Single cell multiplexed assay for proteolytic activity using droplet microfluidics
Single cell multiplexed assay for proteolytic activity using droplet microfluidics
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DOI：
10.1016/j.bios.2016.03.002
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发表时间：
2016-07-15
影响因子：
12.6
通讯作者：
Chen, Chia-Hung
中科院分区：
文献类型：
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作者：
Ng, Ee Xien;Miller, Miles A.;Chen, Chia-Hung
细胞酶以翻译后调节的方式相互作用以控制单个细胞的行为，但当前的平台技术在单个细胞中同时测量多种酶活性的能力受到限制。在这里，我们开发了基于多色福斯特共振能量转移（FRET）的酶底物，并将其用于微流体平台中，以同时测量含有单细胞的油包水滴的多种特定蛋白酶活性。通过将微流体平台与计算分析方法蛋白水解活性矩阵分析 (PrAMA) 相结合，我们能够以高通量方式推断来自单个细胞的六种不同的蛋白酶活性信号（类似于 100 个细胞/实验运行）。我们使用活细胞和原位细胞裂解测定形式，以单细胞分辨率表征了几种癌细胞系的蛋白酶活性谱，包括乳腺癌细胞 MDA-MB-231、肺癌细胞系 PC-9 和白血病细胞系 K-562，特别关注在转移中重要的金属蛋白酶。测量单个细胞分泌或表达的多种蛋白酶的能力使我们能够表征细胞异质性，并具有潜在的应用，包括系统生物学、药理学、癌症诊断和干细胞生物学。 (C) 2016 Elsevier B.V. 保留所有权利。
Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Forster resonance energy transfer (FRET) based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (similar to 100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. (C) 2016 Elsevier B.V. All rights reserved.