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.

中文翻译：

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幻灯片检测选定的单个细胞中的酶活性

随着越来越多地认识到解决复杂的生物系统对解决细胞间异质性的重要性，对能够进行单细胞分析的测定法的需求日益增长。在当前的研究中，我们描述了从载玻片上的混合种群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，该载玻片用抗CD44抗体和特异性DNA引物双重功能化。捕获的CD44阳性细胞在玻片上的裂解导致人拓扑异构酶I的释放，从而使该酶环化添加到玻片上的特定线性DNA底物。产生的圆与锚定的DNA引物杂交，并充当固相支持的滚环扩增反应的模板，从而导致长串联重复产物的产生，在荧光标记探针杂交后，在荧光显微镜中以单分子水平检测到长串联重复产物。载玻片上检测系统被证明对CD44阳性细胞具有直接的定量和特异性。此外，它允许在单个细胞中检测人类拓扑异构酶I活性。