In vitro compartmentalization-based fluorescence-activated cell sorting (IVC-FACS) is a powerful screening tool for directed evolution of enzymes. However, the efficiency of IVC-FACS is limited by the tendency of the fluorescent reporter to diffuse out of the droplets, which decouples the genotype and phenotype of the target gene. Herein we present a new strategy called fluorescence droplet entrapment (FDE) to solve this problem. The substrate is designed with a polarity that enables it to pass through the oil phase, react with the enzyme and generate an oil-impermeable and fluorescent product that remains entrapped inside the droplet. Several FDE substrates were designed, using two distinct substrate engineering strategies, for the detection of phosphotriesterases, carboxylesterases, and glycosidases activities. Model screening assays in which rare phosphotriesterase-active cells were enriched from large excesses of inactive cells showed that the enrichment efficiency achievable using an FDE substrate was as high as 900-fold: the highest yet reported in such an IVC-FACS system. Thus, FDE provides a means to tightly control the onset of the enzymatic reaction, minimize droplet cross-talk, and lower the background fluorescence. It therefore may serve as a useful strategy for the IVC-FACS screening of enzymes, antibodies, and other proteins.

中文翻译：

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基于IVC-FACS的超高通量筛选的基质工程实现荧光液滴捕获

基于体外区室化的荧光激活细胞分选（IVC-FACS）是用于酶的定向进化的强大筛选工具。但是，IVC-FACS的效率受到荧光报告分子从液滴中扩散的趋势的限制，该趋势使靶基因的基因型和表型脱钩。在本文中，我们提出了一种称为荧光滴捕获（FDE）的新策略来解决此问题。底物的极性设计使其能够通过油相，与酶反应并产生不透油的荧光产品，并保留在液滴内部。使用两种不同的底物工程设​​计策略，设计了几种FDE底物，用于检测磷酸三酯酶，羧酸酯酶和糖苷酶的活性。从大量过量的无活性细胞中富集稀有磷酸三酯酶活性细胞的模型筛选试验表明，使用FDE底物可获得的富集效率高达900倍：在这种IVC-FACS系统中报道的最高效率。因此，FDE提供了一种手段来严格控制酶促反应的发生，最小化液滴串扰并降低背景荧光。因此，它可以作为IVC-FACS筛选酶，抗体和其他蛋白质的有用策略。最小化液滴串扰，并降低背景荧光。因此，它可以作为IVC-FACS筛选酶，抗体和其他蛋白质的有用策略。最小化液滴串扰，并降低背景荧光。因此，它可以作为IVC-FACS筛选酶，抗体和其他蛋白质的有用策略。