Distinct Droplet Research Articles

Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity.

Dynamic protein-rich intracellular structures that contain phase-separated intrinsically disordered proteins (IDPs) composed of sequences of low complexity (SLC) have been shown to serve a variety of important cellular functions, which include signalling, compartmentalization and stabilization. However, our understanding of these structures and our ability to synthesize models of them have been limited. We present design rules for IDPs possessing SLCs that phase separate into diverse assemblies within droplet microenvironments. Using theoretical analyses, we interpret the phase behaviour of archetypal IDP sequences and demonstrate the rational design of a vast library of multicomponent protein-rich structures that ranges from uniform nano-, meso- and microscale puncta (distinct protein droplets) to multilayered orthogonally phase-separated granular structures. The ability to predict and program IDP-rich assemblies in this fashion offers new insights into (1) genetic-to-molecular-to-macroscale relationships that encode hierarchical IDP assemblies, (2) design rules of such assemblies in cell biology and (3) molecular-level engineering of self-assembled recombinant IDP-rich materials.

Microelectrode Test Configurations for Measurement of Galvanic Current Under Saline Drops in Simulated Atmospheric Conditions

This work investigates galvanic corrosion of UNS A97075 (AA7075-T6) coupled with a stainless steel UNS S13800 (CRES 13-8) in a simulated atmospheric environment under electrolyte droplets. Three different electrochemical test configurations were compared to understand galvanic corrosion in droplet electrolytes. Micro-pipets were utilized to electrochemically examine alloys and determine galvanic corrosion current from droplet polarization scans. Additionally a novel galvanic corrosion cell with two distinct droplet half cells joined by a micro-pipet salt bridge was constructed to observed galvanic atmospheric corrosion rates in dynamic environmental conditions. The last configuration examined a galvanic couple in contact with a single drop and exhibited corrosion behavior distinct from the latter design to due to reaction product interactions.

Some Lipid Droplets Are More Equal Than Others: Different Metabolic Lipid Droplet Pools in Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) are professional lipid-storing cells and are unique in their property to store most of the retinol (vitamin A) as retinyl esters in large-sized lipid droplets. Hepatic stellate cell activation is a critical step in the development of chronic liver disease, as activated HSCs cause fibrosis. During activation, HSCs lose their lipid droplets containing triacylglycerols, cholesteryl esters, and retinyl esters. Lipidomic analysis revealed that the dynamics of disappearance of these different classes of neutral lipids are, however, very different from each other. Although retinyl esters steadily decrease during HSC activation, triacylglycerols have multiple pools one of which becomes transiently enriched in polyunsaturated fatty acids before disappearing. These observations are consistent with the existence of preexisting “original” lipid droplets with relatively slow turnover and rapidly recycling lipid droplets that transiently appear during activation of HSCs. Elucidation of the molecular machinery involved in the regulation of these distinct lipid droplet pools may open new avenues for the treatment of liver fibrosis.

What factors determine the stability of a weak protein-protein interaction in a charged aqueous droplet?

Maintaining the interface of a weak transient protein complex transferred from bulk solution to the gaseous state via evaporating droplets is a critical question in the detection of the complex association (dissociation) constant by using electrospray ionization mass spectrometry (ESI-MS). Here we explore the factors that may affect the stability of a protein-protein interaction (PPI) using atomistic molecular dynamics (MD) modelling of a complex of ubiquitin (Ub) and the ubiquitin-associated domain (UbA) (RCSB PDB code ) and a non-covalent complex of diubiquitin (RCSB PDB code ) in aqueous droplets. A general method is presented to determine the protonation states of the complexes we investigate in particular, and that of a protein in general, under various pH conditions that an evaporating droplet acquires due to its change in size. We find that the combination of high temperature and high charge states of the protein complexes may destabilize the interface by creating new interfaces instead of a direct rupture of the initial stable interface. We provide evidence that highly charged protein complexes are found in droplets that form conical extrusions of the solvent on the surface due to charge-induced instability. This distinct droplet morphology leads to a higher solvent evaporation rate that assists in transferring the complex in the gaseous state without dissociation. The conical solvent protrusions expose on the droplet surface certain amino acids that otherwise would be solvated in a droplet with the protein complex of low charge states. The new vapor-protein interface does not have a direct effect on the stability of the PPI. A common way in experiments to stabilize the protein complexes in droplets is to reduce the protonation state of the proteins. Here we find that weakly bound protein complexes even at high protonation states can be stabilized by the presence of a small number of counterions, without affecting the protonation state of the protein. Our findings may provide guiding principles in ESI-MS experiments to stabilize weak transient PPIs.

