention that the sensitivity of the enzyme activity-sensing capillary is dependent on the type of immobilized peptidyl-MCA. This divergence in sensitivity from one peptidylMCA to another using the same protease is the basis of patterned recognition of a certain protease over its preferred peptidyl-MCA substrate. On the contrary, this divergence in sensitivity means that the quantitative discussion using a standard curve should be carefully done when the potential cross-sensitive proteases coexist in sample solution. Concerning the ion-sensing RRC, the characteristic sigmoidal response of the binding among the molecular probes and metal ions was achieved. Figure 4b showed a representative example of such plot with a zinc ion-sensing capillary (FluoZn-3 immobilized capillary), and its selectivity against calcium and magnesium ions was also presented. The Fluo-4 and Mag-Fluo-4 immobilized capillaries used for Ca2+ and 912 Analytical Chemistry, Vol. 79, No. 3, February 1, 2007

Figure 4. (a.) Thrombin activity-sensing capillary response to various thrombin concentrations. Inset of the figure shows the fluorescence response profiles for each enzyme concentration using the Boc-Asp(OBzl)-Pro-Arg-MCA immobilized capillary. (b.) Zincsensing capillary response toward different zinc ion concentrations and its selectivity over calcium and magnesium ions.

Mg2+ sensing, respectively, displayed sensitivity similar to that of the zinc-sensing capillary at a similar concentration range (10-3-10-6 M). However, the Mag-Fluo-4 immobilized RRC did not show strict selectivity with Mg2+ since its fluorescence response changed with varying calcium ion concentration as well. This is likely to occur since the binding of aminophenol triacetic acid-based molecular probes, like Mag-Fluo-4, with Ca2+ becomes significant when the Ca2+ concentration exceeds 1 µM.33 Generally, the detection capability of these ion-sensing RRCs could cover some physiologically important ion concentration ranges34-35 except for the Mag-Fluo-4 immobilized capillary, which will be saturated in the presence of interfering Ca2+ in a given biological fluid. However, other molecular probes with higher selectivity to magnesium ions like the coumarin derivative36 can be exploited to yield a magnesium-selective RRC. In addition, the pH-sensing capillary prepared by simply immobilizing the fluorescein molecule also showed a typical sigmoidal response to pH in the same manner as the ion-sensing capillaries described above. The pH response range was ∼4-9, which covers the physiologically important pH range. These typical responses of various RRCs from (33) Koss, K. L.; Putman, R. W.; Grubbs, R. D. Am. J. Physiol. Cell Physiol. 1993, 264, C1259-C1269. (34) Maret, W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12325-12327. (35) Miyake, R.; Gross, R. Biochim. Biophys. Acta 1992, 1165, 167-176. (36) Suzuki, Y.; Komatsu, H.; Ikeda, T.; Saito, N.; Araki, S.; Citterio, D.; Hisamoto, H.; Kitamura, Y.; Kubota, T.; Nakagawa, J.; Oka, K.; Suzuki, K. Anal. Chem. 2002, 74, 1423-1428.

specific analytes suggest that this could be used for quantitative analysis. A table of the performance data of all the RRCs tested is shown in S-0 of the Supporting Information. Drop-and-Sip Sample Introduction. In order to demonstrate the simultaneous multiparametric sensing using the RRC-based CAs-CHIP, development of a sample introduction method is indispensable because the conventional sample filling leads to reagent diffusion and leakage from the RRC into the PDMS channel. Here, the novel sample introduction method using the drop-and-sip technique with the CAs-CHIP is developed and fluid flow behavior is investigated. Previously, other groups have demonstrated a chip designbased microliter/nanoliter fluid handling wherein the microchannel’s surface property, dimension, and even the PDMS bulk property like air permeability were exploited to realize the whole microchip design for fluid manipulation.37-40 The fluid handling inside a microfluidic device is dictated mainly by the overall microchip design. Here, our simple manner of sample introduction, the drop-and-sip technique, involves a microliter “drop” of sample solution on the inlet hole and “sip” by the conventional micropipette, via negative pressure, from the outlet hole. In this case, the sample fluid passed through the PDMS channel as a long sample plug. The short contact time between the sample plug and the RRC allocate part of the sample solution plug to flow into each RRC by capillary force. The sample solution is stopped at the end of the RRC by surface tension, and then the remaining fluid goes into the micropipette on the outlet hole, leaving the sample solutions in each RRC. It should be very interesting to note that this type of sample introduction can be demonstrated only in the case when the hydrophilic glass capillary and the hydrophobic material such as PDMS are combined. Assuming that all the CAs-CHIPs’ microchannel is hydrophilic, i.e., the contact angle of an aqueous sample on the microchannel’s surface is