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A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

  • 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 Forster 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 luminescenceL-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 Forster 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). (C) 2015 Elsevier B.V. All rights reserved.show moreshow less

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Metadaten
Author:Piotr J. Cywinski, Lydia OlejkoORCiDGND, Hans-Gerd LöhmannsröbenGND
DOI:https://doi.org/10.1016/j.aca.2015.06.045
ISSN:0003-2670 (print)
ISSN:1873-4324 (online)
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=26320804
Parent Title (English):Analytica chimica acta : an international journal devoted to all branches of analytical chemistry
Publisher:Elsevier
Place of publication:Amsterdam
Document Type:Article
Language:English
Year of first Publication:2015
Year of Completion:2015
Release Date:2017/03/27
Tag:Aptamer; FRET; Fluoroassay; L-selectin; Lanthanide; Luminescence spectroscopy
Volume:887
Pagenumber:7
First Page:209
Last Page:215
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie
Peer Review:Referiert