Department of Molecular Biology and Genetics, C. F. Møllers Allé 3, Bldg. 1131, Aarhus University, 8000 Aarhus C, Denmark.
Nanoscale. 2017 Sep 21;9(36):13546-13553. doi: 10.1039/c7nr05125e.
With increasing recognition of the importance in addressing cell-to-cell heterogeneity for the understanding of complex biological systems, there is a growing need for assays capable of single cell analyses. In the current study, we describe the measurement of human topoisomerase I activity in single CD44 positive Caco2 cells specifically captured from a mixed population on glass slides, which were dual functionalized with anti-CD44-antibodies and specific DNA primers. On-slide lysis of captured CD44 positive cells, resulted in the release of human topoisomerase I, allowing the enzyme to circularize a specific linear DNA substrate added to the slides. The generated circles hybridized to the anchored DNA primers and acted as templates for a solid support rolling circle amplification reaction leading to the generation of long tandem repeat products that were detected at the single molecule level in a fluorescent microscope upon hybridization of fluorescent labelled probes. The on-slide detection system was demonstrated to be directly quantitative and specific towards CD44 positive cells. Moreover, it allowed reproducible detection of human topoisomerase I activity in single cells.
随着人们越来越认识到在理解复杂生物系统时解决细胞间异质性的重要性，人们越来越需要能够进行单细胞分析的检测方法。在本研究中，我们描述了在玻璃载玻片上从混合群体中特异性捕获的单个 CD44 阳性 Caco2 细胞中人类拓扑异构酶 I 活性的测量，这些细胞通过抗 CD44 抗体和特异性 DNA 引物双重功能化。捕获的 CD44 阳性细胞在载玻片上的裂解导致人类拓扑异构酶 I 的释放，从而使酶能够使添加到载玻片上的特定线性 DNA 底物环化。生成的环与锚定的 DNA 引物杂交，并充当用于固体支持滚环扩增反应的模板，导致长串联重复产物的生成，在荧光显微镜下通过荧光标记探针的杂交在单分子水平上检测到这些产物。该载玻片上的检测系统被证明对 CD44 阳性细胞具有直接的定量和特异性。此外，它允许在单细胞中重复检测人类拓扑异构酶 I 活性。
