硫醇时间分辨比率荧光探针的设计、合成及其应用于细胞成像研究

Fluorescent probe for real-time and quantitative detection of cellular thiols has become a hot topic of research. The reported fluorescent probes can eliminate the interference of fluorescence artifact by the use of a ratiometric fluorescence approach. However, the probes still suffer from the severe interferences of background fluorescence and light scattering, which hampers the real-time and quantitative detection of cellular thiols. To solve this problem, based on our previous research on ratiometric fluorescent probe for thiols, we will design and develop time-resolved ratiometric fluorescent probe by means of modulating the luminescence resonance energy transfer (LRET) efficiency between the luminescent lanthanide complexes and organic fluorescence dye. As a result, taking advantage of time-resolved ratiometric fluorescence imaging technique, the novel fluorescent probe will deeply suppress the interferences of fluorescence artifact, background fluorescence and light scattering simultaneously. Thus, the sensitivity of the probe will enhance significantly, contributing to achieving real-time and quantitative detection of cellular thiols. Meanwhile, through the study of time-resolved ratiometric fluorescent probe for thiols, we will further develop the method of designing time-resolved ratiometric fluorescent probes based on the principle of LRET, which provides theoretical and empirical guidance for the development of time-resolved ratiometric fluorescent probes for other biological analytes.

荧光探针实时定量识别细胞内硫醇已成为当前的一个热点研究。已报道的硫醇荧光探针在实际应用过程中，尽管能够通过比率荧光技术消除荧光假象的影响，但仍然受到背景荧光和光散射的严重干扰，极大地影响了细胞内硫醇的实时定量识别。本项目在申请人前期开发的硫醇比率荧光探针的研究基础上，将首次采用调节稀土发光配合物与有机荧光染料之间发光共振能量转移（LRET）效率的方法，设计、开发出硫醇的时间分辨比率荧光探针。该新型的荧光探针利用时间分辨比率荧光成像技术，从根本上同时消除荧光假象、背景荧光以及光散射的干扰，大大提高了荧光探针的灵敏度，有助于实现实时定量识别细胞内硫醇。同时，通过对硫醇时间分辨比率荧光探针的研究，进一步发展出基于LRET原理设计时间分辨比率荧光探针的方法，为开发出其它生物分子的时间分辨比率荧光探针提供理论和经验指导。

本项目集中于高选择性、高灵敏度荧光探针的开发及相关理论研究，已进行了硫醇时间分辨比率荧光探针的制备和测试工作。同时，在低背景荧光探针的研究、新型荧光信号机理的构建及其应用于开发荧光探针的研究、以及比率型荧光探针的研究等几个主要相关研究方向上取得了一系列研究成果。已完成的研究成果如下：（1）通过调控探针分子内电荷转移方向，开发出具有极低背景荧光的硫醇荧光探针；首次基于多荧光淬灭基团的协同作用，开发出具有零背景荧光的Cu2+荧光探针；首次利用氰化物与探针作用后所生成氰基的强吸电子性质，改变探针分子内电荷转移方向，使自身没有荧光的探针分子产生荧光增强响应，开发出氰化物荧光探针。（2）首次合理地证明了乙酰肼基团内N-N单键旋转能够使荧光染料的荧光发生淬灭。调控这种N-N单键的旋转，能够实现荧光染料荧光强度的调节，因此可以作为一种新型荧光信号机理用于开发荧光探针。（3）基于双荧光染料同时响应机理、分子内电荷转移机理分别开发出次氯酸比率荧光探针、酸性pH比率荧光探针、氰化物比率荧光探针以及Fe3+比率荧光探针。通过本项目的资助，培养了7名研究生（毕业4名，在读3名），发表了9篇SCI检索论文（其中IF > 3.0 论文8篇）。