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 μM (A549, n_cell = 14228), 2.34 ± 0.70 μM (MCF 10A, n_cell = 2455), and 0.98 ± 0.65 μM (Hep G2, n_cell = 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.

中文翻译：

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微流式细胞仪可对单细胞胞内蛋白进行绝对定量

单细胞蛋白质组学的定量分析提供了对细胞异质性的重要见解，而传统的流式细胞术由于缺乏校准方法而无法提供单细胞的细胞内蛋白的绝对定量分析。本文提出了一种基于收缩通道（横截面积小于细胞）的微流式细胞仪，能够收集特定细胞内蛋白的拷贝数。在这个平台上，被荧光标记抗体染色的单细胞被迫通过荧光强度被定量的收缩通道挤压，并且由于细胞在挤压过程中完全充满了收缩通道，因此将带有荧光标记抗体的溶液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准曲线，实现了细胞内蛋白质的绝对定量。作为演示，单个肿瘤细胞的β-肌动蛋白的拷贝数被定量为0.90±0.30μM（A549，n个_单元格= 14228），2.34±0.70μM（MCF 10A，n个_单元格= 2455）和0.98±0.65μM（Hep G2，n个_单元格= 6945）。单个单元格的行进时间被量化为大约10毫秒，因此可以实现100个单元格/秒的吞吐量。该微流体系统可以用于以高通量的方式定量细胞内蛋白质的拷贝数，其可以用作单细胞蛋白质组学领域的使能技术。