聚集/单体型同分子双靶标荧光探针：原位同时分辨活细胞质膜中两种超微结构

Recent researches show that identical molecule/two targets fluorescent probes for imaging cells are new and important biological fluorescent probes. The fluorescent probes that can in-situ and simultaneously and discriminatingly image raft and non-raft in plasma membrane are in strong demand today. According to our theoretical analysis, the intrinsic difference between raft and non-raft lies in their lipid packing state: the lipids in raft are highly packed and ones in non-raft are loosely packed. On the other hand, some organic compounds exhibit different emission colors in the aggregation and monomer states, respectively. Based on the two facts, applicant firstly proposed a new idea to develop an aggregation/monomer identical molecule/two targets fluorescent probe for imaging raft/non-raft with dual colors. As a result, in-situ and simultaneously and discriminatingly imaging both two supramicrostructures of plasma membrane in living cells has been realized by two colors in spectral laser scanning confocal microscopes confocal microscopes, and the in-situ fluorescent spectra of the probe have been recorded.. In this project, we would systematically develop the new kind of probes mentioned above: study the aggregation states of probes in various environment as well as their monomer states in different conditions, compare the absorption and emission spectra of probes in aggregation and monomer states, test the aggregation patterns of probes, particularly, measure the in-situ fluorescent spectra of probes in intracellular raft and non-raft simultaneously in spectral laser scanning confocal microscopes, and demonstrate interaction modes between probe molecules and two kinds of supramicrostructures. We would study the relationship between the chemical structure of probes and imaging ability and break the bottleneck of probes to visualize raft phases and non-raft domains in plasma membrane. And the guiding researches based on the new probes for intracellular aggregation of lipid rafts from stress response will be performed also.

同分子双靶标荧光探针是新型、重要的生物荧光探针。目前生物学界对在活细胞质膜内原位、同时、区别成像脂筏与非脂筏两种超微结构的同分子双靶标荧光探针有强烈需求。利用脂筏结构中磷脂堆砌紧密、非脂筏结构中磷脂堆砌松散的本征区别，结合在聚集态和单体态发光颜色不同的有机荧光分子，申请人首次开发了一种聚集/单体型同分子双靶标荧光探针，并利用光谱型共焦显微镜实现了活细胞质膜内脂筏/非脂筏的原位、同时、区别成像，获取了探针在脂筏/非脂筏的原位荧光光谱。.本项目将系统研究上述新型探针，研究其在各种条件下的聚集状态以及在各种分散系中的单体状态、比较二种状态光学性质、解析聚集态的分子堆砌方式，特别是利用探针在活细胞脂筏与非脂筏中的原位荧光光谱，分析探针分子与两种超微结构的相互作用原理。研究探针分子结构与成像性能的构效关系，突破新型探针的研究瓶颈，并利用新型探针对细胞应激响应诱导的脂筏聚集行为开展先导性研究。

细胞器在细胞的多种生物学过程中起着不可或缺的作用，它们之间既有精细分工，又相互协作，形成细胞器互作网络，实现物质交换和信息交流，维持细胞正常生理功能。为研究两个细胞器间的相互作用，同分子双靶标荧光探针成为新型、重要的生物荧光探针。本项目在执行过程中研制了以下新型荧光探针：.1.成像活细胞质膜上脂筏/非脂筏微区的探针。结合转子效应和分子内电荷转移原理，以转子效应提高保真度，以分子内电荷转移实现对细胞质膜微区的选择性，设计合成了可以高保真区分成像细胞质膜脂筏/非脂筏微区的荧光探针DSPI-18。在人工囊泡的实彩成像中，DSPI-18在脂筏囊泡（DPPC/CL）呈现绿色荧光，在非脂筏囊泡（DOPC）呈现红色荧光。在细胞的实彩成像中，我们发现DSPI-18在SiHa细胞的细胞膜上呈现清晰的绿色和橘红色的荧光，以两种荧光颜色区分成像了脂筏/非脂筏微区。.2.从细胞器间的微环境差异入手，设计了系列单分子双靶标探针：①利用脂滴和内质网含水量的不同，合成了探针PPC、EPC和MNC。其中PPC和EPC对含水量的微小变化极敏感，首次在活样本中实现用单一探针双色成像脂滴和内质网，并首次发现新生脂滴与内质网具有相似的荧光颜色和原位光谱，由此推断脂滴极可能源于内质网。②利用细胞核和线粒体的pH差异，将pH敏感的羟基化半花菁与易结合DNA的苯并噻唑盐结合，合成探针HMBI。HMBI在细胞核和线粒体中分别发绿色和红色荧光。③利用脂滴与溶酶体的pH差异，设计了探针CMHCH。基于环化-开环反应，染色溶酶体时探针在酸性条件下呈开环状态发红色荧光；染色脂滴时探针在中性或碱性下呈关环状态发绿色荧光。该系列探针为研究细胞器间的相互作用提供了便捷工具。.3.在同一荧光骨架分子上修饰不同长度的烷基链，能够大幅度改进探针的孵育浓度和染色速度；进而发现具有长烷基链的分子不仅可以成像细胞膜，也可以高选择性地成像细胞内线粒体等膜性结构，从而首次发现了带有长烷基链的非反应型线粒体追踪探针。

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