基于分子信标自由末端多位点现场标记信号分子的新型DNA传感器

Compared with the traditional linear probe-based DNA biosensor, the molecular beacon (MB)-based hybridization biosensing technology shows the advantages of stronger specificity, higher selectivity, more simple operability and more comprehensive application. However, there are still some disadvantages for this technology, for instance, the labeling and separating processes for the electroactive MB probe are often complicated, the type of the signal molecule labeled on MB probe is few and signal intensity in the classical MB-based biosensor is limited. .To solve these problems, a new strategy that based on the multi-site and in-situ labeling of the electroactive signal molecules on the free terminal of MB probe is established in this project. The main ideas include: (1) Constructing a multi-site platform on the free terminal of the MB probe for several signal molecules to be labeled simultaneously, and therefore the detection signal can be greatly amplified and the analytical sensitivity will be increased. (2) Applying in-situ labeling of the electroactive molecule on the immobilized MB probe to realize the accomplishment of the "labeling-separation-purification" by one step. Thus, the drawbacks of bothering purification steps, time and reagent-consuming that accompanied by the common homogeneous synthesis can be effectively avoided. (3) When the different kinds of multi-site platforms and signal molecules are organically assembled and grafted on the MB probe, the types of the output signals of the DNA biosensor will be dramatically enriched. Also, according to electrochemistry difference of the different signal molecules, the different kinds of target sequences are expected to be analyzed by one biosensor.

基于分子信标的DNA杂交传感技术与传统直线型DNA探针传感器相比，具有特异性强、选择性高、操作简单等优点。但目前该技术还存在信号分子标记-分离过程复杂、探针信号种类单一、信号强度有限等缺点，限制了该类传感器的深入研究和广泛应用。 .为解决上述不足，本课题拟将"多位点平台搭建"和"信号分子现场标记"技术相结合，创建一系列分子信标传感器制备的新方法。主要工作设想包括：(1) 在分子信标自由末端搭建一个多位点平台，用于多个电活性信号分子的同时标记，并通过信号叠加效应提高该类传感器的信号强度；(2)在固定后的分子信标上进行信号分子的现场标记，通过固液相差异简单实现电活性分子信标"标记-分离-纯化" 的同步完成，克服均液相合成中分离、纯化困难的弊端；(3)将不同类型的多位点平台和信号分子进行搭配和组装，丰富该类传感器的信号类型，并根据不同标记分子的信号差异努力实现传感器对多目标序列的同时检测。

1.项目研究背景.与传统DNA传感器中直线型DNA探针不同，分子信标（Molecular Beacon）是一种具有茎环（Stem-loop）结构的发夹型寡聚核苷酸探针。由于分子信标传感器中检测信号的变化完全是由碱基一一配对的热力学过程驱动，因此该类传感器具有特异性强、选择性高、操作简单等优点。但该技术还存在信号分子标记-分离过程复杂、探针信号种类单一、信号强度有限等缺点，限制了该类传感器的深入研究和广泛应用。.2.主要研究内容.为克服上述传统分子信标传感器在制备和应用中的不足，在本课题中，我们主要进行了如下相关研究内容：.（1）高性能电活性信号分子的设计、合成 ；（2）分子信标自由末端多位点标记平台的搭建；（3）信号分子在多位点平台上的现场标记；（4）传感器的杂交检测及性能评价。.3.重要结果及关键数据.以三聚氰胺、纳米金、富勒醇、氧化石墨烯等分子和材料作为多位点平台，以中性红、硫堇、纳米银、系列铜配合物为电活性信号分子，采用共价耦合、配位等层层现场组装的方法，构建多种分子信标电化学传感器，用于了不同序列目标DNA的高灵敏、快速检测，且对不同碱基错配DNA显示出很好的识别性能。研究成果发表在了Scientific Reports, Biosensors and Bioelectronics, Chemical Communication等国际著名期刊，取得了较高的学术影响力。.4.科学意义.（1）解决了传统分子信标DNA传感器电活性标记分子类型单一及单分子标记造成信号有限、检测灵敏度不高的问题。.（2）开辟分子信标寡聚核苷酸在传感界面进行电活性分子现场标记的新方法，解决传统均液相探针合成过程中耗时、耗材、步骤繁琐的弊端，降低该类型DNA传感器的制备成本。