Aqueous Droplets In Oil Research Articles

Suspending droplets beyond the Rayleigh limit: The interplay of acoustic and gravity forces

In this work, we experimentally investigate the suspension behavior of droplets subjected to standing acoustic waves. We focus on the droplet sizes beyond the Rayleigh limit, i.e., when the droplet size is comparable to the wavelength of the acoustic wave. We show that an acoustic field can disrupt the uniform motion of aqueous droplets in oil and cause them to either suspend or settle, depending on the interplay between acoustic and gravity forces. Remarkably, in contrast to droplets within the Rayleigh limit, the critical acoustic power or minimum pressure amplitude required to suspend droplets beyond the Rayleigh limit is dependent on the droplet size. As the droplet size increases, the critical acoustic power increases significantly. Building upon this understanding, a novel sorting method is proposed based on critical acoustic power.

Reconstruction of the Gram-Negative Bacterial Outer-Membrane Bilayer.

The outer membrane (OM) of gram-negative bacteria is highly asymmetric. The outer leaflet comprises lipopolysaccharides (LPS) and the inner leaflet phospholipids. Here, it is shown that the outer membrane lipid bilayer (OMLB) of Escherichia coli can be reconstructed as a droplet interface bilayer (DIB), which separates two aqueous droplets in oil. The trimeric porin OmpF is inserted into the model OMLB and the translocation of the bacteriocin colicin E9 (colE9) through it is monitored. By contrast with LPS-free bilayers, it is found that colE9 made multiple failed attempts to engage with OmpF in an OMLB before successful translocation occurred. In addition, the observed rate for the second step of colE9 translocation is 3-times smaller than that in LPS-free bilayers, and further, the colE9 dissociates when the membrane potential is reversed. The findings demonstrate the utility of the DIB approach for constructing model OMLBs from physiologically realistic lipids and that the properties of the model OMLBs differ from those of a simple lipid bilayer. The model OMLB offers a credible platform for screening the properties of antibiotics.

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions-and perhaps the biological relevance-of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

An experimental study on the non-coalescence exhibited by anchored aqueous droplets in oil

The effect of droplet conductivity on electrocoalescence phenomena is studied experimentally for droplet-in-oil system in an anchored drop setup. Phase diagrams representing the coalescence/non-coalescence behaviour under DC electric field are constructed for three different droplet conductivities. Variation in droplet conductivity and separation distance between droplets do not influence critical electrocapillary number. The critical electrocapillary number varies with oil viscosity. Non-coalescence is not observed for low conductivity (1.5μS/cm) droplets for entire range of s0/R (initial separation gap to radius ratio), therefore cone angle criteria (Bird et al., 2009) does not apply to anchored droplet-in-oil systems. Non-coalescence is observed as bridge vaporization/gas generation due to high current for high s0/R in case of moderate conductivity (1244μS/cm) droplets and low s0/R for high conductivity (89,500μS/cm) droplets. In addition to coalescence and non-coalescence events, subsequent events after first non-coalescence are studied and an effort is made to explain and categorize the behaviour under different regimes.

The effect of demulsifier on the stability of liquid droplets: A study of micro-force balance

An original device was developed by our research group and used for quantitatively and qualitatively studying the stability mechanism of two liquid droplets in another liquid phase. The squeeze time can be employed to detect the stability of liquid droplets. The effect of water soluble emulsifier TX-100 and oil soluble one Span-80 on the squeeze times of both aqueous and kerosene droplets have been investigated. Moreover, the influences of three polyether demulsifiers with different structures, linear PEL, branched PEB and star-type PES, on the stability of liquid droplets have also been studied. Based on experimental results, the hydrophilic head group ethoxylation (EO) chain of TX-100 nearly flats at interface and forms compact interfacial film closed to water phase, which makes kerosene droplets stable at aqueous. On the other hand, two water droplets contain TX-100 will coalesce at once when they contact each other. For oil soluble emulsifier Span-80, the compactness of two sides of interfacial film leads to both stable water droplets and oil droplets. The molecular size of demulsifier may play the crucial role in controlling droplets coalescence: PEL with smaller size can easily destroy aqueous droplets in oil; PEB shows strong effects on both kerosene and aqueous droplets for it has a moderate molecular size; PES with larger size shows strong ability to destroy oil droplets in aqueous but little effects on aqueous droplets in oil.

