Individual Droplets Research Articles

Application of flow cytometry as novel technology in studying lipid oxidation and mass transport phenomena in oil-in-water emulsions

Flow cytometry was used to determine if lipid oxidation products could transfer between individual emulsion droplets. Medium chain triacyclglycerol oil-in-water emulsions containing an oxidizable fluorescent dye, BODIPY665/676, was blended with a soybean oil-in-water emulsion. Results showed that when the concentration of sodium dodecyl sulfonate (SDS) were lower than critical micelle concentration (CMC), lipid oxidation products of triacylglycerols were not able to escape out until emulsions were extremely oxidized. With surfactant micelles, oxidation of BODIPY665/676 was observed. In the presence of free fatty acids, the transfer of prooxidants between droplets was observed even when surfactant concentration was lower than CMC. The decomposition product, 2,4,-decadienal, was also found to be transferred between droplets. The effect of surfactant concentration on prooxidant transfer was investigated using the lipid-soluble radical generator, AMVN. Results showed that surfactants promoted AMVN-triggered degradation of fluorescence at low concentrations but inhibited degradation at high concentration. The CMC of SDS was decreased by NaCl thus affecting the transfer phenomenon. With flow cytometry, the phenomenon of mass transfer between individual droplets was revealed which provides a better understanding of lipid oxidation in emulsion system.

Toward a Spatiotemporal Model of Oxidation in Lipid Dispersions: A Hypothesis‐Driven Review

AbstractUnsaturated lipids are prone to get oxidized through a sequence of reactions known as lipid oxidation. From a phenomenon considered at the beginning as a mere chemical process and described by the triptych of initiation, propagation, and termination, the vision of lipid oxidation has progressively evolved to further integrate the physical dimension of the phenomenon. Despite tremendous research efforts, however, this sequence of reactions is not yet well understood, especially in lipid dispersions where many facts are still inexplicable and unpredictable under the current paradigm. Here, the aim is to suggest that the main reason why a better understanding has not already been achieved is that the reactional network is not yet organized in a coherent spatiotemporal framework. The novel concepts and hypotheses proposed in this article may help redirecting a significant part of research efforts toward the establishment of a spatially and temporally resolved model of oxidation in dispersed lipids.Practical Application: Predicting how oxidation spreads in lipid dispersions represents a goal of crucial importance for academia but also for industry. Such prediction models would indeed greatly help food manufacturers prevent oxidation by using the most adapted antioxidative strategies for their specific products. To achieve such an objective, it is proposed that the first thing to do is to go beyond the extremely reductive and narrow framework in which this chemical process has been locked in. Indeed, while lipid dispersions are composed of a multitude of lipid colloids, researchers usually consider oxidation at the sole level of an individual droplet or membrane. Instead, lipid oxidation is advocated as a dynamic trafficking of chemical species within large communities of different colloidal objects such as droplets, membranes, or micelles dispersed in water—a system that dubbed “colloidal ecosystem”. This might represent a scale complementary to the scales of individual colloids or molecules that were the sole consideration so far to try to represent lipid oxidation. Only then can one hope to effectively apply modeling and “omics” approaches, as is explained in more details in this article.

Auto-affitech: an automated ligand binding affinity evaluation platform using digital microfluidics with a bidirectional magnetic separation method.

The dissociation constant (Kd) is a crucial parameter for characterizing binding affinity in molecular recognition, including antigen-antibody, DNA-protein, and receptor-ligand interactions. However, conventional methods for Kd characterization usually involve a multi-step process and time-consuming operations for incubation, washing, and detection, thus causing problems, such as time delays, microbead loss, degradation of sensitive molecules, and personal errors. Here we demonstrate an automated ligand binding affinity evaluation platform (Auto-affitech) using digital microfluidics (DMF), with individual droplets at the microliter level, programmed to rapidly perform the incubation and separation of target-beads and binding ligands. Because the loss of the beads influences the detection results, we propose a new strategy for magnetic bead separation using DMF, termed the bidirectional separation method. By splitting one droplet into two asymmetric droplets, high bead retention efficiency (89.57% ± 0.05%) and high washing efficiency (99.59% ± 0.17%, with four washings) were obtained. We demonstrate the determination of Kd of an aptamer-protein system (EpCAM and its corresponding aptamer SYL3C) and an antigen-antibody system (H5N1 antigen and antibody), proving the capability and universality of Auto-affitech in various receptor-ligand systems. Integrating all the sample processing procedures, the Auto-affitech not only saves manual labor and minimizes personal errors, but also conserves samples and shortens analysis time. Overall, this platform successfully demonstrates to be an automated approach for dissociation constant evaluation and exhibits great potential for highly efficient screening of ligands.

