无猝灭剂高对比度荧光探针的设计及其在细胞成像中的应用

Fluorescent techniques are powerful tools for chemo/biosensing due to high sensitivity and specificity. Usually, fluorescent analysis relies on analytes-targeted fluorogens and quencher groups, which commonly face with technical difficulties including probe design, operability and background interfering from biological species, thus imposing limitation in practical applications. Herein, we contemplate to synthesize a series of facile fluorescent molecules with emission wavelength of 550-700 nm such as conjugated fluorene (CF) and conjugated polymers (CP). The synthesized fluorescent molecules would circumvent the requirement of quencher groups, and feature with aggregation-caused quench (ACQ) characteristic. Additionally, by virtue the precise paring and structure-tunable capacity of DNA strands, we thus can construct quencher-free and high-contrast complex fluorescent bioprobes through covalently coupling strategy. We endeavor to regulate the hydrophilic/hydrophobic property and aggregation status of the fluorescence bioprobes to reveal the fluorescence response mechanism, and then carry out trials in simulated cell environment to optimize the performance of the fluorescence bioprobes. This will enable us to establish ultrasensitive, specific and high-efficiency fluorescence methods. Furthermore, the designed bioprobes would be introduced to live cells to achieve accurate, high-contrast, real-time and in-situ fluorescence imaging for biologically-significant species. This project holds promise to solve the currently annoyed problem at fluorescence in vivo analysis, and would provide a means for cell imaging and basic biomedical researches.

荧光技术因灵敏度高、特异性好，在生物/化学传感器领域中广泛应用。通常荧光探针需标记荧光基团和猝灭基团，设计难度大、操作繁琐，加之生物材料自体荧光干扰，限制了其在细胞成像中的应用。本项目拟合成最佳发射波长为550-700 nm的共轭荧光小分子（CF）和共轭聚合物（CP），利用其特有的聚集诱导荧光猝灭特性，结合核酸（DNA）的精准配对能力和结构可调控的性质，将CF和CP分别与DNA共价偶联，形成无猝灭剂、高对比度的荧光探针。通过调控探针的聚集状态和亲疏水性，研究并揭示其荧光响应机制；在模拟的细胞复杂体系中，探索出对生物活性物质高灵敏度、高特异性以及高效的检测方法。进一步地，将该探针引入到活细胞中，实现对细胞内特定的生物相关分子准确、实时和原位监测。该类荧光探针由于能克服背景信号干扰，有望解决当前细胞荧光成像研究的一大技术难题，并将为利用荧光探针开展细胞成像及生物医学基础研究提供新的技术思路。

荧光技术因灵敏度高、特异性好，在生物/化学传感器领域中广泛应用。通常荧光探针需标记荧光基团和猝灭基团，设计难度大、操作繁琐，加之生物材料自体荧光干扰，限制了其在细胞成像中的应用。本项目合成出不同分子量大小、不同发光波段的共轭聚合芴（CF和CP）和聚集诱导发光分子（TPEpy），并分别与DNA通过化学反应，共价键结合，形成无猝灭剂荧光、高对比度探针CF-DNA、CP-DNA和TPEpy-DNA。通过调控无猝灭剂、高对比度荧光探针CF-DNA和CP-DNA的聚集状态，实现缓冲溶液和环境水样(海水和湖水)中对重金属离子Pb2+的高灵敏检测，当将该方法引入细胞体系中时，实现在活细胞体系中对Pb2+实时、在线检测；调控无猝灭剂、高对比度荧光探针TPEpy-DNA的聚集状态和亲疏水性，实现在缓冲溶液体系和食物样本(玉米、牛奶和大米）中黄曲霉毒素AFB1的高效检测，当将该方法引入细胞体系中时，实现在活细胞体系中对AFB1实时、在线检测。本项目的顺利实施，为新型实用生物/化学传感器的构建提供重要的理论和实验基础。