Self-Quenching, Dimerization, and Homo-FRET in Hetero-FRET Assemblies with Quantum Dot Donors and Multiple Dye Acceptors

The combination of semiconductor quantum dots (QDs) and Forster resonance energy transfer (FRET) is a powerful tool for bioanalysis and imaging. Through FRET, the dye is able to borrow brightness from the QD, and the FRET efficiency can be tuned through the assembly of multiple acceptor dyes per QD. In principle, the fluorescence intensity from acceptor dyes assembled to a QD donor should always exceed that from the dyes alone, but we observed anomalously low acceptor dye fluorescence intensities in FRET systems with a QD donor and multiple Alexa Fluor 610 (A610) or Alexa Fluor 633 (A633) acceptors. In contrast, fluorescence from Alexa Fluor 555 (A555) or Alexa Fluor 647 (A647) acceptors was well-behaved and agreed with theoretical expectations. The difference between these systems was studied using a combination of UV-visible absorption and fluorescence intensity, lifetime, and anisotropy measurements. Anomalous fluorescence from A610 and A633 arose from the formation of nonfluorescent, H-type dimers of these dyes. The monomer-dimer equilibrium was shifted strongly in favor of the dimer as a result of the locally high concentration of dyes assembled to the QD. Both the lower number of monomeric dyes per QD and the introduction of a competitive energy transfer pathway from the QD to dimeric dyes contributed to the low dye fluorescence. Another consequence of the close proximity between the dyes was homo-FRET, which was particularly evident with A555 and A647 acceptors. Homo-FRET did not appear to lead to significant quenching of dye fluorescence, although there was some evidence of low-efficiency energy transfer to dyes that may act as modest energy sinks. The results of this study help inform the rational design of optimized QD-FRET probes for biological applications.