Anal. Chem. 2003, 75, 388-393
Creation of an On-Chip Enzyme Reactor by Encapsulating Trypsin in Sol-Gel on a Plastic Microchip Kumiko Sakai-Kato,† Masaru Kato,†,‡ and Toshimasa Toyo’oka*,†
Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada Shizuoka, Shizuoka, 422-8526, Japan, and PRESTO, Japan Science and Technology Corporation (JST), Saitama, Japan
Trypsin-encapsulated sol-gel was fabricated in situ onto a plastic microchip to form an on-chip bioreactor that integrates tryptic digestion, separation, and detection. Trypsin-encapsulated sol-gel, which is derived from alkoxysilane, was fabricated within a sample reservoir (SR) of the chip. Fluorescently labeled ArgOEt and bradykinin were digested within the SR followed by electrophoretic separation on the same chip. The plastic microchip, which is made from poly(methyl methacrylate), generated enough electroosmotic flow that substrates and products could be satisfactorily separated. The sol-gel in the SR did not alter the separation efficiency of each peak. With the present device, the analytical time was significantly shortened compared to conventional tryptic reaction schemes. This on-chip microreactor was applicable to the digestion of protein with multiple cleavage sites and separation of digest fragments. Furthermore, the encapsulated trypsin exhibits increased stability, even after continuous use, compared with that in free solution. In the past decade, there have been an explosion of interest in the fields of microfluidic systems.1-15 Microfabricated fluidic * Corresponding author. E-mail:
[email protected]. Fax: +8154-264-5593. Tel: +81-54-264-5656. † University of Shizuoka. ‡ PRESTO, Japan Science and Technology Corp. (1) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (2) Harrison, D. J.; Fluki, F.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (3) Verpoorte, E. Electrophoresis 2002, 23, 677-712. (4) Salimi-Momosavi, H.; Tang, T.; Harrison, D. J. J. Am. Chem. Soc. 1997, 119, 8716-8717. (5) Kopp, M. U.; de Mello, A. J.; Manz, A. Science 1998, 280, 1046-1048. (6) Schilling, E. A.; Kamholz, E.; Yager, P. Anal. Chem. 2002, 74, 1798-1804. (7) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (8) Hadd, A. G.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1999, 71, 52065212. (9) Wang, C.; Oleschuk, R.; Ouchen, F.; Li, J.; Thibault, P.; Harrison, D. J. Rapid Commun. Mass Spectrom. 2000, 14, 1377-1383. (10) Tanaka, Y.; Slyadnev, M. N.; Hibara, A.; Tokeshi, M.; Kitamori, T. J. Chromatogr., A 2000, 894, 45-51. (11) Chiem, N. H.; Harrison, D. J. Clin. Chem. 1998, 44, 591-598. (12) Sato, K.; Tokeshi, M.; Odake, T.; Kimura, H.; Ooi, T.; Nakao, M.; Kitamori, T. Anal. Chem. 2000, 72, 1144-1147. (13) Murakami, Y.; Takeuchi, T.; Yokoyama, K.; Tamiya, E.; Karube, I.; Suda, M. Anal. Chem. 1993, 65, 2731-2735. (14) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636.
