  Fluorescence resonance energy transfer (FRET) from individual, dye-labeled
RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is
carefully compared to FRET from unconfined RNA in solution. The use of
freely-diffusing droplets is a remarkably simple and high-throughput technique
that facilitates a substantial increase in signal-to-noise for
single-molecular-pair FRET measurements. We show that there can be dramatic
differences between FRET in solution and in droplets, which we attribute
primarily to an altered pH in the confining environment. We also demonstrate
that a sufficient concentration of a non-ionic surfactant mitigates this effect
and restores FRET to its neutral-pH solution value. At low surfactant levels,
even accounting for pH, we observe differences between the distribution of FRET
values in solution and in droplets which remain unexplained. Our results will
facilitate the use of nanoemulsion droplets as attoliter volume reactors for
use in biophysical and biochemical assays, and also in applications such as
protein crystallization or nanoparticle synthesis, where careful attention to
the pH of the confined phase is required.
