Studies On Dynamic Endocytosis And High-throughput Single Cell Analysis On A Microchip

Cells are the fundamental unit of life, and studies on cell contribute to reveal the mystery of life. Endocytosis is one of the fundamental processes for the cellular events, in which the cells import selected extracellular molecules, viruses, microorganisms and nanoparticles into their interiors. Until now, studies on cellular uptake of nanoparticles under static conditions are reported. However, the nanoparticles ingested by human are in blood circulation and the traditional cell culture is incapable of simulating dynamic environments of the blood circulation.Determination of the chemical composition in individual cells would greatly improve the probability of discriminating infected cells from healthy ones and provide a solid foundation on study and development in various fields including biochemistry, medicine, and pathology clinic. However, the small amount of analytes present in a single cell makes the analysis challenging. Up to now, to the best of our knowledge, only a few microfludic devices have been reported for single-cell analysis with high-throughput which hindered their applicability.Microchannel with dimensions at micrometer level is ideally suitable for introduction, manipulation, lysis, separation and detection of single cells. Therefore, chip-based systems for endocytosis and single-cell analysis are now attracting broad interests. However, most cells tend to adhere to the surface of plastic or glass materials, which has become a major difficulty in single cell and multiple cell analysis. In another word, cell manipulation has become a critical issue to realize high-throughput for microfluidic cell analysis.An in vitro method by using microfludic chip was reported to investigate the cellular uptake of FITC-doped SiO2 nano-particles to simulate the in vivo blood flowing condition. Cell suspension was delivered from the inlet of the microchannel and immobilized onto the bottom of the channel in static conditions. The microfluidic chip containing adherent cell was placed inside an incubator (37℃and 5% CO2). Culture medium is continuously transported through the microchannel by adjusting the liquid levels of the reservoirs for 1 day. After culturing the cells inside microfluidic channel, FITC-doped SiO2 particles with a diameter of 500 nm as fluorescent markers were added to the culture medium, and perfused through the microchannel via the adhered cells at different flow rates for 6 h. The effect of flow rate on the uptake efficiency of FITC-doped SiO2 particles was determined by fluorescence microscope. Compared to the result (100%) in conventional cell culture flask, the uptake efficiency was significantly decreased from 74.7% to7.1%, when the flow rate increased from 0.022 to 0.74 mm/s.A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33 ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150 cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38 cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance.A novel three-dimensional (3D) hydrodynamic focusing microfluidic device integrated with continuous sampling, rapid dynamic lysis, capillary electrophoretic (CE) separation and detection of fluorescent cytosolic dyes is presented. One of the major difficulties in microfluidic cell analysis for adherent cells is that the cells are prone to attaching to the channel surface. To slove this problem, a cross microfluidic chip with three sheath-flow channels located on both sides of and below the sampling channel was developed, the sheath-flow below the sampling prevents the cells from adhering to the channel surface. Labeled cells were 3D hydrodynamically focused by the sheath-flow streams and swimmingly introduced into the cross section one by one. With electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 400 ms at the entry of the channel by sodium dodecylsulfate (SDS) added in the sheath-flow solution. The maximum rate for introduction of individual cells into the separation channel was about 151 cells/min. The introduction of sheath-flow streams not only ensured single-cell sampling but also avoided blockage of the sampling channel by adherent cells. The microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single HepG2 cells.