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devices are potentially powerful tools for chemical or biological assays. These devices offer rapid analysis and reduced sample consumption and cost.4-7 Specific and efficient biological reactions may benefit greatly with microfluidic devices that offer the means for handling small-volume samples (i.e., nL to µL) in confined reaction zones. Various biological assays have been performed in the devices, such as enzymatic reaction8-10 or immunoassay.11,12 These reactions were performed using a homogeneous enzyme in a channel8,10 or immobilized enzymes9 or antibody12 onto the packed beads or onto the inner surface of a capillary and integrated into a chip.13 We present here a report of the in situ fabrication of trypsinencapsulated gels onto a microchip for an on-chip bioreactor in which integrated tryptic digestion, separation, and detection is performed. This alkoxysilane-derived gel has been used for a decade as a powerful tool for a biomolecule encapsulation technique.16,17 The optical transparency of the material makes it suitable as a matrix for biosensors having a wide range of encapsulated biomolecules. Because the encapsulation process occurs under mild conditions, the biomolecules retain their structures18,19 and biological activity for a prolonged period.20 Furthermore, the in situ gelation process makes it easy to fabricate and interface the materials in a variety of forms such as a thin film,21 bulk form,18,20,22,23 or particles.24 We recently developed a novel protein encapsulation technique using this sol-gel method for the preparation of monolithic capillary columns integrated into a capillary electrophoresis (CE) system.25-27 A variety of proteins were encapsulated into the gel (15) Auroux, P.-A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (16) Livage, J.; Coradin, T.; Roux, C. J. Phys. Condens. Matter 2001, 13, 673691. (17) Jin, W.; Brennan, J. D. Anal. Chim. Acta 2002, 461, 1-36. (18) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, J. I. Sceince 1992, 255, 1113-1115. (19) Edmiston, P. L.; Wambolt, C. L.; Smith, M. K.; Saavedra, S. S. J. Colloid Interface Sci. 1994, 163, 395-406. (20) Braun, S.; Rappoport, S.; Zusman, R.; Avnir, D.; Ottolenghi, M. Mater. Lett. 1990, 10, 1-5. (21) Yao, T.; Harada, I.; Nakahara, T. Bunseki Kagaku 1995, 44, 927-932. (22) Flora, K.; Brennan, J. D. Anal. Chem. 1998, 70, 4505-4513. (23) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A-1127A. (24) Cichna, M.; Knopp, D.; Niessner, R. Anal. Chim. Acta 1997, 339, 241250. (25) Kato, M.; Sakai-Kato, K.; Matsumoto, N.; Toyo’oka, T. Anal. Chem. 2002, 74, 1915-1921. 10.1021/ac026240+ CCC: $25.00
© 2003 American Chemical Society Published on Web 01/03/2003
matrix without loss of protein activities. For example, the encapsulation of bovine serum albumin and ovomucoid in a tetramethoxysilane (TMOS)-based matrix allowed for the separation of drug enantiomers.25 A trypsin-encapsulated capillary column, which was integrated into the CE system, enabled the creation of an on-line enzyme reactor with enhanced reactivity.26,27 Two types of substrates have been typically used for the fabrication of microfabricated devices. The first one is glass.1,2 In most devices, the pumping, valving, and mixing of fluids are achieved via electroosmotic flow (EOF). Therefore, the excellent properties of glass in respect to EOF generation are very suitable for the separation channel. More recently, several plastic substrates have been used.28-35 Among those, poly(dimethylsiloxane) (PDMS)30,31 as a soft plastic and poly(methyl methacrylate) (PMMA)32-35 as a hard plastic are most popular. Because microfluidic devices using these substrates are less fragile and suitable for mass production, they are cost-effective. Taking into account these advantages, we have chosen PMMA as a substrate for our devices. Another reason is that PMMA is reported to be the least hydrophobic of the more common plastic materials available.36 In this report, the microchip, which is commercially available and was originally used for DNA separation, was applied to the determination of fluorescently labeled amino acids, peptides, and proteins. Although the fluorescent derivatization procedure adds to the complexity of sample preparations, it offers a selective and sensitive detection for microfabricated analytical systems. Trypsin was immobilized in the sample reservoir using alkoxysilane-based sol-gel, and labeled samples were digested and separated under optimized conditions. EXPERIMENTAL SECTION Materials and Chemicals. TMOS, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and anhydrous ethylenediamine were purchased from Tokyo Kasei (Tokyo, Japan). Trypsin from porcine pancreas and L-Arg were purchased from Sigma-Aldrich (Milwaukee, WI). Bradykinin was purchased from Peptide Institute, Inc. (Osaka, Japan). Arg ethyl ester dihydrochloride was purchased from Wako Pure Chemicals (Osaka, Japan). 4,4-Difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) casein (BODIPY-casein) was purchased from Molecular Probes, Inc. (Eugene, OR). Water was purified by MilliQ apparatus (Millipore, Bedford, MA). (26) Sakai-Kato, K.; Kato, M.; Toyo’oka, T. Anal. Chem. 2002, 74, 2943-2949. (27) Sakai-Kato, K.; Kato, M.; Toyo’oka, T. Anal. Biochem. 2002, 308, 278284. (28) Soper, S. A.; Ford, S. M.; Qi, S.; McCarley, R. L.; Kelly, K.; Murphy, M. C. Anal. Chem. 2000, 643A-651A. (29) Boone, T. D.; Hugh Fan, Z.; Hooper, H. H.; Ricco, A. J.; Tan, H.; Williams, S. J. Anal. Chem. 2002, 78A-86A. (30) Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A. Anal. Chem. 1997, 69, 34513457. (31) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984. (32) Ford, S. M.; Kar, B.; McWhorter, S.; Davies, J.; Soper, S. A.; Klopf, M.; Calderon, G.; Saile, V. J. Microcolumn Sep. 1998, 10, 413-422. (33) Henry, A. C.; Tutt, T. J.; Galloway, M.; Davidson, Y. Y.; McWhorter, C. S.; Soper, S. A.; McCarley, R. L. Anal. Chem. 2000, 72, 5331-5337. (34) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626-2630. (35) Sassi, A. P.; Paulus, A.; Cruzado, I. D.; Bjornson, T.; Hooper, H. H. J. Chromatogr., A 2000, 894, 203-217. (36) Bayer, H.; Engelhardt, H. J. Microcolumn Sep. 1996, 8, 479-484.
