一种基于微芯片快速生成双层乳化液滴的方法

Rapid generation of double-layer emulsion droplets based on microfluidic chip

摘要:

体外区室化 (In vitro compartmentalization，IVC) 是通过制备微液滴反应小室包裹单个基因 (包含表达体系) 或细胞进行反应和培养，从而建立表现型与基因型的偶联，并借助流式细胞仪 (Fluorescence-activated cell sorting，FACS) 对液滴进行超高通量检测和筛选，进而快速获得目标基因或细胞的一种方法。IVC-FACS筛选方法已被广泛应用于蛋白质工程、酶工程等定向进化研究。但早期利用机械分散法生成的微液滴大小均一性难以控制，严重影响液滴的定量检测，降低了筛选的效率和准确性。随着微流控芯片制备技术的快速发展，在芯片内快速生成微液滴的技术也愈加成熟。本研究首先利用W/O (Water-in-oil) 单层液滴生成芯片高速制备单分散的W1/O液滴，再将W1/O液滴重注入W/O/W (Water-in-oil-in-water) 双层乳化液滴生成芯片制备均一的W1/O/W2双层乳化液滴。通过对油、水相流速与比值的优化，可以生成直径在15.4–23.2 μm的单乳化微液滴，液滴可在培养数天内保持稳定。将单乳化液滴重注入双层乳化液滴芯片，通过调整油相流速，可以获得生成速度在1 000个液滴/s、直径在30–100 μm的双层乳化液滴。利用双层乳化液滴包埋的大肠杆菌细胞能正常进行培养和目标蛋白的诱导表达，为后续建立基于液滴和流式细胞仪的菌株高通量筛选方法奠定基础。

Abstract:

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.