Chemistry and biology in femtoliter and picoliter volume droplets.
Chemistry and biology in femtoliter and picoliter volume droplets.
复制标题
飞升和皮升体积液滴中的化学和生物学。
DOI：
10.1021/ar8002464
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发表时间：
2009-05-19
影响因子：
18.3
通讯作者：
Lorenz, Robert M.
中科院分区：
文献类型：
--
作者：
Chiu, Daniel T.;Lorenz, Robert M.
任何生物系统的基本单位都是细胞，单细胞水平的故障可能导致毁灭性的疾病;例如，在癌症转移中，单个细胞会导致远处肿瘤的形成。细胞虽然很小，但却是高度异质和分区化的结构：蛋白质、脂质、RNA和小分子代谢物不断在细胞内细胞器之间流动。获得有关这些生物分子时空分布的详细信息对于我们理解细胞功能和功能障碍至关重要。为了获取这些信息，我们需要能够从单细胞和亚细胞器中提取全面生化信息的敏感工具。在这个账户中，我们概述了我们的方法，并强调了我们在绘制单细胞中信息流的时空组织方面的进展。我们的技术集中在使用毫微微升和皮升大小的液滴作为纳米实验室来操纵单细胞和亚细胞区室。我们已经开发出一种单细胞纳米外科技术，用于从活细胞中分离出选定的亚细胞结构，这是单细胞高分辨率操作和化学分析所需的能力。我们用于按需生成单个毫微微升大小的液滴的微流体方法包括压力和电场方法;我们还探索了一种按需生成多个水性液滴的设计，以增加吞吐量。液滴的形成只是一系列操作、融合、运输和分析的第一步。光学方法提供了最方便和精确的控制所形成的液滴与我们的技术平台;我们描述了水性液滴操作与光学涡旋陷阱，使显着的能力，动态“调整”的内容物的浓度。热电操纵与这些技术的集成提供了进一步的控制。可以从单个细胞和细胞器中收集的化学信息的量严重依赖于可用于分析液滴内容物的方法。我们描述了我们已经开发的三种技术：（i）液滴封装，快速细胞裂解，和基于荧光的单细胞测定，（ii）液滴中亚细胞器和纳米颗粒的物理尺寸，和（iii）液滴内容物的毛细管电泳（CE）分析。对于生物学研究，我们正在努力将我们技术的不同组成部分整合到一个强大的自动化设备中;我们还在解决对更高通量的预期需求。随着这些领域的进展，我们希望巩固我们的技术作为一种新的工具，用于研究具有前所未有的分子细节的单细胞和细胞器。
The basic unit of any biological system is the cell, and malfunctions at the single-cell level can result in devastating diseases; in cancer metastasis, for example, a single cell seeds the formation of a distant tumor. Although tiny, a cell is a highly heterogeneous and compartmentalized structure: proteins, lipids, RNA, and small-molecule metabolites constantly traffic among intracellular organelles. Gaining detailed information about the spatiotemporal distribution of these biomolecules is crucial to our understanding of cellular function and dysfunction. To access this information, we need sensitive tools that are capable of extracting comprehensive biochemical information from single cells and subcellular organelles. In this Account, we outline our approach and highlight our progress towards mapping the spatiotemporal organization of information flow in single cells. Our technique is centered on the use of femtoliter- and picoliter-sized droplets as nanolabs for manipulating single cells and subcellular compartments. We have developed a single-cell nanosurgical technique for isolating select subcellular structures from live cells, a capability that is needed for the high-resolution manipulation and chemical analysis of single cells. Our microfluidic approaches for generating single femtoliter-sized droplets on demand include both pressure and electric field methods; we have also explored a design for the on-demand generation of multiple aqueous droplets to increase throughput. Droplet formation is only the first step in a sequence that requires manipulation, fusion, transport, and analysis. Optical approaches provide the most convenient and precise control over the formed droplets with our technology platform; we describe aqueous droplet manipulation with optical vortex traps, which enable the remarkable ability to dynamically “tune” the concentration of the contents. Integration of thermoelectric manipulations with these techniques affords further control. The amount of chemical information that can be gleaned from single cells and organelles is critically dependent on the methods available for analyzing droplet contents. We describe three techniques we have developed: (i) droplet encapsulation, rapid cell lysis, and fluorescence-based single-cell assays, (ii) physical sizing of the subcellular organelles and nanoparticles in droplets, and (iii) capillary electrophoresis (CE) analysis of droplet contents. For biological studies, we are working to integrate the different components of our technology into a robust, automated device; we are also addressing an anticipated need for higher throughput. With progress in these areas, we hope to cement our technique as a new tool for studying single cells and organelles with unprecedented molecular detail.
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影响因子：
7.4
作者：
He, MY;Sun, CH;Chiu, DT
通讯作者：
Chiu, DT
影响因子：
6.1
作者：
Hunt, Thomas P.;Issadore, David;Westervelt, R. M.
通讯作者：
Westervelt, R. M.
影响因子：
3.3
作者：
Jeffries, Gavin D. M.;Kuo, Jason S.;Chiu, Daniel T.
通讯作者：
Chiu, Daniel T.
影响因子：
3.3
作者：
Kuyper, Christopher L.;Fujimoto, Bryant S.;Chiu, Daniel T.
通讯作者：
Chiu, Daniel T.
影响因子：
3.9
作者：
Kuo, JS;Spicar-Mihalic, P;Chiu, DT
通讯作者：
Chiu, DT