Figure 1. Diagram of i-chip 3 DNA. The area within a dotted line is one cross-channel: SR, sample reservoir; SW, sample waste; BR, buffer reservoir; BW, buffer waste.
Apparatus. Separations were performed on a microfabricated PMMA chip, i-chip 3 DNA (Hitachi, Tokyo, Japan). The schematic layout is shown in Figure 1. The microchip has dimensions of 85-mm × 50-mm square with three simple cross-channels of 100 µm in width and 30 µm in depth. The distance between the sample reservoir (SR) and the sample waste (SW) was 10 mm, whereas the distance from the buffer reservoir (BR) and the buffer waste (BW) was 44 mm. A small plastic tip was attached to each reservoir and each waste for sample or buffer introduction and removal, respectively. The microchip was placed on the stage of a fluorescence microscope, model IX70 (Olympus, Tokyo, Japan) and could be set up for measurement without any difficulties. Light from a mercury lamp (Olympus) was introduced to the detection point of the microchip after filtering with a fluorescence cube model U-MWIB (Olympus) that contains a 505-nm dichroic mirror and a 460-490-nm band-pass filter and is focused by a 40× objective microscope lens. The fluorescence from the sample going back through the objective lens and the dichroic mirror was passed through a 515-nm interference filter and into a photomultiplier tube (model H5784MOD) from Hamamatsu Photonics (Hamamatsu, Japan). The signal from the photomultiplier tube was amplified and recorded on an 833A data processor (Hitachi). The power supply for the microchip electrophoresis was a model HOPP-3B1-L2 (Matsusada Precision Devices, Kusatsu, Japan), which was computer-controlled by the PC, which was connected to a digital-to-analog converter, JJ Joker (Nippon Filcon, Inagi, Japan) through the RS-232C serial interface. The power supply was driven by a terminal emulation software named KTX (freeware). Sample introduction and separation were controlled through manipulation of the electric field strengths. A 50 mM TrisHCl buffer (pH 7.5) was used as the running buffer. The voltage settings were based on a previously published report.37 In all experiments, the potentials of 100, -90, and -500 V were applied to SR, BR, and SW, respectively, for the 40-s sample introduction, while BW was grounded. This ensured minimal sample introduction bias in the injection cross section. The voltage program for the separation of analytes was 500, 900, and 500 V for SR, BR, and SW, respectively, and BW was grounded. (37) Hashimoto, M.; Tsukagoshi, K.; Nakajima, R.; Konodo, K.; Arai, A. J. Chromatogr., A 2000, 867, 271-279.
