A new route for simple and rapid determination of hydrogen peroxide in RAW264.7 macrophages by microchip electrophoresis |
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Authors: | Xiaocong Gong Qingling Li Kehua Xu Xin Liu Hongmin Li Zhenzhen Chen Lili Tong Bo Tang Hongbo Zhong |
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Institution: | 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, Shandong, P. R. China |
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Abstract: | A new method using MCE with LIF detection was developed for the determination of hydrogen peroxide (H2O2). Bis(p‐methylbenzenesulfonyl)dichlorofluorescein, a new fluorogenic reagent synthesized by our laboratory was employed as a labeling reagent, the derivatization reaction was performed in 0.10 M HEPES buffer (pH 7.4) for 30 min at 37°C. The detection of H2O2 was accomplished in 55 s, using a 40 mM HEPES buffer, 20% mannitol, pH 7.4, on a glass microchip. The RSDs of migration time and peak area were 1.8 and 3.7%, respectively. Method validation showed the linear response ranging from 0.50 to 50 μM with an LOD (S/N=3) of 0.20 μM (19.1 amol). The proposed method was applied to determine H2O2 in phorbol myristate acetate‐stimulated RAW264.7 macrophages, the concentration of H2O2 was found to be 1.86±0.05 μM; recoveries for macrophage samples were from 96.7 to 97.8%, within‐days and between‐days accuracies were 4.5% (n=5) and 6.8% (n=5), respectively. |
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Keywords: | Bis(p‐methylbenzenesulfonyl)dichlorofluorescein Hydrogen peroxide LIF MCE RAW264 7 macrophages |
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