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.