Microfluidic and Biological Engineering, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany;
Mol Cell Proteomics. 2013 Dec;12(12):3898-907. doi: 10.1074/mcp.M113.032821. Epub 2013 Sep 26.
Here, we present the full integration of a proximity ligation assay (PLA) on a microfluidic chip for systematic cell signaling studies. PLA is an in situ technology for the detection of protein interaction, post-translational modification, concentration, and cellular location with single-molecule resolution. Analytical performance advances on chip are achieved, including full automation of the biochemical PLA steps, target multiplexing, and reduction of antibody consumption by 2 orders of magnitude relative to standard procedures. In combination with a microfluidic cell-culturing platform, this technology allows one to gain control over 128 cell culture microenvironments. We demonstrate the use of the combined cell culture and protein analytic assay on chip by characterizing the Akt signaling pathway upon PDGF stimulation. Signal transduction is detected by monitoring the phosphorylation states of Akt, GSK-3β, p70S6K, S6, Erk1/2, and mTOR and the cellular location of FoxO3a in parallel with the PLA. Single-cell PLA results revealed for Akt and direct targets of Akt a maximum activation time of 4 to 8 min upon PDGF stimulation. Activation times for phosphorylation events downward in the Akt signaling pathway including the phosphorylation of S6, p70S6K, and mTOR are delayed by 8 to 10 min or exhibit a response time of at least 1 h. Quantitative confirmation of the Akt phosphorylation signal was determined with the help of a mouse embryonic fibroblast cell line deficient for rictor. In sum, this work with a miniaturized PLA chip establishes a biotechnological tool for general cell signaling studies and their dynamics relevant for a broad range of biological inquiry.
在这里，我们展示了将邻近连接分析（PLA）完全集成到微流控芯片上，用于系统的细胞信号研究。PLA 是一种用于检测蛋白质相互作用、翻译后修饰、浓度和细胞定位的原位技术，具有单分子分辨率。在芯片上实现了分析性能的提升，包括生化 PLA 步骤的完全自动化、目标的多重检测以及相对于标准程序抗体消耗减少 2 个数量级。结合微流控细胞培养平台，该技术可以控制 128 个细胞培养微环境。我们通过在 PDGF 刺激下对 Akt 信号通路进行特征描述，展示了这种组合的细胞培养和蛋白分析芯片的应用。通过监测 Akt、GSK-3β、p70S6K、S6、Erk1/2 和 mTOR 的磷酸化状态以及 FoxO3a 的细胞定位来检测信号转导，同时进行 PLA。单细胞 PLA 结果表明，在 PDGF 刺激下，Akt 和 Akt 的直接靶标最大激活时间为 4 到 8 分钟。Akt 信号通路中向下的磷酸化事件（包括 S6、p70S6K 和 mTOR 的磷酸化）的激活时间延迟 8 到 10 分钟，或者表现出至少 1 小时的反应时间。利用缺乏rictor 的小鼠胚胎成纤维细胞系对 Akt 磷酸化信号进行定量确认。总之，这项微型 PLA 芯片的工作为一般细胞信号研究及其对广泛生物学研究相关的动态建立了生物技术工具。
