Immobilization of Firefly Luciferase on PVA-co-PE Nanofibers Membrane as Biosensor for Bioluminescent Detection of ATP.
Immobilization of Firefly Luciferase on PVA-co-PE Nanofibers Membrane as Biosensor for Bioluminescent Detection of ATP.
复制标题
DOI：
10.1021/acsami.5b07339
复制
发表时间：
2015-08
影响因子：
9.5
通讯作者：
Wenwen Wang;Qinghu Zhao;Mengying Luo;Mufang Li;Dong Wang;Yuedan Wang;Qiongzhen Liu
中科院分区：
文献类型：
--
作者：
Wenwen Wang;Qinghu Zhao;Mengying Luo;Mufang Li;Dong Wang;Yuedan Wang;Qiongzhen Liu
萤火虫荧光素酶催化的生物发光反应已被广泛建立为一个优秀的分析系统，用于测定三磷酸腺苷（ATP）。当在溶液中时，荧光素酶是不稳定的并且不能重复使用。该问题可以通过将荧光素酶固定在固体基质上来部分解决。聚乙烯醇-乙烯共聚物（PVA-co-PE）纳米纤维膜表面具有丰富的活性羟基。首先用三聚氯氰活化聚乙烯-聚乙烯纳米纤维膜。然后将活化的PVA-co-PE纳米纤维膜随后与1，3-丙二胺和生物素反应。将萤火虫荧光素酶固定在1，3-丙二胺和生物素功能化膜的表面上。用红外光谱和扫描电镜对纳米纤维膜的表面化学结构和形貌进行了表征。通过水接触角的测量来测试膜的亲水性。荧光强度检测表明，荧光素酶固定化的PVA-co-PE纳米纤维膜具有较高的催化活性和效率。特别是生物素功能化的荧光素酶固定化生物传感器膜，由于其高的检测灵敏度，有望成为生物发光检测ATP的生物传感器。
The bioluminescent reaction catalyzed by firefly luciferase has become widely established as an outstanding analytical system for assay of adenosine triphosphate (ATP). When in solution, the luciferase is unstable and cannot be reused. The problem can be partially solved by immobilizing the luciferase on solid substrates. The poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibers membrane has abundant active hydroxyl groups on the surface. The PVA-co-PE nanofibers membrane was first activated by cyanuric chloride with triazinyl group. Then the activated PVA-co-PE nanofibers membrane was subsequently reacted with 1,3-propanediamine and biotin. The firefly luciferase was immobilized onto the surface of 1,3-propanediamine- and biotin-functionalized membranes. The surface chemical structure and morphologies of nanofibers membranes were characterized by FTIR-ATR spectra and SEM. The hydrophilicity of membranes was tested by water contact angle measurements. The detection of fluorescence intensity displayed that the firefly-luciferase-immobilized PVA-co-PE nanofibers membranes indicated high catalytic activity and efficiency. Especially, the firefly-luciferase-immobilized nanofiber membrane which was functionalized by biotin can be a promising candidate as biosensor for bioluminescent detection of ATP because of its high detection sensitivity.