Characterization of "Star" Droplet Morphologies Induced by Charged Macromolecules.

"Star" morphologies of charged liquid droplets are distinct droplet conformations that, for a certain charge squared to volume ratio, have lower energy than their spherically shaped analogues. For these shapes to appear, the charge should be carried by a single ionic species. A typical example of a charge carrier that we employ in this study is a fully charged double-stranded oligodeoxynucleotide (dsDNA) in an aqueous and an acetonitrile droplet. We characterize the structure and dynamics of the star-shaped droplets. We find that by increasing the charge squared to volume ratio, the droplet evolves from spherical to "spiky" shapes, by first passing from droplet sizes that undergo enhanced shape fluctuations relative to those of the larger spherical droplets. These fluctuations mark the onset of the instability. We also find that in the spiky droplet, the orientation of the solvent molecules in the first shell about the dsDNA is very close to that in the bulk solution. However, this orientation is substantially different farther away from the dsDNA. With regards to dynamics, the motion of the spikes is reflected in the autocorrelation functions of rotationally invariant order parameters that show a damped oscillator form of decay, indicative of the elastic motion of the spikes. We compare the formation of spikes with that of the ferrofluids and the dielectric materials in an electric field, and we conclude that they represent a different entity that deserves its own characterization. The study provides insight into the manner in which the charge distribution may give rise to well-controlled droplet morphologies and calls for experiments in this direction.

Morphologically and Functionally Distinct Lipid Droplet Subpopulations.

Lipid droplet (LD), a multi-functional organelle, is often found to associate with other cellular membranous structures and vary in size in a given cell, which may be related to their functional diversity. Here we established a method to separate LD subpopulations from isolated CHO K2 LDs into three different size categories. The subpopulation with smallest LDs was nearly free of ER and other membranous structures while those with larger LDs contained intact ER. These distinct subpopulations of LDs differed in their protein composition and ability to recruit proteins. This method was also applicable to LDs obtained from other sources, such as Huh7 cells, mouse liver and brown adipose tissue, et al. We developed an in vitro assay requiring only isolated LDs, Coenzyme A, and ATP to drive lipid synthesis. The LD subpopulation nearly depleted of ER was able to incorporate fatty acids into triacylglycerol and phospholipids. Together, our data demonstrate that LDs in a given cell are heterogeneous in size and function, and suggest that LDs are one of cellular lipid synthetic organelles.

Lipid droplet mobilization: The different ways to loosen the purse strings.

Cytosolic lipid droplets are dynamic lipid-storage organelles that play a crucial role as reservoirs of metabolic energy and membrane precursors. These organelles are present in virtually all cell types, from unicellular to pluricellular organisms. Despite similar structural organization, lipid droplets are heterogeneous in morphology, distribution and composition. The protein repertoire associated to lipid droplet controls the organelle dynamics. Distinct structural lipid droplet proteins are associated to specific lipolytic pathways. The role of these structural lipid droplet-associated proteins in the control of lipid droplet degradation and lipid store mobilization is discussed. The control of the strictly-regulated lipolysis in lipid-storing tissues is compared between mammals and plants. Differences in the cellular regulation of lipolysis between lipid-storing tissues and other cell types are also discussed.

Coexistence of different droplet generating instabilities: new breakup regimes of a liquid filament.

