Ana Jofre

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.

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.

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.

Tuning the optical forces on- and off-resonance in microspherical photonics

Light pressure effect has been discovered long ago and has been used as an optical method to manipulate micro- and nanoparticles. It is usually considered as a nonresonant effect determined by the transfer of the momentum of light. However, recently we have observed that large polystyrene microspheres of 15 - 20 μm diameters supporting high quality whispering gallery resonances can be optically propelled in water at an extraordinary high velocity along tapered fibers under resonant conditions. In this work we compare on- and off-resonant optical forces in microspherical photonics by controlling the detuning between the laser emission line and whispering gallery resonances. Our approach involves manipulation with microspheres using conventional optical tweezers and their advanced spectroscopic characterization in fiber-integrated setups. We demonstrate dramatic difference in the optical forces exerted on microspheres in the on-resonant and off-resonant cases. This method can be used to study spectral properties of the resonantly enhanced forces in microspherical photonics.

Conformational diversity of short DNA duplex.

Two 25 base-pair cDNA strands are encapsulated within an optically trapped nanodroplet, and we observe the kinetics of their hybridization in dynamic equilibrium via single-molecule fluorescence resonance energy transfer (FRET) as a function of temperature and of the solutions NaCl concentration. We have observed the duplex unfolding and refolding, and we have observed quasistable partially unfolded states under low salinity conditions. Furthermore, our measurements reveal that, even in conditions under which the duplex is stable, it undergoes conformational fluctuations in solution.

Hydrosomes: femtoliter containers for fluorescence spectroscopy studies

We report on improvements and innovations in the use of hydrosomes to encapsulate and study single molecules. Hydrosomes are optically-trappable aqueous nanodroplets. The droplets are suspended in a fluorocarbon medium that is immiscible with water and has an index of refraction lower than water, so hydrosomes are stable and optically trapped by a focused laser beam (optical tweezers). Using optical tweezers, we hold the hydrosomes within a confocal observation volume and interrogate the encapsulated molecule by fluorescence excitation. This method allows for long observation times of a molecule without the need for surface immobilization or liposome encapsulation. We have developed a new way for creating hydrosomes on demand by inertially launching them into the fluorocarbon matrix using a piezo-activated micropipette. Time-resolved fluorescence anisotropy studies are carried out to characterize the effects of the hydrosome interface boundary on biological molecules and to determine whether molecules encapsulated within hydrosomes diffuse freely throughout the available volume. We measured the fluorescence anisotropy decay of 20mer DNA duplexes, and enhanced green fluorescent protein (GFP). We conclude that the molecules rotate freely inside the nanodroplets and do not stick or aggregate at the boundary.

Hydrosomes: Optically trapped water droplets as nano-containers

We demonstrate a novel technique for creating, manipulating, and combining femtoliter to attoliter volume chemical containers. Possible uses include creating controlled chemical reactions involving small quantities of reagent, and studying the dynamics of single molecules. The containers, which we call hydrosomes, are surfactant stabilized aqueous droplets in a low index-of-refraction fluorocarbon medium. The index of refraction mismatch between the container and fluorocarbon is such that individual hydrosomes can be optically trapped by single focus laser beams, i.e. optical tweezers. Previous work on single molecules usually involved the tethering of the molecule to a surface, in order to interrogate the molecule for an extended period of time. The use of hydrosomes opens up the possibility for studying free molecules, away from any perturbing surface. We show that this is indeed true in the case of quantitative FRET with RNA. Furthermore, we demonstrate the controlled fusion of two hydrosomes for studying reactions, such as DNA binding kinetics, and single molecule dynamics under non-equilibrium conditions. We also show the applicability of our technique in analytical chemistry, such as for molecule identification and sorting.

Stable and robust nanotubes formed from self-assembled polymer membranes

We create long polymer nanotubes by directly pulling on the membrane of polymersomes using either optical tweezers or a micropipette. The polymersomes are composed of amphiphilic diblock copolymers and the nanotubes formed have an aqueous core connected to the aqueous interior of the polymersome. We stabilize the pulled nanotubes by subsequent chemical cross-linking. The cross-linked nanotubes are extremely robust and can be moved to another medium for use elsewhere. We demonstrate the ability to form networks of polymer nanotubes and polymersomes by optical manipulation. The aqueous core of the polymer nanotubes together with their robust character makes them interesting candidates for nanofluidics and other applications in biotechnology.

Stabilization of amphiphilic block copolymer nanotubes and vesicles by photopolymerization

We create long polymer nanotubes by directly pulling on the membrane of polymersomes using either optical tweezers or a micropipette. The polymersomes are composed of amphiphilic diblock copolymers and the nanotubes formed have an aqueous core connected to the aqueous interior of the polymersome. Stabilized membranes of nanotubes and vesicles were formed by the directed selfassembly of poly(ethylene oxide)-block-polybutadiene, followed by photopolymerization, initiated by UV light, to a maximum double bond conversion of 15%. The photopolymerized nanotubes are extremely robust. The applicability of photopolymerization for biophysics and bioanalytical science is demonstrated by electrophoresing DNA molecules through a stabilized nanotube with an integrated vesicle reservoir.