Microfluidic droplet sorting with a high frequency ultrasound beam.

This paper presents experimental results demonstrating the feasibility of high frequency ultrasonic sensing and sorting for screening single oleic acid (lipid or oil) droplets under continuous flow in a microfluidic channel. In these experiments, hydrodynamically focused lipid droplets of two different diameters (50 µm and 100 µm) are centered along the middle of the channel, which is filled with deionized (DI) water. A 30 MHz lithium niobate (LiNbO(3)) transducer, placed outside the channel, first transmits short sensing pulses to non-invasively determine the acoustic scattering properties of the individual droplets passing through the beam's focus. Integrated backscatter (IB) coefficients, utilized as a sorting criterion, are measured by analyzing the received echo signals from each droplet. When the IB values corresponding to 100 µm droplets are obtained, a custom-built LabVIEW panel commands the transducer to emit sinusoidal burst signals to commence the sorting operation. The number of droplets tested for the sorting is 139 for 50 µm droplets and 95 for 100 µm droplets. The sensing efficiencies are estimated to be 98.6% and 99.0%, respectively. The sorting is carried out by applying acoustic radiation forces to 100 µm droplets to direct them towards the upper sheath flow, thus separating them from the centered droplet flow. The sorting efficiencies are 99.3% for 50 µm droplets and 85.3% for 100 µm droplets. The results suggest that this proposed technique has the potential to be further developed into a cost-effective and efficient cell/microparticle sorting instrument.

Novel plant-derived recombinant human interferons with broad spectrum antiviral activity.

Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.

Particle manipulation in a microfluidic channel using acoustic trap.

A high frequency sound beam was employed to explore an experimental method that could control particle motions in a microfluidic device. A 24 MHz single element lead zirconate titanate (PZT) transducer was built to transmit a focused ultrasound of variable duty factors (pulse duration/pulse repetition time), and its 1-3 piezocomposite structure established a tight focusing with f-number (focal depth/aperture size) of one. The transducer was excited by the Chebyshev windowed chirp signal sweeping from 18 MHz to 30 MHz with a 50% of duty factor, in order to ensure that enough sound beams were penetrated into the microfluidic device. The device was fabricated from a polydimethylsiloxane (PDMS) mold, and had a main channel composed of three subchannels among which particles flowed in the middle. A 60~70 µm diameter single droplet in the flow could be trapped near the channel bifurcation, and subsequently diverted into the sheath flow by releasing or shifting the acoustic trap. Hence, the results showed the potential use of a focused sound beam in microfluidic devices, and further suggested that this method could be exploited in the development of ultrasound-based flow cytometry and cell sorting devices.

Backscattering measurement from a single microdroplet.

Backscattering measurements for acoustically trapped lipid droplets were undertaken by employing a P[VDF-TrFE] broadband transducer of f-number = 1, with a bandwidth of 112%. The wide bandwidth allowed the transmission of the 45 MHz trapping signal and the 15 MHz sensing signal using the same transducer. Tone bursts at 45 MHz were first transmitted by the transducer to hold a single droplet at the focus (or the center of the trap) and separate it from its neighboring droplets by translating the transducer perpendicularly to the beam axis. Subsequently, 15 MHz probing pulses were sent to the trapped droplet and the backscattered RF echo signal received by the same transducer. The measured beam width at 15 MHz was measured to be 120 µ m. The integrated backscatter (IB) coefficient of an individual droplet was determined within the 6-dB bandwidth of the transmit pulse by normalizing the power spectrum of the RF signal to the reference spectrum obtained from a flat reflector. The mean IB coefficient for droplets with a 64 µ m average diameter (denoted as cluster A) was -107 dB, whereas it was -93 dB for 90-µm droplets (cluster B). The standard deviation was 0.9 dB for each cluster. The experimental values were then compared with those computed with the T-matrix method and a good agreement was found: the difference was as small as 1 dB for both clusters. These results suggest that this approach might be useful as a means for measuring ultrasonic backscattering from a single microparticle, and illustrate the potential of acoustic sensing for cell sorting.

Targeted cell immobilization by ultrasound microbeam.

Various techniques exerting mechanical stress on cells have been developed to investigate cellular responses to externally controlled stimuli. Fundamental mechanotransduction processes about how applied physical forces are converted into biochemical signals have often been examined by transmitting such forces through cells and probing its pathway at cellular levels. In fact, many cellular biomechanics studies have been performed by trapping (or immobilizing) individual cells, either attached to solid substrates or suspended in liquid media. In that context, we demonstrated two-dimensional acoustic trapping, where a lipid droplet of 125 µm in diameter was directed transversely toward the focus (or the trap center) similar to that of optical tweezers. Under the influence of restoring forces created by a 30 MHz focused ultrasound beam, the trapped droplet behaved as if tethered to the focus by a linear spring. In order to apply this method to cellular manipulation in the Mie regime (cell diameter > wavelength), the availability of sound beams with its beamwidth approaching cell size is crucial. This can only be achieved at a frequency higher than 100 MHz. We define ultrasound beams in the frequency range from 100 MHz to a few GHz as ultrasound microbeams because the lateral beamwidth at the focus would be in the micron range. Hence a zinc oxide (ZnO) transducer that was designed and fabricated to transmit a 200 MHz focused sound beam was employed to immobilize a 10 µm human leukemia cell (K-562) within the trap. The cell was laterally displaced with respect to the trap center by mechanically translating the transducer over the focal plane. Both lateral displacement and position trajectory of the trapped cell were probed in a two-dimensional space, indicating that the retracting motion of these cells was similar to that of the lipid droplets at 30 MHz. The potential of this tool for studying cellular adhesion between white blood cells and endothelial cells was discussed, suggesting its capability as a single cell manipulator.

