Simultaneous Detection of Reactive Oxygen and Nitrogen Species Released by a Single Macrophage by Triple Potential-Step Chronoamperometry
Simultaneous Detection of Reactive Oxygen and Nitrogen Species Released by a Single Macrophage by Triple Potential-Step Chronoamperometry
复制标题
DOI：
10.1021/ac902486x
复制
发表时间：
2010-02-15
影响因子：
7.4
通讯作者：
Koh, Alaric C. W.
中科院分区：
文献类型：
--
作者：
Amatore, Christian;Arbault, Stephane;Koh, Alaric C. W.
巨噬细胞产生活性氧和氮物种 (ROS/RNS) 以应对免疫挑战。我们之前报道过免疫刺激巨噬细胞使用恒定电位安培法，在四个不同电位下，使用铂碳微电极对释放的 ROS/RNS 进行实时检测和定量。作为这项工作的方法学延伸，我们试图开发一种电分析方法，可以同时监测多个 ROS/RNS。发现铂碳微电极的三电位步长计时电流分析法为此目的提供了令人满意的灵敏度和信噪比。该方法适用于检测单个 IFN-gamma/LPS/PMA 刺激的 RAW 264.7 巨噬细胞内源产生的 ROS/RNS。与未激活的巨噬细胞相比，在刺激的巨噬细胞上检测到显着更高的 H(2)O(2)、ONOO(-) 和 NO（中心点）反应通量，这与一氧化氮合酶 (iNOS) 的诱导同种型和还原型烟酰胺腺嘌呤二核苷酸内源性产生初级 NO（中心点）和 O(2)（中心点-）一致 受刺激细胞中的磷酸盐（NADPH）氧化酶系统。至关重要的是，使用这种方法证明了上述每种物质的释放存在显着的时间变化，而使用恒电位安培法或经典生化方法（例如格里斯测定法）是无法实现这一点的。
Macrophages produce reactive oxygen and nitrogen species (ROS/RNS) in response to immunological challenges. We have previously reported the real-time detection and quantification of released ROS/RNS by immunostimulated macrophages using constant potential amperometry, at four different potentials, with platinized carbon micro-electrodes. As a methodological extension to that work, we sought to develop an electroanalytical method that would allow for the simultaneous monitoring of several ROS/RNS. Triple potential-step chronoamperometry at platinized carbon microelectrodes was found to provide satisfactory sensitivity and signal/noise ratio for this purpose. The title method was applied to the detection of endogenously produced ROS/RNS by single IFN-gamma/LPS/PMA stimulated RAW 264.7 macrophages. Significantly higher fluxes of H(2)O(2), ONOO(-), and NO(center dot) responses were detected over stimulated macrophages as compared to unactivated macrophages, consistent with the endogenous production of primary NO(center dot) and O(2)(center dot-) by both the inducible isoform of nitric oxide synthase (iNOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymatic systems in stimulated cells. Crucially, significant temporal variations in the release of each of the aforementioned species was evidenced using this method, which would not have been achievable with the use of either constant potential amperometry or classical biochemical methods such as the Griess assay.