Discrete-Drop Mode of Ice Accretion on a Cylinder in Transverse Supercooled Flow

A physicomathematical model is developed for spatiotemporal evolution of an ice layer growing upon solid body interaction with individual droplets that slide along the body surface and harden on it, resulting in two-dimensional roughness (lumpy ice). The case of a cross-flow circular cylinder illustrates the calculation results for a set of control parameters typical for ground-based experiments in an aero-refrigeration wind tunnel.

Quantifying the hygroscopic properties of cyclodextrin containing aerosol for drug delivery to the lungs.

Aerosol dynamics is important to quantify in drug delivery to the lungs with the aim of delivering therapeutics to a target location and optimising drug efficacy. The macrocycle (2-hydroxypropyl)-β-cyclodextrin (2-HP-β-CD) is thought to alleviate symptoms associated with neurodegenerative diseases when inhaled but the hygroscopic response is not well understood. Here we measure the hygroscopic growth of individual aqueous aerosol containing 2-HP-β-CD in optical tweezers through analysis of morphology-dependent resonances arising in Raman spectra. Droplets are analysed in the size range of 3-5 μm in radius. The evolving radius and refractive index of each droplet are measured in response to change in relative humidity from 98-20% to determine mass and radius based hygroscopic growth factors, and compared with dynamic vapour sorption measurements. Bulk solution refractive index and density measurements were used in accordance with the self-consistent Lorenz-Lorentz rule to determine melt solute and droplet properties. The refractive index of 2-HP-β-CD was determined to be 1.520 ± 0.002 with a density of 1.389 ± 0.005 g cm-3. To our knowledge, we show the first aerosol measurements of 2-HP-β-CD and determine hygroscopicity. By quantifying the hygroscopic growth and physicochemical properties of 2-HP-β-CD, the impact of aerosol dynamics can be accounted for in tailoring drug formulations and informing models used to predict drug deposition patterns within the respiratory system.

Real-time observation of jumping and spinning nanodroplets.

The manipulation of liquids at nanoscale dimensions is a central goal of the emergent nanofluidics field. Such endeavors extend to nanodroplets, which are ubiquitous objects involved in many technological applications. Here, we employ time-resolved electron microscopy to elucidate the formation of so-called jumping nanodroplets on a graphene surface. We flash-melt a thin gold nanostructure with a laser pulse and directly observe how the resulting nanodroplet contracts into a sphere and jumps off its substrate, a process that occurs in just a few nanoseconds. Our study provides the first experimental characterization of these morphological dynamics through real-time observation and reveals new aspects of the phenomenon. We observe that friction alters the trajectories of individual droplets. Surprisingly, this leads some droplets to adopt dumbbell-shaped geometries after they jump, suggesting that they spin with considerable angular momentum. Our experiments open up new avenues for studying and controlling the fast morphological dynamics of nanodroplets through their interaction with structured surfaces.

Deterministic droplet coding via acoustofluidics.

Droplet microfluidics has become an indispensable tool for biomedical research and lab-on-a-chip applications owing to its unprecedented throughput, precision, and cost-effectiveness. Although droplets can be generated and screened in a high-throughput manner, the inability to label the inordinate amounts of droplets hinders identifying the individual droplets after generation. Herein, we demonstrate an acoustofluidic platform that enables on-demand, real-time dispensing, and deterministic coding of droplets based on their volumes. By dynamically splitting the aqueous flow using an oil jet triggered by focused traveling surface acoustic waves, a sequence of droplets with deterministic volumes can be continuously dispensed at a throughput of 100 Hz. These sequences encode barcoding information through the combination of various droplet lengths. As a proof-of-concept, we encoded droplet sequences into end-to-end packages (e.g., a series of 50 droplets), which consisted of an address barcode with binary volumetric combinations and a sample package with consistent volumes for hosting analytes. This acoustofluidics-based, deterministic droplet coding technique enables the tagging of droplets with high capacity and high error-tolerance, and can potentially benefit various applications involving single cell phenotyping and multiplexed screening.

