Monolithic Bioreactor Immobilizing Trypsin for High-Throughput

Figure 2 Picture of bioreactor and scanning electron micrographs of the monolith prepared using different amounts of PEG: (a) 0.48, (b) 0.53, and (c) ...
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Anal. Chem. 2005, 77, 1813-1818

Monolithic Bioreactor Immobilizing Trypsin for High-Throughput Analysis Masaru Kato,†,‡ Kenji Inuzuka,† Kumiko Sakai-Kato,†,§ and Toshimasa Toyo’oka*,†

Department of Analytical Chemistry, School of Pharmaceutical Sciences and COE Program in the 21st Century, University of Shizuoka, 52-1 Yada Shizuoka, Shizuoka, 422-8526, Japan, PRESTO, Japan Science and Technology Agency (JST), Saitam, Japan, and Faculty of Pharmacy/Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi Nishitokyo-shi, Tokyo, 202-8585, Japan

A miniaturized trypsin reactor was prepared by coating a trypsin-containing gel on a porous silica monolith. The trypsin-encapsulated gel was prepared by the sol-gel method. The sol-gel reaction was optimized so that the sol solution containing trypsin forms a thin film on the sol-gel monolith. The trypsin was encapsulated into the gel matrix without losing its activity. The silica monolith was fabricated to fit into a 96-well microtiter plate well and could then be easily removed. The trypsin-immobilized monolith was reacted in the 96-well microtiter plate. After the reaction, the monolith was removed, and the enzymatic activity was measured. The large surface area of the monolith enabled the immobilized trypsin to achieve a high catalytic turnover rate. Furthermore, the kinetic parameter of the immobilized trypsin indicates the absence of diffusional limitations. The durability and repeatability of the fabricated trypsin-coated monolith was tested and found to be satisfactory. The encapsulated trypsin exhibits an increased stability even after continuous use compared with that in free solution. Furthermore, this onplate bioreactor was applicable to the digestion of protein with multiple cleavage sites. Recently, monolithic materials made of one piece of a porous solid with small-sized skeletons and relatively large through-pores are being used as a column.1-10 Their micrometer-sized pores and * To whom correspondence should be addressed. E-mail: toyooka@ ys2.u-shizuoka-ken.ac.jp. Fax: +81-54-264-5593. Tel: +81-54-264-5656. † University of Shizuoka. ‡ PRESTO, Japan Science and Technology Agency. § Musashino University. (1) Hjerte´n, S.; Li, Y.-M.; Liao, J.-L.; Mohammad, J.; Nakazato, K.; Pettersson, G. Nature 1992, 35, 810-811. (2) Svec, F.; Fre´chet, J. M. J. Science 1996, 273, 205-211. (3) Peters, E. C.; Petro, M.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1997, 69, 3646-3649. (4) Zou, H.; Huang, X.; Ye, M.; Luo, Q. J. Chromatogr., A 2002, 954 5-32. (5) Nakanishi, K.; Soga, N. J. Am., Ceram. Soc. 1991, 74, 2518-1530. (6) Nakanishi, K.; Soga, N. J. Non-Cryst. Solids 1992, 139, 1-13, 14-24. (7) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem. 1996, 68, 3498-3501. (8) Tanaka, N.; Kobayashi, H.; Nakanishi, K.; Minakuchi, H.; Ishizuka, N. Anal. Chem. 2001, 73, 420A-429A. (9) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Nagayama, H.; Hosoya, K.; Tanaka, N. Anal. Chem. 2000, 72, 1275-1280. (10) Dulay, M. T.; Quirino, J. P.; Bennett, B. D.; Kato, M.; Zare, R. N. Anal. Chem. 2001, 73, 3921-3926. 10.1021/ac048388u CCC: $30.25 Published on Web 02/11/2005

