病原体核酸分类检测的集成化芯片技术研究

Pathogen infection is a serious common disease that threatens human's health. And the infectious disease is now the world's largest cause of cancer. However, there were insufficient detection technologies that could exactly, quickly and safely identify pathogen. In this study, based on previous work, a novel integrated chip technology with the ability of multi - level cell separation, nucleic acid extraction, amplification and detection, is proposed. The detection and classification of pathogen nucleic acid information from different sources is implemented, which can provide a new molecular detection technology not only for infectious disease diagnosis in the happening and development, but also for early diagnosis of malignant tumor. In method and technology level, multi - level cell separation principle, chips' surface behavior and surface modification are studied. In chip development level, a multi - level crossflow separation chip, a valve-less type solid phase extraction - isothermal amplification (SPE-LAMP) chip and an integrated chip with microvalves are proposed based on MEMS technology. In systme level, a novel multi-functions and integrated microchip system is developed, which is capable of 3-4 kinds of cell separation, DNA extraction, amplification and detection. In the developed system, the cell separation efficiency is improved to more than 80%. The detection resolution of the proposed system is in the order of tens of pathogens, and the detection processing time is less than 2 hour, approaching the world-leading level. The developed integrated chip technology not only has an important academic significance and realistic value for accurate, fast and safe diagnosis of infectious diseases as well as malignant tumors early diagnosis, but also is a milestone in scientific development of microfluidics with a promising application perspective.

病原体引起感染性疾病严重威胁人们健康，并已成为全球最大的致癌因素。然而，准确、快速、安全的病原体感染检测技术缺乏。本项目在已有研究基础上，创新性地提出研究一种集多种细胞分离、核酸提取、扩增及检测于一体的集成化芯片技术，实现不同来源病原体核酸的分类检测，为判断病原体感染发生、发展及癌变早期诊断提供新的分子检测技术。研究多种细胞分离原理，研制多级错流过滤芯片，实现3-4种细胞的同时分离和收集，分离效率>80%；研究芯片表/界面行为机理，研制无阀式固相萃取-等温扩增芯片，在同一反应微池实现核酸提取和扩增；研究集成化芯片系统，实现死病原体、活病原体、靶细胞、吞噬细胞等不同来源病原体核酸的分类、快速、安全检测，检测灵敏度为几十个病原体，整体耗时<2h，达国际领先水平。本项研究不仅对感染性疾病的准确诊断、恶性肿瘤的早期诊断，具有重要的科学价值和现实意义；而且对微流控芯片的应用和发展具有重要推动作用。

病原体引起感染性疾病严重威胁人们健康，并已成为全球最大的致病、致癌因素。然而，准确、快速、安全的病原体感染检测技术缺乏。本项目在已有研究基础上，研制出一种集核酸提取、等温扩增及实时荧光检测于一体的集成芯片系统，实现Zika病毒核酸的扩增和实时荧光定量检测。检测灵敏度为100个拷贝，扩增体系体积仅需10微升，整体分析时间小于1小时。研究出多种细胞分离芯片，实现3种细胞的同时分离和收集，红细胞分离效率达82.3%。研究出一种新型的细胞捕获芯片技术，实现目标细胞的捕获，CD4+细胞的捕获效率大于>80%。研制出一种无阀式固相萃取-等温扩增（SPE-LAMP）微流控芯片，在同一反应微池中实现了核酸的提取和等温扩增。本项目的研究不仅对病原体检测提供了一种新的芯片技术，同时对于病原体现场检测具有广泛的应用前景。