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After every run, each reservoir was replaced with fresh buffer. A cleaning procedure was performed by flowing the buffer in the channel for 2 min using the same voltage program as that for sample separation. Caution! The electrophoresis uses high voltages and special care should be taken when handling the electrophoresis electrodes. Measurement of EOF. The current-monitoring method was used to measure EOF in the microfabricated channels.38 A single straight channel between BR and BW was used for the measurement. Before the measurement, all the channels and reservoirs except BR was filled with 50 mM Tris-HCl (pH7.5) and BR was filled with 45 mM Tris-HCl (pH7.5). The measurement was performed with open reservoirs during the experiment. Derivatization of Samples with NBD-F. Samples were derivatized with NBD-F according to the procedure of Imai et al.39 Derivatized samples were diluted with the running buffer. Before use, all solutions were filtered through a 0.22-µm membrane (Millipore) and degassed by ultrasonication. Fabrication of Trypsin-Encapsulated Gel onto the Microchip. The procedure for the sol-gel reaction and the trypsin encapsulation is similar to that published in our previous report.26 In this study, 0.1% (w/v) trypsin-containing gel was fabricated for the digestion of NBD-ArgOEt and 10% trypsin-containing gel was for NBD-bradykinin and BODIPY-casein. Briefly, the monomer solution was obtained by mixing the following reagents just prior to use: (1) 761 µL of TMOS, (2) 169 µL of water, and (3) 11 µL of 0.04 N HCl. This monomer solution was stirred for 20 min so that hydrolysis proceeded to form a fully or partially hydrolyzed silane, SiOH4-n(OMe)n. A 120-µL sample of trypsin solution (0.1 or 10% (w/v)) in 50 mM Tris-HCl (pH 7.0) containing 20 mM CaCl2 was added to 20 µL of the hydrolyzed solution. After mixing and ultrasonication for 5 s, 10 µL of the mixture solution was carefully put into the SR and formed into gel. The gelation occurred in ∼15 min. Before the mixture solution was introduced into the SR, all channels were filled with 50 mM Tris-HCl (pH 7.5) and the other three reservoirs except the SR were filled with 50 µL of the buffer to prevent the trypsincontaining sol from permeating into the channels and other reservoirs. After gelation, another 50 µL of the buffer was added to all the reservoirs, including the SR. After 1 h, the channels were flushed with fresh buffer from the BW. Finally, all the reservoirs and channels were filled with the buffer and the reservoirs were sealed. The microchip was stored at 4 °C for more than 4 days.26 Off-Chip Digestion. A 2 mM solution of NBD-ArgOEt was digested off-chip with a soluble trypsin (0.1 mg/mL) in a water bath kept at 37 °C for 5 min. The reaction was stopped by adding methanol, followed by centrifugation (3000 rpm, 5 min) and filtration through a 0.22-µm membrane. The filtrate was injected into a fused-silica capillary, and NBD-ArgOEt and its digest, NBDArg, were determined by CE analysis. On-Chip Tryptic Digestion. Before reaction, the channels were filled with 50 mM Tris-HCl (pH7.5). Also the three reservoirs except SR were filled with 50 µL of the same buffer. A volume of 40 µL of the substrate solution containing an internal standard, NBD-ethylenediamine, was put into the SR and voltage was applied (38) Huang, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 1837-1838. (39) Imai, K.; Fukushima, T.; Hagiwara, K.; Santa, T. Biomed. Chromatogr. 1995, 9, 106-109.
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Figure 2. Electropherograms of NBD-ArgOEt incubated without trypsin (A) or with a soluble trypsin (B) analyzed using a CE system. Conditions: sample, (A) 2 mM NBD-ArgOEt incubated at 37 °C for 5 min without trypsin and (B) The reactant of 2.0 mM NBD-ArgOEt digested by soluble trypsin (0.1 mg/mL) at 37 °C for 5 min; fusedsilica capillary, 34-cm total length; detection, 470 nm.
