Research On The Application Of Microchip-based Capillary Electrophoresis In Cell Analysis

Microchip-based Capillary Electrophoresis (μCE) is an important research areain Miniaturized Total Analysis Systems, which is a new analytical technologydeveloped in recent years,. It offers many potential advantages in analysis, includingspeed, efficiency, portability, automation, solvent/reagent economy, miniaturization,integration and so on. Laser Induced Fluorescence (LIF) is one of the most sensitivedetection techniques, with advantages of high sensitivity, strong specificity and fastresponse. LIF was firstly and still most commonly used as a kind of detectiontechnique in μCE. Nowadays, hot study of microfluidic chip gradually turned to builda variety of different types of lab-on-a-chip, such as chemistry, biological, optics,information, et al. Especially, cell laboratory in chip based on cytobiologysystematical study attracted widely attention.In this paper, μCE-LIF was used to analyse components in cell, the followingfour chapters is included:First, a progress of cell analysis in microfluidic chip, including groups of cellsand single cells, was overviewed.Second, a novel method for simultaneous determintation of intracellular reactiveoxygen species and apoptosis signal was developed by microchip capillaryelectrophoresis with laser induced fluorescence detection. Alexa Fluor488annexinV was used to label phosphatidyl serine in outer surface of the apoptosis cells first,and dihydrorhodamine-123(DHR-123) was used to converted ROS to the fluorescentrhodamine-123(Rh-123) intracellularly second. Cells diluted by PBS to a finaldensity of1.2×106cells/mL, and then crushed by a repetitive freeze thaw method,and the supernatant was separated using a20mM borate buffer (pH9.2) in1min witha separation voltage of1200v after an injection time of60s.Under the optimizedexperimental conditions, the calibration of Rh123was linear in the range of 0.1~2μmol/L with correlation coefficient of0.992. The limit of detection was0.058μmol/L. The established method was tested to measure the relationship of intracellularROS and apoptosis, and ROS in cells was quantified as0.16μmol/L before and1.77μmol/L after apoptosis. An elevation of intracellular ROS was observed inapoptotic HepG2cells induced by Adriamycin.Third, anticancer activitiy of chelerythrine (CHE) and sanguinarine (SAN) werestudied by microchip capillary electrophoresis with laser induced fluorescencedetection. In this experiment, HepG2cells were treated by different concentrations ofChelerythrine (CHE) and Sanguinarine (SAN), after administration of MTT detection,changes of the morphology and refraction of the cells were observed by invertedmicroscope, and intracellular ROS content changes were detected by μCE-LIF.Results showed that, with the prolonged duration of action, the drug concentrationcontinuously improve significantly enhanced the inhibitory effect of cells; the levelsof ROS in apoptosis cell were increased.Fourth, the metabolism of doxorubicin in a single adriamycin-induced humanhepatoma HepG2resistant cell was detected by microfluidic chip electrophoresis.Firstly, the resistant strains of HepG2cells (HepG2/ADR) was established and inducedby intermittently administration of increasing concentrations of doxorubicin. Then,the metabolism of doxorubicin in a single resistant (HepG2/ADR) cell which inducedby the administration concentration (1.0μg/mL) was detected by microchipelectrophoresis. Simultaneously, the problems accompanied with the process ofresistant cell strains cultured was discussed, which will be helpful for exploring thedoxorubicin resistant mechanisms and to seek for drugs to reverse the drug resistance.