原位检测肿瘤细胞外微环境相关生物标志物是肿瘤早期诊断和转移风险评估的前提与基础。针对目前缺乏简便、安全、可靠的细胞外微环境原位检测分析方法的现状，本项目拟以多个疏水性胆固醇分子共同修饰的DNA多面体为细胞膜锚定单元，结合比率荧光探针的高信噪比以及核酸适配体、i-motif DNA和多肽等功能生物探针的特异性分子识别性能，设计并构建基于细胞膜表面的高性能DNA多面体比率荧光传感体系。项目研究拟重点采用共聚焦激光扫描显微镜和流式细胞仪双重技术考察探针在膜上的锚定效率和稳定性，并将其应用于人急性淋巴细胞白血病T淋巴细胞（CCRF-CEM）和人类乳腺癌细胞（MCF-7）外微环境标志物ATP、pH以及MMP-2、MMP-7的原位荧光成像和定量分析。通过本项目研究，可望为相关肿瘤在细胞水平上的早期诊断和转移风险评估提供理论指导并为细胞外微环境原位成像方法的构建提供新思路和新技术。

In situ detection of the tumor extracellular microenvironment（TEM）biomarkers is tremendously important for cancer early diagnosis and metastasis risk assessment. Despite the significance of these TEM biomarkers, unfortunately, their investigation has lagged far behind due to the lack of a desirable analysis technique. In this project, combing the high signal-to-noise ratio of the ratiometric fluorescent probes and the efficient cell membrane anchoring ability of the amphiphilic DNA polyhedron, we propose a simple, safety and reliable strategy for construction of cell membrane anchored polyhedral DNA ratiometric fluorescent probes, in which the DNA aptamer, i-motif DNA and peptide are applied as target recognition elements. The corresponding cell membrane anchoring efficiency and stability of the polyhedral DNA ratiometric fluorescent probes will be investigated by using the confocal laser scanning microscope and flow cytometry, respectively. More importantly, the probes will be applied to in situ imaging and quantitative analysis of ATP, pH in the extracellular microenvironment of human acute lymphatic leukemia cells (CCRF-CEM) and MMP-2 and MMP-7 in the extracellular microenvironment of human breast cancer cells (MCF-7). After this project, it is expected to provide theoretical guidance for early diagnosis and metastatic risk assessment of related tumors at the cellular level and provide novel ideas and techniques for the construction of TEM in situ imaging methods.