(GSH, Cys, Hcy, NO, Ca ^{2 +} ) In The Cell

Cell is the basic unit of organism structure and function, and is also the fundamentalstarting point of the life science research. Cell endogenous small molecules, such aslow-weight biothiols (GSH, Cys, Hcy), nitric oxide (NO), calcium (Ca2+), etc., arerepresentative and important active small molecules in the process of life activity.They not only participate in important physiological processes such as cell activationand regulation, but also closely related with the occurrence and development of manydiseases.For example, GSH not only maintains biological redox homeostasis, but alsoparticipates in the signaling processes associated with cell apoptosis, and at the sametime has the ability of detoxifying of exogenous compounds. The levels of Cys andHcy are also important health indicators: Cys deficiency is often involved in retardedgrowth in children, hematopoiesis decrease, lethargy; Hcy is a risk factor forcardiovascular, atherosclerosis and Alzheimer’s disease. As an important signalmolecule, NO plays an important regulating role in the nervous system,cardiovascular system, and immune system. And the abnormal level of NO isassociated with cardiovascular disease (such as high blood pressure, coronary heartdisease, myocardial infarction, angina pectoris etc.), diabetes, cancer and otherdiseases. Ca2+is an important second messenger, and almost participates in all of thephysiological regulating processes. Meanwhile, the occurrence and development ofsome diseases have been accompanied by significant changes of intracellular Ca2+distribution and the concent varation. On the other hand, small molecules with high reactivity coexist in the cells and theyare related to each other and work together closely. So, the detection technologyshould have the performance of high sensitivity, high selectivity and effectiveseparation of multiple molecules.To reveal the roles of active small molecules in cellregulation, the synergy relationship among them, and quantitative information of themin different physiological processes, new methods with high sensitive,multi-parameter and simultaneous quantitative determination mutiple small molecules(GSH, Cys, Hcy; NO, Ca2+) in cells need to be developed. This thesis mainly includesthe following two parts:In the chapter one, in view of the important roles of low-weight biothiols (GSH,Cys, Hcy) in cells during the redox equilibrium and cancer development, as well astheir close interconvertible relationship, a new LC/ESI-MS method based onN-(Phenylseleno)phthalimide (NPSP) isotope probes was developed. New isotopeprobe NPSP-d5was first designed and synthesized, then the reaction mechanism ofNPSP isotopic probes with bithiols was validated; quantitative comparison ofbiothiols using NPSP isotope probe was characterized; the abblity in avoiding bithiolsoxidation of NPSP isotope probes was proved. Finally, biothiols in normal cells andcancer cells could be compared through the ion signal intensities of biothiols-NPSP-d0and biothiols-NPSP-d5at the same time after the two kinds of cells selectivederivatized using NPSP-d0and NPSP-d5and a series of sample preparation. The newNPSP isotope probe has supplied an effective tool for the simultaneous and accuratedetection of multiple biothiols in different samples.In the chapter two, in view of the fact that NO and Ca2+are important messengermolecule in cells, and the close connection between them, a new method ofsimultaneous determination of NO and Ca2+in single cell based on microfluidic chipelectrophoresis-laser induced fluorescence (MCE-LIF) was developed. NO and Ca2+ were respectively tagged by fluorescent probes3-Amino-4-aminomethyl-2’,7’-difluorescein, diacetate (DAF-FM DA) and1-[2-Amino-5-(2,7-dichloro-6-acetoxymethoxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N’,N’-tetraacetic acid, tetra (acetoxymethyl) ester(Fluo-3AM). Using this method and under the optimized experimental conditions, thecontent of NO and Ca2+within the single cell was detected. The method was furtherextended to study the content of two spieces under different physiological processesand physiological state.Quantitative detection of this active small molecules (GSH, Cys, Hcy; NO, Ca2+) incells contributes to an understanding of their respective physiological functions andthe relationship between them in the process of complex life activities. The twomethods developed in this paper can provide new way in the research of multipleactive small molecules in cells.