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Superhydrophobic paper substrates were patterned with high surface energy black ink using commercially available desktop printing technology. The shape and size of the ink islands were designed to control the adhesion forces on water drops in two directions, parallel (‘drag-adhesion’) and perpendicular (‘extensional-adhesion’) to the substrate. Experimental data on the adhesion forces shows good agreement with classical models for ‘drag’ (Furmidge equation) and ‘extensional’ adhesion (modified Dupré equation). The tunability of the two adhesion forces was used to implement four basic unit operations for the manipulation of liquid drops on the paper substrates: storage, transfer, mixing and sampling. By combining these basic functionalities it is possible to design simple two-dimensional lab-on-paper (LOP) devices. In our 2D LOP prototype, liquid droplets adhere to the porous substrate, rather than absorbing into the paper; as a result, liquid droplets remain accessible for further quantitative testing and analysis, after performing simple qualitative on-chip testing. In addition, the use of commercially available desktop printers and word processing software to generate ink patterns enable end users to design LOP devices for specific applications. [ABSTRACT FROM AUTHOR]

We present a facile approach for the fabrication of low-cost surface biomicrofluidic devices on superhydrophobic paper created by drop-casting a fluoroacrylic copolymer onto microtextured paper. Wettability patterning is performed with a common household printer, which produces regions of varying wettability by simply controlling the intensity of ink deposited over prespecified domains. The procedure produces surfaces that are capable of selective droplet sliding and adhesion, when inclined. Using this methodology, we demonstrate the ability to tune the sliding angles of 10 μL water droplets in the range from 13° to 40° by printing lines of constant ink intensity and varied width from 0.1 mm to 2 mm. We also formulate a simple model to predict the onset of droplet sliding on printed lines of known width and wettability. Experiments demonstrate open-air surface microfluidic devices that are capable of pumpless transport, mixing and rapid droplet sampling (~0.6 μL at 50 Hz). Lastly, post treatment of printed areas with pH indicator solutions exemplifies the utility of these substrates in point-of-care diagnostics, which are needed at geographical locations where access to sophisticated testing equipment is limited or non-existent. [ABSTRACT FROM AUTHOR]

This paper reports a portable viscometer that requires less than 10 μL of sample for a measurement. Using a two-droplet Laplace-induced pumping system on an open microfluidic substrate, the device measures the viscosity of a liquid by determining the time required for one droplet to completely pump into a second droplet. The pumping behaviour follows the Hagen–Poiseuille and Laplace relations where the flow rate, Q, is proportional to the liquid's kinematic viscosity, μ. The progress of pumping is measured by tracking the change in curvature of one of the droplets using a laser that is positioned perpendicular to the microfluidic chip and directed at the "tail" of the shrinking droplet. The angle of incidence and degree of refraction changes depending on the size of the droplet, which is tracked by a linear diode array placed beneath the microfluidic chip. Droplet reservoirs and connecting channels were defined by precise patterning of a glass substrate coated with a commercially available omniphobic coating (Ultra Ever Dry®) using laser micromachining. A 500 μm wide and 20 mm long channel with circular reservoirs (d = 1.5 mm) enabled the measurement of dynamic viscosities in the range of η = 1.0–2.87 mPa s. The materials cost for the entire viscometer (fluidics and electronics, etc.) is <15 USD. [ABSTRACT FROM AUTHOR]

