title: A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements.
abstract: L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10-500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA).
authors: Functional Materials and Devices, Fraunhofer Institute for Applied Polymer Research, Geiselberstr.69, 14476 Potsdam-Golm, Germany; Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany. Electronic address: piotr.cywinski@iap.fraunhofer.de.
authors: Piotr J
authors: PJ
authors: Cywiński
authors: Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany.
authors: Lydia
authors: L
authors: Olejko
authors: Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany.
authors: Hans-Gerd
authors: HG
authors: Löhmannsröben
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D019041:L-Selectin / Q000032:analysis* / Q000097:blood; D057230:Limit of Detection; D008163:Luminescent Measurements / Q000379:methods; D013725:Terbium / Q000737:chemistry
chemical_list: D052157:Aptamers, Nucleotide; D002232:Carbocyanines; D005456:Fluorescent Dyes; C085321:cyanine dye 5; D013725:Terbium; D019041:L-Selectin
keywords: Aptamer; FRET; Fluoroassay; L-selectin; Lanthanide; Luminescence spectroscopy