title: Monolithic bioreactor immobilizing trypsin for high-throughput analysis.
abstract: 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 on-plate bioreactor was applicable to the digestion of protein with multiple cleavage sites.
authors.affiliation: 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.
authors.forename: Masaru
authors.initials: M
authors.lastname: Kato
authors.forename: Kenji
authors.initials: K
authors.lastname: Inuzuka
authors.forename: Kumiko
authors.initials: K
authors.lastname: Sakai-Kato
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
mesh_terms: D019149:Bioreactors; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007700:Kinetics; D044367:Phase Transition; D011092:Polyethylene Glycols / Q000032:analysis; D015203:Reproducibility of Results; D012822:Silicon Dioxide; D014357:Trypsin / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D011092:Polyethylene Glycols; D012822:Silicon Dioxide; D014357:Trypsin