Extensive research has demonstrated the potential of cell viscoelastic properties as intrinsic indicators of cell state, functionality, and disease. For this, several microfluidic techniques have been developed to measure cell viscoelasticity with high-throughput. However, current microchannel designs introduce complex stress distributions on cells, leading to inaccuracies in determining the stress--strain relationship and, consequently, the viscoelastic properties. Here, we introduce a novel approach using hyperbolic microchannels that enable precise measurements under a constant extensional stress and offer a straightforward stress--strain relationship, while operating at a measurement rate of up to 100 cells per second. We quantified the stresses acting in the channels using mechanical calibration particles made from polyacrylamide (PAAm) and found that the measurement buffer, a solution of methyl cellulose and phosphate buffered saline, shows strain-thickening following a power law up to 200 s-1. By measuring oil droplets with varying viscosities, we successfully detected changes in the relaxation times of the droplets and our approach could be used to get the interfacial tension and viscosity of liquid--liquid droplet systems from the same measurement. We further applied this methodology to PAAm microgel beads, demonstrating the accurate recovery of Young's moduli and the near-ideal elastic behavior of the beads. To explore the influence of altered cell viscoelasticity, we treated HL60 human leukemia cells with latrunculin B and nocodazole, resulting in clear changes in cell stiffness while relaxation times were only minimally affected. In conclusion, our approach offers a streamlined and time-efficient solution for assessing the viscoelastic properties of large cell populations and other microscale soft particles. [ABSTRACT FROM AUTHOR]

The transportation and control of microfluidics have an important influence on the fields of biology, chemistry, and medicine. Pump systems based on the electrocapillary effect and room-temperature liquid metal droplets have attracted extensive attention. Flow rate is an important parameter that reflects the delivery performance of the pump systems. In the systems of previous studies, cylindrical structures are mostly used to constrain the droplet. The analysis and quantitative description of the influence of voltage frequency, alternating voltage, direct current voltage bias, and solution concentration on the flow rate are not yet comprehensive. Furthermore, the systems are driven by only one droplet, which limits the increase in flow rate. Therefore, a pump with a cuboid structure is designed and the droplet is bound by pillars, and the flow rate of the pump is increased by more than 200% compared with the cylindrical pump. For this structure, the mechanism of various factors on the flow rate is analyzed. To further enhance the flow rate, a pump system with multi-droplets is proposed. Moreover, the expression of flow velocity of the solution on the surface of each droplet and the relationship between the flow rate, alternating voltage, and the number of droplets are deduced. Finally, the potential of applying the multi-droplet cuboid pump system in drug delivery and analytical chemistry is demonstrated. Additionally, the core of the pump, the droplet area, is modularized, which breaks the overall structural limitations of the liquid metal pump and provides ideas for pump design. [ABSTRACT FROM AUTHOR]

Ultrasonic atomization of liquids into micrometer-diameter droplets is crucial across multiple fields, ranging from drug delivery, to spectrometry and printing. Controlling the size and uniformity of the generated droplets on-demand is crucial in all these applications. However, existing systems lack the required precision to tune the droplet properties, and the underlying droplet formation mechanism under high-frequency ultrasonic actuation remains poorly understood due to experimental constraints. Here, we present an atomization platform, which overcomes these current limitations. Our device utilizes oscillating high aspect ratio micro-channels to extract liquids from various inlets (ranging from sessile droplets to large beakers), bound them in a precisely defined narrow region, and, controllably atomize them on-demand. The droplet size can be precisely dialled from 3.6 μm to 6.8 μm by simply tuning the actuation parameters. Since the approach does not need nozzles, meshes or impacting jets, stresses exerted on the liquid samples are reduced, hence it is gentler on delicate samples. The precision offered by the technique allows us for the first time to experimentally visualise the oscillating fluid interface at the onset of atomization at MHz frequencies, and demonstrate its applications for targeted respiratory drug delivery. [ABSTRACT FROM AUTHOR]

