基于银离子嵌入型四面体和比率型分子机器的肿瘤外泌体电化学传感检测技术研究

The tumors are highly heterogeneous in space, continuously clone and develop under selective pressure, and evolve over time. Noninvasive detection of tumors and longitudinal monitoring of clonal evolution are critical for precise diagnosis and treatment of tumors. However, traditional tissue biopsy can not meet the medical requirement, and thus it is urgent to explore new detection technologies. The emerging liquid biopsy can achieve minimally invasive detection and dynamic monitoring of tumors by continuous sampling and analysis of tumor cells and circulating tumor components, especially rich and stable tumor exosomes. However, exosome detection is currently facing the bottleneck of lacking effective analytical techniques. Electrochemical sensing platform has advantages such as being simple and sensitive, having a broad application prospect in the detection of exosomes. Based on the intersection of disciplines, the emerging DNA nanotechnologies provide nanoscale tools for the construction of electrochemical sensing interfaces. Our previous study found that the insertion of silver ions into the DNA tetrahedron can significantly improve the electron transportability, and it is expected to explore the sensing interface with superior controllability and electron transportability. Meanwhile, DNA molecular machines and aptamers provide new ideas for the construction of sensitive and specific detection systems. Therefore, this project intends to introduce nucleic acid aptamers and DNA nanotechnology into the sensing system, and explore novel techniques for electrochemical sensing detection of tumor exosomes based on silver ion-embedded tetrahedron and ratiometric molecular machines, thereby providing new methods for the diagnosis and monitoring of tumor.

肿瘤在空间上高度异质，并因选择性压力不断克隆进化，随时间发生演变。肿瘤的无创检测和克隆进化的纵向监测对肿瘤精准诊疗至关重要，但传统组织活检不能有效实现，亟待探索新技术。新兴的液体活检通过对肿瘤细胞和循环肿瘤成分的连续取样和分析可实现肿瘤微创检测和动态监测，尤其是丰富且稳定的肿瘤外泌体。然而外泌体检测目前尚面临有效分析技术缺乏的瓶颈。电化学传感平台具有简便灵敏等优势，在外泌体检测中具有广阔应用前景。基于学科交叉，新兴的DNA纳米技术为电化学传感界面的构建提供了纳米级工具。我们前期研究发现银离子嵌入至DNA四面体可显著提高电子传递性，有望探索可控性和电子传递性俱佳的传感界面。同时，DNA分子机器和核酸适体为灵敏特异检测体系的构建提供新思路。由此，本项目拟引入核酸适体，交叉DNA纳米技术，基于银离子嵌入型四面体和比率型分子机器探究肿瘤外泌体电化学传感检测新技术，从而为肿瘤诊断和监测提供新方法。

外泌体含有所来源细胞的蛋白和核酸等信息分子，外泌体检测具有无创、接受度高和可连续采样和实时监测肿瘤等疾病演变的优点。然而，外泌体检测尚面临有效分析技术缺乏的瓶颈。项目围绕外泌体检测的研究目标，交叉引入DNA纳米技术和适体识别技术，在侧流层析试纸条平台、荧光检测平台和电化学传感平台展开研究，探索简便、快速、灵敏、特异的检测新技术，从而为肿瘤等疾病的诊断和监测提供新方法。在试纸条平台探究并建立了基于DNA四面体传感界面的生物传感技术，从提高杂交效率和信号响应性的机理上，并结合比率计原理，探索开发了分析性能佳的外泌体和外泌体microRNA传感检测试纸条。在制备试纸条核酸捕获探针常规方法中，核酸探针须与蛋白质分子连接形成大分子复合物，以避免自身随样品流动。该常规制备方法受限于蛋白分子，设计复杂，限制了核酸探针试纸条的拓展应用。项目提出了利用DNA四面体纳米结构捕获探针构建试纸条传感检测界面的新方法，解决了核酸探针制备受限于其所连接蛋白分子的技术难题。侧流层析试纸条为未经培训人员最方便的生物传感平台之一，DNA纳米结构捕获探针传感界面属于一种通用方法，可应用于核酸标志物检测，进一步引入核酸适体等策略可应用于细胞、外泌体、蛋白和小分子等非核酸标志物检测，具有优良的现场分析或床旁检测应用前景。在荧光平台，建立了单足DNA分子机器，从提高生物识别、空间局域反应效率的策略上，探索开发了灵敏、特异的外泌体检测新方法。在电化学传感平台上，建立了基于多足DNA分子机器的microRNA传感检测新技术，建立了基于CRISPER/Cas介导双链探针特异捕获四面体信号放大标签的外泌体microRNA电化学检测新技术。电化学传感界面上的双链捕获探针避免了单链捕获探针间或单链探针内的非特异杂交，且双链捕获探针具有刚性，有利于解决单链探针倒伏于传感界面的问题，从而提高了检测分析性能。