Single-pair measurements of nonradiative energy transfer efficiency between quantum dots and organic dyes reveals orientational variation

Förster resonance energy transfer (FRET) is a technique that can be used to measure the nanoscale distances and to report on biomolecular dynamics at the single-molecule level. Organic dyes FRET pairs are commonly used in biophysical studies, but this approach suffers from the fact that the organic dyes have short bleaching lifetimes. Quantum dots have longer lifetimes, which makes them ideal for use as FRET donors in longer experiments. However, the energy transfer between quantum dots and organic dye molecule acceptors has not formerly been characterized at the single-molecule level. Therefore, we used TIRF microscopy to observe surface immobilized FRET pairs separated by known lengths of duplex DNA. We used two methods to construct these assemblies. In both cases, we observed broad distributions of FRET efficiencies and little correlation between duplex length and the average FRET efficiency. However, when we probe multiple donor-acceptor distances for a particular quantum dot, we observe the expected decrease in FRET efficiency with increasing duplex length. We attribute these contradictory observations to variations in the orientation factor for each single quantum dot, based on its inherent dipole moment and the location at which the DNA molecule is coupled to its surface.