SAW-driven droplet jetting technology in microfluidic: A review

The introduction of surface acoustic wave (SAW) technology on microfluidics has shown its powerfully controlling and actuating fluid and particle capability in a micro-nano scale, such as fluid mixing, fluid translation, microfluidic pumping, microfluidic rotational motor, microfluidic atomization, particle or cell concentration, droplet or cell sorting, reorientation of nano-objects, focusing and separation of particles, and droplet jetting. The SAW-driven droplet jetting technology enjoys the advantages of simple structure to fabricate with little hindrance, compact size to integrate with other components, high biocompatibility with biological cells or other molecule samples, large force in realizing fast fluidic actuation, and contact-free manipulation with fluid. The realization of this technology can effectively overcome some bottleneck problems in the current micro-injection technology, such as mechanical swear, complicated and bulky structure, and strict limitation of requirements on fluidic characteristics. This article reviews and reorganizes SAW-microfluidic jetting technology from decades of years, referring to the interaction mechanism theory of SAW and fluid, experimental methods of SAW-microfluidic jetting, effects of related parameters on objected pinch-off droplets, and applications of individual structures. Finally, we made a summary of the research results of the current literature and look forward and appraise where this discipline of SAW-microfluidic jetting could go in the future.     

Monodisperse alginate microgel formation in a three-dimensional microfluidic droplet generator

Droplet based microfluidic systems provide an ideal platform for partitioning and manipulating aqueous samples for analysis. Identifying stable operating conditions under which droplets are generated is challenging yet crucial for real-world applications. A novel three-dimensional microfluidic platform that facilitates the consistent generation and gelation of alginate-calcium hydrogel microbeads for microbial encapsulation, over a broad range of input pressures, in the absence of surfactants is described. The unique three-dimensional design of the fluidic network utilizes a height difference at the junction between the aqueous sample injection and organic carrier channels to induce droplet formation via a surface tension enhanced self-shearing mechanism. Combined within a flow-focusing geometry, under constant pressure control, this arrangement facilitates predictable generation of droplets over a much broader range of operating conditions than that of conventional two-dimensional systems. The impact of operating pressures and geometry on droplet gelation, aqueous and organic material flow rates, microbead size, and bead generation frequency are described. The system presented provides a robust platform for encapsulating single microbes in complex mixtures into individual hydrogel beads, and provides the foundation for the development of a complete system for sorting and analyzing microbes at the single cell level.     

Piezoelectric-driven droplet impact printing with an interchangeable microfluidic cartridge

Microfluidic impact printing has been recently introduced, utilizing its nature of simple device architecture, low cost, non-contamination, and scalable multiplexability and high throughput. In this paper, we have introduced an impact-based droplet printing platform utilizing a simple plug-and-play microfluidic cartridge driven by piezoelectric actuators. Such a customizable printing system allows for ultrafine control of droplet volume from picoliters (∼23 pl) to nanoliters (∼10 nl), a 500 fold variation. The high flexibility of droplet generation can be simply achieved by controlling the magnitude of actuation (e.g., driving voltage) and the waveform shape of actuation pulses, in addition to nozzle size restrictions. Detailed printing characterizations on these parameters have been conducted consecutively. A multiplexed impact printing system has been prototyped and demonstrated to provide the functions of single-droplet jetting and droplet multiplexing as well as concentration gradient generation. Moreover, a generic biological assay has also been tested and validated on this printing platform. Therefore, the microfluidic droplet printing system could be of potential value to establish multiplexed micro reactors for high-throughput life science applications.     

