Covalent attachment of trypsin on plasma polymerized allylamine
Covalent attachment of trypsin on plasma polymerized allylamine
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DOI：
10.1016/j.colsurfb.2009.06.007
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发表时间：
2009-10-15
影响因子：
5.8
通讯作者：
Guillochon, Didier
中科院分区：
文献类型：
--
作者：
Abbas, Abdennour;Vercaigne-Marko, Dominique;Guillochon, Didier
本文主要研究了蛋白水解酶--胰酶在等离子体聚合烯丙基胺(PpAA)膜上的固定化。后者是用射频辉光放电的方法沉积在硅衬底上的。酶的共价连接分三步完成：(1)以戊二醛(GA)为连接物的聚合物表面的活化；(2)胰酶的固定化；(3)亚氨基还原处理。用X射线光电子能谱(XPS)和原子力显微镜(AFM)研究了各步骤的效果和效率。利用荧光光谱分析评价固定化过程中生物活性的变化。结果表明，在GA修饰的底物上固定化的酶比吸附的酶活力提高了50%，而亚氨基还原处理的酶保留率比未经处理的样品提高了约30%。用UV-Vis吸收光谱定量固定化酶的量，与XPS和AFM的数据一致，表明烯丙胺等离子体聚合物对胰酶有较高的吸附效率。虽然吸附的酶的活性低于通过GA连接子接枝的酶的活性，但对经历了固定化过程的三个步骤的酶来说，获得的催化活性最高。(C)2009爱思唯尔B.V.保留所有权利。
This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV-vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process. (C) 2009 Elsevier B.V. All rights reserved.