Rheological behavior of high internal phase water-in-oil emulsions: Effects of droplet size, phase mass fractions, salt concentration and aging

The rheological properties of high internal phase emulsions (HIPEs), comprising polydisperse aqueous droplets in oil, have been characterized as a function of emulsification time, salt concentration, phase mass fractions and aging. The droplet size distribution and structural details of the emulsion samples were obtained using cryogenic-scanning electron microscopy (cryo-SEM) and optical microscopy. For rheological characterisation, amplitude sweep tests performed on HIPE samples with a high mass fraction of dispersed phase (93.5wt%) show that the strain behavior, especially the yield strain (εy) and crossover strain (εc), are almost independent of the droplet size and polydispersity. However, emulsions with smaller droplets have higher yield stress (τy) and storage moduli (G′) values; explanations for these observations, based on the physical properties of the systems are suggested. Furthermore, it is observed that, for constant mass fractions of oil and aqueous phases, the strain behavior is also independent of the salt concentration in the dispersed phase. Our findings indicate that, independently of the salt concentration, the energy requirement for the emulsion to start flowing is greater when smaller droplets are present. Aging studies, performed over a period of 6months, show no significant change in the rheological properties of the HIPEs. Experimental rheology data is compared to the Princen and Kiss model, and with modified Mougel model (Ind. Eng. Chem. Res. 2011, 50, 10359–10365), giving insight into the critical effects of non-ideality induced by polydispersity in thickened emulsions.

DMF-MALDI: droplet based microfluidic combined to MALDI-TOF for focused peptide detection

We present an automated droplet microfluidic system (DMF) to generate monitored nanoliter aqueous droplets in oil and their deposition on a commercial stainless steel plate for MALDI-TOF analysis of peptides or protein digests. We demonstrate that DMF-MALDI combination focuses the analyte on the MALDI plate, increasing considerably the homogeneity of the dried material. This results in a 30times enhanced MALDI-TOF MS signal for a model peptide, allowing a significant improvement of the detection sensitivity limit (down to few tens of attomoles). Moreover, positive detection can be achieved from sub-nanomolar peptides solutions and better overall protein sequence coverages are obtained from few tens attomoles of protein digest. These results make DMF-MALDI a promising approach for the treatment of peptides samples as well as a key component for an integrated approach in the proteomic field.

Droplet Manipulation Using Acoustic Streaming Induced by a Vibrating Membrane.

We present a simple method for on-demand manipulation of aqueous droplets in oil. With numerical simulations and experiments, we show that a vibrating membrane can produce acoustic streaming. By making use of this vortical flow, we manage to repulse the droplets away from the membrane edges. Then, by simply aligning the membrane at 45° to the flow, the droplets can be forced to follow the membrane's boundaries, thus steering them across streamlines and even between different oil types. We also characterize the repulsion and steering effect with various excitation voltages at different water and oil flow rates. The maximum steering frequency we have achieved is 165 Hz. The system is extremely robust and reliable: the same membrane can be reused after many days and with different oils and/or surfactants at the same operating frequency.

Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification

Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (10(5)) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.

Interactions of PAMAM dendrimers with negatively charged model biomembranes.

We have investigated the interactions between cationic poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4), a potential gene transfection vector, with net-anionic model biomembranes composed of different ratios of zwitterionic phosphocholine (PC) and anionic phospho-L-serine (PS) phospholipids. Two types of model membranes were used: solid-supported bilayers, prepared with lipids carrying palmitoyl-oleoyl (PO) and diphytanoyl (DPh) acyl chains, and free-standing bilayers, formed at the interface between two aqueous droplets in oil (droplet interface bilayers, DIBs) using the DPh-based lipids. G4 dendrimers were found to translocate through POPC:POPS bilayers deposited on silica surfaces. The charge density of the bilayer affects translocation, which is reduced when the ionic strength increases. This shows that the dendrimer-bilayer interactions are largely controlled by their electrostatic attraction. The structure of the solid-supported bilayers remains intact upon translocation of the dendrimer. However, the amount of lipids in the bilayer decreases and dendrimer/lipid aggregates are formed in bulk solution, which can be deposited on the interfacial layers upon dilution of the system with dendrimer-free solvent. Electrophysiology measurements on DIBs confirm that G4 dendrimers cross the lipid membranes containing PS, which then become more permeable to ions. The obtained results have implications for PAMAM dendrimers as delivery vehicles to cells.

