迈克尔加成-消除可逆型荧光探针对活细胞内GSH的检测性能研究

Improving the sensitivity and accuracy of the reversible fluorescent probes based on Michael addition-elimination mechanism has important scientific and practical value for revealing the production, transfer and mechanism of GSH and evaluating the intracellular redox status. In-depth study of the relationship between the activity of double bonds and the degree and rate of reversible reaction is the key to improve the sensitivity and accuracy of such probes. This project aims to construct different kinds of reversible fluorescent probes by conjugating diverse electron withdrawing groups based on coumarin and BODIPY as fluorescent precursors. Reveal the inherent rules of reaction between GSH and fluorescent probes which containing strong electron-withdrawing groups such as carbonyl, cyano, and nitro groups by testing the degree and rate of reversible reaction through UV and fluorescence methods and analyzing the probe and addition product through theoretical calculation. Improve the sensitivity and accuracy of such probes through the activity of double bonds based on the regulation of electron-withdrawing substituents. Achieve highly sensitive and accurate visual imaging analysis and real-time quantitative monitoring of intracellular GSH. This project will provide guiding significance in developing new reversible fluorescent probes and in-depth understanding of the pathological mechanism of GSH.

提高基于迈克尔加成-消除机理的可逆型荧光探针的灵敏度与精确度对揭示GSH产生、转移及作用机制和评价细胞内的氧化还原态具有重要的科学意义和实用价值。而深入研究双键的活性与可逆反应程度、速率之间的关系是提高荧光探针灵敏度与精确度的关键。本项目拟以香豆素和氟硼荧为荧光母体，通过共轭双键的方式连接吸电子能力不同的取代基，构筑不同结构的可逆型荧光探针；采用紫外、荧光等方法测试可逆反应程度、速率，结合对探针结构与加成产物的理论计算分析，揭示含有羰基、氰基、硝基等强吸电子基团的荧光探针与GSH反应的内在规律；通过吸电子取代基调控双键的活性，提高荧光探针的灵敏度与精确度；在细胞水平上实现对GSH高灵敏、高精确的可视化成像分析与实时定量检测。该项目的成功实施对开发新型可逆荧光探针及深入了解GSH相关的病理机制具有指导意义。

提高基于迈克尔加成-消除机理的可逆型荧光探针的靶向性、灵敏度与精确度，对揭示癌细胞内谷胱甘肽（GSH）产生、转移及其作用机制和评价细胞内的氧化还原态具有重要的科学意义和实用价值。本项目通过共价键连接香豆素与β-环糊精合成了可逆型超分子荧光探针。利用环糊精的空腔、羟基以及主客体相互作用显著提高了荧光探针对GSH的传感效率，同时增加了荧光探针的水溶性和生物相容性；并利用β-环糊精空腔负载金刚烷修饰的靶向环肽，实现了癌细胞内对GSH高灵敏、高精确的实时动态成像分析。通过共价键连接氟硼荧与β-环糊精合成了对谷胱甘肽巯基自由基（GS•）具有高度传感功能的超分子荧光探针，实现了水溶液中硫醇自由基的精准、高效和快速检测，并成功应用于活细胞内GS•的细胞成像分析。此外，采用超分子组装策略构筑了光捕获平台，通过光捕获能量传递对荧光探针的光学信号进行放大，成功应用于活细胞内亚硫酸盐的高灵敏特异性检测。综上结果，通过系列超分子荧光探针的构筑，对拓宽荧光探针的种类及在癌细胞内深入了解GSH、GS•以及亚硫酸盐等生物小分子相关的病理、生理事件具有重要的科学意义。