© 2005 American Chemical Society

large surface area reduce the diffusion path length and provide both low-pressure drop and high column efficiencies. Several examples of such monolithic columns prepared by in situ polymerization in a chromatographic column have been recently reported and proven to be effective for high-speed separation.7,8 Until now there are mainly two materials that have been used for the preparation of a monolith; one is an organic polymer and the other is a nonorganic polymer. However, an organic polymer monolithic may not have adequate mechanical strength, especially for swollen polymer gels. An nonorganic polymer, made of a porous silica rod (monolith) by the hydrolytic polymerization of tetramethoxysilane (TMOS) is rigid and its skeletal structure can resist organic solvents or dryness. The morphology determined by phase separation was solidified by gel formation, resulting in a silica rod with a biporous structure that consists of throughpores and mesopores in the skeletons and have good mechanical strength. The unique structural characteristics of the monolith have advantages for the support of enzyme bioreactors.11-14 Fre`chet et al.11 modified monolithic columns made of an organic polymer prepared by in situ polymerization with covalently bonded trypsin. Vodopivec et al. also used polymeric macroporous materials as a support for an enzyme.12 In both cases, the enzyme was immobilized via covalent bonding, which leads to the loss of enzymatic activity to some extent.12 Recently, the protein encapsulation technique using sol-gel chemistry has been used for affinity chromatography and enzymatic reactions.15-21 The reaction involves the hydrolysis and polycondensation of alkoxysilane monomers. During the process, (11) Xie, S.; Svec, F.; Fre´chet, J. M. J. Biotechnol. Bioeng. 1999, 62, 30-35. (12) Vodopivec, M.; Podgornik, A.; Berovicˇ, M.; Sˇ trancar, A. J. Chromatogr., B. 2003, 795, 105-113. (13) Peterson, D. S.; Rohr, T.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2002, 74, 4081-4088. (14) Ye, M.; Hu, S.; Schoenherr, R. M.; Dovichi, N. J. Electrophoresis 2004, 25, 1319-1326. (15) Sakai-Kato, K.; Kato, M.; Toyo’oka, T. Anal. Chem. 2002, 74, 2943-2949. (16) Kato, M.; Sakai-Kato, K.; Matsumoto, N.; Toyo’oka, T. Anal. Chem. 2002, 74, 1915-1921. (17) Sakai-Kato, K.; Kato, M.; Toyo’oka, T. Anal. Biochem. 2002, 308, 278284. (18) Sakai-Kato, K.; Kato, M.; Toyo’oka, T. Anal. Chem. 2003, 75, 388-393. (19) Sakai-Kato, K.; Kato, M.; Nakakuki, H.; Toyo’oka, T. J. Pharmceut. Biomed. Anal. 2003, 31, 299-309. (20) Cruz-Aguado, J. A.; Chen, Y.; Zhang, Z.; Elowe, N. H.; Brook, M. A.; Brennan, J. D. J. Am. Chem. Soc. 2004, 126, 6878-6879.

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EXPERIMENTAL SECTION Materials and Chemicals. TMOS was purchased from Tokyo Kasei (Tokyo, Japan). Methyltrimethoxysilane was purchased from ShinEtsu Chemicals (Tokyo, Japan). Trypsin from porcine pancreas and NR-benzoyl-arginine-p-nitroanilide (BAPNA) were purchased from Sigma-Aldrich (Milwaukee, WI). Poly(ethylene glycol) (PEG, MW ) 10 000) was purchased from Merck (Schuchardt, Germany). BODIPY FL (4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-propionic acid) casein (BODIPY-casein) was purchased from Molecular Probes, Inc. (Eugene, OR). Water was purified by MilliQ apparatus (Millipore, Bedford, MA). Preparation of Porous Silica Monolith. The preparation of the porous silica monolith was performed as previously described by Ishizuka et al.9 Briefly, the mixed solution (2 mL) of TMOS and methyltrimethoxysilane (MTMS) was added to a solution of PEG (0.53 g, MW ) 10 000) in 0.01 N acetic acid (5.0 mL). This mixture was then stirred at 0 °C for 45 min. The resulting mixture was poured into a 0.6-mL sample tube and allowed to react for 10 days at 40 °C. The formed silica molds were washed with water and then reacted with 0.01 N aqueous ammonium solution for 3 h at 120 °C, followed by a washing with water. The molds were cut to a desired length (=5 mm o.d., 2 mm long). After drying at 40 °C for 3 days, the molds were heated at the gradient

