Simultaneous Determination of Intracellular Reactive Oxygen Species and Apoptosis Signal

KeyWord:microfluidic chip  reactive oxygen species(ROS)  apoptosis

Abstract:

      A novel method for simultaneous determintation of intracellular reactive oxygen species and apoptosis signal was developed by microchip capillary electrophoresis with laser induced fluorescence detection.Alexa Fluor- 488 annexin V was used to label phosphatidyl serine in outer surface of the apoptosis cells,and dihydrorhodamine 123£¨DHR123£© was used to convert ROS to the fluorescent rhodamine 123£¨Rh123£© intracellularly. The cells were diluted with PBS to a final density of 1.2¡Á106 cells/mL,and then crushed by a repetitive freeze thaw method. The supernatant was separated in 1 min using 20 mmol/L borate buffer£¨pH 9.2£© as buffer medium, with a separation voltage of 1.2 kV and an injection time of 60 s. Under the optimized experimental conditions,the calibration of Rh123 was linear in the range of 0.5-3 ¦Ìmol/L with a correlation coefficient (r) of 0.998.The limit of detection£¨S/N=3£© was 0.058 ¦Ìmol/L.The established method was applied in the HepG2 cancer cell,and ROS in cells was quantified as 0.16 ¦Ìmol/L before and 1.77 ¦Ìmol /L after apoptosis.