Abstract
CdSe/ZnS core/shell quantum dots (QDs) are used as efficient Förster Resonance Energy Transfer (FRET) acceptors in a time-resolved immunoassays with Tb complexes as donors providing a long-lived luminescence decay. A detailed decay time analysis of the FRET process is presented. QD FRET sensitization is evidenced by a more than 1000-fold increase of the QD luminescence decay time reaching ca. 0.5 milliseconds, the same value to which the Tb donor decay time is quenched due to FRET to the QD acceptors. The FRET system has an extremely large Förster radius of approx. 100 Å and more than 70% FRET efficiency with a mean donor-acceptor distance of ca. 84 Å, confirming the applied biotin-streptavidin binding system. Time-resolved measurement allows for suppression of short-lived emission due to background fluorescence and directly excited QDs. By this means a detection limit of 18 attomol QDs within the immunoassay is accomplished, an improvement of more than two orders of magnitude compared to commercial systems.
Highlights
CdSe/ZnS semiconductor nanocrystals or quantum dots (QDs) possess unrivalled photophysical properties, such as size tunable emission wavelengths, extremely high extinction coefficients over a broad absorption spectrum and enhanced photostability compared to organic fluorophores [1,2,3,4]
We extend these investigations by a thorough analysis of donor and acceptor luminescence decay times, which are important parameters for understanding the dynamic parameters of the Forster Resonance Energy Transfer (FRET) process [11, 18]
For both QD as well as Tb luminescence decay times, we provide further evidence of efficient QD sensitization by FRET from Tb
Summary
CdSe/ZnS semiconductor nanocrystals or quantum dots (QDs) possess unrivalled photophysical properties, such as size tunable emission wavelengths, extremely high extinction coefficients over a broad absorption spectrum and enhanced photostability compared to organic fluorophores [1,2,3,4]. Journal of Biomedicine and Biotechnology the short-lived background emission and QD fluorescence (from directly excited QDs), and optimizing laser excitation (new laser system with low background emission) and solvent conditions (azide-free solvent leading to decreased luminescence quenching of Tb), a very low detection limit is obtained. This means that a sensitivity improvement of more than two orders of magnitude is accomplished, taking the well established and extensively studied Eu-TBP (Eu3+tris(bypyridine) and APC (allophycocyanin) donor-acceptor system [19,20,21], used within the same streptavidin-biotin assay format, for comparison. The presented results demonstrate the great potential of the Tb to QD FRET system for highly sensitive homogeneous immunoassays for biological as well as clinical and medical applications
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