A microfluidic flow cytometer enabling absolute quantification of single-cell intracellular proteins
A microfluidic flow cytometer enabling absolute quantification of single-cell intracellular proteins
复制标题
能够对单细胞胞内蛋白质进行绝对定量的微流控流式细胞仪
DOI：
10.1039/c7lc00546f
复制
发表时间：
2017-09-21
期刊：
影响因子：
6.1
通讯作者：
Chen, Jian
中科院分区：
文献类型：
--
作者：
Li, Xiufeng;Fan, Beiyuan;Chen, Jian
单细胞蛋白质组学的定量提供了对细胞异质性的关键洞察，而传统的流式细胞术由于缺乏校准方法而不能提供单细胞内蛋白质的绝对定量。本文提出了一种基于微流控流式细胞仪的收缩通道(横截面积小于细胞)，能够采集特定细胞内蛋白质的拷贝数。在该平台中，被荧光标记的抗体染色的单个细胞被强制挤压通过收缩通道，荧光强度被量化，并且由于细胞在挤压过程中充满了收缩通道，带有荧光标记抗体的溶液被冲洗到收缩通道中以获得校准曲线。通过结合原始荧光数据和校正曲线，实现了胞内蛋白质的绝对定量。作为例证，单个肿瘤细胞的β-肌动蛋白拷贝数定量为0.9+/-0.30muM(A549，Ncell=14228)、2.34+/-0.70muM(MCF10A，Ncell=2455)和0.98+/-0.65muM(Hep G2，Ncell=6945)。单个小区的行进时间被量化为大约10ms，因此可以实现每个S 100个小区的吞吐量。这种微流控系统可以高通量地定量细胞内蛋白质的拷贝数，这可能成为单细胞蛋白质组学领域的一种使能技术。
Quantification of single-cell proteomics provides key insights into cellular heterogeneity while conventional flow cytometry cannot provide absolute quantification of intracellular proteins of single cells due to the lack of calibration approaches. This paper presents a constriction channel (with a cross sectional area smaller than cells) based microfluidic flow cytometer, capable of collecting copy numbers of specific intracellular proteins. In this platform, single cells stained with fluorescence labelled antibodies were forced to squeeze through the constriction channel with the fluorescence intensities quantified and since cells fully filled the constriction channel during the squeezing process, solutions with fluorescence labelled antibodies were flushed into the constriction channel to obtain calibration curves. By combining raw fluorescence data and calibration curves, absolute quantification of intracellular proteins was realized. As a demonstration, copy numbers of beta-actin of single tumour cells were quantified to be 0.90 +/- 0.30 mu M (A549, ncell = 14228), 2.34 +/- 0.70 mu M (MCF 10A, ncell = 2455), and 0.98 +/- 0.65 mu M (Hep G2, ncell = 6945). The travelling time for individual cells was quantified to be roughly 10 ms and thus a throughput of 100 cells per s can be achieved. This microfluidic system can be used to quantify the copy numbers of intracellular proteins in a high-throughput manner, which may function as an enabling technique in the field of single-cell proteomics.