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使用毛细管电泳结合激光诱导荧光检测技术对一氧化氮释放进行单细胞测定。

Single cell determination of nitric oxide release using capillary electrophoresis with laser-induced fluorescence detection.

作者信息

Yang Qiao, Zhang Xiaoling, Bao Xiaohui, Lu Hongjuan, Zhang Wenjun, Wu Wenhui, Miao Huinan, Jiao Binghua

机构信息

Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China.

出版信息

J Chromatogr A. 2008 Aug 1;1201(1):120-7. doi: 10.1016/j.chroma.2008.06.001. Epub 2008 Jun 6.

Abstract

Measurements of nitric oxide (NO) release at single cell level are fundamental to understand the diverse physiological functions of this remarkable molecule. To achieve this purpose, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was originally described for the sensitive determination of NO release in individual neuron and mammalian cell after 8-(3,4-diaminophenyl)-2,6-bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (DAMBO-P(H)) was chosen as the fluorescent probe. Various parameters affecting NO trapping in vivo and CE separation were systematically studied. Under the optimal conditions, complete and fast separation of the resulted targeted high-fluorescent triazole (DAMBO-P(H)-T) was achieved in about 3 min (2.89 min), and the relative standard deviations (RSDs) values of migration time and peak area were less than 5% and 9% for intra-day and inter-day assays, respectively. The detection limit was 42 amol (at a signal-to-noise ratio of 3). The feasibility of application of the developed method was validated by successfully applied to the measurements of NO release from four single cell study models. This original application of this method in diverse samples represents a powerful tool to study the kinetics of NO release by neuronal cells during neurotransmission, as well as for the understanding of the pathobiological and therapeutic basis of this molecule for cardiovascular diseases and under oxidative stress.

摘要

在单细胞水平测量一氧化氮(NO)释放对于理解这种重要分子的多种生理功能至关重要。为实现这一目的,最初描述了采用激光诱导荧光检测的毛细管电泳(CE-LIF)用于灵敏测定单个神经元和哺乳动物细胞中的NO释放,其中选用8-(3,4-二氨基苯基)-2,6-双(2-羧乙基)-4,4-二氟-1,3,5,7-四甲基-4-硼-3a,4a-二氮杂-s-茚满(DAMBO-P(H))作为荧光探针。系统研究了影响体内NO捕获和CE分离的各种参数。在最佳条件下,约3分钟(2.89分钟)内实现了目标高荧光三唑(DAMBO-P(H)-T)的完全快速分离,日内和日间测定的迁移时间和峰面积相对标准偏差(RSDs)值分别小于5%和9%。检测限为42 amol(信噪比为3时)。通过成功应用于四个单细胞研究模型的NO释放测量,验证了所开发方法应用的可行性。该方法在不同样品中的这一原始应用是研究神经传递过程中神经元细胞NO释放动力学的有力工具,也是理解该分子在心血管疾病和氧化应激下的病理生物学及治疗基础的有力工具。

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