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

项目名称： 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

项目编号： No.31470220

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 微生物学

项目作者： 马波

作者单位： 中国科学院青岛生物能源与过程研究所

项目金额： 80万元

中文摘要： 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。

中文关键词： 单细胞分析；未培养微生物；拉曼光谱；微流控；高通量筛选

英文摘要： 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

英文关键词： Single cell analysis;Uncultured microorganisms;Raman spectroscopy;Microfluidics;High-throughput screening