On demand nanoliter-scale microfluidic droplet generation,injection, and mixing using a passive microfluidic device

We here present and characterize a programmable nanoliter scale droplet-on-demand device that can be used separately or readily integrated into low cost single layer rapid prototyping microfluidic systems for a wide range of user applications. The passive microfluidic device allows external (off-the-shelf) electronically controlled pinch valves to program the delivery of nanoliter scale aqueous droplets from up to 9 different inputs to a central outlet channel. The inputs can be either continuous aqueous fluid streams or microliter scale aqueous plugs embedded in a carrier fluid, in which case the number of effective input solutions that can be employed in an experiment is no longer strongly constrained (100 s–1000 s). Both nanoliter droplet sequencing output and nanoliter-scale droplet mixing are reported with this device. Optimization of the geometry and pressure relationships in the device was achieved in several hardware iterations with the support of open source microfluidic simulation software and equivalent circuit models. The requisite modular control of pressure relationships within the device is accomplished using hydrodynamic barriers and matched resistance channels with three different channel heights, custom parallel reversible microfluidic I/O connections, low dead-volume pinch valves, and a simply adjustable array of external screw valves. Programmable sequences of droplet mixes or chains of droplets can be achieved with the device at low Hz frequencies, limited by device elasticity, and could be further enhanced by valve integration. The chip has already found use in the characterization of droplet bunching during export and the synthesis of a DNA library.     

Metabolite profiling of microfluidic cell culture conditions for droplet based screening

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast. Metabolite profiling provides a more nuanced estimate of cell state compared to proliferation studies alone. We show that the choice of droplet incubation format impacts cell proliferation and metabolite production. The standard syringe incubation of droplets exhibited metabolite profiles similar to oxygen limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format.     

Enhanced single-cell encapsulation in microfluidic devices: From droplet generation to single-cell analysis

Single-cell analysis to investigate cellular heterogeneity and cell-to-cell interactions is a crucial compartment to answer key questions in important biological mechanisms. Droplet-based microfluidics appears to be the ideal platform for such a purpose because the compartmentalization of single cells into microdroplets offers unique advantages of enhancing assay sensitivity, protecting cells against external stresses, allowing versatile and precise manipulations over tested samples, and providing a stable microenvironment for long-term cell proliferation and observation. The present Review aims to give a preliminary guidance for researchers from different backgrounds to explore the field of single-cell encapsulation and analysis. A comprehensive and introductory overview of the droplet formation mechanism, fabrication methods of microchips, and a myriad of passive and active encapsulation techniques to enhance single-cell encapsulation efficiency were presented. Meanwhile, common methods for single-cell analysis, especially for long-term cell proliferation, differentiation, and observation inside microcapsules, are briefly introduced. Finally, the major challenges faced in the field are illustrated, and potential prospects for future work are discussed.     

A numerical study on the dynamics of droplet formation in a microfluidic double T-junction

In this study, droplet formations in microfluidic double T-junctions (MFDTD) are investigated based on a two-dimensional numerical model with volume of fluid method. Parametric ranges for generating alternating droplet formation (ADF) are identified. A physical background responsible for the ADF is suggested by analyzing the dynamical stability of flow system. Since the phase discrepancy between dispersed flows is mainly caused by non-symmetrical breaking of merging droplet, merging regime becomes the alternating regime at appropriate conditions. In addition, the effects of channel geometries on droplet formation are studied in terms of relative channel width. The predicted results show that the ADF region is shifted toward lower capillary numbers when channel width ratio is less than unity. The alternating droplet size increases with the increase of channel width ratio. When this ratio reaches unity, alternating droplets can be formed at very high water fraction (wf = 0.8). The droplet formation in MFDTD depends significantly on the viscosity ratio, and the droplet size in ADF decreases with the increase of the viscosity ratio. The understanding of underlying physics of the ADF phenomenon is useful for many applications, including nanoparticle synthesis with different concentrations, hydrogel bead generation, and cell transplantation in biomedical therapy.     

A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.     

A Laplace pressure based microfluidic trap for passive droplet trapping and controlled release

Here, we present a microfluidic droplet trap that takes advantage of the net Laplace pressure force generated when a droplet is differentially constricted. Mathematical simulations were first used to understand the working range of the component; followed by finite element modeling using the CFD software package to further characterize the behavior of the system. Controlled release of the trapped droplets is also demonstrated through both a mechanical method and a chemical method that manipulates the total pressure exerted on the trapped droplet. The unique design of this trapping device also provides the capability for selection of a single droplet from a train, as well as droplet fusion.     

