血液中循环游离DNA的电致化学发光分析新方法研究

Because of the relative non-invasive obtaining, simple sample collection and correlating with tumor, circulating DNA in blood had broad application prospect in clinical. For tumor patients, the low level circulating DNA in blood come from the proliferation of necrosis and apoptosis tumor cells and even the release of proliferating tumor cells. The study process on the relationship between circulating DNA in blood and disease was slowly over a long period of time for the lack of high sensitivity and specificity detecting methods..The development of simple, portable and high sensitive electrochemiluminescence (ECL) biosensors have attracted particular interest. The signal amplification strategy based on nanomaterials was one of the research hotspots. This project was based on our previous studies with DNA assembly technology for sensitive detection of cancer biomarkers. To design dual signal amplification strategies, DNA assembly technology and localized surface plasmon resonance of noble metal nanoparticles were both used to enhance the luminescence of quantum dots. Moreover, due to its excellent specificity and good stability to DNA, peptide nucleic acid was applied to capture targets. Combination with ECL detection, a sensitive sensing method for the measurement of circulating DNA in blood was developed. Simple sample collection for the detection of tumor biomarkers realized the early and fast economical diagnosis of diseases, laying the foundation for clinical diagnosis.

由于血液中循环游离DNA的获取相对无创、简便的样本取材方式以及与肿瘤的相关性，使其具有广阔的临床应用前景。对于肿瘤患者，血液中循环游离DNA来自肿瘤细胞坏死凋亡后的扩散以及增生活跃肿瘤细胞的释放，含量较低。由于缺乏高灵敏性和高特异性的检测方法，导致有关血液中循环游离DNA与疾病相关性的研究在较长时期内进展缓慢。.简单、便携、高灵敏的电致化学发光（ECL）生物传感器的发展一直深受关注，而基于纳米材料的信号放大策略是其中的前沿研究热点之一。本项目以我们前期所建立的DNA组装技术实现高灵敏的肿瘤标志物的检测为基础，设计DNA组装技术进行信号分子的富集和贵金属纳米粒子的局域表面等离子体共振增强量子点发光，实现信号的双重放大效应，结合肽核酸优异的稳定性及识别目标的高特异性，其被用来捕获目标物质，通过ECL检测技术，构建血液中循环游离DNA的超灵敏传感检测，简单取样情况下完成肿瘤标志物的检测。

本研究在构建简单、高灵敏、便携的生物传感器方面开展了一系列工作，主要包括以下几个方面：1. 基于目标驱动的DNA步行器体系，以石墨烯功能化的金纳米笼为传感界面，结合DNA步行器和核酸外切酶 (Exo III) 信号放大策略，构建高灵敏的电化学传感器，应用于目标DNA的检测。通过对发夹式DNA探针的巧妙设计，实现目标DNA的序列识别和定量检测。2. 借助NiO@N-doped C的多层介孔结构和催化发卡自组装的信号放大技术，构建DNA电化学传感体系。3. 利用核酸包裹的Ag纳米簇 (Ag NCs) 的导电性、催化性能以及Ag NCs的吸收光谱与CdS QDs的发射光谱之间的重叠，构建一种新型的ECL-RET传感体系，并将其应用于RNA的检测。4. 以二茂铁 (Fc) 和亚甲基蓝 (MB) 为信号标记，基于DNA串联体的信号放大策略，以AuNPs@MoS2功能化纳米材料为传感界面，构建一种灵敏而又准确的电化学生物传感器，实现DNA甲基化的双信号识别。5. 根据目标诱导的电极表面寡核苷酸的构象变化以及循环扩增策略，开发DNA甲基化分析的signal-on电化学DNA (E-DNA) 生物传感平台。本研究极大提高了检测的速度和灵敏度，为构建简便、高灵敏、高准确度的核酸分析提供新型检测平台。