定位线粒体或溶酶体的双态型比率荧光探针的研究

Ratiometric fluorescent probe has captured great attention and plays an important role in quantitative detection in living cells. However, most of ratiometric probes have some shortcomings such as the severe overlapping emission spectra and small change in ratio. More importantly, they can't realize the location detection for the guest molecule in a certain organelle due to weak organell-specificity. The key to solve these problems is to design both organell-targetable and ratiometric fluorescent probes. In this proposal, Severial mitochondrion/lysosome-targetable and ratiometric probes were designed for detecting zinic ion, glutathione and cathepsin C activity, respectively, which is based on the modulation of seminaphthorhodamine fluorophore with characteristic two-state emission spectra. In order to assess their practical application, these fluorescent probes will be further applied in a real-time tracing and quantitative detection of the target molecules in living cells and biological tissues. A new method for designing organell-targetable and ratiometric fluorescent probes is bound to have a major impact in the field of quantitative and targetable detection, and display a promising application.

比率型荧光探针因在定量检测方面发挥着重要作用，而备受关注。目前，绝大多数比率型荧光探针不具有细胞器靶向性，并且其荧光光谱交盖严重、比率值变化范围小，无法满足对细胞器内特定客体分子进行定量检测，解决这一问题的关键在于设计兼具细胞器定位和定量检测功能的比率型荧光探针。本项目中提出利用半萘罗丹明双态所具有的特征双发射，通过双态调控设计比率型荧光探针；整合线粒体或溶酶体靶向性基团和半萘罗丹明比率型探针为一体，设计定位于线粒体或溶酶体中，并检测其中锌离子、谷胱甘肽和蛋白酶C的双态型比率荧光探针。本项目将应用这些荧光探针于活细胞及生物组织中对目标分子进行定位追踪、定量检测，并评估探针的应用价值。本项目为开发兼具细胞器靶向性和比率检测的荧光探针提供了新的设计方法，必将在定量与定位检测领域产生重要影响，并显示良好的应用前景。

细胞器靶向性比率型荧光探针在定量检测和定位检测方面发挥着重要作用，近年来备受关注。本项目按照计划书中的内容和目标稳步开展相应研究工作并取得预期结果：首先，研究了取代基对半萘罗丹明的细胞器靶向性定位的影响以及在细胞膜渗透性、细胞器内保留性等方面的影响，为后续半萘罗丹明的靶向性修饰提供参考，也为设计不同类型细胞器靶向性比率型荧光探针提供新的设计思路。其次，基于半萘罗丹明的双态调控机理，设计了线粒体靶向性硫化氢比色型荧光探针NRS，实现对细胞线粒体内的硫化氢高灵敏度和实时的检测，并且NRS表现出较高的硫化氢选择性，不受生物体内的其他离子和分子的干扰。探针NRS是可定位于线粒体中并检测外源性硫化氢的增强型荧光探针，其必将在基础医学和生命科学领域具有广阔的应用前景。再次，利用氢键对pKa的调控机理，设计了基于罗丹明螺内酰胺的pH荧光探针Rh-Met，该探针在水溶液中的pKa为6.81，是目前已知的罗丹明螺内酰胺探针中pKa最大的。该探针能够同时指示线粒体与脂滴内pH的变化，并且有可能成为揭示线粒体与脂滴生理功能间关系的重要指示剂，其在基础医学和生物学领域将发挥重要作用。最后，依据双态调控机理设计了基于荧光素的双态比率型荧光探针FDI，该探针是将荧光素母体与吲哚阳离子通过乙烯基团进行连接，极大的拓宽了荧光素的光谱范围（360-700nm），为比率检测和线粒体靶向性提供了有利条件。FDI表现出较好的细胞膜通透性和线粒体靶向性，并能够对氯喹刺激下线粒体内pH的变化进行实时监测。探针FDI必定会在线粒体pH实时监测领域发挥重要作用。