The coexistence of multiple droplet breakup instabilities in a Step-emulsification geometry is studied. A liquid filament, which is confined in one dimension by channel walls and surrounded by a co-flowing immiscible continuous phase, decays into droplets when subject to a sudden release of confinement. Depending on the filament aspect ratio and liquid flow rates, an unexpectedly rich variety of droplet breakup regimes is found. All of these breakup regimes are composed of two basic instabilities, i.e. a step- and a jet-instability, that coexist in various combinations on the same filament. Surprisingly, even an asymmetric breakup regime is found, producing droplet families of significantly different diameters, while the filament is subject to a fully symmetric flow field. We suggest key physical principles explaining the spontaneous symmetry breaking and the transitions between individual droplet breakup regimes. The particular ability to produce distinct droplet families from a single filament is demonstrated to allow for simultaneous concentration and encapsulation of particles into one droplet family while excess bulk liquid is released into another family of droplets.

Microbeads and hollow microcapsules obtained by self-assembly of pickering magneto-responsive cellulose nanocrystals.

Cellulose microbeads can be used as immobilization supports. We report on the design and preparation of magneto-responsive cellulose microbeads and microcapsules by self-assembled shells of cellulose nanocrystals (CNC) carrying magnetic CoFe2O4 nanoparticles, that is, a mixture of isotropic and anisotropic nanomaterials. The magnetic CNCs formed a structured layer, a mesh, consisting of CNCs and magnetic particles bound together on the surface of distinct droplets of hexadecane and styrene dispersed in water. Because of the presence of CNCs the highly crystalline mesh was targeted to provide an improved barrier property of the microbead shell compared to neat polymer shells, while the magnetic particles provided the magnetic response. In situ polymerization of the styrene phase led to the formation of solid microbeads (∼8 μm diameter) consisting of polystyrene (PS) cores encapsulated in the magnetic CNC shells (shell-to-core mass ratio of 4:96). The obtained solid microbeads were ferromagnetic (saturation magnetization of ∼60 emu per gram of the magnetic phase). The magnetic functionality enables easy separation of substances immobilized on the beads. Such a functionality was tested in removal of a dye from water. The microbeads were further utilized to synthesize hollow microcapsules by solubilization of the PS core. The CNC-based, magneto-responsive solid microbeads and hollow microcapsules were characterized by electron microscopy (morphology), X-ray diffraction (phase composition), and magnetometry (magnetic properties). Such hybrid systems can be used in the design of materials and devices for application in colloidal stabilization, concentration, separation, and delivery, among others.

Capillary focusing close to a topographic step: shape and instability of confined liquid filaments

Step-emulsification is a microfluidic technique for droplet generation which relies on the abrupt decrease of confinement of a liquid filament surrounded by a continuous phase. A striking feature of this geometry is the transition between two distinct droplet breakup regimes, the “step-regime” and “jet-regime,” at a critical capillary number. In the step-regime, small and monodisperse droplets break off from the filament directly at a topographic step, while in the jet-regime a jet protrudes into the larger channel region and large plug-like droplets are produced. We characterize the breakup behavior as a function of the filament geometry and the capillary number and present experimental results on the shape and evolution of the filament for a wide range of capillary numbers in the jet-regime. We compare the experimental results with numerical simulations. Assumptions based on the smallness of the depth of the microfluidic channel allow us to reduce the governing equations to the Hele-Shaw problem with surface tension. The full nonlinear equations are then solved numerically using a volume-of-fluid-based algorithm. The computational framework also captures the transition between both regimes, offering a deeper understanding of the underlying breakup mechanism .

Lipids and oocyte developmental competence: the role of fatty acids and β-oxidation.