temperature from 25 to 600 °C for 10 h and then 600 °C for 2.5 h. Similarly, the monolith containing MTMS was heated at the gradient temperature from 25 to 300 °C for 5 h and then 300 °C for 4 h. Coating of the Silica Monolith with Trypsin-Encapsulated Gel. The sol-gel reaction basically followed the procedures described in our previous report.15,23 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 forms a fully or partially hydrolyzed silane, SiOH4-n (OMe)n. A 900-µL aliquot of the trypsin solution (10% (w/v)) in 50 mM Tris-HCl (pH 7.0) containing 20 mM CaCl2 was added to 90 µL of the hydrolyzed solution. The silica monolith was carefully put into the mixed solution for 5 min and sonicated for 10 s, so that the trypsin solution soaked through the monolith. After 5 min, the excess solution was removed, and the monolith was held at 4 °C for 2-3 days, so that the remaining sol solution covering the monolith was gelled and became a thin film covering the monolith. Before analysis, the monolith was washed with water. Scanning Electron Microscope. A scanning electron microscope analysis was performed using a SV-5200 (Hitachi, Tokyo, Japan) and a S-2500 (Hitachi). Samples were coated with osmium for the SV-5200 or platinum-palladium for the S-2500. Tryptic Digestion. The tryptic activity was measured using a 96-well microtiter plate (MultiScreen, Millipore). A 150-µL volume of the substrate solution consisting of 0.2 mg/mL BAPNA in 50 mM Tris-HCl (pH 7.5) was put into the microplate. The trypsincoated monolith was put into the well and incubated at 37 °C for 5 min. After the incubation, the monolith was removed, and the increase in absorbance at 405 nm caused by the generation of p-nitroaniline was measured using a microtitler plate reader (Wallac 1420 ARVOSX, Perkin-Elmer, Yokohama, Japan). For the BODIPY-casein digestion, 40 µL of BODIPY-casein was put into the well and incubated 37 °C. After the incubation, the monolith was removed, and the fluorescence intensity (Ex 485 nm, Em 535 nm) was measured using the microtiter plate reader. The removed monolith was washed in water. The enzymatic activity of free trypsin was also measured using a 96-well microtiter plate. The reaction solution containing trypsin and substrates was incubated in a well at 37 °C, and the increase in absorbance at 405 nm or fluorescence intensity at 535 nm was measeured using the microtiter plate reader.

(21) Hodgson, R. J.; Chen, Y.; Zhang, Z.; Tleugabulova, D.; Long, H.; Zhao, X.; Organ, M.; Brook, M. A.; Brennan, J. D. Anal. Chem. 2004, 76, 27802790. (22) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, J. I. Science 1992, 255, 1113-1115. (23) Kato, M.; Sakai-Kato, K.; Jin, H.-M.; Kubota, K.; Miyano, H.; Toyo’oka, T.; Dulay, M. T.; Zare, R. N. Anal. Chem. 2004, 76, 1896-1902. (24) Edmiston, P. L.; Wambolt, C. L.; Smith, M. K.; Saavedra, S. S. J. Colloid Interface Sci. 1994, 163, 395-406. (25) Braun, S.; Rappoport, S.; Zusman, R.; Avnir, D.; Ottolenghi, M. Mater. Lett. 1990, 10, 1-5. (26) Yao, T.; Harada, I.; Nakahara, T. Bunseki Kagaku 1995, 44, 927-932. (27) Flora, K.; Brennan, J. D. Anal. Chem. 1998, 70, 4505-4513. (28) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A-1127A. (29) Cichna, M.; Knopp, D.; Niessner, R. Anal. Chim. Acta 1997, 339, 241250. (30) Schwert, G. W.; Takenaka, Y. Biochim. Biophys. Acta 1995, 16, 570-575.

RESULTS AND DISCUSSION Development of Enzyme-Coated Monolith. In previous studies,15-19 we described the preparation and use of proteinimmobilized reactors in hydrogels made by sol-gel chemistry. Although we demonstrated the usefulness of these materials for the digestion of small molecules, such as peptides, larger molecules, such as proteins, were not able to diffuse through the nanopores of the hydrogel network. To overcome this problem, a macroporous silica monolith was used as the supporting matrix for enzyme immobilization. This technique is advantageous for the preparation of enzyme-immobilized reactors because (1) its micrometer-sized pores allow for the diffusion of large proteins, (2) its large surface area allows for an increase in the number of immobilized trypsin molecules, and (3) the in situ gelation process