A Novel and Robust Single-cell Trapping Method on Digital Microfluidics.

Due to cell heterogeneity, the differences among individual cells are averaged out in bulk analysis methods, especially in the analysis of primary tumor biopsy samples from patients. To deeply understand the cell-to-cell variation in a primary tumor, single-cell culture and analysis with limited amount of cells are in high demand. Microfluidics has been an optimum platform to address the issue given its small reaction volume requirements. Digital microfluidics, which utilizes an electric signal to manipulate individual droplets has shown promise in cell-culture with easy controls. In this work, we realize single cell trapping on digital microfluidic platform by fabricating 3D microstructures on-chip to form semi-closed micro-wells. With this design, 20% of 30 x 30 array can be occupied by isolated single cells. We also use a low evaporation silicon oil and a fluorinated surfactant to lower the droplet actuation voltage and prevent the drop from evaporation, while allowing cell respiration during the long term of culture (24 h). The main steps for single cell trapping on digital microfluidics, as illustrated in this protocol, include 3D microstructures design, 3D microstructures construction on chip and oil film with surfactant for single cell trapping on chip.

Re-evaluation of BioSampler and its improvement for on-line, time-resolved monitoring of environmental coarse aerosol

BioSampler is now being widely used for bioaerosol sampling. However, the sampling efficiency in wide size range especially for nanoparticles as well as the size-dependent retention efficiency have not been well evaluated until now. Through literature review, theoretic analysis and experiments, this paper reviews the sampling process, collection mechanism and sampling performance of commercial BioSampler including pressure drop as a function of sampling flow rate, the mass loss rate and temperature of the collection fluid as a function of sampling time, the variation of retention efficiency with time, and the sampling efficiency in wide size range from nanometers to microns. The effects of low pressure and high relative humidity on determination of sampling efficiency in literature were carefully analyzed. To compensate the collection fluid loss and extract the insoluble/dissolved sample for further analysis, Continuous-Extraction BioSampler (CEBS) is proposed. The retention efficiency for particles of different sizes as well as the collection efficiency were determined and found to be identical with commercial BioSampler when no steam was introduced into CEBS. Finally, the combination uses of CEBS and Inductively-Coupled-Plasma Mass Spectrometer (ICP-MS) was developed. Exponentially Modified Gaussian (EMG) Model was derived and verified to describe the temporal concentration of the dissolved component originating from individual soluble droplet. Using internal standard calibration method and EMG fitting of signal curve, the target element amount in individual droplet could be determined accurately. For continuously-collected soluble droplet with duration of minutes, the observed signal curve can be described by EMG model and the fitted value of area varies linearly with the sample amount introduced. This paper not only provides comprehensive performance evaluation of commercial BioSampler, but also demonstrates that the proposed CEBS-ICPMS is capable of monitoring environmental coarse aerosol with time resolution of ~17 min.

Дискретно-капельный режим обледенения цилиндра в поперечном переохлажденном потоке

It is developed a physicomathematical model of the spatial-temporal evolution of an ice layer growing upon colliding with a solid surface of individual droplets sliding along the surface and solidifying on it, that leads to a two-dimensional roughness (ice hillocks). By the example of a circular cylinder in cross-flow, illustrations of the calculation results for a set of governing parameters typical for the ground-based experiments in an aircooling tunnel are given.

