Issue 8, 2014

Non-competitive aptamer-based quenching resonance energy transfer assay for homogeneous growth factor quantification

Abstract

A non-competitive homogeneous, single-label quenching resonance energy transfer (QRET) assay for protein quantification is now presented using lanthanide-chelate labeled nucleic acid aptamers. A labeled ssDNA aptamer binding to a growth factor has been successfully used to provide luminescence signal protection of the lanthanide label. The QRET technology has previously been applied to competitive assay formats, but now for the first time a direct non-competitive assay is presented. The QRET system is based on the protection of the Eu(III)-chelate from a soluble quencher molecule when the aptamer interacts with a specific target protein. The direct QRET assay is possible as the aptamer structure itself cannot protect the Eu(III)-label from quenching. The dynamic range for the optimized vascular endothelial growth factor (VEGF) assay is 0.25–10 nM. A successful quantification of the basic fibroblast growth factor (bFGF) is also demonstrated using the same QRET assay format with a dynamic range of 0.75–50 nM. These assays evidently show the suitability of the direct QRET technique to simple and efficient detection of large biomolecules. The QRET assay can potentially be applied as a detection platform for any other protein targets with a known aptamer sequence.

Graphical abstract: Non-competitive aptamer-based quenching resonance energy transfer assay for homogeneous growth factor quantification

Supplementary files

Article information

Article type
Paper
Submitted
23 Sep 2013
Accepted
23 Jan 2014
First published
24 Jan 2014

Analyst, 2014,139, 2016-2023

Non-competitive aptamer-based quenching resonance energy transfer assay for homogeneous growth factor quantification

K. Kopra, M. Syrjänpää, P. Hänninen and H. Härmä, Analyst, 2014, 139, 2016 DOI: 10.1039/C3AN01814H

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