A microfluidic platform for size-dependent generation of droplet interface bilayer networks on rails

Droplet interface bilayer (DIB) networks are emerging as a cornerstone technology for the bottom up construction of cell-like and tissue-like structures and bio-devices. They are an exciting and versatile model-membrane platform, seeing increasing use in the disciplines of synthetic biology, chemical biology, and membrane biophysics. DIBs are formed when lipid-coated water-in-oil droplets are brought together—oil is excluded from the interface, resulting in a bilayer. Perhaps the greatest feature of the DIB platform is the ability to generate bilayer networks by connecting multiple droplets together, which can in turn be used in applications ranging from tissue mimics, multicellular models, and bio-devices. For such applications, the construction and release of DIB networks of defined size and composition on-demand is crucial. We have developed a droplet-based microfluidic method for the generation of different sized DIB networks (300–1500 pl droplets) on-chip. We do this by employing a droplet-on-rails strategy where droplets are guided down designated paths of a chip with the aid of microfabricated grooves or “rails,” and droplets of set sizes are selectively directed to specific rails using auxiliary flows. In this way we can uniquely produce parallel bilayer networks of defined sizes. By trapping several droplets in a rail, extended DIB networks containing up to 20 sequential bilayers could be constructed. The trapped DIB arrays can be composed of different lipid types and can be released on-demand and regenerated within seconds. We show that chemical signals can be propagated across the bio-network by transplanting enzymatic reaction cascades for inter-droplet communication.     

The dynamics and stability of lubricating oil films during droplet transport by electrowetting in microfluidic devices

The operation of digital microfluidic devices with water droplets manipulated by electrowetting is critically dependent on the static and dynamic stability and lubrication properties of the oil films that separate the droplets from the solid surfaces. The factors determining the stability of the films and preventing surface fouling in such systems are not yet thoroughly understood and were experimentally investigated in this study. The experiments were performed using a standard digital microfluidic cartridge in which water droplets enclosed in a thin, oil-filled gap were transported over an array of electrodes. Stable, continuous oil films separated the droplets from the surfaces when the droplets were stationary. During droplet transport, capillary waves formed in the films on the electrode surfaces as the oil menisci receded. The waves evolved into dome-shaped oil lenses. Droplet deformation and oil displacement caused the films at the surface opposite the electrode array to transform into dimples of oil trapped over the centers of the droplets. Lower actuation voltages were associated with slower film thinning and formation of fewer, but larger, oil lenses. Lower ac frequencies induced oscillations in the droplets that caused the films to rupture. Films were also destabilized by addition of surfactants to the oil or droplet phases. Such a comprehensive understanding of the oil film behavior will enable more robust electrowetting-actuated lab-on-a-chip devices through prevention of loss of species from droplets and contamination of surfaces at points where films may break.     

Vimentin networks at tunable ion-concentration in microfluidic drops

The structure and function of biological systems, for example, cells and proteins, depend strongly on their chemical environment. To investigate such dependence, we design a polydimethylsiloxane-based microfluidic device to encapsulate biological systems in picoliter-sized drops. The content of each individual drop is tuned in a defined manner. As a key feature of our method, the individual chemical composition is determined and related to the drop content. In our case, the drop content is imaged using microscopy methods, while the drops are immobilized to allow for long-time studies. As an application of our device, we study the influence of divalent ions on vimentin intermediate filament networks in a quantitative way by tuning the magnesium concentration from drop to drop. This way we are able to directly image the effect of magnesium on the fluorescently tagged protein in a few hundreds of drops. Our study shows that with increasing magnesium concentration in the drops, the compaction of the networks becomes more pronounced. The degree of compaction is characterized by different morphologies; freely fluctuating networks are observed at comparatively low magnesium concentrations of 5–10 mM, while with increasing magnesium concentration reaching 16 mM they develop into fully aggregated networks. Our approach demonstrates how a systematic study of interactions in biological systems can benefit from the exceptional controllability of microfluidic methods.     

