On-slide detection of enzymatic activities in selected single cells

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On-slide detection of enzymatic activities in selected single cells. / Keller, Josephine Geertsen; Tesauro, Cinzia; Coletta, Andrea; Graversen, Astrid Damgaard; Ho, Yi-Ping; Kristensen, Peter; Stougaard, Magnus; Knudsen, Birgitta Ruth.

In: Nanoscale, Vol. 9, 2017, p. 13546-13553.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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Keller JG, Tesauro C, Coletta A, Graversen AD, Ho Y-P, Kristensen P et al. On-slide detection of enzymatic activities in selected single cells. Nanoscale. 2017;9:13546-13553. https://doi.org/10.1039/c7nr05125e

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Keller, Josephine Geertsen ; Tesauro, Cinzia ; Coletta, Andrea ; Graversen, Astrid Damgaard ; Ho, Yi-Ping ; Kristensen, Peter ; Stougaard, Magnus ; Knudsen, Birgitta Ruth. / On-slide detection of enzymatic activities in selected single cells. In: Nanoscale. 2017 ; Vol. 9. pp. 13546-13553.

Bibtex

@article{9e4177b922e44dcfbcc0d9d1d2f275cb,
title = "On-slide detection of enzymatic activities in selected single cells",
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.",
keywords = "Journal Article",
author = "Keller, {Josephine Geertsen} and Cinzia Tesauro and Andrea Coletta and Graversen, {Astrid Damgaard} and Yi-Ping Ho and Peter Kristensen and Magnus Stougaard and Knudsen, {Birgitta Ruth}",
year = "2017",
doi = "10.1039/c7nr05125e",
language = "English",
volume = "9",
pages = "13546--13553",
journal = "Nanoscale",
issn = "2040-3364",
publisher = "ROYAL SOC CHEMISTRY",

}

RIS

TY - JOUR

T1 - On-slide detection of enzymatic activities in selected single cells

AU - Keller, Josephine Geertsen

AU - Tesauro, Cinzia

AU - Coletta, Andrea

AU - Graversen, Astrid Damgaard

AU - Ho, Yi-Ping

AU - Kristensen, Peter

AU - Stougaard, Magnus

AU - Knudsen, Birgitta Ruth

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

KW - Journal Article

U2 - 10.1039/c7nr05125e

DO - 10.1039/c7nr05125e

M3 - Journal article

C2 - 28872165

VL - 9

SP - 13546

EP - 13553

JO - Nanoscale

JF - Nanoscale

SN - 2040-3364

ER -