High-throughput experiments involving isolated droplets based on patterned superwettable surfaces are important for various applications related to biology, chemistry, and medicine, and they have attracted a large amount of interest. This paper provides a directional anchoring liquid-infused superamphiphobic surface (DAS), via combining concepts based on the droplet-anchoring behavior of beetle backs with patterned wettability, the directional adhesion of butterfly wings, and the slippery liquid-infused surfaces (SLISs) of pitcher plants. Regularly arranged ">"-shaped SLIS patterns were created on a superamphiphobic (SAM) background through ultrafast-laser-based technology. Improved directional anchoring abilities with a sliding angle difference of 77° were achieved; this is the largest sliding angle difference in a one-dimensional direction achieved using an artificial surface, to the best of the authors' knowledge. Thanks to the directional anchoring abilities, the DAS coupled droplet 'anchoring' and 'releasing' abilities. Furthermore, a high-throughput droplet manipulation device was designed, on which a micro-droplet array with a large number of droplets can be 'captured', 'transferred', or 'released' in a single step. With the addition of lubricant, the DAS can work continuously for even more than 30 cycles without cross-contamination between different droplets. The DAS also shows good stability under an ambient atmosphere and can maintain its functionality when manipulating corrosive droplets. The DAS and corresponding high-throughput droplet manipulation method are excellent candidates for practical applications. [ABSTRACT FROM AUTHOR]

Lab-on-chip electrokinetic focusing and separation techniques are widely used in several scientific fields. In a number of cases, these techniques have been combined with a selective analyte extraction for off-chip analysis. Nevertheless, the usability of the extracts is limited by diffusion which reduces the separation resolution. In this paper we propose the integration of a droplet generator capable of continuous or on-demand generation and extraction of electrokinetically separated and focused analytes. We demonstrate the selective droplet extraction of model analytes separated and concentrated via ion concentration polarization focusing (ICPF). We report extracted droplets with 1000-fold increased concentration. Importantly, the droplet generator does not interrupt the ICPF process making it suitable for integration with the majority of electrokinetic separation techniques. [ABSTRACT FROM AUTHOR]

This reply to the comment by Nakajima on our article that appeared in Lab on a Chip (E. Amstad, M. Chemama, M. Eggersdorfer, L. R. Arriaga, M. Brenner and D. A. Weitz, Lab Chip, 2016, 16, 4163–4172) highlights the differences between the microchannel step emulsification devices developed by the Nakajima group and the millipede device reported by us in Lab on a Chip. [ABSTRACT FROM AUTHOR]

Co-encapsulation of two distinct particles within microfluidic droplets provides the means to achieve various high-throughput single-cell assays, such as biochemical reactions and cell–cell interactions in small isolated volumes. However, limited by the Poisson statistics, the co-encapsulation rate of the conventional co-flow approach is low even under optimal conditions. Only up to 13.5% of droplets precisely contain a pair of two distinct particles, while the rest, either being empty or encapsulating unpaired particles become wastes. Thus, the low co-encapsulation efficiency makes droplet-based assays impractical in biological applications involving low abundant bioparticles. In this paper, we present a highly promising droplet merging strategy to increase the co-encapsulation efficiency. Our method first enriches droplets exactly encapsulating a single particle via fluorescence or scattering-light activated sorting. Then, two droplets, each with a distinct particle, are precisely one-to-one paired and merged in a novel microwell device. This deterministic approach overcomes the Poisson statistics limitation facing conventional stochastic methods, yielding an up to 90% post-sorting particle capture rate and an overall 88.1% co-encapsulation rate. With its superior single-particle pairing performance, our system provides a promising technological platform to enable highly efficient microdroplet assays. [ABSTRACT FROM AUTHOR]

Taking information as material to realize non-electronic physical computing is a promising idea, which facilitates the integration of technologies in different fields such as chemistry, biology, and mechanical control into a new computing platform. Here, we propose a novel, efficient and robust manipulation platform that drives droplet computing by way of inertial force. Combining this with droplet flow path design, we demonstrated multiple basic functions of droplet manipulation, including storage, dosing, interrupts, controllable release and addressing. These basic functions without external control lay the foundation for the realization of droplet calculation. We developed AND, OR, and XOR logic gates of the "liquid circuit" and combined them into a binary adder, which successfully completed the addition of four-digit binary numbers through droplet movement. Moreover, we attempted to perform algorithmic design for biological information under the control of droplets based on synchronous logical operations, developing the possibility of biological applications. This programmable physical computing system exists independently of electronic computing, aiming to supplement and expand the computing methods outside the field of electronic technology and to open a new method for the algorithmic operation of materials after combining new physical computing technologies such as biological or chemical computing. [ABSTRACT FROM AUTHOR]

