Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels
Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels
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DOI：
10.1021/acsami.5b09301
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发表时间：
2015-11-25
影响因子：
9.5
通讯作者：
Walde, Peter
中科院分区：
文献类型：
--
作者：
Kuechler, Andreas;Bleich, Julian N.;Walde, Peter
白无齿菌蛋白酶K（Engyodontium album proteinase K，proK）广泛用于从生物样品中分离核酸过程中降解蛋白质杂质，或用于蛋白质组学和朊病毒研究。针对proK在流动反应器中的应用，发展了一种在玻璃微吸管中稳定固定proK的简单方法。通过树枝状聚合物-酶偶联物从水溶液中吸附实现酶的固定化。该共轭物首先由聚阳离子树枝化聚合物（denpol）和proK合成，并且平均由2000个denpol重复单元和140个proK分子组成，其通过稳定的双芳基腙键沿着denpol链连接。尽管管内proK的固定仅基于非特异性、非共价相互作用，但固定的proK不会从管中泄漏，并且在4摄氏度下长期储存期间以及在25摄氏度和pH = 7.0下连续操作期间保持活性。所开发的程序被成功地应用于proK的固定在玻璃/PDMS（聚二甲基硅氧烷）微芯片，这是一个要求在该领域中的应用程序的proK为基础的蛋白质分析与这种类型的微流控装置。
Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acids from biological samples, or in proteomics and prion research. Toward applications of proK in flow reactors, a simple method for the stable immobilization of proK inside glass micropipette tubes was developed. The immobilization of the enzyme was achieved by adsorption of a dendronized polymer-enzyme conjugate from aqueous solution. This conjugate was first synthesized from a polycationic dendronized polymer (denpol) and proK and consisted, on average, of 2000 denpol repeating units and 140 proK molecules, which were attached along the denpol chain via stable bis-aryl hydrazone bonds. Although the immobilization of proK inside the tube was based on nonspecific, noncovalent interactions only, the immobilized proK did not leak from the tube and remained active during prolonged storage at 4 degrees C and during continuous operation at 25 degrees C and pH = 7.0. The procedure developed was successfully applied for the immobilization of proK on a glass/PDMS (polydimethylsiloxane) microchip, which is a requirement for applications in the field of proK-based protein analysis with such type of microfluidic devices.