Development of microfluidic platform capable of high-throughput absolute quantification of single-cell multiple intracellular proteins from tumor cell lines and patient tumor samples
Development of microfluidic platform capable of high-throughput absolute quantification of single-cell multiple intracellular proteins from tumor cell lines and patient tumor samples
复制标题
开发能够对来自肿瘤细胞系和患者肿瘤样本的单细胞多种细胞内蛋白进行高通量绝对定量的微流体平台
DOI：
10.1016/j.bios.2020.112097
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发表时间：
2020-05-01
影响因子：
12.6
通讯作者：
Chen, Jian
中科院分区：
文献类型：
--
作者：
Liu, Lixing;Yang, Hongyu;Chen, Jian
单细胞蛋白质的定量在细胞异质性中起着关键作用，但由于技术限制，大量单细胞中的多个细胞内蛋白质的绝对数量仍然缺失，导致细胞类型分类的结果受到影响。本文介绍了一种能够高通量绝对定量单细胞多种细胞内蛋白质的微流控平台，其中被荧光标记抗体染色的细胞被吸入收缩微通道，激发的荧光信号被检测到，并根据荧光标记抗体的梯度溶液直接进入收缩微通道形成的校准曲线转换为目标蛋白的结合部位。基于这种方法，我们首先对数万个具有代表性的肿瘤细胞系中的β-肌动蛋白、α-微管蛋白和β-微管蛋白结合部位的单细胞数量进行了定量，得到了83.0+/-7.1%的细胞类型分类率。然后对数千个恶性程度不同的肿瘤细胞株的β-肌动蛋白、生物素和RhoA结合部位的单细胞数量进行了量化，报告的细胞类型分类率为93.7。2.8%。此外，从CAL27、WSU-HN6两个口腔肿瘤细胞系和两个口腔肿瘤患者样本中提取的数千个细胞的RAS、c-Myc和P53结合位点的单细胞数量被定量，有助于肿瘤细胞系(98.6%)和肿瘤患者样本(83.4%)的高分类。总之，开发的微流控平台能够对大量单细胞中的多种细胞内蛋白质进行定量，收集的蛋白质表达数据使有效的细胞类型分类成为可能。
Quantification of single-cell proteins plays key roles in cell heterogeneity while due to technical limitations absolute numbers of multiple intracellular proteins from large populations of single cells were still missing, leading to compromised results in cell-type classifications. This paper presents a microfluidic platform capable of high-throughput absolute quantification of single-cell multiple types of intracellular proteins where cells stained with fluorescent labelled antibodies are aspirated into the constriction microchannels with excited fluorescent signals detected and translated into numbers of binding sites of targeted proteins based on calibration curves formed by flushing gradient solutions of fluorescent labelled antibodies directly into constriction microchannels. Based on this approach, single-cell numbers of binding sites of beta-actin, alpha-tubulin and beta-tubulin from tens of thousands of five representative tumor cell lines were first quantified, reporting cell-type classification rates of 83.0 +/- 7.1%. Then single-cell numbers of binding sites of beta-actin, biotin and RhoA from thousands of five tumor cell lines with varieties in malignant levels were quantified, reporting cell-type classification rates of 93.7. 2.8%. Furthermore, single-cell numbers of binding sites of Ras, c-Myc and p53 from thousands of cells derived from two oral tumor lines of CAL 27, WSU-HN6 and two oral tumor patient samples were quantified, contributing to high classifications of both tumor cell lines (98.6%) and tumor patient samples (83.4%). In conclusion, the developed microfluidic platform was capable of quantifying multiple intracellular proteins from large populations of single cells, and the collected data of protein expressions enabled effective cell-type classifications.