Fusion and sorting of two parallel trains of droplets using a railroad-like channel network and guiding tracks

We propose a robust droplet fusion and sorting method for two parallel trains of droplets that is relatively insensitive to frequency and phase mismatch. Conventional methods of droplet fusion require an extremely precise control of aqueous/oil flows for perfect frequency matching between two trains of droplets. In this work, by combining our previous two methods (i.e., droplet synchronization using railroad-like channels and manipulation of shape-dependent droplets using guiding tracks), we realized an error-free droplet fusion/sorting device for the two parallel trains of droplets. If droplet pairs are synchronized through a railroad-like channel, they are electrically fused and the fused droplets transit to a middle guiding track to flow in a middle channel; otherwise non-synchronized non-fused droplets will be discarded into the side waste channels by flowing through their own guiding tracks. The simple droplet synchronization, fusion, and sorting technology will have widespread application in droplet-based chemical or biological experiments, where two trains of the chemically or biologically treated or pre-formed droplets yield a train of 100% one-to-one fused droplets at the desired outlet channel by sorting all the non-synchronized non-fused droplets into waste outlets.     

流动聚焦型微流控体系中的液滴的图案化排列和转变模式

流体在微流通道中形成剪切流场（低雷诺数）．不同于宏观体系,由于剪切力和表面张力的竞争作用,产生的液滴在微尺度下的微流通道中形成特殊的排列现象---周期性类似“晶格”排列现象．设计了新型流动聚焦型微流控芯片,分析研究在微流体系中液滴周期性图案化排列和转变机理性,液滴排列模式受两方面因素影响：水油两相的流速比值和微通道尺寸．当微通道宽度为250或300 μm时,液滴形成单层分散,双层和单层挤压排列．当微通道宽度为350 μm 时,液滴会形成单层分散到三层排列到双层挤压最后到单层挤压排列．当出口通道宽度增加到400 μm时,甚至出现了液滴四层排列的现象．同时研究了各个液滴排列模式的“转变点”.     

Design of microfluidic channel geometries for the control of droplet volume, chemical concentration, and sorting

Passive microfluidic channel geometries for control of droplet fission, fusion and sorting are designed, fabricated, and tested. In droplet fission, the inlet width of the bifurcating junction is used to control the range of breakable droplet sizes and the relative resistances of the daughter channels were used to control the volume of the daughter droplets. Droplet fission is shown to produce concentration differences in the daughter droplets generated from a primary drop with an incompletely mixed chemical gradient, and for droplets in each of the bifurcated channels, droplets were found to be monodispersed with a less than 2% variation in size. Droplet fusion is demonstrated using a flow rectifying design that can fuse multiple droplets of same or different sizes generated at various frequencies. Droplet sorting is achieved using a bifurcating flow design that allows droplets to be separated base on their sizes by controlling the widths of the daughter channels. Using this sorting design, submicron satellite droplets are separated from the larger droplets.     

Droplet size based separation by deterministic lateral displacement-separating droplets by cell--induced shrinking

We present a novel method for passive separation of microfluidic droplets by size at high throughput using deterministic lateral displacement (DLD). We also show that droplets containing Saccharomyces cerevisiae shrink significantly during incubation while droplets containing only yeast media retain or slightly increase their size. We demonstrate the DLD device by sorting out shrunken yeast-cell containing droplets from 31% larger diameter droplets which were generated at the same time containing only media, present at a >40-fold excess. This demonstrates the resolving power of droplet separation by DLD and establishes that droplets can be separated for a biological property of the droplet contents discriminated by a change of the physical properties of the droplet. Thus suggesting that this technique may be used for e.g. clonal selection. The same device also separates 11 μm from 30 μm droplets at a rate of 12,000 droplets per second, more than twofold faster than previously demonstrated passive hydrodynamic separation devices.     

