| With the development of molecular biology, biomedical and biological analysis techniques, it is possible to understand biological processes and research the physiological activities under cell and subcellular level. For the individual cells, concentration and distribution of some important signaling molecules in different subcellular area are critical to maintain normal morphology and physiological activities. Especially the distribution of intracellular pH and Ca2+ are closely related to many life activities. So the real-time and dynamic monitoring of signaling molecules within organelle has important significance for life science and biomedical research.Among the analytical methods, fluorescence analysis has some distinguished advantages, such as high sensitivity, various characteristic parameters, simple operation and other merits to promote the widely used in chemistry analysis and bioimaging. At present, fluoremetry mainly depends on a variety of organic fluorescent probes. However, most of them suffer from some drawbacks such as photobleaching, complex steps of synthesis and cytotoxicity. And how to realize the targeted modification is another problem preventing it’s further application in bioassay.With the continuous development of nanotechnology, fluorescence nanoprobes, severing as a new type of probe, have attracted extensive attention. They are easily synthesized, nontoxic and easy to be modified with unique optical properties. Compared with the traditional fluorescent probes, fluorescence nanoprobes are brighter with better photostability, biocompatibility and dispersibility. Meanwhile, as the preparation of nano materials is further improved, precise control of the scale of nanoparticles and functionalization of the particles method is perfected. These make fluorescent nanoprobes can meet the requirements in the field of chemical sensors and biological probes.Based on the research and development of DNA nanomachines and silicon dioxide nanoparticles, we designed and synthesized several fluorescent nanoprobes. And works are carried out by the following four aspects respectively:(1) Synthesis and application of a pH-sensitive DNA nanomachine. A pH-sensitive DNA nanomachine named t-switch, based on DNA duplex-triplex transition and FRET, is developed for motoring intracellular pH gradient. The results indicate that t-switch is an efficient reporter of pH from pH 5.3 to 6.0 with a fast response of a few seconds. And the uptake of t-switch is speedy. The excellent performance laid a foundation for the real-time dynamic analysis of pH gradient.(2) Synthesis and application of pH-sensitive PEI/DNA complexes. Lysosome targeted PEI/DNA complexes are designed and synthetised based on electrostatic interaction. After complexed with PEI, the dynamic range is extended to pH 4.6-7.8. On the other hand, PEI/DNA complexes are packaged by endocytosis pathway into lysosomes and PEI could protect DNA nanomachine from enzymatic degradation. PEI/DNA complexes are used to monitoring real-time and dynamic pH changes during endocytosis successfully.(3) Synthesis and application of mitochondrial targeted Ca2+ fluorescence nanoprobes(FSiNPs-Peptide). The FSiNPs-Peptide is preared with water-in-oil microemulsion technique, then modified with signal peptide. The results showed that FSiNPs-Peptide has perfect function of mitochondrial targeting.(4) Synthesis of mitochondrial targeted Ca2+ fluorescence nanoprobe(CFNP-M) and endoplasmic reticulum targeted ratiometric Ca2+ fluorescence nanoprobe(CFNP-E) and simultaneous monitoring of Ca2+ concentration changes both in mitochondrial and endoplasmic reticulum. Based on Ca2+ probe(STDBT), CFNP-M and CFNP-E are prepared with water-in oil microemulsion technique and modified with signal peptide. Then CFNP-E and CFNP-M are successfully used in the real-time monitoring of Ca2+ concentration changes both in mitochondria and endoplasmic reticulum. A new method for exploring the role of calcium ion in the maintenance of cell function is provided. |
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