Technical advances in electrowetting-on-dielectric (EWOD) over the past few years have extended our attraction to three-dimensional (3D) devices capable of providing more flexibility and functionality with larger volumetric capacity than conventional 2D planar ones. However, typical 3D EWOD devices require complex and expensive fabrication processes for patterning and wiring of pixelated electrodes that also restrict the minimum droplet size to be manipulated. Here, we present a flexible single-sided continuous optoelectrowetting (SCOEW) device which is not only fabricated by a spin-coating method without the need for patterning and wiring processes, but also enables light-driven 3D droplet manipulations. To provide photoconductive properties, previous optoelectrowetting (OEW) devices have used amorphous silicon (a-Si) typically fabricated through high-temperature processes over 300 °C such as CVD or PECVD. However, most of the commercially-available flexible substrates such as polyethylene terephthalate (PET) and polyethylene naphthalate (PEN) experience serious thermal deformation under such high-temperature processes. Because of this compatibility issue of conventional OEW devices with flexible substrates, light-driven 3D droplet manipulations have not yet been demonstrated on flexible substrates. Our study overcomes this compatibility issue by using a polymer-based photoconductive material, titanium oxide phthalocyanine (TiOPc) and thus SCOEW devices can be simply fabricated on flexible substrates through a low-cost, spin-coating method. In this paper, analytical studies were conducted to understand the effects of light patterns on static contact angles and EWOD forces. For experimental validations of our study, flexible SCOEW devices were successfully fabricated through the TiOPc-based spin-coating method and light-driven droplet manipulations (e.g. transportation, merging, and splitting) have been demonstrated on various 3D terrains such as inclined, vertical, upside-down, and curved surfaces. Our flexible SCOEW technology offers the benefits of device simplicity, flexibility, and functionality over conventional EWOD and OEW devices by enabling optical droplet manipulations on a 3D featureless surface. [ABSTRACT FROM AUTHOR]

Increases in complexity attainable in molecular self-assembly necessitates both advanced molecular design as well as microenvironmental control. Such control is offered by microfluidics, where precise chemical compositions and gradients can be readily established. A droplet microfluidic platform combining upstream step emulsification with downstream hydrodynamic microtraps has been designed to facilitate molecular self-assembly. The step emulsification rapidly generates uniform droplets which act as reaction chambers. The hydrodynamic microtraps hold droplets against the flow ensuring they are exposed to a continuous supply of fresh fluid for constant reagent extraction and/or delivery. Additionally, the droplet immobilization permits real-time droplet characterization and reaction monitoring. Subsequently, droplets can be released from the traps through flow reversal, allowing post-process characterization. The microfluidic system was demonstrated by the phase separation of lyotropic droplets. Ethanol/water droplets were created in a continuous ambient squalene/monoolein microflow, causing the continuous extraction of ethanol from the droplets and delivery of monoolein from the ambient microflow. Unlike conventional bulk techniques and continuous microfluidics, where finite microchannel lengths necessarily impose limits to the extent to which slow processes can proceed, this approach allows extended duration reactions whilst enabling real time process monitoring. [ABSTRACT FROM AUTHOR]

Picoinjection is a robust method for reagent addition into microfluidic droplets and has enabled the implementation of numerous multistep droplet assays. Although serial picoinjectors allow to screen many conditions in one run by injecting different combinations of reagents, their use is limited because it is complex to accurately control each injector independently. Here, we present a novel method for flexible, individual picoinjector control that allows to inject a predefined range of volumes by controlling the flow rate of the injector as well as turning off injection by setting the equilibrium pressure, which resulted in a stable interface of the injector liquid with the main microfluidic channel. Robust setting of the equilibrium pressure of an injector was achieved by applying accurate (R2 > 0.94) linear models between the injector and oil pressure in real-time. To illustrate the flexibility of this method, we performed several proof-of-concepts using 1, 2 or 3 picoinjectors loaded with fluorescent dyes. We successfully demonstrated picoinjection approaches using time-invariant settings, in which an injector setting was applied for prolonged times, and time-variant picoinjection, in which the injector settings were continuously varied in order to sweep the injected volumes, both resulting in monodisperse (CV < 3.4%) droplet libraries with different but reproducible fluorescent intensities. To illustrate the potential of the technology for fast compound concentration screenings, we studied the effect of a concentration range of a detergent on single-cell lysis. We anticipate that this robust and versatile methodology will make the serial picoinjection technology more accessible to researchers, allowing its wide implementation in numerous life science applications. [ABSTRACT FROM AUTHOR]

