(71e) Cross-Linking Polymerization for Preparation of Enzyme-Immobilized Micro-Reactor

Microreactors are potentially powerful tools in chemistry and biotechnology fields. The excellent performance of microreaction systems is achieved by rapid heat and mass transfers, and larger surface/interface area. These attractive features are favorable to catalytic reactions through catalysts immobilized on the reactor. Enzyme is one of the catalysts, which is useful in substance production and has high potential for analysis applications. There have been demands for enzymatic microreaction devices in several fields, especially for bioanalyses. Several enzyme-immobilized microreactors have been developed. We have developed modified sol-gel techniques for surface modification of microchannel and their application for preparation of microreactor [1]. Although it is necessary to reduce cost, effort, and time for production of microreactor, the developed methods of microreactor preparations require high-level techniques and/or multi-step procedure which could lead to low cost performance. Also, disposability of the reactor is considered essential in bioanalyses. The present study was designed to develop a facile and inexpensive method to immobilize enzyme on a microreactor. We report on the procedure for immobilizing enzyme on the internal surface of the microchannel by forming an enzyme-polymer membrane through cross-linking polymerization method. A typical procedure for the enzyme-membrane embedded microreactor is as follows. Commercially available polytetrafluoroethylene (PTFE) tube (500 ?Ým inner diameter and 6 cm length) was used for microreactor preparation. Glutaraldehyde (GA) and paraformaldehyde (PA) were employed as bifunctional cross-linker agents to facilitate enzyme-enzyme covalent binding. The enzyme was dissolved in PBS (pH 8.0) and the final concentration equivalent to 10 mg ml-1 was filtered (pore size 0.2 ?Ým). The enzyme and the aldehyde reagents containing PA and GA solutions were separately loaded into a PTFE tube from 1 ml syringe equipped with a syringe pump, to form enzyme-immobilized membrane on microchannel wall. The resulting microreactor showed excellent reaction performance. Also, we examined stability of enzymes against denaturating agents. The microreactor showed good stability against urea and DMSO treatment. This method may also be of interest in various micro-analysis system involving the immobilized biomolecules such as enzyme, antibody, glycoprotein, and other functional proteins.

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