title: Green fluorescent protein in inertially injected aqueous nanodroplets.
abstract: 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.
authors: Physics Laboratory and Electronics and Electrical Engineering Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899, USA.
authors: Jianyong
authors: J
authors: Tang
authors: Ana M
authors: AM
authors: Jofre
authors: Geoffrey M
authors: GM
authors: Lowman
authors: Rani B
authors: RB
authors: Kishore
authors: Joseph E
authors: JE
authors: Reiner
authors: Kristian
authors: K
authors: Helmerson
authors: Lori S
authors: LS
authors: Goldner
authors: Mark E
authors: ME
authors: Greene
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D013053:Spectrophotometry; D013816:Thermodynamics
chemical_list: D049452:Green Fluorescent Proteins