immediately. For casein digestion, 40 µL of BODIPY-casein was put into the SR and voltage was applied after a 1-h digestion period. In all experiments, the voltage setting and timing were the same as those mentioned in the Apparatus section. CE Equipment CE experiments were carried out on a Hewlett-Packard 3DCE system (Palo Alto, CA) equipped with a diode-array detector. Substrates were introduced electrokinetically at the anodic side (4 kV, 10 s). For separation, a voltage of 4 kV was applied and the temperature was kept at 25 °C in all experiments. The running buffer was 50 mM Tris-HCl (pH 7.5) in all experiments. Substrates were diluted in the mobile phase. Before use, all solutions were filtered through a 0.22-µm membrane (Millipore) and degassed by ultrasonication. RESULTS AND DISCUSSION Characterics of the PMMA Microchip. In this study, we used the PMMA microchip, i-chip 3 DNA, which is commercially available at a relatively low price. As a result, it is expected that mass production for commercial applications can be easily achieved. The i-chip 3 DNA was originally designed for DNA separations; however, there are no reports on the use of these chips for the separation of other types of samples. Therefore, we studied the characteristics of the microchip before applying this chip to a bioreactor. EOF in the microchannels was determined using the current-monitoring method with Tris-HCl (pH 7.5) as running buffer. The mobility was 1.4 × 10-4 cm2/V‚s on average, which is similar to the reported value, 1.2 × 10-4 cm2/V‚s.32 The run-to-run relative standard deviation (RSD, n ) 3) of the EOF velocity calculated from separate runs made on the same microfluidic channel was 3%. An NBD derivative of ArgOEt was used as our fluorescent substrate. NBD-ArgOEt was digested using a soluble form of trypsin and the production of NBD-Arg was confirmed by CE. Figure 2 shows the electropherogram of NBD-ArgOEt that was incubated at 37 °C for 5 min without trypsin (Figure 2A) and with
Figure 3. (A) Electropherogram of NBD-ArgOEt, NBD-Arg, and NBD-ethylenediamine as an internal standard separated on a microchip without gel. Conditions: sample, 0.1 mM IS, 0.5 mM NBDArgOEt, and 0.25 mM NBD-Arg; detection point, 16 mm; internal standard (I.S.), NBD-ethylenediamine. (B) Electropherogram of an on-chip tryptic reaction using NBD-ArgOEt as a substrate. Conditions: sample, 0.1 mM I.S., 1 mM NBD-ArgOEt; detection point, 16 mm; I.S., NBD-ethylenediamine.
a soluble trypsin (Figure 2B). In the blank sample where no trypsin was added, digestion of NBD-ArgOEt was not observed (Figure 2A), whereas NBD-ArgOEt was digested into NBD-Arg by the soluble form of trypsin (Figure 2B). The separation conditions of NBD-ArgOEt and NBD-Arg were examined using the microchip without gel. NBD-ethylenediamine was used as internal standard for the quantitative determination of the digest. In the preliminary experiment, it was confirmed that NBD-ethylenediamine did not inhibit the tryptic reaction. As shown in Figure 3A, three peaks were successfully separated in ∼1 min. The migration times of NBD-ArgOEt and NBD-Arg were 35 and 65 s, respectively, and with the same elution order observed in CE using a fused-silica capillary (Figure 2). The migration times in the microchip, however, were one-tenth shorter compared with those in the CE system. Design of the Microchip Enzyme Reactor. Figure 1 is a schematic illustration of the microchip used as enzyme reactor. Trypsin-encapsulated gel was fabricated within the SR. The substrates are introduced onto the gel with a micropipet. When the voltage was applied in all reservoirs, the substrates diffused into the gel and were converted into products by encapsulated trypsin during the sample loading procedure. Finally, the unreacted substrates and products, which were loaded at the cross section, were injected and separated by electrophoresis. Tryptic Reaction Using NBD-ArgOEt. The tryptic reaction using NBD-ArgOEt as a substrate was studied using the microchip with trypsin-encapsulated gel. A 0.1% trypsin solution was encapsulated in the gel. Figure 3B illustrates the typical electropherogram of NBD-ArgOEt and NBD-Arg along with the internal standard, when 1.0 mM NBD-ArgOEt was introduced into the microchip. Encapsulated trypsin successfully hydrolyzed NBDArgOEt to NBD-Arg without stopping the flow. Using the same
Figure 4. (A) Activity of the encapsulated trypsin depending on substrate concentrations. (B) Double-reciprocal plot of the enzymatic activity of encapsulated trypsin. Conditions: substrates, NBD-ArgOEt. Other conditions are as in Figure 3B.
