On-slide detection of enzymatic activities in selected single cells

Josephine Geertsen Keller, Cinzia Tesauro, Andrea Coletta, Astrid Damgaard Graversen, Yi-Ping Ho, Peter Kristensen, Magnus Stougaard, Birgitta Ruth Knudsen

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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.

OriginalsprogEngelsk
TidsskriftNanoscale
Vol/bind9
Nummer36
Sider (fra-til)13546-13553
Antal sider8
ISSN2040-3364
DOI
StatusUdgivet - 2017

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