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On-slide detection of enzymatic activities in selected single cells

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Abstract

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.

Graphical abstract: On-slide detection of enzymatic activities in selected single cells

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Publication details

The article was received on 14 Jul 2017, accepted on 25 Aug 2017 and first published on 28 Aug 2017


Article type: Paper
DOI: 10.1039/C7NR05125E
Citation: Nanoscale, 2017, Advance Article
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    On-slide detection of enzymatic activities in selected single cells

    J. G. Keller, C. Tesauro, A. Coletta, A. D. Graversen, Y. Ho, P. Kristensen, M. Stougaard and B. R. Knudsen, Nanoscale, 2017, Advance Article , DOI: 10.1039/C7NR05125E

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