| Cells are the basic unit of organism structure and functions. Therefore, cell studies must be done to explore the laws and essence of cell life activities for the organism. The bulk cellular analysis does not offer information concerning cell to cell differences. However, the analysis of single cells could tackle the issue. It is a frontier field of sciences that has been developed from the interdisciplinary collaborations of analytical chemistry, biology and medicine. Especially analysis of intracellular contents helps to understand basic cell functions and the cell's internal and external contact, as well as detect and identify the unnormal cell in a large population of cells. Superoxide anion, a precursor for other reactive oxygen species (ROS), its excess production not only harms many biological molecules, but also could convert into other more toxic radicals such as HO·, H2O2. Therefore, superoxide is of great interest for research on cell metabolism and cytotoxic pathogenesis.A microfluidic chip, also called a micro total analysis system or lab on a chip was presented by Manz and Widmer. Recently, microchips have attracted much attention because of several advantages including cell-compatible sizes, low sample consumption and rapid analysis. Microchip development goals are miniaturization, integration, automation and simplification. Currently, microchips integrated with micropumps and microvalves have been applied to single-cell analysis, but the system was tough to establish due to micrometer-scale chip channels. Intracellular components analysis on the simple microchip was commonly accomplished by hydraulic pressure generated by difference of liquid level combined with electric fileds or electric fileds. Simplest injection, pinched injection and gated injection mode were usually companied. Cell sampling, cell loading and cell trapping were two-steps by means of these injection modes. Besides, A cell just controlled at the channel intersection was not easy due to the tiny channel. In conclusion, operation procedures were troublesome and time-consuming for single-cell analysis using injection modes mentioned above. So far, only one piece of paper on determination of superoxide in individual cells on microchips has appeared. Also, a method combining hydraulic pressure and electric fileds with pinched injection mode was employed. In the paper, microchips-based gated combined with pinched injection scheme was used for determination of superoxide in individual cells. 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) was utilized as the fluorescent probe. It exhibites high selectivity. Drawbacks stated above were addressed in the previous work. We made two aspects of investigation as follows:First, gated (loading step) and pinched injection (dispensing step) based on a simple microchip was proposed for the automated determination of superoxide (O2-.) in individual HepG2 cells. A fluorescent probe 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) synthesized can specifically react with O2-.. Gated injection mode (loading step) was supported by the flow profile of DBZTC oxide (DBO). Cell sampling, cell loading and cell trapping were completed in one step. Trapping a single cell used 15 s. Cell viability assay indicated the electric field strength applied was appropriate. The linear range of the method was 2.1- 102.8 amol with a detection limit (S/N = 3) of 0.82 amol (8 nM) for DBO. The average content of O2-. in individual HepG2 cells was 6.22±3.49 amol (RSDs = 56%, n = 18). The average separation efficiencie for O2-. in cell lysates was 4.73×10~4 theoretical plates. This method enable fast achievement of the process of single-cell analysis.Second, hydraulic pressure generated by the difference of liquid level gated injection (loading step) combined with electrokinetic pinched injection (dispensing step) on microchips was applied to determination of superoxide (O2-.) in single HepG2 cells. A fluorescent probe 2-chloro-1, 3-dibenzothiazolinecyclohexene (DBZTC) synthesized was used. The type of gated injection mode (loading step) was demonstrated through the flow profile of fluorescent dye Rhodamine-6G (Rh-6G). A wider cell sample population of 1×10~5-1.0×10~6 cells?mL and cell-injection time of 4 s were obtained. Cell sampling, cell loading and cell trapping were completed in one step. The linear range of the method was 2.1- 103 amol with a detection limit (S/N = 3) of 1.0 amol (10 nM) for DBZTC oxide (DBO).The average content of O2-. in individual HepG2 cells was found to be 7.82±3.87 amol (RSDs = 49%, n = 14). This method was simple, fast and accurate. |
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