层层解装：等离子体纳米探针用于从细胞表面到细胞质内肿瘤相关生物分子的逐层解析

Different types of tumor-related biomolecules are distributed at various parts of cells. Some of them locate on the membrane, and some of them exist in the cytoplasm. However, most of the reported detection methods focus on the analysis of a certain type of tumor-related molecules, thus fail to obtain the multilayer information from cell surface to cytoplasm. Therefore, this project proposes a composite plasmonic nanoprobe for the layer-by-layer analysis of tumor-related biomolecules from cell surface glycans to intracellular tumor markers. Gold nanoparticles (AuNPs) are used as the substrates, and a glycan-specific fluorescent molecule and a molecular beacon (MB) for specific recognition of intracellular target molecules are functionalized on the AuNP surface. The obtained nanoprobes enter cells through the interactions with cell surface glycans with the glycan-specific fluorescent molecules leaving from the probe surface and binding with the target glycans on cell membrane. After the probes enter into the cytoplasm, the MB can specifically recognize the target tumor-related molecule in cytoplasm and depart from the probe surface to produce fluorescent signals. At the same time, the changes in color and scattering spectra of AuNPs can be observed under a dark-field microscope, which are used for the localization of the intracellular probes and the monitoring of MB departure processes on probe surface.

不同类型的肿瘤相关生物分子分布于细胞的不同部位，有些分布在细胞膜上，有些分布在细胞质内。然而，大部分已有的检测方法主要集中于检测单一种类的肿瘤相关分子，无法得到从细胞表面到细胞质内多层次的相关信息。因此，本项目提出一种可层层解析细胞表面肿瘤相关聚糖以及细胞质内肿瘤相关生物分子的复合等离子体纳米探针。以金纳米粒子为基底，在其表面功能化可特异性识别细胞膜糖基的荧光分子，同时将荧光标记的可靶向识别细胞内肿瘤相关的分子信标修饰在纳米粒子表面。探针通过与细胞表面糖基末端的相互作用进入细胞，其表面的糖基识别荧光分子解离并与细胞膜糖基末端结合，标记在细胞表面。探针进入细胞后，其表面修饰的分子信标可特异性结合细胞质内目标分子，脱离金纳米粒子表面，产生荧光信号。同时在暗场显微镜下，通过观察金纳米颗粒颜色及散射光谱的变化，可定位监测探针进入细胞后的分布情况以及探针表面分子信标反应过程。

本项目按照计划内容顺利进行，完成了预期研究成果。3年来，在本项目资助下，申请人围绕构建等离子体纳米探针的多通道检测体系，对多种肿瘤相关分子进行了成像分析并研究其信号通路，取得系列创新性成果。通过设计多功能复合纳米探针，对细胞不同位点肿瘤相关分子进行了成像分析；此外还利用电化学分析对细胞功能分子进行了超灵敏检测与修饰，并实现了细胞膜张力的原位检测。本项目共发表一作/通讯SCI论文11篇，申请发明专利3项，授权1项。参加学术会议10次并作报告5次。