Chiu Daniel T, Lorenz Robert M
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195-1700, USA.
Acc Chem Res. 2009 May 19;42(5):649-58. doi: 10.1021/ar8002464.
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 toward 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.
任何生物系统的基本单位都是细胞，单细胞水平的功能故障可能导致毁灭性疾病；例如在癌症转移中，单个细胞会引发远处肿瘤的形成。细胞虽小，却是高度异质且分隔化的结构：蛋白质、脂质、RNA和小分子代谢物在细胞内细胞器之间不断运输。获取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细胞器中提取全面生化信息的灵敏工具。在本综述中，我们概述了我们的方法，并强调了在绘制单细胞信息流时空组织方面取得的进展。我们的技术以使用飞升和皮升大小的液滴作为用于操纵单细胞和亚细胞区室的纳米实验室为核心。我们开发了一种单细胞纳米手术技术，用于从活细胞中分离选定的亚细胞结构，这是对单细胞进行高分辨率操纵和化学分析所必需的能力。我们用于按需生成单个飞升大小液滴的微流控方法包括压力和电场方法；我们还探索了一种用于按需生成多个水滴以提高通量的设计。液滴形成只是一系列操作、融合、运输和分析步骤中的第一步。光学方法在我们的技术平台上为所形成的液滴提供了最方便和精确的控制；我们描述了利用光学涡旋阱对水滴进行操纵，这使得能够显著动态“调节”内容物浓度。将热电操纵与这些技术相结合可提供进一步的控制。从单细胞和细胞器中能够收集到的化学信息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分析，（ii）对液滴中亚细胞细胞器和纳米颗粒进行物理尺寸测量；（iii）对液滴内容物进行毛细管电泳（CE）分析。对于生物学研究，我们正在努力将技术的不同组件集成到一个强大的自动化设备中；我们也在满足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，使其成为一种以前所未有的分子细节研究单细胞和细胞器的新工具。