Emulsion Templating of Poly(lactic acid) Particles: Droplet Formation Behavior

Monodisperse poly(dl-lactic acid) (PLA) particles of diameters between 11 and 121 μm were fabricated in flow focusing glass microcapillary devices by evaporation of dichloromethane (DCM) from emulsion droplets at room temperature. The dispersed phase was 5% (w/w) PLA in DCM containing 0.1-2 mM Nile Red and the continuous phase was 5% (w/w) poly(vinyl alcohol) in reverse osmosis water. Particle diameter was 2.7 times smaller than the diameter of the emulsion droplet template, indicating very low particle porosity. Monodisperse droplets have only been produced under dripping regime using a wide range of dispersed phase flow rates (0.002-7.2 cm(3)·h(-1)), continuous phase flow rates (0.3-30 cm(3)·h(-1)), and orifice diameters (50-237 μm). In the dripping regime, the ratio of droplet diameter to orifice diameter was inversely proportional to the 0.39 power of the ratio of the continuous phase flow rate to dispersed phase flow rate. Highly uniform droplets with a coefficient of variation (CV) below 2% and a ratio of the droplet diameter to orifice diameter of 0.5-1 were obtained at flow rate ratios of 4-25. Under jetting regime, polydisperse droplets (CV > 6%) were formed by detachment from relatively long jets (between 4 and 10 times longer than droplet diameter) and a ratio of the droplet size to orifice size of 2-5.     

Analysis of small droplets with a new detector for liquid chromatography based on laser-induced breakdown spectroscopy

The miniaturization of analytical techniques is a general trend in speciation analytics. We have developed a new analytical technique combining high pressure liquid chromatography (HPLC) with laser-induced breakdown spectroscopy (LIBS). This enables a molecule-specific separation followed by an element-specific analysis of smallest amounts of complex samples. The liquid flow coming from a HPLC pump is transformed into a continuous stream of small droplets (diameter 50–100 μm, volume 65–500 pl) using a piezoelectric pulsed nozzle. After the detection of single droplets with a droplet detector, a Q-switched Nd:YAG Laser is triggered to emit a synchronized laser pulse that irradiates a single droplet. The droplets are evaporated and transformed to the plasma state. The spectrum emitted from the plasma is collected by a spherical mirror and directed through the entrance slit of a Paschen–Runge spectrometer equipped with channel photomultipliers. The spectrometer detects 31 elements simultaneously covering a spectral range from 120 to 589 nm. Purging the measurement chamber with argon enables the detection of vacuum–UV lines. Since the sample is transferred to the plasma state without dilution, very low flow rates in the sub-μl/min range can be realised.     

A unified platform for optoelectrowetting and optoelectronic tweezers

A platform capable of seamlessly unifying both optoelectrowetting and optoelectronic tweezers is presented. This enables the user to manipulate aqueous droplets (with electrowetting) as well as individual particles within those droplets (with dielectrophoresis). The device requires no photolithography and droplet/particle manipulation can occur continuously over the entire surface of the device. Droplet and 10 μm polystyrene particle speeds of up to 8 mm s(-1) and 60 μm s(-1), respectively, are demonstrated. Particle concentration within, and subsequent splitting of, a droplet is performed resulting in average concentration efficiencies of 93%. Serial concentration is also demonstrated resulting in exponentially increasing particle concentrations and a 10× concentration increase. Finally, the platform is used to select a single cell out of a cohort and subsequently encapsulate it in its own aqueous droplet.     

Effect of droplet size on the phosphine depression of calcium atomic emission signals in flame spectrometry

The effect of the droplet size on the magnitude of the phosphine depression of calcium signals is discussed. The droplet size governs the time required for droplet desolvation in the flame. It is during the desolvation process that the combustion products of phosphine, PO_x, diffuse into the droplet and form a refractory compound with the analyte. The longer the desolvating droplet resides in the PO_x-contaminated flame, the more severe the depression. A uniform droplet generator, a pneumatic nebulizer spray chamber system, and a glass-frit nebulizer spray chamber system were the sample introduction systems studied. Droplet sizes of (55 μm, 0.5–10 μm (2 μm mean), and <0.1–2 μm (0.1 μm mean) were produced by these systems, respectively, and depressions of the calcium signals of 57%, 55%, and 4% were noted. These results suggest that the phosphine depression on calcium signals may be alleviated by using a glass-frit nebulizer (or modifying a commercial nebulizer) to remove droplets greater than 2 μm in diameter.     

High-speed, clinical-scale microfluidic generation of stable phase-change droplets for gas embolotherapy

In this study we report on a microfluidic device and droplet formation regime capable of generating clinical-scale quantities of droplet emulsions suitable in size and functionality for in vivo therapeutics. By increasing the capillary number-based on the flow rate of the continuous outer phase-in our flow-focusing device, we examine three modes of droplet breakup: geometry-controlled, dripping, and jetting. Operation of our device in the dripping regime results in the generation of highly monodisperse liquid perfluoropentane droplets in the appropriate 3-6 μm range at rates exceeding 10(5) droplets per second. Based on experimental results relating droplet diameter and the ratio of the continuous and dispersed phase flow rates, we derive a power series equation, valid in the dripping regime, to predict droplet size, D(d) approximately equal 27(Q(C)/Q(D))(-5/12). The volatile droplets in this study are stable for weeks at room temperature yet undergo rapid liquid-to-gas phase transition, and volume expansion, above a uniform thermal activation threshold. The opportunity exists to potentiate locoregional cancer therapies such as thermal ablation and percutaneous ethanol injection using thermal or acoustic vaporization of these monodisperse phase-change droplets to intentionally occlude the vessels of a cancer.     

