A comparative study of different protein immobilization methods for the construction of an efficient nano-structured lactate oxidase-SWCNT-biosensor.
A comparative study of different protein immobilization methods for the construction of an efficient nano-structured lactate oxidase-SWCNT-biosensor.
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DOI：
10.1016/j.bios.2014.08.072
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发表时间：
2015-02-15
影响因子：
12.6
通讯作者：
Griebenow, Kai
中科院分区：
文献类型：
--
作者：
Pagan, Miraida;Suazo, Damaris;del Toro, Nicole;Griebenow, Kai
关键词：
我们通过将乳酸氧化酶（LOx）固定到单壁碳纳米管（SWCNT）电极上构建了乳酸生物传感器。传感器构建的第一步是将氧化的 SWCNT 固定到用 4-氨基苯硫酚 (4-ATP) 修饰的铂电极上。采用两种酶固定方法构建生物传感器，即使用1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDC)共价固定和物理吸附。原子力显微镜 (AFM) 实验证实了生物传感器构建过程中 SWCNT 的固定，X 射线光电子能谱 (XPS) 实验证实了第一种方法中 LOx 共价固定到 SWCNT 上。基于共价酶固定化的生物传感器的灵敏度为5.8 μA/mM，L-乳酸线性度高达0.12 mM，检测限为4.0 μM。基于蛋白质吸附的生物传感器的灵敏度为 9.4 μA/mM，L-乳酸保持高达 0.18 mM 的线性，检测限为 3.0 μM。生物传感器响应的差异可归因于共价固定期间蛋白质构象或动力学变化。在不同温度和不同储存期后测试了生物传感器的稳定性。 60°C 孵育后生物传感器的热稳定性表明，与吸附的蛋白质相比，共价固定 LOx 的生物传感器保留了更高的响应。长期稳定性实验表明，储存 25 天后，共价固定蛋白的残余活性为 40%，而吸附蛋白的残余活性为 20%。与吸附相比，共价蛋白质固定在长时间保持生物传感器功能方面优于吸附。
We constructed lactate biosensors by immobilization of lactate oxidase (LOx) onto a single-walled carbon nanotube (SWCNT) electrode. The first step of the sensor construction was the immobilization of oxidized SWCNT onto a platinum electrode modified with 4-aminothiophenol (4-ATP). Two enzyme immobilization methods were used to construct the biosensors, i.e., covalent immobilization using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and physical adsorption. Atomic force microscopy (AFM) experiments confirmed the immobilization of SWCNT during the biosensor construction and X-ray photoelectron spectroscopy (XPS) experiments confirmed covalent immobilization of LOx onto the SWCNT in the first method. The biosensor based on covalent enzyme immobilization showed a sensitivity of 5.8 μA/mM, a linearity up to 0.12 mM of L-lactate, and a detection limit of 4.0 μM. The biosensor based on protein adsorption displayed a sensitivity of 9.4 μA/mM, retaining linearity up to 0.18 mM of L-lactate with a detection limit of 3.0 μM. The difference in the biosensor response can be attributed to protein conformational or dynamical changes during covalent immobilization. The stability of the biosensors was tested at different temperatures and after different storage periods. The thermostability of the biosensors after incubation at 60°C demonstrated that the biosensor with covalently immobilized LOx retained a higher response compare with the adsorbed protein. Long-term stability experiments show a better residual activity of 40% for the covalently immobilized protein compared to 20% of residual activity for the adsorbed protein after 25 d storage. Covalent protein immobilization was superior compared to adsorption in preserving biosensor functionality over extended time period.
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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DOI：
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发表时间：
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期刊：
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通讯作者：
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作者：
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影响因子：
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作者：
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