the proteins are incorporated into the matrix. The encapsulation process proceeds in one step, which is under mild conditions so that the biomolecules can retain their structures22,23 and biological activity.24 Furthermore, the in situ gelation process makes it easy to fabricate and interface the materials in a variety of forms, such as film,25,26 bulk form,26-29 or particles.30 Trypsin was encapsulated into the gel matrix for preparation of the on-line enzyme reactor. The enzymatic reaction and separation of the unreacted substrates and products were simultaneously performed in a capillary or microchip. The reactors had excellent enzymatic activity, which was ∼700 times higher than that in free solution.15 It was anticipated that coating the surface of the highly permeable silica monolith with the enzyme-containing gel would increase the conversion rates and decrease the diffusional limitations, which are often observed in the immobilized enzymes. In this paper, we report the development of a coating procedure for preparing a trypsin-encapsulated sol-gel as a thin film on the silica monolith. We chose the microtiter plate as the analytical platform for development of the high-throughput systems, and the monolith was fabricated to fit into the 96-well microtiter plate.

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Figure 2. Picture of bioreactor and scanning electron micrographs of the monolith prepared using different amounts of PEG: (a) 0.48, (b) 0.53, and (c) 0.58 g of PEG.

Figure 1. Effect of preparatory composition of the bioreactors on their tryptic activity. (a) PEG content; (b) MTMS and optimum composition.

makes it easy to fabricate the materials in a variety of forms, such as a thin film,25,26 bulk,26-29 or particles.30 Several reaction parameters for the fabrication of the silica monoliths were studied in order to optimize the performance of the trypsin-immobilized monolith.The preparatory procedures for the silica monolith basically followed by those previously reported. The starting composition can be chosen to obtain the desirable size and volume of the macropores. Effect of PEG Content. Water-soluble polymer additives, PEG, that induce phase separation, control the size and volume of the macropores in the monolith because the glycol forms strong hydrogen bonds with the silanols of the growing silicate polymers.31 The PEG/silica ratio affects the size of the phase-separated domains, and the increase in the ratio leads to a smaller pore size, macroporous structure and an increase in the surface area of the monolith.31 The various amounts of PEG were investigated for the fabrication of a silica monolith, and it was optimized by measuring the tryptic activity. We added 0.42, 0.48, 0.53, and 0.58 g of PEG in 2 mL of an alkoxysilane, based on a previously reported procedure.9 The monolith prepared using 0.42 g of PEG was fragile and not appropriate for the enzymatic assay. As shown in Figure 1a), the tryptic activity was the highest when 0.48 g of PEG was used for the preparation of the silica monolith. Also, the enzymatic stability of the repeated analyses was the best when 0.48 g of PEG was used (data not shown). Figure 2 shows a (31) Motokawa, M.; Kobayashi, H.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Jinnai, H.; Hosoya, K,; Ikegami, T.; Tanaka, N. J. Chromatogr., A 2002, 961, 53-63.

photograph of a prepared bioreactor and SEM images of the monolith prepared using the various amounts of PEG. In all cases, the formation of a porous structure by the phase separation reaction was observed. It is known that the amount of PEG determines the surface area of the monolith.31 The increase in the amount of PEG increased the surface area and resulted in a fine skeletal structure and through-pore.31 To increase the tryptic activity, bigger through-pores are preferable for substrates to readily approach the trypsin that is immobilized on the surface of the monolith. This is probably the reason the monolith prepared using 0.48 g of PEG showed the highest activity. Although the activity of the monolith prepared using 0.58 g of PEG was slightly higher than that using 0.53 g, this is probably because the increase in the amount of PEG increased the surface area and, as a result, increased the immobilized trypsin. Effect of MTMS Ratio. Conventionally, TMOS is the most popular alkoxysilane used as a sol-gel starting material. One reason is that the elimination of the methoxy group is faster than that of other alkoxy groups during the process of hydrolysis or condensation. One of the advantages of the sol-gel technique is that the physical or chemical characteristics of the silica monolith are readily changed by the combination of various alkoxysilanes. By incorporating MTMS into TMOS at various ratios (0-50%), we tried to control the characteristics of the monolith to induce a high tryptic activity. Figure 1b shows that the silica monolith prepared using TMOS containing 50% MTMS induces the highest enzymatic activity in addition to its stability of the enzymatic activity (data not shown). The monolith prepared using 20 and 30% MTMS was fragile. Figure 3 shows the SEM images of the silica monolith prepared using TMOS containing different ratios of MTMS. By adding a smaller amount of MTMS, the silica skeleton and the through-pore became finer. On the contrary, the addition of 50% MTMS resulted in a larger skeleton size and through-pore. This indicates that the mechanism of the monolithic structure formation in 50% MTMS is different from that of the other percentages of MTMS. When a smaller amount of MTMS was used, the hydrogen PEG-silica oligomer was predominantly formed and the solubility decreased. On the other hand, when Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

