Ultrasensitive determination of intracellular superoxide in individual HepG2 cells by microfluidic chip electrophoresis

• Published in: Talanta (Oxford) Vol. 75; no. 5; pp. 1227 - 1233
• Main Authors: Zhu, Lanlan, Lu, Min, Yin, Xuefeng
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.06.2008; Oxford Elsevier
• Subjects: Analytical chemistry; Carcinoma, Hepatocellular - chemistry; Carcinoma, Hepatocellular - metabolism; Carcinoma, Hepatocellular - pathology; Cations; Cell Line, Tumor; Chemistry; Chromatographic methods and physical methods associated with chromatography; Dihydroethidium; Electrophoresis, Microchip - instrumentation; Electrophoresis, Microchip - methods; Ethidium - analogs & derivatives; Ethidium - analysis; Exact sciences and technology; Fluorescent Dyes - chemistry; Humans; Light; Microfluidic Analytical Techniques - instrumentation; Microfluidic Analytical Techniques - methods; Microfluidic chip; Microscopy, Fluorescence - methods; Other chromatographic methods; Oxidation-Reduction; Oxygen - chemistry; Oxygen - isolation & purification; Photochemistry; Sensitivity and Specificity; Single-cell analysis; Spectrometric and optical methods; Superoxide; Superoxides - analysis; Dihydroethidium; Microfluidic chip; Superoxide; Single-cell analysis; Fluid mechanics; Oxygen; System on a chip; Fluorescence spectrometry; Fluorescent labelling; Detection limit; Electrophoresis; Cations; Oxidation; Intracellular; Photooxidation; Microfluidics
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2008.01.017

A microchip electrophoresis method was established for the determination of intracellular superoxide (O 2 −) in individual HepG2 cells. Dihydroethidium (DHE) was used as the specific fluorescent probe to react with intracellular O 2 − to form the fluorescent 2-hydroxyethidium. Excellent resolution between 2-hydroxyethidium and ethidium cation (E +) can be achieved within 20 s. E + was reported to be generated from photochemical oxidation of DHE and interfere the determination of O 2 − with fluorescence microscopic technique. An extremely low detection limit of 2.0 amol was achieved owing to the minute sample volume and insignificant dispersion effect during microfluidic chip-based electrophoretic separation. Furthermore, only 2-hydroxyethidium peak was detected with the suggested single-cell analysis method, which indicates the photooxidation of DHE to E + could be blocked by isolating either oxygen or light from them.