Droplet microfluidics has emerged as a prospering field for lab-on-a-chip devices, where droplets serve as liquid vessels e.g. for biochemical reagents. Key to the fluid processing in droplet format are the controlled droplet handling and movement on the microscale. Hence this paper proposes droplet handling by combining droplet microfluidics with bulk acoustic wave (BAW) acoustophoresis. BAW acoustophoresis has formerly focused on cell and particle handling, whereas here we determine the various abilities of this method for the field of droplet microfluidics. In silicon microdevices, water-in-oil droplets of 200 μm size were generated for a set of unit operations including droplet fusion, focusing, sorting and medium exchange around 0.5–1 MHz acoustic frequency. Compared to existing droplet handling methods, the shown method is simple in fabrication, robust in operation and versatile to meet the needs of various droplet processing microfluidic devices. [ABSTRACT FROM AUTHOR]

Crystallization is one of the most crucial steps in the process of pharmaceutical formulation. In recent years, emulsion-based platforms have been developed and broadly adopted to generate high quality products. However, these conventional approaches such as stirring are still limited in several aspects, e.g., unstable crystallization conditions and broad size distribution; besides, only simple crystal forms can be produced. In this paper, we present a new flow-free droplet-based formation process for producing highly controlled crystallization with two examples: (1) NaCl crystallization reveals the ability to package saturated solution into nanoliter droplets, and (2) glycine crystallization demonstrates the ability to produce polymorphic crystallization forms by controlling the droplet size and temperature. In our process, the saturated solution automatically fills the microwell array powered by degassed bulk PDMS. A critical oil covering step is then introduced to isolate the saturated solution and control the water dissolution rate. Utilizing surface tension, the solution is uniformly packaged in the form of thousands of isolating droplets at the bottom of each microwell of 50–300 μm diameter. After water dissolution, individual crystal structures are automatically formed inside the microwell array. This approach facilitates the study of different glycine growth processes: α-form generated inside the droplets and γ-form generated at the edge of the droplets. With precise temperature control over nanoliter-sized droplets, the growth of ellipsoidal crystalline agglomerates of glycine was achieved for the first time. Optical and SEM images illustrate that the ellipsoidal agglomerates consist of 2–5 μm glycine clusters with inner spiral structures of ~35 μm screw pitch. Lastly, the size distribution of spherical crystalline agglomerates (SAs) produced from microwells of different sizes was measured to have a coefficient variation (CV) of less than 5%, showing crystal sizes can be precisely controlled by microwell sizes with high uniformity. This new method can be used to reliably fabricate monodispersed crystals for pharmaceutical applications. [ABSTRACT FROM AUTHOR]

This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated. [ABSTRACT FROM AUTHOR]

In this paper, we develop a strategy to form on-demand droplets with specific pH values. The pH control is based on electrolysis of water in microfluidics, and the produced hydrogen and hydroxyl ions are separated and confined in individual containers during the droplet generation, triggered by a pressure pulse. By tuning the applied voltages and pressure pulses, we can control on demand the pH value in a droplet. [ABSTRACT FROM AUTHOR]

This paper reports a droplet-based microfluidic approach to fabricate a large number of monodisperse, portable microtissues, each in an individual drop. We use water–water–oil double emulsions as templates and spatially assemble hepatocytes in the core and fibroblasts in the shell, forming a 3D liver model in a drop. [ABSTRACT FROM AUTHOR]

