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Enrichment of rare events using a multi-parameter high throughput microfluidic droplet sorter.
Lab on a Chip ( IF 6.1 ) Pub Date : 2020-01-24 , DOI: 10.1039/c9lc00790c Sheng-Ting Hung 1 , Srijit Mukherjee 2 , Ralph Jimenez 2
Lab on a Chip ( IF 6.1 ) Pub Date : 2020-01-24 , DOI: 10.1039/c9lc00790c Sheng-Ting Hung 1 , Srijit Mukherjee 2 , Ralph Jimenez 2
Affiliation
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High information content analysis, enrichment, and selection of rare events from a large population are of great importance in biological and biomedical research. The fluorescence lifetime of a fluorophore, a photophysical property which is independent of and complementary to fluorescence intensity, has been incorporated into various imaging and sensing techniques through microscopy, flow cytometry and droplet microfluidics. However, the throughput of fluorescence lifetime activated droplet sorting is orders of magnitude lower than that of fluorescence activated cell sorting, making it unattractive for applications such as directed evolution of enzymes, despite its highly effective compartmentalization of library members. We developed a microfluidic sorter capable of selecting fluorophores based on fluorescence lifetime and brightness at two excitation and emission colors at a maximum droplet rate of 2.5 kHz. We also present a novel selection strategy for efficiently analyzing and/or enriching rare fluorescent members from a large population which capitalizes on the Poisson distribution of analyte encapsulation into droplets. The effectiveness of the droplet sorter and the new selection strategy are demonstrated by enriching rare populations from a ∼108-member site-directed mutagenesis library of fluorescent proteins expressed in bacteria. This selection strategy can in principle be employed on many droplet sorting platforms, and thus can potentially impact broad areas of science where analysis and enrichment of rare events is needed.
中文翻译：
使用多参数高通量微流体液滴分选仪富集罕见事件。
高信息含量的分析、丰富以及从大量群体中选择稀有事件对于生物学和生物医学研究非常重要。荧光团的荧光寿命是一种独立于荧光强度且与荧光强度互补的光物理特性，已通过显微镜、流式细胞术和液滴微流体技术被纳入各种成像和传感技术中。然而，荧光寿命激活液滴分选的通量比荧光激活细胞分选低几个数量级，这使得它对于酶的定向进化等应用没有吸引力，尽管它对文库成员进行了高效的划分。我们开发了一种微流体分选器，能够根据两种激发和发射颜色的荧光寿命和亮度以 2.5 kHz 的最大液滴速率选择荧光团。我们还提出了一种新颖的选择策略，用于有效分析和/或富集大量群体中的稀有荧光成员，该策略利用分析物封装到液滴中的泊松分布。通过从细菌表达的荧光蛋白定点诱变库中富集稀有种群，证明了液滴分选器和新选择策略的有效性。这种选择策略原则上可以在许多液滴分选平台上采用，因此可能会影响需要分析和富集罕见事件的广泛科学领域。
更新日期：2020-02-19
中文翻译：
使用多参数高通量微流体液滴分选仪富集罕见事件。
高信息含量的分析、丰富以及从大量群体中选择稀有事件对于生物学和生物医学研究非常重要。荧光团的荧光寿命是一种独立于荧光强度且与荧光强度互补的光物理特性，已通过显微镜、流式细胞术和液滴微流体技术被纳入各种成像和传感技术中。然而，荧光寿命激活液滴分选的通量比荧光激活细胞分选低几个数量级，这使得它对于酶的定向进化等应用没有吸引力，尽管它对文库成员进行了高效的划分。我们开发了一种微流体分选器，能够根据两种激发和发射颜色的荧光寿命和亮度以 2.5 kHz 的最大液滴速率选择荧光团。我们还提出了一种新颖的选择策略，用于有效分析和/或富集大量群体中的稀有荧光成员，该策略利用分析物封装到液滴中的泊松分布。通过从细菌表达的荧光蛋白定点诱变库中富集稀有种群，证明了液滴分选器和新选择策略的有效性。这种选择策略原则上可以在许多液滴分选平台上采用，因此可能会影响需要分析和富集罕见事件的广泛科学领域。
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