On-slide detection of enzymatic activities in selected single cells.
Nanoscale
; 9(36): 13546-13553, 2017 Sep 21.
Article
em En
| MEDLINE
| ID: mdl-28872165
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Topoisomerases Tipo I
/
Análise de Célula Única
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Nanoscale
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Dinamarca