Construction and Manipulation of Functional Three-Dimensional Droplet Networks

Previously, we reported the manual assembly of lipid-coated aqueous droplets in oil to form two-dimensional (2D) networks in which the droplets are connected through single lipid bilayers. Here we assemble lipid-coated droplets in robust, freestanding 3D geometries: for example, a 14-droplet pyramidal assembly. The networks are designed, and each droplet is placed in a designated position. When protein pores are inserted in the bilayers between specific constituent droplets, electrical and chemical communication pathways are generated. We further describe an improved means to construct 3D droplet networks with defined organizations by the manipulation of aqueous droplets containing encapsulated magnetic beads. The droplets are maneuvered in a magnetic field to form simple construction modules, which are then used to form larger 2D and 3D structures including a 10-droplet pyramid. A methodology to construct freestanding, functional 3D droplet networks is an important step toward the programmed and automated manufacture of synthetic minimal tissues.

Constructing droplet interface bilayers from the contact of aqueous droplets in oil

We describe a protocol for forming an artificial lipid bilayer by contacting nanoliter aqueous droplets in an oil solution in the presence of phospholipids. A lipid monolayer forms at each oil-water interface, and when two such monolayers touch, a bilayer is created. Droplet interface bilayers (DIBs) are a simple way to generate stable bilayers suitable for single-channel electrophysiology and optical imaging from a wide variety of preparations, ranging from purified proteins to reconstituted eukaryotic cell membrane fragments. Examples include purified proteins from the α-hemolysin pore from Staphylococcus aureus, the anthrax toxin pore and the 1.2-MDa mouse mechanosensitive channel MmPiezo1. Ion channels and ionotropic receptors can also be reconstituted from membrane fragments without further purification. We describe two approaches for forming DIBs. In one approach, a lipid bilayer is created between two aqueous droplets submerged in oil. In the other approach, a membrane is formed between an aqueous droplet and an agarose hydrogel, which allows imaging in addition to electrical recordings. The protocol takes <30 min, including droplet generation, monolayer assembly and bilayer formation. In addition to the main protocol, we also describe the preparation of Ag/AgCl electrodes and sample preparation.

Self-organization of the bacterial cell-division protein FtsZ in confined environments

We report a microfluidic approach to generate aqueous droplets in oil of different dimensionality, stabilized by a lipid monolayer, to systematically probe the polymerization of bacterial cell-division protein FtsZ into fibrous networks as a function of the concentrations of crowding agent, FtsZ, and GTP. FtsZ bundles confined in droplets were dynamic, and their distribution depended on the intrinsic properties of the system and restrictions imposed by the spatial boundaries.

Self-Regulated, Droplet-Based Sample Chopper for Microfluidic Absorbance Detection

Akin to optical beam chopping, we demonstrate that formation and routing of aqueous droplets in oil can chop a fluidic sample to permit phase sensitive detection. This hand-operated microfluidic sample chopper (μChopper) greatly reduces the detection limit of molecular absorbance in a 27 μm optical path. With direct dependence on path length, absorbance is fundamentally incompatible with microfluidics. While other microfluidic absorbance approaches use complex additions to fabrication, such as fiber coupling and increased optical paths, this self-regulated μChopper uses opposing droplet generators to passively alternate sample and reference droplets at ~10 Hz each. Each droplet's identity is automatically locked-in to its generator, allowing downstream lock-in analysis to nearly eliminate large signal drift or 1/f noise. With a lock-in time constant of 1.9 s and total interrogated volume of 59 nL (122 droplets), a detection limit of 3.0 × 10(-4) absorbance units or 500 nM bromophenol blue (BPB) (29 fmol) was achieved using only an optical microscope and a standard, single-depth (27 μm) microfluidic device. The system was further applied to nanoliter pH sensing and validated with a spectrophotometer. The μChopper represents a fluidic analog to an optical beam chopper, and the self-regulated sample/reference droplet alternation promotes ease of use.

