A double-emulsion microfluidic platform for in vitro green fluorescent protein expression
A double-emulsion microfluidic platform for in vitro green fluorescent protein expression
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DOI：
10.1088/0960-1317/21/5/054032
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发表时间：
2011-05-01
影响因子：
2.3
通讯作者：
Zhu, Y.
中科院分区：
文献类型：
--
作者：
Wu, N.;Oakeshott, J. G.;Zhu, Y.
微流体液滴技术由于优于常规乳液技术的优点和用于广泛应用的能力而得到普及。在本文中，一个简单的基于微流体的双乳液系统的发展报告。这种系统可以潜在地用于体外蛋白质合成。该系统涉及两步法以制备水包油包水（W/O/W）乳液。PMMA微芯片用于形成油包水（W/O）单乳液液滴。然后，将单乳液液滴输送到PDMS/玻璃微芯片以制备W/O/W双乳液液滴。该系统的特点是首先检测荧光素钠盐作为模型染料在内部的水液滴，使用激光诱导荧光。研究了内水相和外连续水相流速对双乳液滴形成的影响，为系统优化提供信息。还研究了双乳液液滴的芯片上存储，以允许使用商业体外转录和翻译试剂盒或粗大肠杆菌S30提取物从PCR产生的DNA模板合成蛋白质。在体外表达的绿色荧光蛋白成功地证明在这个系统。
Microfluidic droplet technology has gained popularity due to the advantages over conventional emulsion techniques and capabilities for a wide range of applications. In this paper, the development of a simple microfluidic-based double-emulsion system is reported. Such a system could be potentially used for in vitro protein synthesis. The system involves a two-step process to make water-in-oil-in-water (W/O/W) emulsions. A PMMA microchip is used for the formation of water-in-oil (W/O) single-emulsion droplets. Then, the single-emulsion droplets are transported to a PDMS/glass microchip to make the W/O/W double-emulsion droplets. The system was first characterized by detecting fluorescein sodium salt as a model dye in the internal aqueous droplets using laser-induced fluorescence. The effect of the flow rates of the internal aqueous phase and outer continuous aqueous phase on the formation of the double-emulsion droplets is investigated to provide information for system optimization. On-chip storage of double-emulsion droplets is also investigated to allow for protein synthesis from a PCR-generated DNA template using either commercial in vitro transcription and translation kits or crude Escherichia coli S30 extracts. In vitro expression of the green fluorescent protein is successfully demonstrated in this system.