Behavior of liquid plugs at bifurcations in a microfluidic tree network

Flows in complex geometries, such as porous media or biological networks, often contain plugs of liquid flowing within air bubbles. These flows can be modeled in microfluidic devices in which the geometric complexity is well defined and controlled. We study the flow of wetting liquid plugs in a bifurcating network of micro-channels. In particular, we focus on the process by which the plugs divide as they pass each bifurcation. The key events are identified, corresponding to large modifications of the interface curvature, the formation of new interfaces, or the division of a single interface into two new ones. The timing of the different events and the amplitude of the curvature variations are analyzed in view of the design of an event-driven model of flow in branching micro-networks. They are found to collapse onto a master curve dictated by the network geometry.     

Millifluidics as a simple tool to optimize droplet networks: Case study on drop traffic in a bifurcated loop

We report that modular millifluidic networks are simpler, more cost-effective alternatives to traditional microfluidic networks, and they can be rapidly generated and altered to optimize designs. Droplet traffic can also be studied more conveniently and inexpensively at the millimeter scale, as droplets are readily visible to the naked eye. Bifurcated loops, ladder networks, and parking networks were made using only Tygon^® tubing and plastic T-junction fittings and visualized using an iPod^® camera. As a case study, droplet traffic experiments through a millifluidic bifurcated loop were conducted, and the periodicity of drop spacing at the outlet was mapped over a wide range of inlet drop spacing. We observed periodic, intermittent, and aperiodic behaviors depending on the inlet drop spacing. The experimentally observed periodic behaviors were in good agreement with numerical simulations based on the simple network model. Our experiments further identified three main sources of intermittency between different periodic and/or aperiodic behaviors: (1) simultaneous entering and exiting events, (2) channel defects, and (3) equal or nearly equal hydrodynamic resistances in both sides of the bifurcated loop. In cases of simultaneous events and/or channel defects, the range of input spacings where intermittent behaviors are observed depends on the degree of inherent variation in input spacing. Finally, using a time scale analysis of syringe pump fluctuations and experiment observation times, we find that in most cases, more consistent results can be generated in experiments conducted at the millimeter scale than those conducted at the micrometer scale. Thus, millifluidic networks offer a simple means to probe collective interactions due to drop traffic and optimize network geometry to engineer passive devices for biological and material analysis.     

An integrated microfluidic platform to fabricate single-micrometer asymmetric giant unilamellar vesicles (GUVs) using dielectrophoretic separation of microemulsions

We present a microfluidic technique that generates asymmetric giant unilamellar vesicles (GUVs) in the size range of 2–14 μm. In our method, we (i) create water-in-oil emulsions as the precursors to build synthetic vesicles, (ii) deflect the emulsions across two oil streams containing different phospholipids at high throughput to establish an asymmetric architecture in the lipid bilayer membranes, and (iii) direct the water-in-oil emulsions across the oil–water interface of an oscillating oil jet in a co-flowing confined geometry to encapsulate the inner aqueous phase inside a lipid bilayer and complete the fabrication of GUVs. In the first step, we utilize a flow-focusing geometry with precisely controlled pneumatic pressures to form monodisperse water-in-oil emulsions. We observed different regimes in forming water-in-oil multiphase flows by changing the applied pressures and discovered a hysteretic behavior in jet breakup and droplet generation. In the second step of GUV fabrication, an oil stream containing phospholipids carries the emulsions into a separation region where we steer the emulsions across two parallel oil streams using active dielectrophoretic and pinched-flow fractionation separations. We explore the effect of applied DC voltage magnitude and carrier oil stream flow rate on the separation efficiency. We develop an image processing code that measures the degree of mixing between the two oil streams as the water-in-oil emulsions travel across them under dielectrophoretic steering to find the ideal operational conditions. Finally, we utilize an oscillating co-flowing jet to complete the formation of asymmetric giant unilamellar vesicles and transfer them to an aqueous phase. We investigate the effect of flow rates on properties of the co-flowing jet oscillating in the whipping mode (i.e., wavelength and amplitude) and define the phase diagram for the oil-in-water jet. Assays used to probe the lipid bilayer membrane of fabricated GUVs showed that membranes were unilamellar, minimal residual oil remained trapped between the two lipid leaflets, and 83% asymmetry was achieved across the lipid bilayers of GUVs.     

