Generation and mixing of subfemtoliter aqueous droplets on demand.

We describe a novel method of generating monodisperse subfemtoliter aqueous droplets on demand by means of piezoelectric injection. Droplets with volumes down to 200 aL are generated by this technique. The droplets are injected into a low refractive index perfluorocarbon so that they can be optically trapped. We demonstrate the use of optical tweezers to manipulate and mix droplets. For example, using optical tweezers we bring two droplets, one containing a calcium sensitive dye and the other calcium chloride, into contact. The droplets coalesce with a resulting reaction time of about 1 ms. The monodispersity, manipulability, repeatability, small size, and fast mixing afforded by this system offer many opportunities for nanochemistry and observation of chemical reactions on a molecule-by-molecule basis.

Droplet confinement and fluorescence measurement of single molecules.

We describe a method for molecular confinement and single-fluorophore sensitive measurement in aqueous nanodroplets in oil. The sequestration of individual molecules in droplets has become a useful tool in genomics and molecular evolution. Similarly, the use of single fluorophores, or pairs of fluorophores, to study biomolecular interactions and structural dynamics is now common. Most often these single-fluorophore sensitive measurements are performed on molecules that are surface attached. Confinement via surface attachment permits molecules to be located and studied for a prolonged period of time. For molecules that denature on surfaces, for interactions that are transient or out-of-equilibrium, or to observe the dynamic equilibrium of freely diffusing reagents, surface attachment may not be an option. In these cases, droplet confinement presents an alternative method for molecular confinement. Here, we describe this method as used in single-fluorophore sensitive measurement and discuss its advantages, limitations, and future prospects.

Green fluorescent protein in inertially injected aqueous nanodroplets.

We inertially inject and study the contents of optically trappable aqueous nanodroplets (hydrosomes) emulsified in a perfluorinated matrix. A new piezoelectric actuated device for production of single hydrosomes on demand is introduced. Hydrosomes containing enhanced green fluorescent protein (EGFP) were injected, optically trapped, and held at the focus of an excitation laser in a confocal microscope, and single-molecule photobleaching events were observed. The rotational diffusion time of EGFP in trapped hydrosomes was measured using time-resolved fluorescence anisotropy. In free solution, the mean rotational diffusion time was determined to be 13.8 +/- 0.1 ns at 3 microM and 14.0 +/- 0.2 ns at 10 microM. In hydrosomes, the mean rotational diffusion time was similar and determined to be 12.6 +/- 1.0 ns at 3 microM and 15.5 +/- 1.6 ns at 10 microM. We conclude that the rotational motion inside the nanodroplets is consistent with rotation in free solution and that the protein therefore does not aggregate at the water-oil interface. Protein can be confined in hydrosomes with high efficiency using this technique, which provides an alternative to surface attachment or lipid encapsulation and opens up new avenues of research using single molecules contained in fluid nanovolumes.