Metabolism and ATP levels within the oocyte and adjacent cumulus cells are associated with quality of oocyte and optimal development of a healthy embryo. Lipid metabolism provides a potent source of energy and its importance during oocyte maturation is being increasingly recognised. The triglyceride and fatty acid composition of ovarian follicular fluid has been characterised for many species and is influenced by nutritional status (i.e. dietary fat, fasting, obesity and season) as well as lactation in cows. Lipid in oocytes is a primarily triglyceride of specific fatty acids which differ by species, stored in distinct droplet organelles that re-localise during oocyte maturation. The presence of lipids, particularly saturated vs unsaturated fatty acids, in in vitro maturation systems affects oocyte lipid content as well as developmental competence. Triglycerides are metabolised by lipases that have been localised to cumulus cells as well as oocytes. Fatty acids generated by lipolysis are further metabolised by β-oxidation in mitochondria for the production of ATP. β-oxidation is induced in cumulus-oocyte complexes (COCs) by the LH surge, and pharmacological inhibition of β-oxidation impairs oocyte maturation and embryo development. Promoting β-oxidation with l-carnitine improves embryo development in many species. Thus, fatty acid metabolism in the mammalian COC is regulated by maternal physiological and in vitro environmental conditions; and is important for oocyte developmental competence.

Natural discharge after pulse and cooperative electrodes to enhance droplet velocity in digital microfluidics

Digital Microfluidics (DMF) is a promising technology for biological/chemical micro-reactions due to its distinct droplet manageability via electronic automation, but the limited velocity of droplet transportation has hindered DMF from utilization in high throughput applications. In this paper, by adaptively fitting the actuation voltages to the dynamic motions of droplet movement under real-time feedback monitoring, two control-engaged electrode-driving techniques: Natural Discharge after Pulse (NDAP) and Cooperative Electrodes (CE) are proposed. They together lead to, for the first time, enhanced droplet velocity with lower root mean square voltage value.

Modeling and Optimization of Superhydrophobic Condensation

Superhydrophobic micro/nanostructured surfaces for dropwise condensation have recently received significant attention due to their potential to enhance heat transfer performance by shedding water droplets via coalescence-induced droplet jumping at length scales below the capillary length. However, achieving optimal surface designs for such behavior requires capturing the details of transport processes that is currently lacking. While comprehensive models have been developed for flat hydrophobic surfaces, they cannot be directly applied for condensation on micro/nanostructured surfaces due to the dynamic droplet-structure interactions. In this work, we developed a unified model for dropwise condensation on superhydrophobic structured surfaces by incorporating individual droplet heat transfer, size distribution, and wetting morphology. Two droplet size distributions were developed, which are valid for droplets undergoing coalescence-induced droplet jumping, and exhibiting either a constant or variable contact angle droplet growth. Distinct emergent droplet wetting morphologies, Cassie jumping, Cassie nonjumping, or Wenzel, were determined by coupling of the structure geometry with the nucleation density and considering local energy barriers to wetting. The model results suggest a specific range of geometries (0.5–2 μm) allowing for the formation of coalescence-induced jumping droplets with a 190% overall surface heat flux enhancement over conventional flat dropwise condensing surfaces. Subsequently, the effects of four typical self-assembled monolayer promoter coatings on overall heat flux were investigated. Surfaces exhibiting coalescence-induced droplet jumping were not sensitive (<5%) to the coating wetting characteristics (contact angle hysteresis), which was in contrast to surfaces relying on gravitational droplet removal. Furthermore, flat surfaces with low promoter coating contact angle hysteresis (<2 deg) outperformed structured superhydrophobic surfaces when the length scale of the structures was above a certain size (>2 μm). This work provides a unified model for dropwise condensation on micro/nanostructured superhydrophobic surfaces and offers guidelines for the design of structured surfaces to maximize heat transfer. Keywords: superhydrophobic condensation, jumping droplets, droplet coalescence, condensation optimization, environmental scanning electron microscopy; micro/nanoscale water condensation, condensation heat transfer.