Ignition of Individual Droplets in a Reactive Fuel/Air Mixture behind Reflected Shock Waves

<div class="section abstract"><div class="htmlview paragraph">Multiphase-induced ignition is frequently discussed as a trigger for early ignition in internal combustion engines. In this context, we investigated the ignition process of single lubricant-oil droplets and their interaction with the bulk air/fuel mixture in a high-pressure shock tube, mimicking oil-fuel interaction in turbocharged internal combustion engines at the end of the compression stroke. A fast micro-dispensing injector released single fuel or lubricant oil droplets with a diameter of 200±50 µm into shock-heated fuel/air mixtures consisting of PRF95 and synthetic air. The injector was flush-mounted in the sidewall of the shock tube. The droplets were released into the gas after the passage of the reflected shock waves at post-shock conditions of 2 MPa and 750-950 K. With a high-frame-rate color camera, the entire evolution of droplet injection and ignition was traced in space and time through a large sapphire window in the endwall of the shock tube. A high-repetition-rate laser at 532 nm was used to illuminate the droplets. The light scattered by the droplets was detected in the green channel of the camera, whereas ignition and soot luminosity were detected in the blue and red channel, respectively. From the camera images the time for the appearance of first detected kernel related to local ignition (originating from either droplet or gaseous ignition) was determined. Additionally, the volume ignition that proceeds after an ignition delay time was determined from the OH* emission time-trace from a photomultiplier tube. It was found that the oil-droplet injection had an influence on the local ignition whereas the ignition delay time was reduced in the low temperature range only.</div></div>

87 Analyzing metabolomic profile of bovine IVF and somatic cell nuclear transfer embryos through Raman spectroscopy

While knowledge of early embryo development and viability has continually increased, there is still a need for noninvasive methods to identify embryos with the highest chances of development to term when transferred. The most widely used technique, morphological assessment, is highly subjective and limited by personnel experience. Spent media from invitro culture could be used as a valuable noninvasive marker for embryo quality assessment. Raman spectroscopy has proven to be a powerful tool for identifying the molecular characteristics of spent culture media by measuring vibration of chemical bonds, allowing for metabolomic profiling of varying stages and quality of embryos. It is well documented that embryos produced through somatic cell nuclear transfer (SCNT) result in lower pregnancy rates and higher incidences of pregnancy loss when compared with embryos produced through IVF (Heyman et al. 2002 Biol. Reprod. 66, 1-13). This study was designed to examine differences in spent media between bovine embryos produced by IVF and SCNT. The SCNT embryos with a metabolomic profile more similar to IVF embryos may have a higher developmental competence. Bovine cumulus-oocyte complexes (COCs) were retrieved from abattoir-derived ovaries and matured for 21h in maturation medium. After IVM, COCs were either used for IVF or SCNT and cultured in 50μL droplets of synthetic oviductal fluid medium + fetal bovine serum in groups of 45 from Day 0-5. On Day 5, embryos that had reached morula stage were placed in individual droplets of 13μL of synthetic oviductal fluid + bovine serum albumin until Day 7. All embryos were cultured at 38.5°C and 5% CO2. On Day 7 embryos were assessed for developmental stage and quality and 10μL of medium from individual culture drops was collected for Raman spectroscopy. Samples were loaded on an MgF2 optical window, dried, and analysed using a 785nm near-infrared laser in the spectral range of 600 to 1800cm−1. Raw Raman data were first pre-processed by baseline correction (asymmetric least-squares smoothing) and normalization. Principal component analysis and partial least squares were then applied to reduce data dimensions. The score of the most significant principal components from principal component analysis and the optimum number of scores from partial least squares were used for linear discriminant analysis. Spent media samples from 4 SCNT embryos, 3 IVF embryos, and 3 empty media samples were analysed, with 50 spectra obtained from each sample. Preliminary data showed grouping of medium containing embryos developed to blastocyst from medium containing embryos arrested at morula or empty medium. We also saw grouping of medium containing SCNT embryos from medium containing IVF embryos within both the morula and blastocyst stage from empty medium. This shows evidence of metabolomic differences between embryos of different developmental potential and embryos produced by IVF and SCNT. Further investigation of the Raman profile of these groups can display specific differences in chemical components and help to identify candidate genes causing differing metabolism of these groups.

Novel Platform for Droplet Detection and Size Measurement Using Microstrip Transmission Lines.

