仿生智能纳米孔道的构筑及其在肝癌相关miRNA逻辑检测中的应用

Nanopore-based analytical technique plays an important role in research of bionanotechnology. It has a great potential application for nucleic acid sequencing and high sensitive detection of biological molecules. However, the sensitivity of the conventional solid-state nanopore sensors is not high enough due to the lack of effective signal amplification means, restricting the application of nanopore sensors in the field of analytical chemistry. In this project, we select a solid-state nanopore modified with polyethyleneimine and hemin as the sensing platform. The high-performance sensor is based upon the reversible interaction between the surface charge of the nanopore and ionic current rectification, which is resulted from DNA cascade signal amplification in solution. The main studies are to design and construct nanopore logic sensor and multiplex target recognition system, taking simplifying protocols and improving assay performance as the starting point. Depending on the nucleic acid’s hybridization and configuration transform, the designed nanopore sensing systems are expected to realize the intelligent detection of multiple liver cancer related miRNA integrating with molecular logic diagnostic techniques. On this basis, the signal amplification rules of nanopore logic sensor will be mastered for creating new biosensing method to intelligently detect multiple liver cancer related miRNAs in complex biological samples. The proposed project provides theoretical guidance and technical support for clinical diagnosis and therapeutic evaluation of liver cancer.

纳米孔分析技术是生物纳米技术研究的重要内容之一，对于核酸快速测序和生物分子的高灵敏检测有着潜在的应用价值。然而，常规固态纳米孔传感器缺乏有效的信号扩增手段，导致纳米孔传感器灵敏度不足，成为纳米孔传感领域应用的瓶颈。本项目拟选用聚乙烯亚胺和氯化血红素功能化的固态纳米孔作为传感平台, 利用信号级联放大反应在溶液中组装DNA长链，探索锥形纳米孔表面电荷的变化机制与离子整流之间的关系，制备高性能仿生纳米孔传感器；以传感平台精简和性能提升为关键出发点，利用核酸杂交配对、立体构象转变等性质，并结合分子逻辑诊断技术，构建纳米孔逻辑传感体系，完成对多种肝癌相关miRNA的智能化检测。在此基础上，解析纳米孔逻辑传感体系的信号扩增规律，建立复杂生物样品中肝癌相关miRNAs的多元化检测新方法，为肝癌的早期诊断和疗效评价提供理论指导与技术支持。

本项目选用聚乙烯亚胺和锆离子功能化的聚合物纳米孔作为传感平台，利用信号级联放大反应在溶液中组装DNA长链，探索锥形纳米孔表面电荷的变化机制与离子整流之间的关系，制备高性能仿生纳米孔传感器。项目实施过程中借助氧化石墨烯对单链DNA/RNA的选择性吸附作用，并结合分子逻辑诊断技术，构建纳米孔逻辑传感体系，完成了对多种肝癌相关miRNA、甲基转移酶、OTA和四环素等检测。另外还利用滚环扩增和DNA步行器完成了Dam和CpG甲基转移酶的检测。多篇研究论文发表在国内外权威刊物上，如：Analytical Chemistry，Talanta，Analytical Methods，Journal of Chromatography A，Dyes and Pigments，Journal of Chemical and Engineering Data，Journal of Chemical Technology and Biotechnology等，发表SCI收录论文9篇，其中一区二区论文6篇。授权国家发明专利2项，获省部级奖项2项。