细胞与活体中功能蛋白糖基化与磷酸化位点识别与互作实时原位荧光成像

Cellular or in vivo functional proteins glycosylation and phosphorylation and interactions are closely related with the occurrence and development of the disease. Therefore, it is very important to reveal the interaction between glycosylation and phosphorylation of functional proteins for the diagnosis and treatment of diseases. In this project, nano-fluorescent probes with specific recognition properties for glycoproteins and phosphorylated proteins were prepared by using epitope-based imprinting technique and signal amplification characteristics of fluorescent conjugated polymers. Real time fluorescence imaging of protein glycosylation and phosphorylation in vivo or in vivo was realized by using the spatiotemporal resolution properties of fluorescence spectra. The polyfluorene conjugated polymers are selected as the light-emitting cells, and chosen specific functional units for glycosylation and phosphorylation as recognition sites, and then the polymer fluorescent nanoprobe was prepared by molecular imprinting. This method combines the advantages of epitope-based imprinting technique and conjugate polymer signal amplification, effectively reduces background fluorescence interference, and improves the sensitivity and reliability of detection and imaging methods. The research of this project is expected to achieve the high selectivity and high sensitivity detection and imaging analysis of glycoproteins and phosphorylated proteins in cells or living organisms, and then reveal the interaction between glycosylation and phosphorylation of functional proteins, which has important theory and application value.

细胞或活体中功能蛋白糖基化和磷酸化的互相作用与疾病的发生发展有着密切关系。因此，揭示功能蛋白糖基化与磷酸化的互作对于疾病的诊断及治疗有着非常重要的意义。本项目利用抗原决定基印迹技术，结合荧光共轭聚合物的信号放大特性，制备对糖蛋白、磷酸化蛋白具有特异性识别性能的纳米荧光探针，利用荧光光谱的时空分辨特性，实现细胞或活体中蛋白糖基化与磷酸化识别与互作实时原位荧光成像。项目研究拟选择聚芴类共轭聚合物作为发光单元，选用对糖基化与磷酸化位点有特异性识别功能单元，结合分子印迹技术制备聚合物荧光纳米探针。该方法融合了抗原决定基印迹技术与共轭聚合物的信号放大作用的优点，有效降低背景荧光干扰，提高检测与成像方法的灵敏度与可靠性。本项目研究有望实现细胞或活体中糖蛋白、磷酸化蛋白的高选择性、高灵敏性的检测与成像分析，进而揭示功能蛋白糖基化与磷酸化的互作对相关疾病的发生、发展研究有着重要的理论意义与实际应用价值。

以细胞与活体内蛋白质糖基化与磷酸化位点为研究对象，发展了几种新型的纳米荧光探针，成功的用于细胞与活体内蛋白质糖基化与磷酸化位点识别与互作原位成像分析。采用Zr(IV)为活性中心的金属有机框架（MOFs）材料，利用其荧光特性实现了磷酸化位点的特异性识别。同时在MOFs结构中修饰硼酸基团，利用茜素红调控配体的荧光，通过茜素红与糖基的竞争作用实现了糖基化位点的识别与检测。成功的用于细胞及活体中蛋白质糖基化与磷酸化位点互作研究。由1,8-萘酰亚胺和硼酸酯分别作为荧光团和识别基团组成ROS识别单元，同时利用Zr(IV)与磷酸基团的特异性相互作用，实现炎症模型中磷酸化与ROS位点的原位荧光成像。通过在Zr(IV)为活性中心的MOFs中修饰pH识别基团，构建了对pH和蛋白质磷酸化水平的同时检测与成像新方法，评估小鼠疾病模型进程。总之，该项目发展了几种细胞与活体内蛋白质糖基化与磷酸化检测新方法，为细胞与活体内蛋白质翻译后修饰相关疾病标志物的检测与机理研究提供了适宜的荧光分析工具。