We demonstrate the feasibility of trapping and manipulating individual macromolecules such as globular proteins (~ 5
nm in diameter) in free solution using a flow-based, microfluidic confinement method. This new method enables
confinement of small nanoscale objects in free solution by utilizing a planar extensional flow created at a microchannel
junction. The fluid flow based confinement method described in this work expands the micro/nanomanipulation toolbox
by offering a powerful and versatile platform for non-perturbative observation and analysis of single macromolecules in
free solution without force fields (electrical, magnetic, optical and acoustic) and surface immobilization.
Lasing from spherical microdroplets ejected into a liquid medium with lower refractive index is observed in a
microchannel. A microfabricated device that combines the droplet production and excitation/detection has been
utilized. Droplets of 50 µm-diameter containing a fluorescent dye were first detected and then excited through
multimode fibers after their production at a T-junction. Images show intense lasing emission around the droplet rim.
Spectra from the droplets exhibit morphology dependent resonances (MDRs) which are red shifted relative to the
bulk fluorescence emission from the dyes. The dependence of resonant peak intensities on the pump beam power is
nonlinear.
A microdroplet can act as a high quality factor optical cavity that supports Morphology Dependent Resonances(MDRs). Enhanced radiative energy transfer through these optical resonances can also be utilized as a transduction mechanism for chemical and biological sensing. Enhancement in radiative energy transfer is observed when a donor/acceptor pair is present in the resonant medium of a microcavity. Here, we demonstrate avidin-biotin binding and its detection through a FRET pair as a potential application for ultra-sensitive detection for fluoroimmunoassays. The binding interaction between the biotinylated donor molecules and streptavidin-acceptor conjugate was used to observe the energy transfer between the dye pairs. The radial modes of MDRs extend to approximately 0.6 r0 inside the droplet. As a result, the fluorescent emission around the center is not coupled to the optical resonances losing sensitivity. To address this problem, we prepared water-in-oil emulsions of avidin and biotin containing solutions. The water phase contains the streptavidin-Alexa Fluor 610 and the oil phase contains biotinylated fluorescent bead. Streptavidin-biotin binding reaction occurs at the water-oil interface. The water phase accumulates at the droplet air interface due to higher specific density enhancing the resonance coupling. Water and oil phase are index-matched to avoid scattering problems. As a result, a large portion of the avidin-biotin complex was localized at the pendant droplet and air interface. Strong coupling of acceptor emission into optical resonances shows that the energy transfer is efficiently mediated through these resonances.
A microdroplet or a latex microsphere often acts as an optical cavity that supports Morphology Dependent Resonances (MDRs) at wavelengths where the droplet circumference is an integer multiple of the emission wavelength. Enhanced radiative energy transfer through these optical resonances can also be utilized as a transduction mechanism for chemical and biological sensing. Enhancement in radiative energy transfer is observed when a donor/acceptor pair is present in the resonant medium of a microcavity. Here, we demonstrate avidin-biotin binding and its detection through a FRET pair as a potential application for ultra-sensitive detection for fluoroimmunoassays. The binding interaction between the biotinylated fluorescent beads (donor) and streptavidin-Alexa Fluor 555 (acceptor) conjugate was used to observe the energy transfer between the dye pairs. Strong coupling of acceptor emission into optical resonances shows that the energy transfer is efficiently mediated through these resonances.
The detection of single bacterial cells and novel absorbing labels has been demonstrated through optical resonances in microdroplets. The setup enables high throughput detection of single Escherichia coli (E. Coli) cells without any direct labeling although Rhodamine 6G (R6G) was used as the signal transduction mechanism. A micro droplet acts as an optical cavity that supports Morphology Dependent Resonances (MDRs) at wavelengths where the droplet circumference is an integer multiple of the emission wavelength. The cells inside the droplet have a direct effect on the fluorescence lasing spectrum of R6G fluorescence by means of scattering and local refractive index change. The change in the lasing spectrum can be observed at the concentrations where each droplet has as little as one cell. C60 fluorescence quenching has also been demonstrated in microdroplets. R6G in ethanol (10μM) was used for the fluorescence spectrum measurements. Quenching of the optical resonances was observed when C60 dissolved in ethanol was mixed with the R6G-Ethanol solution. Quenching can be observed at C60 concentrations of 1μM in the final solution. The background signal was also checked by repeating the experiment with only R6G and only C60 in the solvent, assuring that the signal reduction was due to the addition of C60 in to the solution. This quenching mechanism may have many applications in multiplexing in bioassays.
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