Protein
Quantification Using Resonance Energy Transfer
between Donor Nanoparticles and Acceptor Quantum Dots
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Abstract
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 Eu<sup>3+</sup> 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
Förster 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