Temperature-induced droplet coalescence in microchannels

This paper reports a technique for temperature-induced merging of droplets in a microchannel. The multiphase system consists of water droplet and oil as the dispersed phase and the carrying continuous phase. A resistive heater provides heating in a rectangular merging chamber. The temperature of the chamber is controlled by the voltage applied to the heater. The merging process of two neighboring droplets was investigated with different applied voltage, flow rate ratio between water and oil and total flowrate. Merging is found to be effective at high flow rate ratio, high temperature, and low total flowrate. The presented technique could be used for merging and mixing in droplet-based lab-on-a-chip platforms.     

Focusing-enhanced mixing in microfluidic channels

A focusing-based microfluidic mixer was studied. The micromixer utilizes the focusing process required for cytometry to reduce the diffusion distance of molecules to be mixed in order to facilitate the passive diffusion-controlled mixing process. It was found that both the high flow rate ratio of the sheath flow to the flows to be mixed and the low flow rate of the mixing fluids resulted in the short mixing length required within the microfluidic channel. It was shown that a complete mixing was achieved within a distance of 4 mm in the micromixer for the focused mixing fluids at a flow rate of 2 μl∕min and a flow rate ratio of the sheath flow to the flows to be mixed at 4:1. The mixer described here is simple and can be easily fabricated and controlled.     

Osteocyte culture in microfluidic devices

This paper presents a microfluidic device (poly-dimethylsiloxane micro channels bonded with glass slides) enabling culture of MLO-Y4 osteocyte like cells. In this study, on-chip collagen coating, cell seeding and culture, as well as staining were demonstrated in a tubing-free manner where gravity was used as the driving force for liquid transportation. MLO-Y4 cells were cultured in microfluidic channels with and without collagen coating where cellular images in a time sequence were taken and analyzed, confirming the positive effect of collagen coating on phenotype maintaining of MLO-Y4 cells. The proliferating cell nuclear antigen based proliferation assay was used to study cellular proliferation, revealing a higher proliferation rate of MLO-Y4 cells seeded in microfluidic channels without collagen coating compared to the substrates coated with collagen. Furthermore, the effects of channel dimensions (variations in width and height) on the viability of MLO-Y4 cells were explored based on the Calcein-AM and propidium iodide based live/dead assay and the Hoechst 33258 based apoptosis assay, locating the correlation between the decrease in channel width or height and the decrease in cell viability. As a platform technology, this microfluidic device may function as a new cell culture model enabling studies of osteocytes.     

Field-free particle focusing in microfluidic plugs

Particle concentration is a key unit operation in biochemical assays. Although there are many techniques for particle concentration in continuous-phase microfluidics, relatively few are available in multiphase (plug-based) microfluidics. Existing approaches generally require external electric or magnetic fields together with charged or magnetized particles. This paper reports a passive technique for particle concentration in water-in-oil plugs which relies on the interaction between particle sedimentation and the recirculating vortices inherent to plug flow in a cylindrical capillary. This interaction can be quantified using the Shields parameter (θ), a dimensionless ratio of a particle’s drag force to its gravitational force, which scales with plug velocity. Three regimes of particle behavior are identified. When θ is less than the movement threshold (region I), particles sediment to the bottom of the plug where the internal vortices subsequently concentrate the particles at the rear of the plug. We demonstrate highly efficient concentration (∼100%) of 38 μm glass beads in 500 μm diameter plugs traveling at velocities up to 5 mm/s. As θ is increased beyond the movement threshold (region II), particles are suspended in well-defined circulation zones which begin at the rear of the plug. The length of the zone scales linearly with plug velocity, and at sufficiently large θ, it spans the length of the plug (region III). A second effect, attributed to the co-rotating vortices at the rear cap, causes particle aggregation in the cap, regardless of flow velocity. Region I is useful for concentrating/collecting particles, while the latter two are useful for mixing the beads with the solution. Therefore, the two key steps of a bead-based assay, concentration and resuspension, can be achieved simply by changing the plug velocity. By exploiting an interaction of sedimentation and recirculation unique to multiphase flow, this simple technique achieves particle concentration without on-chip components, and could therefore be applied to a range of heterogeneous screening assays in discrete nl plugs.