Glutamine biosensors for biotechnology applications, with suppression of the endogenous glutamate signal
Glutamine biosensors for biotechnology applications, with suppression of the endogenous glutamate signal
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DOI：
10.1021/ac970173f
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发表时间：
1997-09-15
影响因子：
7.4
通讯作者：
Woodward, JR
中科院分区：
文献类型：
--
作者：
Madaras, MB;Spokane, RB;Woodward, JR
描述了用于安培测量的谷氨酰胺膜。通过在具有共同固定的谷氨酰胺酶和谷氨酸氧化酶的谷氨酰胺敏感层的顶部使用由固定化的谷氨酸氧化酶和过氧化氢酶组成的补充的抗谷氨酸层，大大减少了内源性谷氨酸的干扰。介绍了用具有不同渗透特性的聚碳酸酯膜来控制底物进入酶层的方法，以及酶层密度对膜灵敏度的影响。所制备的膜具有良好的操作稳定性(至少5天)和很好的线性(可达10 mM谷氨酰胺)。采用适当的膜选择和交联条件，制备了对谷氨酸具有良好截留率的膜(对于含有5 mM谷氨酸的5 mM谷氨酰胺样品，相对标准偏差小于6%)。这些膜适合于监测哺乳动物细胞培养中的谷氨酰胺水平，而不需要单独测量谷氨酸。
Glutamine membranes for amperometric measurements are described. The interference of the endogenous glutamate is greatly diminished by using a supplementary ''anti-glutamate'' layer consisting of immobilized glutamate oxidase and catalase on top of the glutamine-sensitive layer having co-immobilized glutaminase and glutamate oxidase. The use of polycarbonate membranes with different permeability characteristics for the control of the substrate's access to the enzyme layers is presented, as well as the effect of the density of the enzyme layer on the sensitivity of these membranes. The fabricated membranes have good operational stability (at least 5 days) and very good linearity (up to 10 mM glutamine). Using an appropriate choice of membranes and cross-linking conditions, membranes with good rejection of glutamate have been fabricated (less than 6% RSD for a 5 mM glutamine sample containing 5 mM glutamate as interferent). These membranes are suitable for monitoring of glutamine levels in mammalian cell cultures without the need of a separate measurement for glutamate.