Modulation of emulsion rheology through electrostatic heteroaggregation of oppositely charged lipid droplets: influence of particle size and emulsifier content

The influence of electrostatically-induced heteroaggregation of oppositely charged lipid droplets on the rheology and stability of emulsions has been studied. 20 wt.% oil-in-water emulsions (pH 6) containing oppositely charged droplets were fabricated by mixing cationic lactoferrin-coated lipid droplets with anionic β-lactoglobulin-coated lipid droplets. Emulsions containing mixtures of droplets with different charges (positive or negative) and sizes (large or small) were prepared, and then their overall particle characteristics (ζ-potential and size) and rheology were measured. Emulsions formed by mixing positive droplets and negative droplets that were both relatively small (d(43) ≈ 0.3 μm) exhibited extensive flocculation and had paste-like properties at intermediate positive-to-negative particle ratios. On the other hand, emulsions formed by mixing positive droplets and negative droplets that were both relatively large (d(43) ≈ 3 μm) exhibited little aggregation and had relatively low viscosities at all particle ratios. Emulsions with small negative droplets and large positive droplets (or vice versa), exhibited some aggregation and viscosity enhancement at intermediate particle ratios. The presence of relatively high levels of protein in the aqueous phase of mixed emulsions reduced the level of droplet aggregation and viscosity enhancement observed, which was attributed to the ability of protein molecules to bind to droplet surfaces and neutralize their charges. Electrostatically-induced heteroaggregation of lipid droplets may be a useful means of controlling the physicochemical properties of emulsion-based products in the food, personal care, pharmaceutical and cosmetic industries.     

Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells

The synthesis and secretion of lipids by mammary epithelial cells is a highly ordered process that involves several distinct steps. Triacylglycerols are synthesized in the endoplasmic reticulum and incorporated into microlipid droplets which coalesce into cytoplasmic lipid droplets. These are vectorially transported to the apical plasma membrane where they are secreted into the milk surrounded by a membrane bilayer. The origin of this membrane as well as the mechanism by which cytoplasmic lipid droplets form and become surrounded by membrane is poorly understood. Proteomic analysis of the protein composition of milk fat globules and cytoplasmic lipid droplet has revealed that the endoplasmic reticulum is not only involved in the synthesis of the lipid but also potentially contributes to the membrane component of milk fat globules. The proteins identified suggest possible mechanisms of multiple steps during this process. Completion of the proteome of milk fat globule membranes and cytoplasmic lipid droplets will provide the necessary reporter molecules to follow and dissect the mechanisms of the sorting and ultimate secretion of cytoplasmic lipid droplets.     

A dielectrophoretic continuous flow sorter using integrated microelectrodes coupled to a channel constriction

In this article, we propose a novel dielectrophoretic continuous flow sorter using planar micro electrodes coupled to a channel constriction. This design enables a high particle sorting efficiency at low voltages while relying on a simple fabrication and integration process. We have numerically simulated the AC electrokinetic effects and the fluid behavior to predict particle trajectories. Simulation results are in accordance with experimental data: 10 and 5 μm polystyrene beads were continuously sorted with <2% errors at flow speeds of 100 μm/s. We were also able to change the particle buffer while sorting beads. Finally, to demonstrate the interest of our device for cell sorting, we also sorted dead and living yeast cells according to their different dielectric properties. Living cell concentration was enriched by a factor of 4 versus dead cell concentration after passing the sorting device.     

