Eklund Sven E, Kozlov Eugene, Taylor Dale E, Baudenbacher Franz, Cliffel David E
Department of Chemistry, Vanderbilt University, Nashville, TN, USA.
Methods Mol Biol. 2005;303:209-23. doi: 10.1385/1-59259-901-X:209.
A technique for simultaneously measuring changes in extracellular glucose, lactate, and oxygen concentrations in conjunction with acidification rates on a Cytosensor Microphysiometer is described. Platinum electrodes are inserted into the standard Cytosensor plunger head and modified with enzymes and biocompatible polymeric films. The lactate and glucose oxidase enzymes catalyze the reaction of lactate and glucose. An end product of these catalyses, H2O2, is measured amperometrically. Extracellular oxygen is also measured amperometrically, while the acidification rate is measured potentiometrically by the Cytosensor. Useful information is obtained during the Cytosensor stop-flow cycles, which produce increasing or decreasing peaks, owing to the production of lactic and carbonic acid and consumption of glucose and oxygen by the cells. Fabrication of the modified sensor head and deposition of the electrode films is detailed, and the operation of the technique is described and illustrated by the simultaneous measurement of all four analytes during the addition of 20 mM fluoride to mouse fibro blast cells.
本文描述了一种利用细胞传感器微生理仪同时测量细胞外葡萄糖、乳酸和氧浓度变化以及酸化速率的技术。将铂电极插入标准的细胞传感器柱塞头中，并用酶和生物相容性聚合物膜进行修饰。乳酸氧化酶和葡萄糖氧化酶催化乳酸和葡萄糖的反应。这些催化反应的终产物H2O2通过安培法进行测量。细胞外氧也通过安培法测量，而酸化速率则由细胞传感器通过电位法测量。在细胞传感器的停流循环过程中可获得有用信息，由于细胞产生乳酸和碳酸以及消耗葡萄糖和氧，会产生上升或下降的峰值。详细介绍了修饰传感器头的制作和电极膜的沉积，并通过向小鼠成纤维细胞中添加20 mM氟化物时对所有四种分析物的同时测量来描述和说明该技术的操作。
