Two 25 base-pair complementary DNA strands are encapsulated within an optically trapped nano-droplet, 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 solution's NaCl concentration. We have observed binding and
unbinding events between the two freely diffusing DNA strands, and our measurements reveal that the duplex can exist
in multiple conformational states at elevated temperatures and low concentrations of NaCl.
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.
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.
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.
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.