We propose a novel platform for detecting as well as measuring the size of individual droplets in microfluidic channels using microstrip transmission lines. The most outstanding feature of our platform is that, as opposed to previous related works, its design allows for the droplet to flow in a microfluidic channel fabricated between the top strip and the ground plane of a microstrip transmission line. This provides enhanced interaction of the electromagnetic field with the detected droplets. The proposed design allows us to measure droplet size directly from the phase of the microwave signal, without the need for a resonator. The platform is based on low temperature co-fired ceramic (LTCC), which makes it more compatible with Radiofrequency (RF) and microwave technology than platforms used in previous works. With this platform, we are able to measure droplets as small as 150 µm in radius. It is worth pointing out that our device could also be used for detection, counting and measurement of other microscopic objects.

Connecting the Drops: Observing Collective Flow Behavior in Emulsions

Thoroughly mixing immiscible fluids creates droplets of one phase dispersed in a continuum of the other phase. In such emulsions, the individual droplets have rather mundane mechanical behavior. However, densely confining these suspended droplets generates a packing of particles with a spectacular diversity of mechanical behavior whose origins we are only beginning to understand. This mini review serves to survey a non-exhaustive range of experimental dense slow flow emulsion work. To embed these works in the context of the flow behavior of other structured fluids, we also discuss briefly the related non-local flow modeling attempts as one of the approaches that has been used successfully in describing emulsion flow properties and other materials.

Microfluidic method for determining drop-drop coalescence and contact times in flow

Coalescence kinetics is an important parameter when describing the stability of dispersions. A number of methods to measure coalescence time are available, however they often take long time and do not allow working with small droplets or coalescence in flow. Here we present a new microfluidic method for recording and analysing hundreds or thousands of individual droplet interactions in flow. Our method allows to extract information about both the coalescence and the contact times of drops. In addition, we can distinguish the coalescence events by the droplet size, as well as the approach velocity of the colliding droplets. We validated the proposed methodology by systematically changing a number parameters of the experiment (salinity, oil composition, presence of surfactant, temperature). The increase of salinity lead to compression of the double layer and decreased coalescence time. A difference was found when studying coalescence of heptane, xylene and dodecane, which was attributed to their hydrophobicity and viscosity. The addition of surfactant caused a significant increase of coalescence time through additional repulsion and Marangoni effect, while higher temperature caused faster coalescence of droplets. We also found that increased approach velocity generally reduced both contact and coalescence times, up to certain (critical) value. Beyond that, no coalescence was observed.

Aerosol Optical Tweezers Constrain the Morphology Evolution of Liquid-Liquid Phase-Separated Atmospheric Particles

Summary Chemical models of atmospheric particles are vital in understanding the role of aerosol particles in atmospheric chemistry, air pollution, human health, and climate change. Advancing these models requires new frameworks that can realistically predict how critical particle properties evolve. We present such a framework for predicting particle phase separation and which morphology will prevail; this controls how each particle interacts with and affects the atmosphere. We studied the mixing behavior of α-pinene secondary organic aerosol (SOA) with different organic phases, as relative humidity was varied to determine the interplay between polarity, miscibility, interfacial tension, and the resulting morphology. Using measurements from aerosol optical tweezers experiments and literature data, a general trend in morphology with increasing atmospheric oxidation was observed, from biphasic partially engulfed (where both phases are immediately accessible to the gas phase) to biphasic core shell (where the organic shell conceals the core) and finally to a single-phase, homogeneous morphology.

Analysis of the LIF/Mie Ratio from Individual Droplets for Planar Droplet Sizing: Application to Gasoline Fuels and Their Mixtures with Ethanol

In this work, the possibility of using planar droplet sizing (PDS) based on laser-induced fluorescence (LIF) and Mie scattering was investigated within the framework of measuring the droplet Sauter mean diameter (SMD) of direct-injection spark-ignition (DISI) spray systems. For this purpose, LIF and Mie signals of monodisperse fuel droplets produced by a droplet generator were studied at engine relevant diameters (20–50 µm). The surrogate gasoline fuel Toliso (consisting of 65 vol. % isooctane, 35 vol. % toluene) and the biofuel blend E20 (consisting of 80 vol. % Toliso, 20 vol. % ethanol) were used and which were doped with the fluorescence dye “nile red”. The effects of ethanol admixture, dye concentration, laser power, and temperature variation on the LIF/Mie ratio were studied simultaneously at both macroscopic and microscopic scale. The deduced calibration curves of the LIF and Mie signals of both fuels showed volumetric and surface dependent behaviors, respectively, in accordance with the assumptions in the literature. The existence of glare points and morphology-dependent resonances (MDRs) lead to slightly higher LIF and Mie exponents of E20 in comparison to Toliso. In principle, these calibration curves enable the determination of the SMD from LIF/Mie ratio images of transient fuel sprays.

