亚细胞靶向的比例计量型双光子荧光探针

In the proposed project, subcellular (mitochondrial, lysosomal) targeted groups will be introduced into two-photon fuorescent probes. Based on fluorescence resonance energy transfer (FRET) and intramolecular charge transfer (ICT) mechanism,the merits of the two-photon fluorescence(deep penetration of the excitation light into the biological tissues, high spatial resolution) and the merits of ratiometric detection(small environmental interference, high sensitivity) are combined to design the subcellular targeted ratiometric two-photon fluorescent probes. The efficient C-C, C-N coupling reactions and "Click" reaction will be used to quickly build flurorescent probe library based on coumarin and rhodamine fluorophores. Fluorescence, ultrafast laser spectroscopy, laser confocal imaging technology will be used to systemically evaluate the properties of the new prboes,including optical property, cytotoxicity and subcellular targeted detection performance. The electron transfer and energy transfer mechanism between the bulding blocks of probe molecules and the structure-properity relationship of the probes will be explored. According to these results, the structure of the probe will be further optimized.The aim of the project is to develop subcellular targeted two-photon fluorescent probes with large two-photon absorption cross section, and use this probes to achieve the real-time subcelluar detection of bioactive substances (such as ions, small signal molecules, etc.) with high selectivity and sensitivity.

本项目拟将亚细胞（线粒体、溶酶体）靶向基团引入到双光子荧光探针分子的设计中，将双光子荧光的激发光生物组织穿透力强、空间分辨率高和比例计量检测的环境干扰小、灵敏度高的优点结合起来，根据荧光共振能量转移（FRET）、分子内电荷转移（ICT）机理设计具有亚细胞靶向功能的比例计量型双光子荧光探针。利用高效C-C、C-N偶联反应以及"Click"反应，快速构建基于香豆素、罗丹明荧光团的探针分子库。利用荧光、超快激光光谱，激光共聚焦成像等技术，系统比较各类新合成的探针分子的光学性质、亚细胞靶向检测性能，探讨探针分子各构建基元之间的电子转移、能量传递机制，理解探针分子的构效关系。并根据这些构效关系进一步优化探针分子设计，开发出具有亚细胞靶向功能、大双光子吸收截面比例计量型双光子荧光探针分子，实现在亚细胞体中对生物活性物质（如离子，信号小分子等）的高选择性、高灵敏度实时原位检测。

项目将线粒体（三苯磷、吡啶盐）、溶酶体（吗啉）的靶向基团引入到双光子荧光探针分子的设计中，构建了一系列基于香豆素、罗丹明荧光团的双光子荧光探针具有线粒体、溶酶体定位作用的双光子荧光探针，利用双光子的激发光生物组织穿透力强、空间分辨率高和比例计量检测的环境干扰小、灵敏度高的优点为相关科学研究提供了可靠的实时成像工具。 在此基础上，项目组利用构建了可以实时监测溶酶体、线粒体内微环境（极性、粘度）变化的双光子荧光探针，项目组通过利用这些探针对细胞自噬过程中溶酶体、线粒体内微环境（极性、粘度）变化的实时监测，结合细胞自噬过程中溶酶体、线粒体内微环境会发生变化这一特点，成功发展出一种全新的细胞自噬的监测方法，克服了传统细胞自噬检测方法（电镜观测、western-blot法）不能对活细胞自噬过程实时原位检测的缺点。截止目前，项目组已在包括J.Am.Soc.Chem., Chem.Commun., Biosensors and Bioelectronics, Dalton Trans. J.Org.Chem. J. Mater. Chem. B等学术期刊发表SCI收录论文26篇，申请国家发明专利8项，其中授权发明专利5项。培养博士1名，硕士10名。