Abstract：
We report quantitative determination of extracellular H2O2 released from single COS-7 cells with high spatial resolution, using scanning electrochemical microscopy (SECM). Our strategy of depth scan imaging in vertical x-z plane was conveniently utilized to a single cell for obtaining probe approach curves (PACs) to any positions on the membrane of a live cell by simply drawing a vertical line on one depth SECM image. This SECM mode provides an efficient way to record a batch of PACs, and visualize cell topography simultaneously. The H2O2 concentration at the membrane surface in the center of an intact COS-7 cell was deconvoluted from apparent O2, and determined to be 0.020 mM by overlapping the experimental PAC with the simulated one having a known H2O2 release value. The H2O2 profile determined in this way gives insight into physiological activity of single live cells. In addition, intracellular H2O2 profile was demonstrated using confocal microscopy by labelling the cells with a luminomphore, 2\',7\'-dichlorodihydrofluorescein diacetate. The two methodologies have illustrated complementary experimental results of H2O2 detection, indicating that H2O2 generation is centered at endoplasmic reticula.
摘要：
我们报告了高空间分辨率的单个COS-7细胞释放的细胞外H2O2的定量测定，使用扫描电化学显微镜（SECM）。我们在垂直x-z平面中的深度扫描成像策略可方便地用于单个细胞，以通过简单地在一个深度SECM图像上绘制垂直线来获得到活细胞膜上任何位置的探针接近曲线（PAC）。这种SECM模式提供了一种记录一批PAC的有效方法，同时可视化细胞形貌。完整的COS-7细胞中心的膜表面处的H2O2浓度从表观O2解卷积，并通过将实验PAC与具有已知H2O2释放值的模拟PAC重叠来确定为0.020mM。以这种方式确定的H2O2谱提供了对单个活细胞的生理活性的洞察。此外,使用共聚焦显微镜通过用荧光体标记细胞来证明细胞内H2O2谱，2\',7'-二氯二氢荧光素二乙酸酯。这两种方法说明了H2O2检测的互补实验结果，表明H2O2的产生集中在内质网。
