循环肿瘤源性外泌体高灵敏光学传感探针构建与应用研究

The tumor-derived or tumor-associated exosome is an important mechanism to control the development of tumor, tumor metastasis, immune response inhibition, angiogenesis and other processes. Analysis and detection of tumor exosome may assist the early diagnosis, curative effect evaluation and prognosis of tumor. In this project, with the analysis of the circulating tumor exosome in the body fluid, the accurate and rapid extraction and purification techniques of the exosome will be developed. By combining the technologies of biological nanotechnology, molecular engineering and single molecular imaging, a series of highly sensitive sensing probes with different signal acquisition modes will be constructed based on nucleic acid signal amplification technology and nanoparticle signal enhancement effect. Moreover, the technology platform for the highly sensitive detection and single molecular imaging of circulating tumor exosome and its specific components in blood samples from the clinical patients will be developed. On this basis, the in vivo tumor diagnosis and efficacy monitoring will be performed. This study holds great promise to provide new methods and technologies for the early and accurately clinical diagnosis of tumor. And this project is also an innovative subject that is expected to promote the development of modern life analysis science and clinical biomedicine in the cross field of molecular engineering and biological nanotechnology.

肿瘤来源或肿瘤相关外泌体是调控肿瘤发生发展、肿瘤转移、免疫反应抑制、血管生成等过程的重要机制，对肿瘤外泌体的分析和检测可以辅助肿瘤的早期诊断、疗效评估和预后分析。本项目拟以体液中循环肿瘤外泌体为分析检测研究对象，开发外泌体的准确、快速分离提取及纯化技术，并结合生物纳米技术、分子工程技术原理以及单分子成像技术等，构建一系列基于核酸信号放大技术和纳米颗粒信号增强效应的具有不同信号获取模式的高灵敏光学传感探针，发展临床病人血液等体液样品中循环肿瘤外泌体及其特异成分的高灵敏分析检测和单分子成像技术平台，并在此基础上，开展活体肿瘤诊断和疗效监测研究，为实现临床肿瘤早期精确诊断提供新方法和新技术。这将是一项在分子工程技术和生物纳米技术等前沿交叉领域推动生命分析科学和临床生物医学发展的创新性课题。

本项目从肿瘤早期诊断出发，以循环肿瘤外泌体为研究对象，结合生物纳米技术、分子工程设计原理以及单分子成像分析技术，设计了多种识别信号发生单元、信号增强机制和信号获取方式，构建了一系列基于核酸信号放大技术和纳米颗粒信号增强作用的肿瘤外泌体光学传感探针，实现了血液等体液中循环肿瘤外泌体及其特异成分的高灵敏放大检测和单颗粒荧光成像分析，为肿瘤早期诊断与治疗监测提供了新原理与新方法。主要成果包括：（1）发展了基于四氧化三铁磁性纳米颗粒和微流控技术的肿瘤外泌体快速分离与提取技术；（2）发展了多种性能优良的肿瘤外泌体miRNAs高灵敏检测探针，如基于DNAzyme的级联催化放大检测方法，基于杂交链置换反应的无酶信号放大检测方法，基于Y字形探针的肿瘤外泌体miRNAs多目标检测分析等；（3）发展了基于核酸适配体靶向识别的肿瘤外泌体膜蛋白高灵敏检测探针，如基于核酸适配体-肿瘤外泌体特异性识别的三维多足DNA步行器，基于激活式核酸适配体和滚环放大技术的外泌体放大检测探针，基于核酸适配体-肿瘤外泌体-抗体三明治杂交原理的外泌体检测探针；（4）发展了基于单颗粒成像分析技术的肿瘤外泌体特异性miRNAs和膜蛋白定量与化学计量分析平台。通过该项目，发表SCI学术论文24篇，授权发明专利1项，培养博士/硕士研究生10名。