耦合液滴单细胞培养的快速微生物鉴定质谱接口关键技术研究

Rapid identification is the key to the control of (and the response to) pathogen transmission. Mass spectroscopy (M.S.) is a highly sensitive method for rapid identification of microbes, but its strength of “rapid identification” is far from being fully exploited in present applications. The bottleneck in improving the speed and throughput of M.S.-based microbial identification has been the reliance of its sample preparation on traditional spreading plate culture and manual sampling. To tackle this challenge, we propose an innovative solution that directly and seamlessly couples droplet-based single cell cultivation with mass spectroscopy identification. As a single cell can be encapsulated and cultivated into a microcolony (consisting of thousands of cells) in a microdroplet, the cellular biomass is large enough to meet the sensitivity requirements of MALDI-TOF-MS, which can be thoroughly spotted on the MALDI chip automatically. This strategy thus remove the need for cultivating single cells into visible colonies followed by manual sampling. As the result, the duration of sample preparation can be significantly reduced, from one to several days to only several hours, and the throughput can also be improved by several orders of magnitudes. In this project we will develop a series of key technologies for MS microbial identification, including single-cell droplet culture, microbial droplet optical identification and sorting, and droplet dispenser on the MALDI chip. Together these will lay the principle and key technological basis for developing a high throughput MS-based rapid microbial identification system that can facilitate scientific research and industrial application in clinical diagnostics, food safety, disease prevention, and other strategic or social needs that involve rapid detection and identification of microbes.

快速鉴定是有效控制与应对致病微生物传播的关键。质谱是一种高灵敏的微生物快速鉴定手段，但其快速鉴定优势在实际应用中远未充分体现，而制约其速度和通量的关键瓶颈在于样品依赖于传统涂布培养增菌和手动点样过程。针对上述瓶颈，本项目提出直接耦合液滴单细胞培养与质谱鉴定的创新思路。由于液滴能将单细胞培养成微克隆（数千到数万个细胞）且能全部无损自动地点样在MALIDI芯片上满足质谱灵敏度要求，因此无需单细胞培养成肉眼可见克隆再手动点样，样品增殖周期有望从1到数天大幅缩短至数小时，且容易实现自动化。基于上述原理，本项目拟重点研制基于微生物单细胞液滴培养、微生物液滴光学识别与分选及微生物液滴-MALDI芯片点样等微生物质谱鉴定接口关键技术和方法，为高通量自动化的微生物质谱快速鉴定系统的研制奠定原理和关键技术基础。本项目的实施将最终服务于临床微生物医学、食品微生物等领域，科学和现实意义兼具。

快速鉴定是有效控制与应对致病微生物传播的关键。质谱是一种高灵敏的微生物快速鉴定手段，但其快速鉴定优势在实际应用中远未充分体现，而制约其速度和通量的关键瓶颈在于样品依赖于传统涂布培养增菌和手动点样过程。本项目自实施以来，已成功发展了基于液滴单细胞培养的系统、微生物液滴识别与分选系统、液滴-MALDI芯片点样系统。成功实现基于液滴包裹的单细胞培养微克隆的直接自动质谱进样和检测，从而为高通量自动化的微生物质谱鉴定系统研制奠定原理和关键技术基础。微生物单细胞和所需的培养基被分散包裹在水包油液滴中，液滴单独收集完成离线培养经过约3-6小时的培养后可在显微镜下能观测到液滴内光学透明度发生变化。同时构建液滴分选系统将培养后的液滴再注射进入芯片光学检测通道，从而完成对微生物包裹液滴的光学识别与分选，分选效率可达90%以上。发展两种微液滴-MALDI芯片点样接口，其中动态接口可与实现90%左右的点样效率，静态液滴阵列导出可达到95%以上。导出到MALDI靶板后，对大肠杆菌和阿氏肠杆菌进行质谱的验证。本项目的实施为高通量自动化的微生物质谱快速鉴定系统的研制奠定原理和关键技术基础。本项目的实施将最终服务于临床微生物医学、食品 微生物等领域，科学和现实意义兼具。

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