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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.

Concentric Forster resonance energy transfer (cFRET) is an emerging application of semiconductor quantum dots (QDs) that takes advantage of their excellent photoluminescence (PL) properties, small size, and versatile surface area. To date, cFRET configurations have combined QDs with only two fluorescent dyes, limiting multiplexed bioanalysis to two targets. Here, we expanded cFRET to create a triply multiplexed configuration that comprised a central QD assembled with multiple copies of three different peptide sequences labeled with one of three different fluorescent dyes. These dyes were Alexa Fluor 555 (A555), either Cyanine 3.5 (Cy3.5) or Atto 594 (At594), and Alexa Fluor 647 (A647). The expanded cFRET configuration had double the number of FRET pathways of previous systems, with three competitive pathways from the QD to the dyes and three sequential pathways between the dyes. The quenching efficiencies for QD and dye PL in the full three-acceptor cFRET configuration were successfully predicted from rate analyses of the simpler one-and two-acceptor configurations. This capability provides a basis for a priori design of cFRET probes with new luminescent materials or for new applications. Importantly, combinations of the A555/QD, Cy3.5/QD or At594/QD, and A647/QD PL intensity ratios were unique to particular combinations of the number of each acceptor dye per QD, permitting calculation of the number of each dye per QD from the measured PL ratios. This uniqueness was the basis of quantitative biosensing and enabled multiplexed and selective assays of the activities of three proteases (trypsin, chymotrypsin, and enterokinase) in parallel. The three-acceptor cFRET configuration is a potentially powerful multifunctional probe vector that challenges the current paradigm of N colors of QD for N biological targets. It is anticipated to be adaptable to many types of multiplexed bioanalysis and bioimaging applications.

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