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

doi:10.1039/c7nr05125e

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 avis › Tidsskriftartikel › Forskning › peer review

7 Citationer (Scopus)

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.

Originalsprog	Engelsk
Tidsskrift	Nanoscale
Vol/bind	9
Nummer	36
Sider (fra-til)	13546-13553
Antal sider	8
ISSN	2040-3364
DOI	https://doi.org/10.1039/c7nr05125e
Status	Udgivet - 28 sep. 2017

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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.",

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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",

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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 - 2017/9/28

Y1 - 2017/9/28

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

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U2 - 10.1039/c7nr05125e

DO - 10.1039/c7nr05125e

M3 - Journal article

C2 - 28872165

SN - 2040-3364

VL - 9

SP - 13546

EP - 13553

JO - Nanoscale

JF - Nanoscale

IS - 36

ER -

On-slide detection of enzymatic activities in selected single cells

Abstract

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