Mechanisms of InAs/GaAs quantum dot formation during annealing of In islands

We have examined the formation mechanisms of InAs quantum dots (QDs) via annealing In islands under As flux. We report two distinct mechanisms, droplet epitaxy (DE) and solid phase epitaxy (SPE), which depend on As surface coverage. On c(4 × 4) GaAs surfaces, QDs form by DE. For c(4 × 4)α, one-to-one conversion from In islands to InAs QDs is observed. For c(4 × 4)β, lower densities of larger QDs are observed, presumably due to enhanced In surface diffusion in the absence of metastable Ga-As dimers. For the As capped surface, In deposition leads to an amorphous film, from which QDs nucleate by SPE during annealing.

Film thickness measurements in liquid–liquid slug flow regimes

At present there is significant interest in the development of small scale medical diagnostic equipment. These devices offer faster processing times and require smaller sample volumes than equivalent macro scale systems. Although significant attention has been focused upon their outputs, little attention has been devoted to the detailed fluid mechanics that govern the flow mechanisms within these devices. Conventionally, the samples in these small scale devices are segmented into distinct discrete droplets or slugs which are suspended in an organic carrier phase. Separating these slugs from the channel wall is a very thin film of the organic carrier phase.The magnitude of this film is the focus of the present study and the effects of sample slug length and carrier phase fluidic properties on the film are examined over a range of Capillary numbers. A non-intrusive optical technique was used to capture images of the flow from which the magnitude of the film was determined.The experimental results show that the film is not constant along the length of the slug; however above a threshold value for slug length, a region of constant film thickness exists. When compared with existing correlations in the literature, the experimental data showed reasonable agreement with the Bretherton model when the Capillary number was calculated based on the mean two phase flow velocity. However, significant differences were observed when the Capillary number was redefined to account for the mean velocity at the liquid interface, i.e., the mean slug velocity.Analysis of the experimental data revealed that it fell into two distinct flow regimes; a visco-capillary regime and a visco-inertial regime. A modified Taylor expression is presented to estimate the magnitude of the film for flows in the visco-capillary regime while a new model is put forward, based on Capillary and Weber numbers, for flows in the visco-inertial regime. Overall, this study provides some novel insights into parameters, such as aqueous slug length and carrier phase fluidic properties, that affect the thickness of the film in liquid–liquid slug flow regimes.

FSP27 and PLIN1 interaction promotes the formation of large lipid droplets in human adipocytes

Human adipocytes express high levels of two distinct lipid droplet proteins, fat specific protein 27 (FSP27; also called CIDEC), a member of the CIDE family, and perilipin1 (PLIN1), a member of the PAT family. Both proteins play a role in fat metabolism in adipocytes, but how they interact is not known. Our present study demonstrates that FSP27 and PLIN1 co-localize and interact in cultured human primary adipocytes. We also found that the C-terminal domain of FSP27, aa 120–220, interacts with PLIN1. Individual expression of exogenous FSP27 or PLIN1 increased triglyceride content and decreased glycerol release (a measure of lipolysis), but co-expression of both proteins did not further increase triglyceride content or decrease lipolysis in human adipocytes. However, the combination of PLIN1 and FSP27 increased the average size of lipid droplets or caused the formation of unilocular adipocytes. Our data suggest that FSP27 interacts with PLIN1 to regulate lipid droplet size in human adipocytes in a concerted manner.

Surface morphology of magnesium alloy irradiated by high-intensity pulsed ion beam

Surface morphology of magnesium alloy irradiated by high-intensity pulsed ion beam (HIPIB) at energy density of 1.1–3.4J/cm2 with shot number from one to ten has been investigated by using scanning electron microscope and profilometer in order to explore the interaction mechanism between HIPIB and metallic materials. Three groups of samples with different initial surface roughness (Ra) were prepared to determine the effect of surface state on surface morphology. It was found that for the medium-roughness and high-roughness samples the irradiated surface represents a wavy feature and the formation of crater in local region due to selective ablation of the microprotrusions deriving from the rougher initial surface, however, no obvious craters were observed for the low-roughness sample, which exhibited texture feature and relatively mild irradiation damage. The mean spacing of surface profile irregularities (Sm) increases as shot number increases for various initial surface roughness samples. For low-roughness and medium-roughness samples Ra increases with increasing shot number, whereas Ra first increases slightly, and then decreases with increasing shot number for the high-roughness sample. It is also demonstrated that increasing energy density led to the increase in both Ra and Sm at all the shot number investigated, and the corresponding surface morphology altered from obvious craters at the lower energy density of 1.1J/cm2 to distinct droplet ejection feature owing to more intense energy deposition at the higher energy density of 2.3 and 3.4J/cm2.

