Wang Chenyu, Liu Wenwen, Wei Qingquan, Ren Lufeng, Tan Manqing, Yu Yude
Biomicrofluidics. 2018 May 7;12(3):034103. doi: 10.1063/1.5030203. eCollection 2018 May.
Conventional cell-sized well arrays have advantages of high occupancy, simple operation, and low cost for capturing single-cells. However, they have insufficient space for including reagents required for cell treatment or analysis, which restricts the wide application of cell-sized well arrays as a single-cell research tool alone. Here, we present a novel dual-well array chip, which integrates capture-wells (20 m in diameter) with reaction-wells (100 m in diameter) and describe a flow method for convenient single-cell analysis requiring neither complicated infra-structure nor high expenditure, while enabling highly efficient single cell trapping (75.8%) with only 11.3% multi-cells. Briefly, the cells are first loaded into the dual-wells by gravity and then multi-cells in the reaction-wells are washed out by phosphate buffer saline. Next, biochemical reagents are loaded into reaction-wells using the scraping method and the chip is packed as a sandwich structure. We thereby successfully measured intracellular β-galactosidase activity of K562 cells at the single-cell level. We also used computational simulations to illustrate the working principle of dual-well structure and found out a relationship between the wall shear stress distribution and the aspect ratio of the dual-well array chip which provides theoretical guidance for designing multi-wells chip for convenient single-cell analysis. Our work produced the first dual-well chip that can simultaneously provide a high occupancy rate for single cells and sufficient space for reagents, as well as being low in cost and simple to operate. We believe that the feasibility and convenience of our method will enhance its use as a practical single-cell research tool.
传统的细胞尺寸孔阵列在捕获单细胞方面具有高占有率、操作简单和成本低的优点。然而，它们用于容纳细胞处理或分析所需试剂的空间不足，这限制了细胞尺寸孔阵列作为单一的单细胞研究工具的广泛应用。在此，我们展示了一种新型的双孔阵列芯片，它将捕获孔（直径20μm）与反应孔（直径100μm）集成在一起，并描述了一种流动方法，用于便捷的单细胞分析，既不需要复杂的基础设施，也不需要高额费用，同时能够实现高效的单细胞捕获（75.8%），多细胞率仅为11.3%。简而言之，细胞首先通过重力加载到双孔中，然后用磷酸盐缓冲盐水冲洗掉反应孔中的多细胞。接下来，使用刮涂法将生化试剂加载到反应孔中，并将芯片包装成三明治结构。我们从而成功地在单细胞水平上测量了K562细胞的细胞内β-半乳糖苷酶活性。我们还使用计算模拟来说明双孔结构的工作原理，并发现了双孔阵列芯片壁面剪应力分布与纵横比之间的关系，这为设计用于便捷单细胞分析的多孔芯片提供了理论指导。我们的工作制造出了首个双孔芯片，它能够同时为单细胞提供高占有率以及为试剂提供充足空间，而且成本低、操作简单。我们相信我们方法的可行性和便利性将增加其作为实用单细胞研究工具的应用。
