Ultrasensitive determination of intracellular superoxide in individual HepG2 cells by microfluidic chip electrophoresis.
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.
Zhu
Lanlan
L
Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, 310027 Hangzhou, China.
Lu
Min
M
Yin
Xuefeng
X
D006528:Carcinoma, Hepatocellular / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D002412:Cations; D045744:Cell Line, Tumor; D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D004996:Ethidium / Q000031:analogs & derivatives / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008027:Light; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000302:isolation & purification; D010777:Photochemistry; D012680:Sensitivity and Specificity; D013481:Superoxides / Q000032:analysis*
C506392:2-hydroxyethidium; D002412:Cations; D005456:Fluorescent Dyes; D013481:Superoxides; D004996:Ethidium; D010100:Oxygen