Simultaneous determination of reactive oxygen and nitrogen species in mitochondrial compartments of apoptotic HepG2 cells and PC12 cells based on microchip electrophoresis-laser-induced fluorescence

Zhenzhen Chen, Qingling Li, Qianqian Sun, Hao Chen, Xu Wang, Na Li, Miao Yin, Yanxia Xie, Hongmin Li, Bo Tang

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the first application of a microchip electrophoresis-laser-induced fluorescence (MCE-LIF) method for concurrent determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), i.e., superoxide (O2-•) and nitric oxide (NO) in mitochondria, was developed using fluorescent probes 2-chloro-1,3- dibenzothiazolinecyclohexene (DBZTC) and 3-amino,4-aminomethyl-2′, 7′-difluorescein (DAF-FM), respectively. Potential interference of intracellular dehydroascorbic acid (DHA) and ascorbic acid (AA) for NO detection with DAF-FM was eliminated through oxidation of AA with the addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescent products of O2-• and NO, DBZTC oxide (DBO), and DAF-FM triazole (DAF-FMT) showed excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric field of 500 V/cm. The levels of DBO and DAF-FMT in mitochondria isolated from normal HepG2 cells and PC12 cells were evaluated using this method. Furthermore, the changes of DBO and DAF-FMT levels in mitochondria isolated from apoptotic HepG2 cells and PC12 cells could also be detected. The current approach was proved to be simple, fast, reproducible, and efficient. Measurement of the two species with the method will be beneficial to understand ROS/RNS distinctive functions. In addition, it will provide new insights into the role that both species play in biological systems.

Original languageEnglish (US)
Pages (from-to)4687-4694
Number of pages8
JournalAnalytical Chemistry
Volume84
Issue number11
DOIs
StatePublished - Jun 5 2012
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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