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Protein quantification using resonance energy transfer between donor nanoparticles and acceptor quantum dots

  • A homogeneous time-resolved luminescence resonance energy transfer (TR-LRET) assay has been developed to quantify proteins. The competitive assay is based on resonance energy transfer (RET) between two luminescent nanosized particles. Polystyrene nanoparticles loaded with Eu3+ chelates (EuNPs) act as donors, while protein-coated quantum dots (QDs), either CdSe/ZnS emitting at 655 nm (QD655-strep) or CdSeTe/ZnS with emission wavelength at 705 nm (QD705-strep), are acceptors. In the absence of analyte protein, in our case bovine serum albumin (BSA), the protein-coated QDs bind nonspecifically to the EuNPs, leading to RET. In the presence of analyte proteins, the binding of the QDs to the EuNPs is prevented and the RET signal decreases. RET from the EuNPs to the QDs was confirmed and characterized with steady-state and time-resolved luminescence spectroscopy. In accordance with the Forster theory, the approximate average donor acceptor distance is around 15 nm at RET efficiencies, equal to 15% for QD655 and 13% for QD705 acceptor,A homogeneous time-resolved luminescence resonance energy transfer (TR-LRET) assay has been developed to quantify proteins. The competitive assay is based on resonance energy transfer (RET) between two luminescent nanosized particles. Polystyrene nanoparticles loaded with Eu3+ chelates (EuNPs) act as donors, while protein-coated quantum dots (QDs), either CdSe/ZnS emitting at 655 nm (QD655-strep) or CdSeTe/ZnS with emission wavelength at 705 nm (QD705-strep), are acceptors. In the absence of analyte protein, in our case bovine serum albumin (BSA), the protein-coated QDs bind nonspecifically to the EuNPs, leading to RET. In the presence of analyte proteins, the binding of the QDs to the EuNPs is prevented and the RET signal decreases. RET from the EuNPs to the QDs was confirmed and characterized with steady-state and time-resolved luminescence spectroscopy. In accordance with the Forster theory, the approximate average donor acceptor distance is around 15 nm at RET efficiencies, equal to 15% for QD655 and 13% for QD705 acceptor, respectively. The limits of detection are below 10 ng of BSA with less than a 10% average coefficient of variation. The assay sensitivity is improved, when compared to the most sensitive commercial methods. The presented mix-and-measure method has potential to be implemented into routine protein quantification in biological laboratories.show moreshow less

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Metadaten
Author:Harri Harma, Sari Pihlasalo, Piotr J. Cywinski, Piia Mikkonen, Tommy Hammann, Hans-Gerd LöhmannsröbenGND, Pekka Hanninen
DOI:https://doi.org/10.1021/ac303586n
ISSN:0003-2700 (print)
Parent Title (English):Analytical chemistry
Publisher:American Chemical Society
Place of publication:Washington
Document Type:Article
Language:English
Year of first Publication:2013
Year of Completion:2013
Release Date:2017/03/26
Volume:85
Issue:5
Pagenumber:6
First Page:2921
Last Page:2926
Funder:Marie Curie European Reintegration grant; QUANTUMDOTIMPRINT [PERG05-GA-2009-247825]; FP7 Collaborative Project, NANO-GNOSTICS [HEALTH-F5-2009-242264]
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie
Peer Review:Referiert