基于指数级恒温非酶扩增模式的miRNA检测传感器及其在肿瘤分子诊断中运用

Circulating miRNA-based molecular diagnosis assay possesses great potential in the early diagnosis and non-invasive liquid biopsy of tumor. However, the peculiarity of circulating miRNA obviously limited its clinical application, such as the easy degradation, short sequence and difficult to amplify. Thus, developments of the high-efficiency protection, extraction, amplification and detection technology for circulating miRNAs have become as the key problem that need to be addressed. The applicant early constructed the graphene oxide-based RNA protection and extraction method which provide a long-acting protection in the extraction and storage process, but its mechanism and standardization still need the further explorations. Furthermore, we developed the enzyme-free amplification mode that could accommodate the peculiarity of miRNA, and didn’t rely on the enzymatic amplification process such as PCR, and its sensitivity still has a large potential to be ascended. On the basis of existing research, this project aims to explore the mechanism of circulating miRNA protection and extraction, develop the new enzyme-free amplification mode that possesses the exponential amplification capability, and construct the novel miRNA detection sensor by integrating the efficient photoelectric sensor and paper-based chip. The successful proceeding of this project will acquire a simple, quick and low-cost miRNA protection, extraction, amplification and detection methods which could provide a novel strategy for early diagnosis and non-invasive liquid biopsy of tumor based on the exponential enzyme-free detection pattern.

基于循环miRNA水平的分子诊断方法在肿瘤早期诊断及无创性液体活检领域具有重大潜力，但循环miRNA易降解、序列短小且较难扩增等特性明显地限制了其临床应用。因此，研发新型样品保护、提取、扩增及检测新技术已成为亟需解决的关键问题。申请人前期已构建了基于石墨烯纳米材料的样品保护及提取方法，为其提取及储存过程提供长效保护，但保护机制及流程标准化仍需进一步探索。此外，还发展了适应miRNA特性且不依赖于PCR等酶促扩增过程的非酶扩增模式，简化了扩增条件及操作，其灵敏度尚具有较大提升潜力。本项目拟在已有基础上，探索循环miRNA保护、提取机制，发展具有指数级扩增性能的新非酶扩增模式，并结合高效光电传感及纸基芯片技术以构建新型检测传感器。本项目的顺利开展，将研发得到简单、快速且成本低廉的样品保护、提取、扩增及检测新技术，以期为肿瘤早期诊断和无创性液体活检提供基于指数级非酶扩增检测模式。

miRNA表达水平能够作为肿瘤以及病毒、虫媒相关疾病的重要生理/生化标志物，由此衍生的基于循环miRNA水平的分子诊断技术将在肿瘤早期诊断领域发挥重要作用。本项目通过探索提出了循环miRNA保护与提取新机制，开发了具有指数扩增性能的非酶扩增模式，并将其整合至电化学发光检测平台得到了新型循环miRNA检测传感器。本项目探索提出了基于石墨烯的miRNA保护及提取方法，能够为体外核糖核酸提供较长时间高效的保护，在液相氧化石墨烯负载核酸保护下，暴露于空气中96h依然能够维持80%以上的完整性；构建不依赖酶促PCR扩增过程的指数扩增模式，极大地简化了扩增反应条件、降低了检测成本等；通过非酶扩增步骤间的线性链接和互作方式构建高灵敏度的非酶扩增体系，并完成了检测限、特异性等相关效能评估；本项目研发得到集样品采集、检测及其信号给出系列技术体系，并成功应用于血液、尿液和唾液等不同复杂系统的寨卡病毒检测，为临床样品和无创性液体活检提供技术支持。在本项目的支持下，团队共发表SCI论文4篇（IF>10，3篇），申请发明专利2项，获批1项。