氮杂环类单荧光探针对溶酶体内氧化还原过程的动态监测

The dynamic balance of the oxidizing and reducing substances in the lysosome plays an important role in the maintenance of cell metabolism and normal physiological function. The oxidation and reductive materials of subcellular organelles in real-time, synchronous visualization monitoring usually requires the use of two fluorescent probes, which exists overlap of fluorescent signals, transport and positioning inconsistent difficulties. This project aims to develop with a single fluorescent probe with specific lysosomal targeting, two active sites for hypochlorous acid and reductive hydrogen sulfide, to realize the dynamic monitoring of the lysosomes of the redox process. (1) Screening the specific targeting lysosomal N-heterocyclic molecules, and design with fluorescent groups which can be distinguished on the spectral response of hypochlorite and hydrogen sulfide, on this basis, construct lysosomes target double signal response single fluorescent probe. (2) Investigate the sensitivity and selectivity of the probe for the detection of hypochlorite and hydrogen sulfide. (3) Establish the relationship between the changes of hypochlorite and hydrogen sulfide signal response in the redox process of lysosome and the spectral response, and to further reveal the relationship between the signal changes and cell proliferation, apoptosis and injury. The expected results of this project will provide a powerful means for understanding the biological significance of redox equilibrium at the subcellular level.

溶酶体内氧化和还原性物质的动态平衡对维持细胞代谢及正常生理功能具有重要作用。目前对亚细胞器中氧化和还原性物质的实时、同步可视化监测通常需要使用两种荧光探针，而双荧光探针存在信号交叉、细胞输运与定位不一致等困难。本项目拟开发具有溶酶体特异性靶向，并对氧化性次氯酸和还原性硫化氢产生荧光“点亮”响应的双活性单荧光探针，实现对溶酶体内氧化还原过程的动态监测。（1）筛选特异性靶向溶酶体的氮杂环类结构，同时设计对次氯酸和硫化氢具有可区分的光谱响应型荧光基团，在此基础上，合成靶向溶酶体的双重信号响应单荧光探针。（2）探讨该探针对次氯酸与硫化氢检测的灵敏性与选择性，实现基于荧光共振能量转移的双通道荧光同步分析。（3）建立溶酶体内氧化还原过程中次氯酸和硫化氢变化与光谱响应的关系，并进一步揭示其信号变化与细胞增殖、凋亡及损伤之间的内在联系。本项目预期结果将为理解亚细胞水平氧化还原平衡的生物学意义提供有力手段。

针对目前同时使用双荧光探针存在信号交叉、细胞输运与定位不一致等困难，本项目发展了一系列单荧光探针，筛选了靶向溶酶体的氮杂环类荧光探针，完成了对细胞内氧化还原性小分子及酶活性的检测方面的研究工作，探索了探针的结构与目标分子的响应性能之间的关系，建立了双通道荧光同步分析方法，实现了对溶酶体长期标记、细胞内不同区域中的还原性小分子硫化氢（溶酶体、细胞质内和细胞核）、以及细胞质中脂肪颗粒和还原性小分子二氧化硫及其衍生物的快速检测和动态示踪，为理解亚细胞水平氧化还原平衡的生物学意义提供有力手段。同时还开发了一系列特异性靶向细胞质中端粒酶活性及端粒酶RNA的单荧光探针，可以有效区分肿瘤细胞和正常细胞。相关研究成果在高影响力的ACS nano，Nanoscale，Sensors and Actuators B: Chemical，ACS sensors, ACS Applied Bio Materials等国际期刊杂志上发表SCI论文8篇，申请国家发明专利6项，授权1项。