基于血晶素标记探针的高灵敏核酸检测方法研究

Highly sensitive and quantitative detection of sequence-specific nucleic acid fragment shows its great significance in the areas of disease diagnosis, food safety, environmental monitoring and judicial identification. The proposed project employs a small-molecule organocatalyst namely hemin to covalently label nucleic acid probe. The recognition of probe to target nucleic acid can be transduced to activation of hemin catalytic activity, thus produce amplified detection signal through catalytic reaction of hemin. In order to further amplify the detection signal of hemin labeled probe and improve its sensitivity, two feasible pathways are described in the project. The number of hemin activated by the recognition of target nucleic acid can be increased by introducing molecular biology recycling technique such as catalyzed hairpin assembly. On the other hand, the catalytic activity of activated hemin can be improved by formation of hemin/G4 DNAzyme. The structure-activity relationship of hemin labeled probe, the catalytic system of hemin as well as the combination of signal amplification techniques are under planned investigation and optimization to sufficiently exert the potential of hemin catalysis. A series of simple, rapid and also highly sensitive and selective nucleic acid detection methods are expected to be established through the project. The proposed project paves a promising approach for developing highly sensitive nucleic acid probe by small-molecule organocatalyst labeling.

特定序列核酸片段的高灵敏定量检测在疾病诊断、食品安全、环境监测和司法鉴定等领域具有重大意义。本项目提出以小分子催化剂作为新型核酸探针标记物，选用血晶素（hemin）分子共价标记核酸探针，建立目标核酸片段激活血晶素催化活性的信号转导模式，通过血晶素的催化反应输出检测信号。为了进一步提高血晶素标记核酸探针的检测灵敏度，本项目拟从引入分子生物学循环技术和提高血晶素的催化性能两个角度入手，一方面通过联用催化发卡组装等分子生物学信号放大技术提高每分子目标核酸激活的血晶素分子数目，另一方面通过形成hemin/G4 DNAzyme等高效催化结构提高激活后血晶素分子的催化性能，在对血晶素标记探针构效关系、血晶素催化体系及信号放大技术联用方式进行系统研究和优化的基础上，充分发挥血晶素的催化潜能，最终建立一系列高灵敏、高特异性且简单高速的核酸检测策略。本项目将为小分子催化剂标记高灵敏核酸探针的发展奠定基础。

催化是化学对现代社会的重要贡献之一。在生物分析领域，天然酶（如辣根过氧化物酶等）或模拟酶（如hemin-G4 DNAzyme等）都可以放大检测信号，实现生物分子的灵敏检测。以hemin分子为代表的卟啉类化合物是天然酶的活性中心，具有优异的本征催化活性。本项目通过分子设计和化学合成，利用核酸识别或构筑有机纳米结构等形式精确控制卟啉类化合物的空间分布并调控其催化及光学性能，用于设计生物分析检测体系。.通过设计催化小分子hemin共价标记且hemin催化性能自抑制的核酸探针，联用催化发卡组装放大技术，提高了每分子目标核酸激活的hemin分子数目，建立一分子目标核酸激活多分子双hemin探针催化活性的信号转导模式，通过双重催化效应提高了hemin标记核酸探针的检测灵敏度，对靶标核酸的均相检测下限为0.01 nM。利用卟啉分子作为结构构筑块合成卟啉基多孔有机框架化合物纳米酶，有效防止因卟啉分子聚集导致的催化活性降低，作为纳米探针开发了对葡萄糖的比色检测方法以及对抗氧化剂抗氧化能力的评估方法。通过设计了具有双齿配体结合位点的有机框架材料纳米探针，利用Cu2+对纳米探针荧光高效且高选择性的光致电子转移猝灭，发展了对Cu2+的荧光检测方法，检测限为0.076 μM。.本项目的研究为设计催化能力优异的卟啉基催化体系，并用于构建高灵敏的生物分析体系提供借鉴。

内容获取失败，请点击重试