| In recent years, the field of fluorescent probe is developing very fast. It was widely applied in chemical sensing, optical materials and biology. However, there are no targeting and quantitative information of the distribution of object can be recieved from most probes. So there is an urgent demand of the fluorescent probes which have the cell organelle positioning function and provided the quantitative information of the object distribution. It plays a vital role for viscosity in physiological processes, so it is important for people to develop the probes which can detect the distribution of intracellular viscosity and real-time monitor the viscosity changes in vivo. The mechanism of the most viscosity probes is based on the twisted intramolecular charge transfer mechanism (TICT). But there are some disadvantages. For example, signal insensitivity, susceptible interference by dye concentration, poor cell permeability and so on. Photoinduced electron transfer (PET) is one of the typical methods which are used to design fluorescent probe. PET usually has the advantages of high sensitivity and fast response time. So we developed two types of fluorescence probes with specific function in cells. One probe, based-on PET mechanism and FRET mechanism, can quantitatively be detected the viscosity distribution of total cells, the other can be located in lysosome to detect H2S.1. We designed and synthesized a ratiometric viscosity probe VN-1based on PET and FRET mechanism. Aniline was used as an electron donor,1,8-naphthalimide fluorophore and anthracene fluorophores were used as electron acceptor and as acceptor and donor for FRET. Probe VN-1performed strong fluorescence enhancement as the increasing of solvent viscosity and decreasing of temperature. The probe showed the ratiometric changes. Compared with compounds VN-2and VN-3, fluorescence enhancement of VN-1probe is mainly owed to PET and TICT inhibition. Through the analysis of the probe crystal and measurements at different excitation wavelength, we deduce that efficient FRET happened. Probe VN-1was insensitive with pH, metal ions and biomolecules which can provide its application in cells. Fluorescence lifetime studies and theoretical calculations about VN-1can be explained the PET mechanism in the viscosity of the environment from another point of view. 2. The probe VN-1was well used in cells to reflect the intracellular viscosity distribution. Probe VN-1can reflect the different viscosity distribution by a fluorescent signal in different types of cells. The colocalization experiments showed that brightest region is lysosomes, followed by the mitochondria. The probe VN-1can be used with two-photon ratio image to detect the viscosity distribution. It also showed the highest viscosity of lysosomes. We applied VN-1in MCF-7cells to focus on viscosity changes during the process of apoptosis. The fluorescence intensity and lifetime increased of the total cells in the process of cell apoptosis observed by two-photon ratio imaging and fluorescence lifetime imaging, which means the viscosity within cells increased. But the phenomenon in lysosome was opposite from the above which means its viscosity decreased.3. We designed and synthesized two hydrogen sulfide-detecting reaction-based probes (NI-NHS and lyso-NHS). Probe lyso-NHS is composed of morpholine ring which used as lysosomal orientation. Comparing with NI-NHS, Lyso-NHS showed stable fluorescence signal at lysosome pH range. They performed good hydrogen sulfide detecting in the bovine serum. Two-photon absorption cross-section of both NI-NSH and lyso-NSH increased to175GM and186GM respectively after detecting hydrogen sulfide. In colocalization experiments compared with commercial dyes, it showed that lyso-NHS detected hydrogen sulfide located in lysosomes. |
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