College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, People's Republic of China.
Anal Chem. 2009 Mar 15;81(6):2193-8. doi: 10.1021/ac801777c.
A method for the first time to simultaneously determine superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) was developed. 2-Chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and bis(p-methylbenzenesulfonyl) dichlorofluorescein (FS), two probes that can be specifically derivatized by superoxide and hydrogen peroxide, respectively, were synthesized and used. Parameters influencing the derivatization and on-chip separation were optimized. With the use of a HEPES (20 mM, pH 7.4) running buffer, a 50 mm long separation channel, and a separation voltage of 1800 V, baseline separation was achieved within 48 s for the two derivatization products, DBZTC-oxide (DBO) and 2,7-dichlorofluorescein (DCF). The linearity ranges of the method were 0.08-5.0 and 0.02-5.0 microM with detection limits (signal-to-noise ratio = 3) of 10 nM (1.36 amol) and 5.6 nM (0.76 amol) for superoxide and hydrogen peroxide, respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 2.0% and 5.0%, respectively. The recoveries of the cell extract samples spiked with 1.0 microM standard solutions were 96.1% and 93.0% for superoxide and hydrogen peroxide, respectively. With the use of this method, superoxide and hydrogen peroxide in phorbol myristate acetate (PMA)-stimulated macrophage RAW 264.7 cell extracts were found to be 0.78 and 1.14 microM, respectively. The method has paved a way for simultaneously determining two or more reactive oxygen species (ROS) in a biological system with high resolution.
首次建立了利用微芯片电泳激光诱导荧光检测（MCE-LIF）同时测定巨噬细胞 RAW 264.7 细胞提取物中超氧化物和过氧化氢的方法。合成了两种探针，即 2-氯-1,3-二苯并噻唑啉环庚烯（DBZTC）和双（对甲基苯磺酰）二氯荧光素（FS），它们可以分别与超氧化物和过氧化氢特异性衍生化。优化了影响衍生化和芯片分离的参数。使用 HEPES（20 mM，pH 7.4）运行缓冲液，50 mm 长分离通道和 1800 V 分离电压，两种衍生化产物 DBZTC-氧化物（DBO）和 2,7-二氯荧光素（DCF）在 48 s 内实现了基线分离。该方法的线性范围分别为 0.08-5.0 和 0.02-5.0 μM，超氧化物和过氧化氢的检测限（信噪比=3）分别为 10 nM（1.36 amol）和 5.6 nM（0.76 amol）。迁移时间和峰面积的相对标准偏差（RSD）分别小于 2.0%和 5.0%。用 1.0 μM 标准溶液对细胞提取物样品进行加标回收试验，超氧化物和过氧化氢的回收率分别为 96.1%和 93.0%。该方法为同时测定生物体系中两种或两种以上活性氧（ROS）提供了一种高分辨率的方法。
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