微滴微流控技术在单细胞分析、抗体发现和定向进化等领域中被广泛应用，从而彻底改变了定量高通量生物测定和筛选的方法。然而，针对表型的荧光读出和分选的微滴微流体平台仍然主要用于专门实验室，由于它们的设置复杂。与传统的荧光激活细胞分选技术相辅相成，微流控方法可用于筛选细胞库中的分泌因子，甚至针对分泌或表面表达的因子对第二个细胞类型的影响进行筛选。此外，它们还可以通过PCR激活微滴分选来分离携带特定标记的遗传物质。在本文中，我们详细介绍了一种高通量微滴分析仪和分选器的构建方法，这可以由非专业人员在约45个工作小时内完成。所得的仪器配备有三个激光器，用于激发微滴中的荧光染料和光传感器，以在蓝色（425-465纳米）、绿色（505-545纳米）和红色（580-630纳米）光谱下获取荧光信号。此外，该仪器还允许通过分析通过微滴的亮场光强度来进行透射激活的微滴分选。该设置通过将荧光珠分选速度达到200赫兹，准确性达到99.4％的实验加以验证。我们展示了一个实验的结果，其中包含了单个细胞的微滴，根据基质金属蛋白酶活性的增加进行了分选，以展示我们的工作站在单个细胞分子生物学等领域中的应用，例如分析癌症转移的分子决定因素。© 2023. Springer Nature Limited.

Droplet microfluidics has revolutionized quantitative high-throughput bioassays and screening, especially in the field of single-cell analysis where applications include cell characterization, antibody discovery and directed evolution. However, droplet microfluidic platforms capable of phenotypic, fluorescence-based readouts and sorting are still mostly found in specialized labs, because their setup is complex. Complementary to conventional FACS, microfluidic droplet sorters allow the screening of cell libraries for secreted factors, or even for the effects of secreted or surface-displayed factors on a second cell type. Furthermore, they also enable PCR-activated droplet sorting for the isolation of genetic material harboring specific markers. In this protocol, we provide a detailed step-by-step guide for the construction of a high-throughput droplet analyzer and sorter, which can be accomplished in ~45 working hours by nonspecialists. The resulting instrument is equipped with three lasers to excite the fluorophores in droplets and photosensors that acquire fluorescence signals in the blue (425-465 nm), green (505-545 nm) and red (580-630 nm) spectrum. This instrument also allows transmittance-activated droplet sorting by analyzing the brightfield light intensity transmitting through the droplets. The setup is validated by sorting droplets containing fluorescent beads at 200 Hz with 99.4% accuracy. We show results from an experiment where droplets hosting single cells were sorted on the basis of increased matrix metalloprotease activity as an application of our workstation in single-cell molecular biology, e.g., to analyze molecular determinants of cancer metastasis.© 2023. Springer Nature Limited.