Deformation and breakup of micro- and nanoparticle stabilized droplets in microfluidic extensional flows

Using a microfluidic flow-focusing device, monodisperse water droplets in oil were generated and their interface populated by either 1 μm or 500 nm amine modified silica particles suspended in the water phase. The deformation and breakup of these Pickering droplets were studied in both pure extensional flow and combined extensional and shear flow at various capillary numbers using a microfluidic hyperbolic contraction. The shear resulted from droplet confinement and increased with droplet size and position along the hyperbolic contraction. Droplet deformation was found to increase with increasing confinement and capillary number. At low confinements and low capillary numbers, the droplet deformation followed the predictions of theory. For fully confined droplets, where the interface was populated by 1 μm silica particles, the droplet deformation increased precipitously and two tails were observed to form at the rear of the droplet. These tails were similar to those seen for surfactant covered droplets. At a critical capillary number, daughter droplets were observed to stream from these tails. Due to the elasticity of the particle-laden interface, these drops did not return to a spherical shape, but were observed to buckle. Although increases in droplet deformation were observed, no tail streaming occurred for the 500 nm silica particle covered droplets over the range of capillary numbers studied.     

Stratified morphology of a polypropylene/elastomer blend following channel flow

A thermoplastic olefin blend consisting of isotactic polypropylene (PP) and an ethylene‐butene copolymer (EBR) impact modifier (25 wt % EBR) was subjected to a short, high‐shear pulse within the flow channel of a pressure‐driven microextruder following low‐shear channel filling from a reservoir of the melt. The resulting morphology was examined by laser scanning confocal fluorescence microscopy (LSCFM), with contrast provided by a fluorescent tracer in the EBR minor phase. Shear experiments were performed under isothermal conditions with a known wall shear stress for a specified duration, providing a well‐defined thermal and flow history. Low‐shear channel filling produces small droplets across the central region of the channel and large droplets, consistent with steady‐state shear, in the regions near the channel walls. After cooling the molten blend to a crystallization temperature of 153 °C, a brief interval (5 s ～ 1/2000 of the quiescent crystallization time) of high shear (wall shear stress: 0.1 MPa) induces rapid, highly oriented crystallization and a stratified morphology. Ex situ LSCFM reveals a “skin” at the channel walls (～70 μm) in which greatly elongated fiberlike droplets, oriented along the flow direction, are embedded in highly oriented crystalline PP. Further from the walls but directly beside the skin layers are surprising zones in which EBR domains show no deformation or orientation. Several zones of intermediate deformation and orientation at an angle to the flow direction are located closer to the center of the channel. At the center of the channel, EBR droplets are spherical, as expected for channel flow. The various strata are explained by the interplay of droplet deformation, breakup, and coalescence with the shear‐induced crystallization kinetics of the matrix. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 2842–2859, 2002     

A microfluidic chip capable of switching W/O droplets to vertical laminar flow for electrochemical detection of droplet contents

Analysis of droplet contents is a key function involved in droplet-based microfluidic systems. Direct electrochemical detection of droplet contents suffers problems such as relatively poor repeatability, interference of capacitive current and relatively poor detectability. This paper presents a novel hybrid polydimethylsiloxane-glass chip for highly sensitive and reproducible amperometric detection of droplet contents. By wettability-patterning of the channel surface of the hybrid chip, water in oil droplets generated in the upstream part of the central channel can be switched to a two-phase vertical laminar flow (i.e., a continuous oil stream flowing atop a continuous aqueous stream) in the downstream part of the channel. The vertical laminar flow keeps the analyte in the underneath-flowing aqueous stream in direct contact with the sensing electrodes located on the bottom surface of the channel. Therefore, steady-state current signals with high sensitivity (1.2 A M⁻¹ cm⁻² for H₂O₂), low limit of detection (0.12 μM, S/N = 2), and good reproducibility (RSD 1.1% at 0.3 mM H₂O₂) were obtained. The methods for patterning of the inner channel surface are presented, and the behaviors of the microchip in flow profile switching and amperometric detection are discussed. The application of the developed microchip to enzyme kinetics study is also demonstrated.     

Electrohydrodynamic deposition of polymeric droplets under low-frequency pulsation

Circularly shaped polymeric droplets with diameter of about 20 μm have been intermittently ejected and deposited in an orderly manner on a collector from a syringe needle by means of near-field, electrohydrodynamic reactions using pulsating voltages at around 2.25 kV. The needle has an inner diameter of 100 μm and was placed 1 mm above a silicon conductor substrate to have location control for droplet depositions. Under low-frequency operation of less than 100 Hz, the deposition frequency of droplets, f(dep), has been observed to be equal to the frequency of the applied driving voltage divided by an integer, N, as small as 1. Furthermore, the diameter of the deposited droplets has been found to be linearly dependent on (Q/f(dep))(1/3), where Q is the polymer solution supply rate at around 30 nL/s. These experimentally observed droplet ejection rules under low-frequency pulsation provide useful design guidelines for controllable deposition of polymer droplets in various potential applications, including electrohydrodynamic printing.     