voltage program as that for the microchip without gel, three peaks were completely separated and the migration times did not change in the presence of the gel in the SR (Figure 3). The migration times of NBD-ethylenediamine were 31.4 ( 2.6 s for chips without gel (n ) 3) and 32.4 ( 1.8 s for chips with the gel (n ) 3). These results indicate that the gel fabricated in SR has little influence on electroosmotic mobility. Figure 4A depicts the change of NBD-Arg production, which depends on the substrate concentrations. Production was increased with an increase in the substrate concentrations. Figure 4B shows the double-reciprocal plot (Lineweaver-Burk plot) of the enzymatic activity of encapsulated trypsin. The Km calculated using the plot has a value of 3.59 mM,26 which is 19 times higher than the value measured for free trypsin (0.19 mM). This increase is ascribed to the decrease in mass transfer within the gel matrix, and this is often observed with immobilized enzymes. 40,41 The variability of tryptic activity by run-to-run analysis was acceptable, and RSD was less than 5% (n ) 3). On the other hand, RSD for tryptic activity by chip-to-chip analysis was 14% (n ) 3), which was almost the same level as that for conventional tryptic reaction schemes using free trypsin (RSD ) 12%, n ) 3).26 Tryptic Reaction Using Peptide. We investigated the availability of this system for peptide digestion using NBD-bradykinin as a substrate. NBD-bradykinin was digested at the peptide bond of N-terminal Arg and NBD-Arg was produced. The tryptic reaction using NBD-bradykinin as a substrate was studied on the microchip with trypsin-encapsulated gel. Figure 5 illustrates the typical electropherogram of NBD-bradykinin and (40) Xie, S.; Svec, F.; Fre´chet, J. M. J. Biotechnol. Bioeng. 1999, 62, 30-35. (41) Chibata, I. Immobilized enzymes; Kodansha: Tokyo, 1978.
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Figure 5. Electropherogram of an on-chip tryptic reaction using NBD-bradykinin. Conditions: sample, 0.1 mM I.S., 0.18 mM NBDbradykinin; detection point, 16 mm; I.S., NBD-ethylenediamine.
Figure 6. Electropherogram of tryptic digest of BODIPY-casein after 1-h digestion period. Conditions: sample, 0.2 mg/mL BODIPY-casein; detection point, 30 mm.
Table 1. Run-to-Run Repeatability of Migration Timea migration time (s)
average (s) SD RSD (%)
I.S.
NBD-bradykinin
NBD-Arg
26.2 0.42 1.6
39.1 0.70 1.8
54.6 0.99 1.8
a Conditions: sample, 0.1 mM IS, 0.18 mM NBD-bradykinin; detection point, 8 mm; I.S., NBD-ethylenediamine.
NBD-Arg. NBD-bradykinin was successfully hydrolyzed on a chip, and the separation of substrates and products was satisfactory. The gel gave no undesirable effects on the separation efficiency including peak broadening or delay in the migration time, which were serious problems in the bioreactor using the trypsinencapsulated capillary integrated into a CE system.26 This difference is due to the fact that trypsin-encapsulated gel was not fabricated in the separation channels in this microchip bioreactor, whereas the gel was fabricated in the same capillary columns as that for separation in a CE system.26 Peak shapes and migration times were reproducible for 10 runs (Table 1). This result indicates that the adsorption of analytes onto the inner surface of microchannel was negligible. Tryptic Reaction Using Protein. To demonstrate that this microchip reactor is a powerful tool for proteomic research, it is essential that larger substrates with multiple cleavage sites be digested. As a substrate, we used casein (MW 12 000-24 000) that was labeled with BODIPY dye. Upon binding of five molecules of dye to a single casein, the fluorescence of the dye is intramolecularly self-quenched. Digestion of the BODIPY-labeled and quenched casein by an unlabeled trypsin yields smaller peptide fragments in which the fluorescence of the associated BODIPY tags is restored.42,43 Figure 6 shows the electropherogram of the trypsin digest of BODIPY-casein after 1 h of digestion. Many digest fragments were observed and the peak intensity increased with an increase in a 392 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003
reaction period, which showed that the digestion proceeded successfully. In fact, in the electropherogram using a microchip without gel, undigested labeled casein was seen as a wide, lowintensity peak due to quenching of the BODIPY dye (data not shown). Because the separation channel was relatively short and many fragments were produced, complete separation was not achieved. However, it should be noted that this on-line protein digestion and separation were achieved for the first time by fabricating the trypsin encapsulation sol-gel on a microchip, but it was not possible in a capillary format.26 No peaks were observed when protein was used as a substrate in the trypsin digestion reaction in a capillary. This observation may be caused by several factors. First, in the microchip, the substrate can be in contact for long periods of time with trypsin on the gel surface, which has a much larger surface area than that in a capillary (∼3000 times). A small digested peptide can more easily penetrate into the gel as compared to a large digested peptide. Second, the gel length along the channel is shortened in a microchip (