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Figure 3. Scanning electron micrographs of the monolith prepared using the different MTMS compositions: (a) 0, (b) 10, (c) 20, and (d) 50% MTMS.

50% MTMS was added, the effect of unreacted methyl groups derived form MTMS was significant, and the methyl groups decreased the solubility of the monolith. The resulting large through-pores with 50% MTMS presumably caused the biggest activity just the same as the effect of PEG. Optimum Monolith Skeleton. Based on these investigations, the monolith was prepared using 0.48 g of PEG and 50% MTMS, which gave the highest enzymatic activity. The prepared monolith showed a 146% higher activity than that prepared using the previously reported conditions (Figure 1b). The combination of the optimum PEG composition and MTMS led to the optimum monolithic skeleton. Surface Treatment. It is reported that the alkali treatment of the monolith forms mesopores on the surface, and the strength of the used alkali, treatment time, and temperature affect the size of these mesopores.32 The monolith was treated with 0.01 M NH3 for 3 h at 120 °C. Polymerization of the silica monolith before the alkali treatment was not sufficient, and the surface structure was not clearly observed by SEM. On the other hand, the treated monolith acquired a rough surface structure, which means an increase in the surface area, as well as rigidness (Figure 4a). Figure 4b shows an SEM image of the monolith, which was covered with trypsin embedded in the sol-gel. In a previous paper, we reported that the coating by the protein-containing gel covered only the surface of the monolith and did not affect the (32) Nakanishi, K.; Shikata, H.; Ishizuka, N.; Koheiya, N.; Soga, N. J. High Resolut. Chromatogr. 2000, 23, 106-110.

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Figure 4. Scanning electron micrographs of the optimum monolith skeleton: (a) before coating; (b) after coating.

size of the through-pore.23 In this report, we could observe the fine surface structure using a high-resolution SEM. As a result, it was found that a cover with a protein-containing gel reduced the roughness of the surface (Figure 4b). This indicates that the sol solution containing trypsin covered the surface of the monolith, and the mesopore was filled with the resulting gel. Because trypsin is a ∼4-nm spherical enzyme, and the osmium-coating has a thickness of 2 nm, the trypsin coating was expected to be observed as a 5-10-nm spherical clod. In fact, a 7-nm roughness was observed in Figure 4b, and this was probably derived from the trypsin that was immobilized on the surface of the monolith. Kinetic parameters of the immobilized trypsin. Figure 5a and Figure 6a depict the change in the initial velocity depending on the substrate concentration using the immobilized trypsin and free trypsin. Both systems show a similar increase in the initial velocity at the lower substrate concentration, followed by the gradual slope saturation. Double-reciprocal plots of the tryptic activity (Lineweaver-Burk plot) are shown in Figure 5b and 6b. The Km and Vmax values were obtained from these doublereciprocal plots and the following eq 1,

1/v ) Km/Vmax × 1/[S] + 1/Vmax

(1)

where v is the velocity of the enzymatic activity, Km is the Michaelis constant, Vmax is the maximum velocity, and [S] is the concentration of the substrates. The Km value of BAPNA for the monolithic bioreactor was 0.156 on average, which is almost the same as that of the free trypsin (0.136). This indicates the absence

Figure 7. Stability of the bioreactor activity.

Figure 5. (a) Velocity of the immobilized trypsin depending on substrate concentration. (b) Double-reciprocal plot of the enzymatic activity of immobilized trypsin. Conditions: substrates, BAPNA; temperature, 37 °C; reaction time, 5 min; detection, UV (405 nm).

