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

Abstract

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.

Spectral-Time Multiplexing in FRET Complexes of AgInS2/ZnS Quantum Dot and Organic Dyes

“…The latter comes from the existence of a set of exciton energy states inside QDs band-gap, the luminescent transitions from which with different decay times form the broad PL band of t-QDs [ 30 , 31 ]. This fact and large PL bandwidth of ternary QDs allow to potentially realize simultaneously several effective FRET channels from QDs to different acceptors with different absorption and radiation wavelengths, as was recently shown for Mn (II): CdS/ZnS QDs [ 29 ] and discussed in [ 27 , 32 , 33 ]. This yields FRET-induced luminescence of acceptors with different wavelengths and lifetimes, which opens up the possibility of LT coding of the labels.…”

Light microscopy of proteins in their ultrastructural context

“…Fluorescence microscopy, unlike electron microscopy, is however fundamentally prohibited from resolving the ultrastructural context of the cell: the smallest fluorescent labels, organic dyes, are ~1 nm in diameter, a size comparable to the distance between proteins in the densely crowded cellular interior 2 . Many binding sites are therefore masked or unreachable and neighboring fluorescent labels sterically hinder or self-quench each other via electron transfer or dipole-dipole interactions 3 . This limits the achievable fluorophore density and thereby the contrast and sampling necessary to resolve the crowded and complex ultrastructural context of the cell 4 .…”

Improvement of Optical Properties for Semiconductor Nanoparticles by the Precise Control of Electron and Energy Transfer

“…PL quenching at condense state is often explained in relation to the nonluminescent associates in case of molecular dyes, in which they behave as energy sinks as a result of energy transfer. 40,58 Although semiconductor NPs are not likely to make associates, those prepared by a current technique inevitably contain nonluminescent impurities. In addition, the "blinking" of semiconductor NPs are very common phenomena that is usually observed in single particle analyses due to the Auger recombination of charged NPs.…”

“…The FRET occurs between semiconductor NP fluorophores and acceptors and have been used for the detection of antibody-antigen interaction as well as other kinds of biological reactions. 39,40 However, besides these examples for sensing, homo-FRET is known to occur between well-passivated semiconductor NPs due to their small Stokes shifts peculiar to the band edge transition radiation. [40][41][42] In terms of developing good fluorophores, not only individual NPs but also ensemble should be cared.…”

“…39,40 However, besides these examples for sensing, homo-FRET is known to occur between well-passivated semiconductor NPs due to their small Stokes shifts peculiar to the band edge transition radiation. [40][41][42] In terms of developing good fluorophores, not only individual NPs but also ensemble should be cared. Even individual fluorophores possess high PL quantum yield, they decrease the QY upon condensation, which is well known as the concentration (or self ) quenching.…”