Monolithic bioreactor immobilizing trypsin for high-throughput analysis.
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.
Kato
Masaru
M
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.
Inuzuka
Kenji
K
Sakai-Kato
Kumiko
K
Toyo'oka
Toshimasa
T
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*
D004800:Enzymes, Immobilized; D011092:Polyethylene Glycols; D012822:Silicon Dioxide; D014357:Trypsin