Stable, biocompatible lipid vesicle generation by solvent extraction-based droplet microfluidics.

In this paper, we present a microfluidic platform for the continuous generation of stable, monodisperse lipid vesicles 20-110 µm in diameter. Our approach utilizes a microfluidic flow-focusing droplet generation design to control the vesicle size by altering the system's fluid flow rates to generate vesicles with narrow size distribution. Double emulsions are first produced in consecutive flow-focusing channel geometries and lipid membranes are then formed through a controlled solvent extraction process. Since no strong solvents are used in the process, our method allows for the safe encapsulation and manipulation of an assortment of biological entities, including cells, proteins, and nucleic acids. The vesicles generated by this method are stable and have a shelf life of at least 3 months. Here, we demonstrate the cell-free in vitro synthesis of proteins within lipid vesicles as an initial step towards the development of an artificial cell.

PLGA micro/nanosphere synthesis by droplet microfluidic solvent evaporation and extraction approaches.

In this paper, we present two approaches for the synthesis of poly(lactide-co-glycolide) (PLGA) micro/nanospheres using non-toxic organic solvents in droplet-based microfluidic platforms. Solvent evaporation and solvent extraction methods were employed to enable the controlled generation of monodisperse PLGA particles that range from 70 nanometres to 30 microns in diameter. Determination of particle size was carried out with dynamic light scattering (DLS) and image analysis to show less than 2% variation in particle size. Sizes of the PLGA microspheres were controlled by the PLGA concentration in solvent and by the relative flow rates of oil and aqueous phases in the system. A penetration imaging assay was performed to determine the depth of diffusion of a model drug molecule fluorescein, out of the PLGA nanoparticles into corneal tissue. With the ability to prepare high quality, monodisperse, biodegradable particles, our methods have great potential to benefit drug delivery applications.

Transverse acoustic trapping using a gaussian focused ultrasound.

The optical tweezer has become a popular device to manipulate particles in nanometer scales and to study the underlying principles of many cellular or molecular interactions. Theoretical analysis was previously carried out at the authors' laboratory, to show that similar acoustic trapping of microparticles may be possible with a single beam ultrasound. This article experimentally presents the transverse trapping of 125 microm lipid droplets under an acoustically transparent mylar film, which is an intermediate step toward achieving acoustic tweezers in three-dimension. Despite the lack of axial trapping capability in the current experimental arrangement, it was found that a 30 MHz focused beam could be used to laterally direct the droplets toward the focus. The spatial range within which acoustic traps may guide droplet motion was in the range of hundreds of micrometers, much greater than that of optical traps. This suggests that this acoustic device may offer an alternative for manipulating microparticles in a wider spatial range.

Single beam acoustic trapping.

A single beam acoustic device, with its relatively simple scheme and low intensity, can trap a single lipid droplet in a manner similar to optical tweezers. Forces in the order of hundreds of nanonewtons direct the droplet toward the beam focus, within the range of hundreds of micrometers. This trapping method, therefore, can be a useful tool for particle manipulation in areas where larger particles or forces are involved.

Droplet microfluidics.

Droplet-based microfluidic systems have been shown to be compatible with many chemical and biological reagents and capable of performing a variety of "digital fluidic" operations that can be rendered programmable and reconfigurable. This platform has dimensional scaling benefits that have enabled controlled and rapid mixing of fluids in the droplet reactors, resulting in decreased reaction times. This, coupled with the precise generation and repeatability of droplet operations, has made the droplet-based microfluidic system a potent high throughput platform for biomedical research and applications. In addition to being used as microreactors ranging from the nano- to femtoliter range; droplet-based systems have also been used to directly synthesize particles and encapsulate many biological entities for biomedicine and biotechnology applications. This review will focus on the various droplet operations, as well as the numerous applications of the system. Due to advantages unique to droplet-based systems, this technology has the potential to provide novel solutions to today's biomedical engineering challenges for advanced diagnostics and therapeutics.

Thermodynamic and mechanical properties of model mitochondrial membranes.

Cardiolipin is a unique four-tailed, doubly negatively charged lipid found predominantly within the inner mitochondrial membrane, and is thought to be influential in determining membrane potential and permeability. To determine the role of cardiolipin in modulating the properties of membranes, this study investigates the thermodynamics of mixed cardiolipin and phosphatidylcholine monolayers and bilayers. Gibbs free energy analysis of mixed monolayers indicates that at low cardiolipin concentrations (5-10 mol%), there is a positive deviation from ideality on a pure water subphase, while at physiological salt concentrations a negative deviation from ideality is observed. The mechanical properties of bilayers containing cardiolipin were measured using micropipette aspiration. Both apparent area compressibility modulus, as well as lysis tension, decrease with increasing cardiolipin content. This destabilization indicates a decrease in the cohesive energy of the membrane. This interplay between interactions of lipids in monolayers and bilayers, suggests cardiolipin plays a dual role in modulating membrane properties. Cardiolipin enhances lateral interactions between lipids within monolayer leaflets, while simultaneously decreasing the cohesive energy of membranes at physiologically relevant concentrations. Taken together, these findings correlate with the decreased permeability and creation of folds in the inner mitochondrial membrane.