Intracellular protein determination using droplet-based immunoassays.
Intracellular protein determination using droplet-based immunoassays.
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使用基于液滴的免疫测定法测定细胞内蛋白质。
DOI：
10.1021/ac200876q
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发表时间：
2011
影响因子：
7.4
通讯作者：
Martino C
中科院分区：
文献类型：
--
作者：
Martino C
本文描述了一种使用微滴技术开发的用于分析细胞内蛋白质的灵敏的芯片上免疫分析方法的实现。该系统提供了许多分析功能，使低细胞数量的裂解以及蛋白质的检测和定量集成在一个单一的工艺流程中。将细胞以悬浮液形式引入装置，并在原位进行电溶解。细胞裂解物随后与抗体功能化的珠子一起被封装成稳定的油包水小滴，这些小滴被储存在芯片上。荧光检测到细胞内蛋白质与微球的结合。通过分析许多单独的液滴并针对标准添加获得的数据进行量化，我们测量了两种细胞内蛋白的水平，即在HEK-293细胞中表达的HRAS-mCitine和在MCF-7细胞中表达的肌动蛋白-EGFP。我们测定了这些蛋白质的浓度超过5个数量级，从∼50 PM到1μM。将这种半自动方法的结果与使用Western blotts进行测定的结果进行了比较，发现不仅速度更快，而且需要的细胞数量更少。
This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysedin situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ∼50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.
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影响因子：
7.4
作者：
Courtois, Fabienne;Olguin, Luis F.;Abell, Chris
通讯作者：
Abell, Chris
DOI：
10.1126/science.1188302
发表时间：
2010-06-25
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Huh D;Matthews BD;Mammoto A;Montoya-Zavala M;Hsin HY;Ingber DE
通讯作者：
Ingber DE
影响因子：
16.6
作者：
Joensson, Haakan N.;Samuels, Michael L.;Andersson-Svahn, Helene
通讯作者：
Andersson-Svahn, Helene
影响因子：
3.9
作者：
Zagnoni, Michele;Le Lain, Guillaume;Cooper, Jonathan M.
通讯作者：
Cooper, Jonathan M.
影响因子：
6.1
作者：
Zagnoni, Michele;Cooper, Jonathan M.
通讯作者：
Cooper, Jonathan M.