基于阵列介电捕获的微生物单细胞高通量拉曼流式分选方法学研究

Microorganisms are the most important reservoir of functional genes for synthetic biology and biotechnology industry. Identification and sorting of single cells with specific phenotype is the premise of breakthroughs in fundamental questions such as "phenotypic heterogeneity " and "uncultured microbes". Compared with fluorescence-activated cell sorting (FACS), Raman-activated cell sorting (RACS) is a promising technology and holds several significant advantages as it is label-free, non-invasive, in-situ and able to detect "unknown" phenotypes. High-throughput RACS technology will open the door for numerous new scientific applications. Yet the inability to capture and identify single cells under a high flowing speed is the key bottleneck for high-throughput RACS development. This project proposes a solution to solve this key obstacle by using dielectrophoretic single-cell capture/release for Raman signal acquisition and identification. The cell with targeted Raman signal could be sorted by utilizing a solenoid valve suction based microfluidic single cell isolation technology. A Raman cytometry platform suitable for most of microorganisms will be established and high-throughput (from several cells/minute at present to 50 cells/second) and high accuracy (>90%) sorting efficiency will be demonstrated for microorganisms. This platform will provide single cells with a specific phenotype or function for downstream single cell sequencing or culture. Finally, this project will provide a method for the characterization of microbial communities at single-cell resolution

微生物是合成生物学及生物技术产业最重要的资源库。特定表型单细胞分选是突破 "微生物功能异质性和表型多样性"、"大多微生物尚难培养"等重大科学与技术屏障的前提。与荧光分选（FACS）相比，基于拉曼分选单细胞(RACS)的创新和优势为：免标记、原位和非侵害性检测、能探测未知表型等。高通量拉曼分选技术将带来上述共性科学问题的重大突破。但高速流动状态下单细胞快速捕获识别是目前制约拉曼高通量分选的关键技术瓶颈。本项目拟针对该瓶颈发展基于阵列介电单细胞捕获/释放的快速拉曼识别技术，并耦合基于电磁阀吸吮的微流控细胞分离技术，建立通用的拉曼流式分选方法学平台，实现微生物细胞高通量（从目前数个/分钟提高到50个/秒）和高准确度（准确率>90%）的非标记原位表型功能分选，同时将可为下游微生物单细胞测序和培养提供特定表型的细胞源。项目的实施将为微生物群落功能解析提供方法学平台，为仪器开发奠定原理和关键技术基础。

微生物是合成生物学及生物技术产业最重要的资源库。特定表型单细胞分选是突破 “微生物功能异质性和表型多样性”、“大多微生物尚难培养”等重大科学与技术屏障的前提。与荧光分选（FACS）相比，基于拉曼分选单细胞(RACS)的创新和优势为：免标记、原位和非侵害性检测、能探测未知表型等。高通量拉曼分选技术将带来上述共性科学问题的重大突破。但高速流动状态下单细胞拉曼光谱的识别及目标细胞的准确分离是目前制约拉曼高通量分选的关键技术瓶颈。针对该瓶颈问题，本项目首先发展了（i）基于阵列介电单细胞捕获/释放的快速拉曼识别技术，并耦合基于电磁阀吸吮的微流控细胞分离技术，实现了色素酵母的高通量分选（~60个细胞每分钟）；（ii）基于尖角阵列电极的精确捕获技术，捕获准确率达95%；（iii）基于液滴微流控的目标细胞分离技术，实现了高产虾青素雨生红球藻细胞的高通量分选（260个细胞每分钟）、高准确率（98%）分选，而且分选后仍有92%细胞保持活性，与未分选细胞相比无明显差异。最终将基于尖角阵列电极的介电单细胞捕获/释放的快速拉曼识别技术与基于液滴微流控的高通量单细胞分离技术耦合，并以石英为基材，建立了通用的拉曼流式分选方法学平台，在实现基于共振拉曼信号的单细胞分选的同时，进一步实现了具有低拉曼信号，如产油酵母的分选，其通量可达120个细胞每分钟。本项目建立的高通量拉曼流式分选平台利于实现微生物细胞高通量和高准确度的非标记原位表型功能分选，同时将可为下游微生物单细胞测序和培养提供特定表型的细胞源。项目的实施为微生物群落功能解析提供方法学平台，为仪器开发奠定原理和关键技术基础。