For Development And Application Of New Fluorescent Probes For Cell Physiological Characteristics Of The Study

The progress of life is a total result of many relative chemical reactions which a number of biological active substances take part in. Many active substances can combine with amino acid, protein or other biological molecules, and play important biological action, special physiological function and high biochemical effects. With the progress of biochemistry and molecular biology and the using of chemistry, physics and biology, the course of chemical reaction in organism was gradually elucidated. But more and more experimental evidences suggest that the results obtained from the reaction occurred in test tubes cannot reflect the instance in living object. So it is necessary to explore the real time changes of active substances in order to comprehend the life. Among of them, H+ and ROS are two important active substances.H+ concentration, as an important metabolic and cellular parameter, plays a central modulating role in many cellular events such as cell growth, calcium regulation, enzymatic activity, receptor-mediated signal transduction, endocytosis, chemotaxis, and other cellular processes. Reports have suggested a connection between some pathological states, notably cancers, renal failure, neural ceroid lipofuscinoses, and chronic obstructive pulmonary disease and abnormal pH values in cytoplasm or acidic organelles. As a result, the precise measurement for intracellular pH is of great importance. A number of methods are currently available for the measurement of pH including microelectrodes, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. Among these methods, fluorescence spectroscopy offers significant advantages due to its nondestructive character, high sensitivity and specificity, and the availability of a wide range of indicator dyes. Moreover, fluorescence microscopic imaging technique allows us to map the spatial and temporal distribution of H+ within living cells.Nowadays, two classes of fluorescent pH probes have been developed, that is probes for cytosol which work at a pH of 6.8-7.4, and probes for acidic organelles such as lysosomes and endosomes which function over the pH range of 4.5–6.0. As minor variations of intracellular pH may induce cellular dysfunction, desirable pH fluorescent probes should be able to sensitively respond to a minor change of pH, and can avoid interfering from native cellular species. However, limitations of the currently available pH probes include low sensitivity, and/or excitation profiles in the ultraviolet region which can damage living samples and cause interfering autofluorescence from native cellular molecules. Near-infrared fluorescence probe was widely used in biological science field because of their special photophysics and photochemical characters, high sensitivity, wide response range and appropriate condition. Additionally, very few acidic fluorescent probes are considered to be desirable ones for studying acidic organelles, which is really a bottleneck in cell biological or medical studies. Thus, the focus of our study is to design near-infrared fluorescent probe with good selectivity, high sensitivity, good photostability and working within the acidic pH range.In addition, various reactive oxygen species (ROS) are generated in the course of biological metabolism, such as superoxide (O₂^-·), hydrogen peroxide (H₂O₂), hydroxyl radical (HO·), nitric oxide (NO) and peroxynirte (ONOO^-); these ROS play a vital role in physiology. They are important mediators for the pathological conditions of various diseases. A rapid rise in intracellular oxidant levels under oxidative stress could cause damage to biological molecules and result in various diseases such as carcinogenesis, inflammation, ischemia-reperfusion injury. To date, several methods for ROS detection include electron spin resonance spectroscopy (ESR) or electron paramagnetic resonance spectrometry (EPR), HPLC, chemiluminescence (CL), fluorescence and electrochemistry method. However, the short live and low concentration of ROS mean the determination difficult, especially the determination in vivo and in situ. At present, fluorescence imaging methods have good sensitivity, selectivity and take full advantage of benefits provided by confocal laser scanning microscopy (CLSM). CLSM is a type of high-resolution fluorescence microscopy that can give the fluorescence image of cells and tissues, where ROS are detected by fluorescent probe. Fluorescence imaging method challenges the traditional fluorescent method and realizes the detecting of ROS in vivo and in situ. There is an exigent need for researchers to develop fast-respond probes to trap ROS for investigation of the mechanisms of the production, metabolism and trafficking. Therefore, considering the applicable probes, it is of great importance to improve reaction rate and selectivity to avoid potential side reactions with other ROS under some conditions. If novel fluorescence probes that overcome the above problems were available, they would contribute greatly to the elucidation of the roles of ROS in living cells.In summary, fluorescence analysis and imaging techenology was widely used in biological analysis field. Effective probes for H+, ROS should feature good compatibility with biological samples, low toxicity, and resistance to oxidation, visible-wavelength excitation and emission profiles to minimize sample damage and native cellular autofluorescence, and a fluorescence response for mapping spatial resolution.Based on the changes in spectrum characters of the fluorescent probes reacting with H+, reactive oxygen species, we have carried out two aspects of investigation:(一) A dual near-infrared pH fluorescent probe, AP-Cy, was synthesized through a one-step reaction of tricarbocyanine with 3-aminophenol under mild conditions with a large throughput. AP-Cy exhibits high sensitivity, good photostability, excellent cell membrane permeability and strong pH dependence. The pH titrations indicate more than 10-fold increase in fluorescence intensity within the pH range of 4.0-6.5 with pKa value of 5.14 and pKa value of 11.31 within the pH range of 10.5-11.8. The pH fluorescent probe firstly realized dual measurements for H+ within different pH ranges at different excitation and emission wavelengths. Furthermore, as the first near-infrared fluorescent probe for intracellular acidic pH imaging, it is especially suitable for studying acidic organelles. We have demonstrated the value of AP-Cy by monitoring intracellular H+ within HepG2 cells.(二) A novel fluorescent probe for the determination of superoxide anion radical (O2-·) was designed and synthesized through the reaction of fluorescein with triphenylgermanium bromide. The probe can rapidly respond to O2-·, and has high selectivity, sensitivity and good photostability. Furthermore, as one of organic germanium compounds, the probe is low even no toxic, which will have important application in detecting O2-·in biological systems.