Detection, Based On The Multi-component Microfluidic Chip Electrophoresis Of Single Cells And Mitochondria Active Substances While

Earth is a planet with a magnificent variety of plants and animals, they are diverse inmorphology and structure. But they are all consist of cells. Cell is the structural andfunctional unit of an organism, i.e., it is the basic unit of structure. A cell in itself is thesmallest part of an organism, which is capable of functioning independently and can carryout the fundamental duties of life like reproduction and metabolism. Under normalphysiological conditions and specific pathological conditions, the expression of organismat the cellular level is different. Awareness of the various types of reactive species, theirlevels and changes within the plant and animal cells will contribute to the understanding ofthe basic physiological functions of plant and animal cells.Affection of cells by endogenous or exogenous factors will result in the production ofreactive oxygen species and reactive nitrogen species, whose levels are closely related tophysiology and pathology. On one hand, appropriate levels of free radicals in the organismare required to maintain normal functions. They are involved in signal transduction,regulation of gene expression and cellular physiological function. On the other hand, whenfree radicals are overproduced in the organism, they will result in many pathologicalchanges because of their high reactivity. What's more, production of free radicals is in aconstant, instantaneous transformation state in the organism. Accompanied by theformation of free radicals, there exists a scavenging, antioxidant defense systems, whichwill protect cells from oxidative injury. Among them, glutathione (GSH) system is animportant part in maintaining cellular microenvironmental redox state. GSH protects cellsfrom oxidative damage by depleting free radicals and maintaining plant and animalintracellular redox balance.Research on the formation of free radicals within cells, their scavenging andtransformation mechanism are far from being reached. But developments in the lifesciences today still require precise and accurate information on them. The reason thatdeeper research on cells'oxidative stress is difficult to carry out is mainly because oflacking of effective analytical methods for determination of free radicals and glutathione.Based on this, we choose microchip electrophoresis technology as our research platform, and selected HepG2 cells and PC12 cells as our research cell targets, aiming at establishinga simultaneous determination method of cellular reactive species (free radicals, GSH). Thestudy is of great scientific importance, not only for the research on free radical cell biology,particularly for cellular oxidative stress, but also provides new strategies and methods forstudy on oxidative stress in plants and animals at the cellular level. Three parts areincluded as follows:The application of microchip electrophoresis with laser-induced fluorescence(MCE-LIF) detection to simultaneously determine GSH and H₂O₂in mitochondria extractwas described. Organoselenium probe Rh-Se-2 and bis(p-methylbenzenesulfonate)dichloro- fluorescein (FS) synthesized in our laboratory were utilized as fluorescent probesfor GSH and H₂O₂, respectively. Rh-Se-2, which is non-fluorescent, reacts with GSH toproduce rhodamine 110 (Rh110) with high quantum yield. Similarly, non-fluorescent FSreacts with H₂O₂and produces dichlorofluorescein (DCF) accompanied by drasticfluorescence enhancement. Both probes exhibit good sensitivity toward their respectivetarget molecule determination. Fast, simple and sensitive determination of GSH and H₂O₂was realized within 37 s using a running buffer of 50 mM mannitol, 40 mM HEPES (pH7.4) and an electric field of 360V/cm for separation. The MCE-LIF assay was utilized toinvestigate the levels of GSH and H₂O₂in mitochondria extract isolated from HepG2 cells.The method was further extended to observe situations of the two species in mitochondriaextract of HepG2 cells experiencing cell apoptosis that were induced by doxorubicin andphotodynamic therapy.Next we applied MCE-LIF method for concurrent determination of reactive oxygenspecies (ROS) and reactive nitrogen species (RNS), i. e. superoxide (O2-·) and nitric oxide(NO) in mitochondria, using fluorescent probes 2-chloro-1,3- dibenzothiazoline-cyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM),respectively. Potential interference of intracellular dehydroascorbic acid (DHA) andascorbic acid (AA) for NO detection with DAF-FM were eliminated through oxidation ofAA on addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescentproducts of O₂^·-and NO, that is, DBZTC oxide (DBO) and DAF-FM triazole (DAF-FMT)realized excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric-field of 500 V/cm. Using the method, weinvestigated the levels of DBO and DAF-FMT in mitochondria extract isolated fromnormal HepG2 cells and PC12 cells, and further extended the method to detect DBO andDAF-FMT level change in mitochondria extract isolated from apoptotic HepG2 cellsinduced by reseveratrol, and PC12 cells induced by amyloidβpeptide, which was provedto be simple, fast, reproducible and efficient.Based on the electrokinetic gated injection, we established the automation andintegration of multiple single-cell operations, including cell sampling, single cell loading,single cell cytolysis, electrophoresis separation and ROS/RNS simultaneous detection, on asimple cross microchip with assistant channel using a a microfluidic system. Fluid shapesof fluorescent dyes and microsphere confirms the validity of the method, and we nextinvestigated the possibility of single-cell continuous injection. With the method, wedetermined O₂^·-and NO in single cells.Using microchip electrophoresis technology as our research platform, we realizedsimultaneous determination of GSH and H₂O₂, O₂^·-and NO in mitochondria extractisolated from apoptotic cells, then we attempted to determine O₂^·-and NO in single cellsconcurrently. Determination of intracellular bioactive species will afford beneficialinformation related to cell metabolism, signal transduction, cell function and diseasetreatment. Simultaneous measurement of those species with the method will helpunderstand their distinctive functions in addition to the interaction between them, and willprovide a new insight into the role that those species play in biological systems. Ourresearch realized determination of biological samples from cellular extracts to single cell,and investigated the factors that influence cellular redox state and the basic relationshipbetween ROS and RNS. The research work has not been reported previously and hasprovided new strategy and new method for free radical biology in life science field.