Based On Microfluidic Analysis System Of Single-cell Injection, The Dissolved Membrane Separation And Reactive Oxygen Species Detection

Hydrogen peroxide (H₂O₂) plays a pivotal role as a second messenger in cellular signaling, and mounting evidence supports its role as a small molecule mediator of physiology, aging, and diseases in organisms. Additionally, H₂O₂ is also closely linked to other free radicals, such as superoxide (O₂^–) and hydroxyl radical (·OH). Therefore, to determine H₂O₂ at single cell level is deemed to be important for biochemistry, molecular cell biology, drug discovery and diagnosis of diseases at early stage.In this paper, a versatile programmable eight-path-electrode power supply (PEPS) and a laser-induced fluorescence detector (LIFD)-based microfluidic system for detecting H₂O₂ in single cells was developed by using a home-synthesized fluorescent probe bis(p-methylbenzenesulfonyl)dichloro?uorescein (FS) and electrokinetic gated injection. Two chapters are included as follows:In the chapter one, first of all, an overview of recent research achievements in single-cell component analysis was provided. The review focused on chip-based single cell manipulation, derivation, cytolysis technology and detection methods. Then the application of microfluidic chip in the single-cell component analysis was reviewed in detail. Finally, the work for single cell high-throughput analysis on the microfluidic chip was summarized.In the chapter two, a microfluidic system for the first time to determine H₂O₂ in individual HepG2 cells based on the electrokinetic gated injection was developed. The fluorescent probe FS, was employed to label intracellular H₂O₂ in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection (cell sampling, single cell loading), cytolysis, electrophoresis separation and detection of H₂O₂, were automatically carried out using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). This method contained three steps: In single cell injection step, single cells from reservoir S could enter into the sample channel, S-C, pass through the cross, C, and flow toward reservoir SW; In single cell loading step, one cell would be loaded into the separation channel, C-BW. Cytolysis and electrophoresis separation were immediately carried out under the high electric field in the third step. With the use of this method, the average content of H₂O₂ in single HepG2 cells was found to be 16.09±9.84 amol (n =15). This developed method was simple, fast, and sensitive. It potentially allows determination of other intracellular constituents at single-cell level.