A heatable and evaporation-free miniature reactor upon superhydrophobic pedestals

Superhydrophobic material-based miniature reaction systems (MRSs) show great potential in the effective use of valuable reagents and solvents, but suffer from a water evaporation problem. Here we report a novel MRS utilizing superhydrophobic materials to freely manipulate aqueous droplets in oil, which provides a confined space with the capability to perform a heatable (80 °C) and time-consuming (24h) miniature reaction inside the coalesced droplet reactor.

Formation of droplet networks that function in aqueous environments.

Aqueous droplets in oil that are coated with lipid monolayers and joined through interface bilayers are useful for biophysical measurements on membrane proteins. Functional networks of droplets that can act as light sensors, batteries and electrical components can also be made by incorporating pumps, channels and pores into the bilayers. These networks of droplets mimic simple tissues, but so far have not been used in physiological environments because they have been constrained to a bulk oil phase. Here, we form structures called multisomes in which networks of aqueous droplets with defined compositions are encapsulated within small drops of oil in water. The encapsulated droplets adhere to one another and to the surface of the oil drop to form interface bilayers that allow them to communicate with each other and with the surrounding aqueous environment through membrane pores. The contents in the droplets can be released by changing the pH or temperature of the surrounding solution. The multicompartment framework of multisomes mimics a tissue and has potential applications in synthetic biology and medicine.

Droplet Tracking for Single Molecule Confinement

We report on a robust system for tracking femtoliter and smaller aqueous droplets in oil over tens of minutes with a few nanometers accuracy in a 150 Hz bandwidth. Tracking permits the droplet position to be maintained in the detection volume of a confocal microscope without need for physical or optical trapping, permitting detailed optical analysis of droplet contents. In addition, droplet tracks can be used to determine droplet size. As an alternative to surface attachment or liposomal confinement, we use droplets to confine individual biomolecules for study using single-molecule fluorescence trajectories. Back focal plane imaging onto a position sensitive detector (PSD), similar to the configuration frequently used to determine the position of a particle in an optical tweezer, is used to determine droplet position with respect to the confocal detection volume. Our tracking system, inspired in part by the work in Ref. (1), utilizes a dedicated microprocessor to implement a feedback loop between the PSD and a fast piezoelectric flexure stage. Motion of the stage keeps the particle in the confocal detection volume. Here we present data characterizing the system and also report on progress in using tracked droplets in single-molecule studies of RNA. 1. Desai, K. V., T. G. Bishop, L. Vicci, E. T. O'Brien, R. M. Taylor, and R. Superfine. 2008. Agnostic particle tracking for three-dimensional motion of cellular granules and membrane-tethered bead dynamics. Biophys. J. 94:2374–2384.

Picoliter-volume aqueous droplets in oil: Electrochemical detection and yeast cell electroporation

An electrochemical detection method was introduced for aqueous droplet analysis in oil phase of microfluidic devices. This method is based on the electrochemical signal difference between aqueous and oil. Applying a low alternating current (AC) voltage to a couple of Au microelectrodes, this method can offer size information and ion concentration range from 0.02 mmol/L to 1 mol/L of tens of picoliter to nanoliter aqueous droplets. Alternatively, applying a relative high AC voltage (18 Vpp) at a frequency of 1 kHz leads to electroporation of yeast cells encapsulated into picoliter droplets. We believe that this simple technique is useful for a number of aqueous droplet-based chemical and biological analyses as well as cell electroporation.

Electro-generation of single femtoliter- and picoliter-volume aqueous droplets in microfluidic systems

This letter presents a new method to generate individual femtoliter- to picoliter-volume aqueous droplets in oil using single voltage pulses. With high-speed imaging, we characterized the displacement of the water-oil interface as a function of the time and amplitude of the voltage pulse. At high voltages where jetting and droplet formation occurred, we observed the ratio of droplet-to-jet diameter to be ∼1.84, which suggested Rayleigh instability as the primary mechanism responsible for droplet breakup. Droplets with volumes ranging from 14 fl to 8 pl were produced using this method.