Synthesis And Application Of Fluorescent Probes Based On Carbazole And Triphenylamine

Recently, two-photon fluorescence microscopy (TPM), employing two lower energy photon as the excitation source, is superior to traditional confocal microscopy when imaging thick, opaque and living specimen. TPM produces high detection sensitivity, no image distortion, low photodamage and reduced photobleaching. With above advances, biologists can observe and study the life processes in living cells and tissues. Fluorescence microscopy and fluorescent probes are complementary to each other, new technology need new probe and new probe will promote the development of the fluorescence detection technology. Two-photon fluorescent probes have continuously developed with the development of the two-photon microscope. But lack of the two-photon fluorescent probes restricts the wide application of TPM. Therefore, to develop two-photon fluorescent probes is very important and significative.Intracellular pH plays an essential role in regulating many cellular processes, including cellular metabolism, signal transduction, enzyme activity, ion transport, cell growth, proliferation, apoptosis, and autophagy. Abnormality of the intracellular pH may lead to dysfunction of the organelles, and is a phenotype of many diseases such as cancer, stroke, and Alzheimer’s disease. Imaging and quantitative measurement of pH values inside living cells are crucial for understanding the mechanism of physiological functions. Among numerous methods (such as H+-permeable microelectrodes,31PNMR spectroscopy, and optical microscopy) to measure intracellular pH, the fluorescence technique is a simple, sensitive, and powerful tool for assessing and imaging pH in living cells. However, the only detection changes of signals in fluorescence intensity such as turn-on/off are limited to photo-bleaching, probes concentration, probes distribution nonuniform in cells or between groups of cells, variable cell thickness, photo-stability, excitation path length, and other environmental conditions. By contrast, the ratiometric fluorescent probes allow the measurement of emission intensities at different wavelengths, which can eliminate most or all ambiguities by self-calibration and can also increase the dynamic range of fluorescence measurement. And the ratiometric fluorescent probes are highly preferred for monitoring in complex system and better resistance to variations of interference factors.In this thesis, we developed a novel wonderful membrane permeability, high selectivity, excellent sensitivity one/two-photon ratiometric fluorescent pH probe PCBT with single excitation dual-emission peaks that can quantitative measure intracellular pH values using TPM and confocal microscopy. PCBT was prepared in44%yields by heck reaction, and was characterized by NMR, high resolution mass spectra. Firstly, photophysical properties of PCBT in various solvents were investigated, We propose that these photophysical properties variations are due to a intramolecular charge transfer (ICT) mechanism, which is suitable to desigin ratiometric probes. Study of photophysical properties in Acidic and alkaline solution indicate the ability of PCBT as a polarity probe. pH titration on SPEF and TPEF have been surveyed, and all results displayed that PCBT possess pH-dependent optical properties. All above results suggested that PCBT can serve as an excellent one/two-photon pH ratiometric probe. On account that the amines may bind many metal cations expressed in cytoplasm and intracellular environment is very complex, an additional important work of the probe was performed to determine whether other ions were potential interferents. PCBT showed excellent selectivity response to H+in the presence of metal cations and anions. MTT assay clearly indicated that PCBT is low cytotoxicity and good biocompatibilityImages on one/two-photon confocal microscope were obtained of SiHa cells using405nm and800nm excitation and choosing two optical windows. PCBT was well-distributed in whole cytoplasm, and DIC image confirmed the viability of the cells, indicating excellent membrane permeability. The ratiometric fluorescence images were obtained from two optical windows. The standard intracellular calibration experiment was performed in SiHa cells with H+/K+ionophore nigericin. And the various ratiometric fluorescent imagings, displayed a characteristic pH-dependent signal, which showed specific pseudocolor at different pH values. On the basis of these standard pseudocolor, the real intracellular pH value at different area of intact Siha and HeLa cells is quantitative measured using PCBT as a one/two-photon ratiometric pH probe.At last, the imaging depth in living tissue slice was investigated by utilizing TPM. And the maximum depth of imaging in the same region was66μm in liver tissue and42μm in kidney tissue. Moreover, we studied the relationship between imaging depth and the staining concentrations/time. The detection depth and concentration was positively proportional relationship.2、Two mitochondrial fluorescent probe (APPI and ECPI) have been synthesized by simple organic reactions based on carbazole and triphenylamine. Photophysical properties of them in various solvents were investigated, We propose that these photophysical properties variations are due to a photoinduced charge transfer (PET) mechanism. APPI and ECPI have no response to the change of pH value by studying the emission spectrum of two molecules under condition of acid and alkali. MTT assay indicated that APPI and ECPI are low cytotoxicity and good biocompatibility. Images on confocal microscope were obtained of SiHa cells using405nm.3. Fluorescent dyes based on carbazole and pyridine derivatives were designed and synthesized. Photophysical properties in different pH buffer solution were studied. MTT assay showed that these molecules were low toxicity and high biocompatibility for living cell. Lliving cell imaging staining with these dyes were obtained by confocal microscopy using405nm. ECP and CP can be used for living cell ratiometric imaging because of their two peaks emissions. And the pseudo color located at different positions in the cytoplasm. Combined with the molecular structure, we proposed that they may be associated with hydrogen ion in the cell. These studies can make a suggestion for design living cell fluorescence probe using carbazole and triphenylamine derivatives in the future.In summary, we developed a novel wonderful membrane permeability, high selectivity, excellent sensitivity one/two-photon ratiometric fluorescent pH probe PCBT with single excitation dual-emission peaks that can quantitative measure intracellular pH values using TPM and confocal microscopy. Two mitochondrial living cell fluorescent probes were synthesized and living cell imagings were investigated using them. Finally, fluorescent dyes based on carbazole and triphenylamine derivatives have carried on the beneficial exploration.