具有识别功能荧光聚合物ATP比率探针的设计及在体外磷酸化过程动态检测中的应用

Among biological anions, nucleoside polyphosphates such as ATP play pivotal roles in various cellular events, both as the ubiquitous energy currency in all living organisms and as activated substrates in many enzymatic processes. Therefore, selective detection and quantification of these polyphosphate species under biological conditions would help elucidate their roles and related physiological events. In recent decades, a number of fluorescent probes for nucleoside polyphosphates have been reported. However, despite significant effort, there are few probes capable of ratiometric detection of nucleoside polyphosphates. To develop novel ratiometric fluorescent probe for ATP, this project will focus our efforts on three aspects as follows: ① Introduction of the characteristic structure model of "Fluorophore-Spacer- Receptor unit" in fluorescent probes for ATP based on organic small molecules into the polymer backbone as the repeating unit of the polymer chain to obtain novel fluorescent polymer with highly selective recognition ability for ATP; ② The receptor unit in polymer consists of two recognition sites, namely, using boronic acid and zinc (II) complex as the binding motif, which would help greatly improve sensitivity and selectivity of fluorescent polymer probe for ATP by precisely controlling the distance between the recognition sites; ③ Novel sensing mechanism will be exploited to realize ratiometric detection of ATP by using different fluorescence signals. The binding of analytes will induce changes in the polymer backbone conformation, leading to either preferentially folding to, or unfolding from, fluorophore dimers. Hence, the binding event is translated into a shift in the unimer to dimmer equilibrium of the fluorophore in polymer main chain that can be conveniently visualized by UV/vis and fluorescence spectroscopy and thus be used for nucleotides sensing. This project has important scientific significance in the field of novel functional polymers, biochemistry and clinical applications.

ATP是一种具有重要生物学意义的阴离子，在生理条件下选择性地检测和定量ATP将有助于深入理解其在生命过程中的重要作用。本项目拟以设计合成对ATP具有选择性识别功能的荧光聚合物为出发点，以获得高灵敏度高选择性的ATP比率荧光探针为目标：① 将有机小分子ATP荧光探针经典的"荧光基团-连接基团-受体单元"的结构模式引入到聚合物中，构成聚合物链的重复单元；② 采用双识别位点构成受体单元，通过精确调控识别位点之间的距离，提高聚合物对ATP识别的灵敏度和选择性；③ 利用聚合物对ATP选择性识别能力，通过ATP与聚合物的结合来诱导聚合物链构象变化，进而调控聚合物主链上荧光基团之间的距离和取向，使体系具有荧光基团单体及其激基缔合物双重荧光发射能力，利用二者荧光强度比值随ATP含量变化的规律，实现对ATP高灵敏度高选择性的比率检测。该课题具有较强的学科交叉性，在材料、生物化学及临床应用等领域均有重要意义。

ATP是一种具有重要生物学意义的阴离子，在生理条件下高灵敏度高选择性地检测和定量ATP，将有助于深入理解其在生命过程中的重要作用。本项目设计合成了一系列基于共轭聚噻吩、聚苝二酰亚胺、聚双吡啶盐的ATP荧光探针，利用紫外-可见吸收光谱、荧光光谱、圆二色光谱、激光光散射和DFT分子模拟等分析方法和手段，系统研究了探针与ATP的相互作用，探究了探针识别传感的机理；发展了自促进聚集、FRET偶合聚集诱导荧光淬灭等新的传感机制，实现了生理环境下ATP浓度的灵敏定量检测；结合荧光显微成像技术，实现了荧光探针的细胞器定位、活细胞中ATP浓度的原位检测以及活细胞中多种酶活性的间接检测，包括ATP水解酶，肌酸激酶，蛋白激酶等。重要成果如下：设计合成了新型的基于FRET偶合聚集诱导的荧光淬灭（ACQ）机理的ATP比率探针。该探针可用于生理pH条件下ATP的裸眼和荧光双模式检测，以及体外ATP水解酶活性的检测。该成果发表在Polymer Chemistry杂志上（Polym. Chem., 2017, 8, 1138-1145，受编辑邀请作为内封面发表）；针对聚噻吩ATP探针灵敏度低的问题，提出在其侧链上引入蒽基团，通过侧链上蒽基团的-堆积促进聚噻吩主链聚集的策略，大大提高了探针的检测限（低至10-9 M）。该成果发表在Chemical Communication上（Chem. Commun., 2015, 51, 8544-8546, 他引15次）。在基金的资助下，本项目已发表高水平学术论文12篇，培养硕士研究生10名，出站博士后1名，6人次参加2015和2017年的全国高分子学术论文报告会，并提交会议论文2篇，申请发明专利5项，获得授权1项。