Immobilization of enzymes on microchannel surface through cross-linking polymerization

摘要

The enzyme-microreactor has a strong potential for applications such asbiotechnological syntheses and analyses. At present, interesting microreactors with variousenzymes have been reported. However, the developed methods of microreactor preparationsrequire high-level techniques and/or multi-step procedure which could lead to low costperformance. Also, disposability of the reactor is considered essential in bioanalyses.Simplifying the procedure of enzyme-immobilization in microreactor has been attractive. This isachievable by concertedly utilizing the reaction for enzyme-immobilization and the characteristicof microfluidics. We have designed an enzyme-immobilized microreactor possessing asubstrate membrane that covers the internal surface of the micro-channel. The cylindricalmembrane is composed of cross-linked polymerized enzyme product. This carrier-free methodbased on enzyme-cross-linked aggregation (CLEA) realized a facile and inexpensivepreparation of an enzyme-immobilized microreactor. In this study, α-chymotrypsin was usedas a model enzyme for protein digestion. Recently, microreactors capable of protein digestionhas been attractive in proteomics studies. By utilizing the optimized condition in CLEAformation, α-chymotrypsin-immobilized tubing microreactor was successfully prepared bycross-linking polymerization in a concentric laminar flow. The resulting microreactor showedhigher stability against heat and organic solvent than those of the free enzyme. These resultsshow that this preparation method leads to a flexible technology platform for screening anddesigning a potential enzyme-microreactor for broad applications.