基于循环放大技术的高灵敏度核酸线性阈值门和 ncqds应用于fe3检测的研究-study on application of high sensitivity nucleic acid linear threshold gate and ncq ds to fe3 detection based on cyclic amplification technology.docxVIP

基于循环放大技术的高灵敏度核酸线性阈值门和NCQDs 应用于 Fe3+检测的研究摘 要本论文的研究工作围绕生物化学分析和纳米分析开展，在癌症的早期预诊与 检测和临床研究具有重要的实际意义。主要内容包括：构建了一种新型 DNA 剪 切聚合-滚环扩增的双重循环放大技术，基于此种技术，实现了目标 DNA 的高效 灵敏检测；基于前述循环放大技术，构建了一种新型的核酸线性阈值门体系，实 现肿瘤原发组织判断；制备了一种荧光性能稳定的氮掺杂量子点，通过与三价铁 离子发生荧光猝灭反应作为响应信号，实现了对三价铁离子的高灵敏检测。详细 研究内容如下：1、以 SERS 检测技术为手段建立了一种新型 DNA 剪切聚合-滚环扩增的双重 循环放大技术，实现了目标 DNA 的高效灵敏检测。为此，首先制备了一种由纳 米金生物条码合成的“金海绵”配合自制银片构成的新型表面拉曼增强基底。设 计构建了剪切聚合-滚环扩增反应的双重循环扩大，采用表面增强拉曼散射分析方 法，实现了对目标 DNA 的高灵敏检测。利用这种方法可使目标 DNA 的检测限达 到 10-16 M 级别。2、基于上述放大机制，构建了一种新型的核酸线性阈值门系统，通过集成 多种核酸反应，简化了传统核酸线性阈值门的设计，并附加了信号放大功能，从 而能够用较简单的系统设计和操作以及较短的时间来实施输入为微量的肿瘤原 发组织标志物（miRNA 组合）的核酸阈值门响应，从而实现某些条件下的肿瘤原 发组织判断。3、以碳化二乙基三胺五乙酸（DTPA）为主要原料，制备了一种新型氮掺杂 碳量子点。此量子点具有稳定荧光性能和高荧光强度，且与 Fe3+作用时，荧光被 高效猝灭，基于此种现象为以后构建一种简单稳定灵敏的生物传感器，用于检测 人血清中 Fe3+的含量提供了依据。关键词：表面增强拉曼散射 酶促循环放大 核酸线性阈值门 碳量子点 荧光 猝灭IIISTUDIES ON HIGH-SENSITIVITY NUCLEIC ACID LINEAR THRESHOLD GATE BASED ON ENZYMATIC CYCLING AMPLIFICATION AND THE NCQDs FOR THE DETECTION OF Fe3+ABSTRACTIn this thesis, a novel dual amplification mechanism based on the nicking-polymerization cycling and rolling-circle amplification was performed. High-sensitivity detection of DNA was realized with such amplification mechanism. At the same time, a novel nucleic acid linear threshold gate was constructed for the discrimination of the origin of the cancer tissue based on the aforementioned mechanism. Finally, a nitrogen-doped quantum dot with stable fluorescence emission was developed. The emission of this QD can be quantitatively quenched by Fe3+, which can be used for the highly-sensitive detection of Fe3+.The research was summarized as follows:A high-sensitive DNA assay was developed using dual nucleic acid amplification and novel SERS enhancement substrate. The SERS substrate was constructed featuring the “gold sponge” (constructed with the bio-bar-code functioned gold nanoparticles) on home-made silver layer on the copper slips; the amplification mechanism was developed by combining nicking-polymerization cycle and rolling circle amp