Na-caseinate/oil/water systems: Emulsion morphology diagrams

The concentrated (dispersed phase 50–70wt%) composition space of Na-caseinate, a family of milk proteins, stabilised emulsions was investigated for three different oils: soybean oil, palm olein and tetradecane with pH 6.8 phosphate buffer continuous phase. The variation of emulsion stability and microstructure were explored using static light scattering, diffusion nuclear magnetic resonance, cryo-scanning electron microscopy, rheology and the time varying macroscopic phase separation of the emulsions. For soybean oil and palm olein a rich diversity of emulsion microstructures and stabilities are realised. Five emulsion domains, each having a different microstructure and macroscopic stability have been identified within the composition space probed. For the lowest concentrations of emulsifier bridging flocculation is evident and emulsions are of low stability. Increasing Na-caseinate concentration leads to an increased stability and the existence of distinct individual oil droplets, visualised using cryo-scanning electron microscopy. Further increases in Na-caseinate concentration reduce emulsion stability due to depletion flocculation. Na-caseinate self-assembly is then initiated. At sufficiently high Na-caseinate and/or oil concentrations the continuous phase of the emulsion is a three-dimensional protein network and emulsion stability is again enhanced. At the limits of the emulsion composition space a gel-like paste is formed. The diversity of emulsion microstructure is reduced when tetradecane is the discrete phase. Na-caseinate self-assembly is limited and there is no evidence for formation of a protein network.

Transient analysis of intermittent multijet sprays

This paper analyzes the transient characteristics of intermittent sprays produced by the single-point impact of multiple cylindrical jets. The aim is to perform a transient analysis of the intermittent atomization process to study the effect of varying the number of impinging jets in the hydrodynamic mechanisms of droplet formation. The results evidence that hydrodynamic mechanisms underlying the physics of ligament fragmentation in 2-impinging jets sprays also apply to sprays produced with more than 2 jets during the main period of injection. Ligaments detaching from the liquid sheet, as well as from its bounding rim, have been identified and associated with distinct droplet clusters, which become more evident as the number of impinging jets increases. Droplets produced by detached ligaments constitute the main spray, and their axial velocity becomes more uniformly distributed with 4-impinging jets because of a delayed ligament fragmentation. Multijet spray dispersion patterns are geometric depending on the number of impinging jets. Finally, an analysis on the Weber number of droplets suggests that multijet sprays are more likely to deposit on interposed surfaces, thus becoming a promising and competitive atomization solution for improving spray cooling.

Synergistic combustion of droplets of ethanol, diesel and biodiesel mixtures

Freely-falling droplets of ethanol, diesel, (castor oil) biodiesel, and their mixtures were experimentally examined in a high-temperature combustion chamber. The combustion characteristics including the burning rate, microexplosion, and sooting propensity are reported. Results show that adding biodiesel to diesel significantly reduces the extent of soot formation and slightly reduces the burning rate. In addition, higher soot production of methyl stearate than castor oil biodiesel is observed suggesting strong oxidation propensity of the OH function group in castor oil biodiesel. Furthermore, by adding ethanol to diesel and biodiesel, microexplosion is observed, with the biodiesel/ethanol mixtures exhibiting stronger propensity, leading to significantly reduced gasification time and extent of soot formation. The burning of these mixtures occurs in a three-staged, liquid-phase diffusion-limited manner previously observed for distinct bi-component droplets with highly disparate volatilities.