Ultrasimple Single-Cell Detection of Multiple Cytokines by a Nanowell Chip Integrated with Encoded Microarrays
Ultrasimple Single-Cell Detection of Multiple Cytokines by a Nanowell Chip Integrated with Encoded Microarrays
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DOI：
10.1021/acssensors.9b00765
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发表时间：
2019-09-01
期刊：
影响因子：
8.9
通讯作者：
Wang, Jun
中科院分区：
文献类型：
--
作者：
Abdullah, Mohammed A. A.;Wang, Jun
细胞因子的产生通常被认为是免疫细胞活化状态的标志。细胞因子的谱和时间分泌在细胞表型之间并且甚至在相同表型内显著变化。单个免疫细胞的细胞因子分泌的多参数分析一直是一个具有挑战性和复杂的过程，需要在实验室环境中的特殊设施。在此，我们提出了一种超简单的方法，具有高灵敏度和高鲁棒性，以单细胞分辨率定量细胞因子表达。微芯片是基于胶带上的聚二甲基硅氧烷微细胞开发的，而每个微细胞与DNA-抗体可转换微阵列集成。整个单细胞分析过程只需要移液。通过测量六种重组细胞因子蛋白的不同浓度来评价测定的灵敏度，发现其与常规方法相当。一旦将单细胞加载到细胞中并在那里孵育，则使用Fluorinert FC-40分离细胞;因此，来自这些细胞的细胞因子被单独的微阵列捕获。夹心酶联免疫吸附测定检测过程的其余部分也简单地通过移液各种试剂来执行。该方法通过测量来自数百个单细胞的细胞因子产生来验证。它将典型的复杂的多重单细胞测定简化为无仪器的检测点技术，因此可以在临床诊断中找到广泛的实用性。
Cytokine production is often regarded as the marker of immune cells' activation status. The spectrum and temporal secretion of cytokines are dramatically varied between cell phenotypes and even within the same phenotype. Multiparameter analysis of individual immune cell's cytokine secretion has always been a challenging and complicated process that needs special facilities in a laboratory setting. Herein, we present an ultrasimple method with high sensitivity and high robustness to quantify cytokine expression at the single-cell resolution. A microchip is developed based on poly(dimethylsiloxane) nanowells on sticky tape, while each nanowell is integrated with a DNA-antibody convertible microarray. Only pipetting is needed for the whole single-cell analysis process. The sensitivity of the assay is evaluated by measuring various concentrations of six recombinant cytokine proteins, which was found comparable to conventional methods. Once single cells are loaded to nanowells and incubated there, a Fluorinert FC-40 is used to isolate nanowells; so, cytokines from those cells are captured by separate microarrays. The rest of the sandwich enzyme-linked immunosorbent assay detection process is also executed simply by pipetting of various reagents. This method is validated by measuring cytokine production from hundreds of single cells. It has simplified a typically sophisticated multiplex single-cell assay into an instrument-free, point-of-detection technology, and thus it may find a broad utility in clinical diagnostics.