Passive droplet trafficking at microfluidic junctions under geometric and flow asymmetries

When droplets enter a junction they sort to the channel with the highest flow rate at that instant. Transport is regulated by a discrete time-delayed feedback that results in a highly periodic behavior where specific patterns can continue to cycle indefinitely. Between these highly ordered regimes are chaotic structures where no pattern is evident. Here we develop a model that describes droplet sorting under various asymmetries: branch geometry (length, cross-section), droplet resistance and pressures. First, a model is developed based on the continuum assumption and then, with the assistance of numerical simulations, a discrete model is derived to predict the length and composition of the sorting pattern. Furthermore we derive all unique sequences that are possible for a given distribution and develop a preliminary estimation of why chaotic regimes form. The model is validated by comparing it to numerical simulations and results from microfluidic experiments in PDMS chips with good agreement.     

Thermophoresis in Nanoliter Droplets to Quantify Aptamer Binding

Biomolecule interactions are central to pharmacology and diagnostics. These interactions can be quantified by thermophoresis, the directed molecule movement along a temperature gradient. It is sensitive to binding induced changes in size, charge, or conformation. Established capillary measurements require at least 0.5 μL per sample. We cut down sample consumption by a factor of 50, using 10 nL droplets produced with acoustic droplet robotics (Labcyte). Droplets were stabilized in an oil–surfactant mix and locally heated with an IR laser. Temperature increase, Marangoni flow, and concentration distribution were analyzed by fluorescence microscopy and numerical simulation. In 10 nL droplets, we quantified AMP‐aptamer affinity, cooperativity, and buffer dependence. Miniaturization and the 1536‐well plate format make the method high‐throughput and automation friendly. This promotes innovative applications for diagnostic assays in human serum or label‐free drug discovery screening.     

High-throughput sorting of nanoliter droplets enabled by a sequentially addressable dielectrophoretic array

Droplet microfluidics has emerged as a powerful tool for a diverse range of biomedical and industrial applications such as single-cell analysis, directed evolution, and metabolic engineering. In these applications, droplet sorting has been effective for isolating small droplets encapsulating molecules, cells, or crystals of interest. Recently, there is an increased interest in extending the applicability of droplet sorting to larger droplets to utilize their size advantage. However, sorting throughputs of large droplets have been limited, hampering their wide adoption. Here, we report our demonstration of high-throughput fluorescence-activated droplet sorting of 1 nL droplets using an upgraded version of the sequentially addressable dielectrophoretic array (SADA), which we reported previously. The SADA is an array of electrodes that are individually and sequentially activated/deactivated according to the speed and position of a droplet passing nearby the array. We upgraded the SADA by increasing the number of driving electrodes constituting the SADA and incorporating a slanted microchannel. By using a ten-electrode SADA with the slanted microchannel, we achieved fluorescence-activated droplet sorting of 1 nL droplets at a record high throughput of 1752 droplets/s, twice as high as the previously reported maximum sorting throughput of 1 nL droplets.     

Formulation and acoustic studies of a new phase-shift agent for diagnostic and therapeutic ultrasound

Recent efforts in the area of acoustic droplet vaporization with the objective of designing extravascular ultrasound contrast agents has led to the development of stabilized, lipid-encapsulated nanodroplets of the highly volatile compound decafluorobutane (DFB). We developed two methods of generating DFB droplets, the first of which involves condensing DFB gas (boiling point from -1.1 to -2 °C) followed by extrusion with a lipid formulation in HEPES buffer. Acoustic droplet vaporization of micrometer-sized lipid-coated droplets at diagnostic ultrasound frequencies and mechanical indices were confirmed optically. In our second formulation methodology, we demonstrate the formulation of submicrometer-sized lipid-coated nanodroplets based upon condensation of preformed microbubbles containing DFB. The droplets are routinely in the 200-300 nm range and yield microbubbles on the order of 1-5 μm once vaporized, consistent with ideal gas law expansion predictions. The simple and effective nature of this methodology allows for the development of a variety of different formulations that can be used for imaging, drug and gene delivery, and therapy. This study is the first to our knowledge to demonstrate both a method of generating ADV agents by microbubble condensation and formulation of primarily submicrometer droplets of decafluorobutane that remain stable at physiological temperatures. Finally, activation of DFB nanodroplets is demonstrated using pressures within the FDA guidelines for diagnostic imaging, which may minimize the potential for bioeffects in humans. This methodology offers a new means of developing extravascular contrast agents for diagnostic and therapeutic applications.