具有荧光增强特性的磁性微球对循环肿瘤DNA高灵敏检测

The levels of circulating tumor DNA (ctDNA) in the peripheral blood have been associated with tumor burden and malignant progression. Therefore, the analysis of trace ctDNA plays a vital role in cancer early diagnosis and treatment. Currently, the reliable methods for ctDNA sensing are DNA sequencing and PCR, but they are costly and time-consuming. In this project, a liquid chip for rapid detection of ctDNA with high sensitivity will be explored by combination of plasmonic fluorescence enhancement technology and fluorescence amplification technology of hybridization chain reaction (HCR). In order to realize this proposal, the following three parts of research will be carried out orderly. (1) By precise controlling the noble metal nanostructure on the surface of magnetic microbeads, plasmonic magnetic microbeads with the property of fluorescence enhancement to specific dye will be prepared. (2) By optimizing the spatial distribution of HCR on the surface of plasmonic magnetic microbeads, make sure that the fluorescence enhancement effect on the surface of plasmonic magnetic microbeads is compatible with fluorescence amplification of HCR. (3) By designing relevant DNA molecules and optimizing detection condition, a dual-fluorescence amplification method based on the combination of plasmonic magnetic microbeads and HCR will be developed for ctDNA rapid analysis with high sensitivity. The obtained results in this project will provide technical support for the development of high-sensitive liquid biochip.

循环肿瘤DNA（ctDNA）在外周血液中的含量水平直接反映患者体内肿瘤负荷和恶性进展程度，因此ctDNA的痕量分析对肿瘤的早期诊断和治疗具有极其重要的意义。ctDNA分析方法主要是DNA测序和PCR技术，但这些方法分析周期长，费用昂贵。本项目拟探索将等离子荧光增强技术与杂交链式反应（HCR）荧光放大技术相结合，发展针对ctDNA高灵敏快速分析检测的液态芯片。具体研究内容包括：（1）精确调控磁性微球表面贵金属纳米岛膜的微观结构，合成对特定荧光染料具有荧光增强特性的等离子磁性微球；（2）优化等离子磁性微球表面HCR的空间分布，实现贵金属纳米表面荧光增强效应与HCR荧光放大效应之间的兼容性设计；（3）相关DNA分子的设计和检测条件的优化，发展基于等离子磁性微球和HCR的荧光双重放大特性的检测原理和方法，实现对ctDNA的高灵敏快速检测。本项目的预期成果有望为高灵敏生物液态芯片的开发提供技术基础。

等离子荧光增强技术与固态芯片技术的耦合在高灵敏生物分析领域展现出了巨大的应用前景。然而，固态芯片技术面临反应动力学低，高通量检测程度化不高等缺陷，无法满足现实中对多种目标物的高通量同时分析检测需求。本项目拟在不改变传统液态芯片技术操作流程的情况下，制备具有等离子荧光增强特性的磁性微球，实现对传统有机荧光信号分子荧光的极大增强，从而实现生物分子的高灵敏分析检测。具体研究内容如下：（1）探索在微球表面构建对有机荧光染料具有荧光增强效应的贵金属纳米岛膜，并实现其表面的生物功能化修饰。（2）发展基于等离子荧光增强技术的液态微球对肿瘤标志物的高灵敏检测。（3）制备多尺寸的具有荧光增强效应的磁性微球，实现对多种肿瘤标志物的高通量同时检测。本项目的预期成果将为肿瘤标志物的高通量高灵敏检测提供科学依据和技术途径。