Simultaneous detection of two growth factors from human single-embryo culture medium by a bead-based digital microfluidic chip

The measurement of growth factors released in a culture medium is considered to be an attractive non-invasive approach, apart from the embryo morphology, to identify the condition of an embryo development after fertilization in vitro (IVF), but the available embryo culture medium in the current method is only a few microlitres. This small sample volume, also of small concentration, makes difficult the application of a conventional detection method, such as an enzyme-linked immunosorbent assay (ELISA). A reliable detection of the growth factor from each embryo culture medium of such a small concentration hence remains a challenge. Here for the first time we report the results of measurement of not just one, but two, growth factors, human IL-1β and human TNF-α, from an individual droplet of embryo culture medium with a bead-based digital microfluidic chip. The required sample volume for a single measurement is only 520 nL; the total duration of the on-chip process is less than 40 min. Using the culture media of human embryos with normal morphologic features, we found that the concentrations of TNF-α change little from day 3 to day 5–6, but the concentrations of IL-1β for some embryos might double from day 3 to day 5–6. For other embryos even with similar normal morphologic features, some growth factors, such as IL-1β, might exhibit different expressions during the culture period. Those growth factors could serve to distinguish the development conditions of each embryo, not merely from an observation of embryo morphology.

A High-performance Homogeneous Droplet Routing Technique for MEDA-based Biochips

Recent advancement of microelectrode-dot-array (MEDA)-based architecture for digital microfluidic biochips has enabled a major enhancement in microfluidic operations for traditional lab-on-chip devices. One critical issue for MEDA-based biochips is the transportation of droplets. MEDA allows dynamic routing for droplets of different size. In this article, we propose a high-performance droplet routing technique for MEDA-based digital microfluidic biochips. First, we propose the basic concept of droplet movement strategy in MEDA-based design together with a definition of strictly shielded zones within the layout in MEDA architecture. Next, we propose transportation schemes of droplets for MEDA architecture under different blockage or crossover conditions and estimate route distances for each net in offline. Finally, a priority-based routing strategy combining various transportation schemes stated earlier has been proposed. Concurrent movement of each droplet is scheduled in a time-multiplexed manner. This poses critical challenges for parallel routing of individual droplets with optimal sharing of cells formulating a routing problem with higher complexity. The final compaction solution satisfies the timing constraint and improves fault tolerance. Simulations are carried out on standard benchmark circuits, namely, Benchmark suite I and Benchmark suite III. Experimental results show satisfactory improvements and prove a high degree of robustness for our proposed algorithm.

The Decarburization Kinetics of Metal Droplets in Emulsion Zone

A mathematical model has been developed to predict the decarburization rate within individual droplets in the emulsion zone. All the chronological events pertaining to the life cycle of a metal droplet in the emulsion zone like oxygen supply (from slag), external and internal decarburization have been modeled dynamically and validated against experimental data available in open literature. The bloating behavior of metal droplets in the emulsion was represented theoretically by incorporating an escape function dependent on internal CO gas generation. The model is able to predict the onset of bloating and the residence time of metal droplets in the emulsion zone. The residence time of droplet containing 2.6 wt pct C and 0.007 wt pct S is in the range of 10 to 13 seconds. The contribution of decarburization rate in the emulsion zone to the overall decarburization rate is studied using the industrial data reported by Cicutti et al. The model predicts 5 to 75 pct of total decarburization takes place in the emulsion zone. It is found that the extent of decarburization of a metal droplet depends on its initial carbon content rather than its oxygen content for slag containing FeO greater than 10 wt pct.