Figure 6. (a) Velocity of the free trypsin depending on substrate concentration. (b) Double-reciprocal plot of the enzymatic activity of free trypsin. Conditions: substrates, BAPNA; temperature, 37 °C; reaction time, 5 min; detection, UV (405 nm).

of any diffusional limitation in this bioreactor. This enables the higher conversion rates and shorter reaction times. The Vmax for the encapsulated trypsin was 34.0 pmol min-1 (mg of protein)-1 on average, which is almost the same as that of the free trypsin (38.8 pmol min-1 (mg of protein)-1). These features make this bioreactor an ideal support for enzyme immobilization and show

the effectiveness of this trypsin-encapsulated technique for the assay of enzyme activity. Repeatability and Reproducibility. The variability of tryptic activity based on a run-to-run analysis was acceptable, and RSD was less than 3.1% (n ) 3). On the other hand, the RSD for tryptic activity by a batch-to-batch analysis was 7.8% (n ) 3), which was almost the same level as that for the conventional tryptic reaction schemes using free trypsin (RSD ) 12%, n ) 3).15 The monolith was prepared to fit in a well of a microtiter plate by cutting with a knife before complete drying. Therefore, the variability of the enzymatic activity could be partially ascribed to the variability of the size of the prepared monolith. This variability can be reduced by automation of the preparatory procedures. Stability. This bioreactor can be used for at least several days with no perceptible loss in activity (Figure 7). The monolith was not destroyed during the repeated measurements. Furthermore, subsequent storage at 4 °C for 2 months did not lead to any significant decrease in enzyme activity. On the other hand, a soluble trypsin, which was stored in the same running buffer of 50 mM Tris-HCl buffer (pH 7.5) at 25 °C, almost completely lost its activity within 1 day.15 The stability of trypsin is enhanced when it is immobilized in a sol-gel matrix because the matrix prevents autolysis of the enzyme. This result means that this reactor has superior characteristics as a high-throughput analytical system. Tryptic Reaction Using Protein. As a substrate, we used casein (MW 12 000-24 000) that was labeled with BODIPY dye. Upon the binding of five molecules of this 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.33,34 Figure 8 shows the trypsin digest of BODIPY-casein by the immobilized bioreactor. A gradual increase in the fluorescent intensity with reaction time shows that the substrates were digested by the trypsin, and the peptide fragments were produced. This result shows that this immobilized enzyme can digest not only small molecules but also proteins, which indicates that this system can be used for proteome studies. CONCLUSION We developed an immobilized trypsin bioreactor by the solgel method. This bioreactor can be integrated in the microtiter (33) Jones, L. J.; Upson, R. H.; Haugland, R. P.; Panchuk-Voloshina, N.; Zhou, M. J. Anal. Biochem. 1997, 251, 144-152. (34) Welder, F.; McCorquodale, E. M.; Colyer, C. L. Electrophoresis 2002, 23, 1585-1590.

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The encapsulated trypsin exhibited increased stability during continuous use compared to that in free solution, rendering this bioreactor suitable for prolonged use. Furthermore, this system enabled the removal of an enzyme with ease. This sol-gel encapsulation technique is applicable for a wide variety of biomolecules. Therefore, the combination of this technique with the microtiter plates opens a new pathway for application to automated and high-throughput analytical schemes for a multianalyte device or drug screening platform. Figure 8. Change in fluorescent intensity depending on the reaction time. Conditions: substrate, 10 µg/mL BODIPY-casein; reaction solution, 10 mM Tris-HCl buffer (pH 7.5); temperature, 37 °C; detection, fluorescence (Ex 485 nm, Em 535 nm).

plate, which is frequently used for a variety of high-throughput assays. The simple in situ encapsulation procedure, which is carried out under mild conditions, was shown to be suitable for the immobilization of trypsin, while still maintaining its catalytic activity. The advantage of this technique is the ease with which the materials can be fabricated and interfaced to analytical devices with good control.

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ACKNOWLEDGMENT This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.. The authors aknowledge Dr. Nakanishi, Dr. Minakuchi and Dr. Ishizuka for advice on the fabrication of the silica monolith. A part of this work was supported by “Nanotechnology Support Project” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Received for review December 30, 2004. AC048388U

November

1,

2004.

Accepted