Sharma Bineet, Takamura Yuzuru, Shimoda Tatsuya, Biyani Manish
Department of Bioscience and Biotechnology, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Center for Single Nanoscale Innovative Devices, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan.
Sci Rep. 2016 May 20;6:26257. doi: 10.1038/srep26257.
The extreme miniaturization of biological and chemical assays in aqueous-droplet compartments enables spatiotemporal control for large-scale parallel experimentation and can thus permit new capabilities for "digitizing" directed molecular evolution methodologies. We report a remarkably facile bulk method to generate mega-scale monodisperse sub-femtoliter aqueous droplets by electrospray, using a prototype head with super-fine inkjet technology. Moreover, the electrostatic inkjet nozzle that injects the aqueous phase when immersed within an immiscible phase (an optimized oil/surfactant mixture) has the advantage of generating cell-like sub-femtoliter compartments for biomolecule encapsulation and successive biological and chemical reactions. Sub-femtoliter droplets of both liquid (water-in-oil, volumes ranging from 0.2 to 6.4 fL) and gel bead (agarose-in-oil, volume ranging from 0.3 to 15.6 fL) compartments with average sizes of 1.3 μm and 1.5 μm, respectively, were successfully generated using an inkjet nozzle at a speed of more than 10(5) droplets per second. We demonstrated the applicability of this system by synthesizing fluorescent proteins using a cell-free expression system inside electrosprayed sub-femtoliter droplets at an accelerated rate, thereby extending the utility of in vitro compartmentalization with improved analytical performance for a top-down artificial cellular system.
将生物和化学分析方法极度微型化至水滴隔室中，能够实现对大规模平行实验的时空控制，从而为“数字化”定向分子进化方法带来新的能力。我们报道了一种极为简便的批量方法，利用具有超精细喷墨技术的原型喷头，通过电喷雾产生百万级规模的单分散亚飞升水滴。此外，当浸入不混溶相（优化的油/表面活性剂混合物）中时注入水相的静电喷墨喷嘴，具有生成类似细胞的亚飞升隔室用于生物分子封装以及后续生物和化学反应的优势。使用喷墨喷嘴以每秒超过10⁵个液滴的速度成功生成了平均尺寸分别为1.3μm和1.5μm的液体（油包水，体积范围为0.2至6.4fL）和凝胶珠（油包琼脂糖，体积范围为0.3至15.6fL）隔室的亚飞升液滴。我们通过在电喷雾亚飞升液滴内使用无细胞表达系统加速合成荧光蛋白，证明了该系统的适用性，从而扩展了体外分隔的效用，并为自上而下的人工细胞系统提供了改进的分析性能。
