Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis
Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis
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DOI：
10.1002/elps.200500232
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发表时间：
2005-12-01
期刊：
影响因子：
2.9
通讯作者：
Fang, ZL
中科院分区：
文献类型：
--
作者：
Ling, YY;Yin, XF;Fang, ZL
采用微芯片电泳法同时测定单个红细胞中的活性氧(ROS)和还原型谷胱甘肽(GSH)。该方法将细胞采样、单细胞装载、对接、裂解、毛细管电泳分离和激光诱导荧光检测集成在一个交叉通道的微流控芯片上。ROS在完整细胞中用二氢罗丹明123标记，而GSH在芯片上用分离介质中的2，3-萘二甲醛标记。芯片上的电裂解，其特点是极快地破坏细胞膜(<；40ms)，在测定过程中被用来最大限度地减少酶对分析物浓度的影响。优化了微流控网络，以防止在分离阶段从样品库(S)泄漏到分离中。对S的结构进行了修改，以避免沉积细胞堵塞其出口。ROS和GSH的检出限分别为0.5和6.9amol。细胞平均吞吐能力为25个/h，可同时测定单个细胞内GSH和ROS含量以及细胞内GSH和ROS含量随外界刺激的变化。
A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (< 40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.