A novel trypsin-encapsulation technique using the sol-gel method was developed for the preparation of an on-line enzyme reactor integrated into capillary electrophoresis. Trypsin was encapsulated in tetramethoxysilane-based hydrogel, and its enzymatic activity was evaluated using alpha-N-benzoyl-L-arginine ethyl ester and two peptides (bradykinin and [Ter8]-bradykinin). The enzyme encapsulation was carried out in a single step under mild conditions within a capillary, and 1.5-cm gel was formed at the inlet of the capillary. The resultant monolithic reactor showed excellent enzymatic activity, which was approximately 700 times higher than that in free solution, without stopping the flow. Separation of the unreacted substrates and products in the same capillary also showed high selectivity, and sample size in this system decreased 3 orders of magnitude from conventional tryptic reaction schemes. The encapsulated trypsin maintains its substrate specificity even in a sol-gel matrix. Furthermore, the encapsulated trypsin exhibits increased stability even after continuous use compared to that in free solution.

中文翻译：

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通过溶胶-凝胶法将胰蛋白酶包裹的在线酶反应器集成到毛细管电泳中。

开发了一种使用溶胶-凝胶法的新型胰蛋白酶包囊技术，用于制备整合到毛细管电泳中的在线酶反应器。将胰蛋白酶封装在基于四甲氧基硅烷的水凝胶中，并使用α-N-苯甲酰基-L-精氨酸乙酯和两种肽（缓激肽和[Ter8]-缓激肽）评估其酶活性。在温和条件下在毛细管内一步进行酶包封，并在毛细管入口处形成1.5厘米长的凝胶。所得的整体反应器显示出优异的酶活性，其比游离溶液中的酶活性高约700倍，而没有停止流动。在同一毛细管中分离未反应的底物和产物也显示出高选择性，与传统的胰蛋白酶反应方案相比，该系统中的样品量减少了3个数量级。包囊的胰蛋白酶即使在溶胶-凝胶基质中也能保持其底物特异性。此外，与游离溶液相比，包封的胰蛋白酶甚至在连续使用后也显示出增加的稳定性。