This study presented a convenient method of gathering, splitting, merging, and sorting microdroplets by dynamic pneumatic rails in double-layered microfluidic devices. In these devices, the pneumatic rails were placed below the droplet channel, with a thin elastic polydimethylsiloxane (PDMS) film between them. The PDMS film would sag down to the rail channel, forming a groove pattern at the bottom of the droplet channel, when the fluid pressure in the droplet channel was higher than the air pressure in the rail channel. The groove could capture the flattened droplets and guide the flow path of them due to the lowered surface energy when they extended into the groove. We have designed different components consisting of pneumatic rails to split, merge and sort droplets, and demonstrated that the components maintained good performance in manipulating droplets only by controlling the air pressure. Furthermore, a pneumatic rail-based sorter has been successfully used to sort out single-cell droplets. The pneumatic rail can be integrated into pneumatic valve-based microfluidic devices to be a flexible tool for droplet-based biological and chemical analysis. [ABSTRACT FROM AUTHOR]

Microfluidic chips have demonstrated unparalleled abilities in droplet generation, including precise control over droplet size and monodispersity. And yet, their rather complicated microfabrication process and operation can be a barrier for inexperienced researchers, which hinders microdroplets from unleashing their potential in broader fields of research. Here, we attempt to remove this barrier by developing an integrated and modular revolving needle emulsion generator (RNEG) to achieve high-throughput production of uniformly sized droplets in an off-chip manner. The RNEG works by driving a revolving needle to pinch the dispersed phase in a minicentrifuge tube. The system is constructed using modular components without involving any microfabrication, thereby enabling user-friendly operation. The RNEG is capable of producing microdroplets of various liquids with diameters ranging from tens to hundreds of micrometres. We further examine the principle of operation using numerical simulations and establish a simple model to predict the droplet size. Moreover, by integrating curing and centrifugation processes, the RNEG can produce hydrogel microparticles and transfer them from an oil phase into a water phase. Using this ability, we demonstrate the encapsulation and culture of single yeast cells within hydrogel microparticles. We envisage that the RNEG can become a versatile and powerful tool for high-throughput production of emulsions to facilitate diverse biological and chemical research. [ABSTRACT FROM AUTHOR]

The dynamic control of the chemical concentration within droplets is required in numerous droplet microfluidic applications. Here, we propose an acoustofluidic method for the generation of a library of aqueous droplets with the desired chemical concentrations in a continuous oil phase. Surface acoustic waves produced by a focused interdigital transducer interact with two parallel laminar streams with different chemical compositions. Coupling the acoustic waves with the flow streams results in the controlled acoustofluidic mixing of the aqueous solutions through the formation of acoustic streaming flow-induced microvortices. The mixed streams are split at a bifurcation, and one of the streams with a precisely controlled chemical concentration is fed into a T-junction to produce droplets with tunable chemical concentrations. The periodic acoustofluidic mixing of the aqueous streams enables the generation of a droplet library with a well-defined inter-droplet concentration gradient. The proposed method is a promising tool for the on-chip dynamic control of in-droplet chemical concentrations and for next-generation droplet microfluidic applications. [ABSTRACT FROM AUTHOR]

Droplet microfluidics systems hold great promise in their ability to conduct high-throughput assays for a broad range of life science applications. Despite their promise in the field and capability to conduct complex liquid handling steps, currently, most droplet microfluidic systems used for real assays utilize only a few droplet manipulation steps connected in series, and are often not integrated together on a single chip or platform. This is due to the fact that linking multiple sequential droplet functions within a single chip to operate at high efficiency over long periods of time remains technically challenging. Considering sequential manipulation is often required to conduct high-throughput screening assays on large cellular and molecular libraries, advancements in sequential operation and integration are required to advance the field. This current limitation greatly reduces the type of assays that can be realized in a high-throughput droplet format and becomes more prevalent in large library screening applications. Here we present an integrated multi-layer droplet microfluidic platform that can handle large numbers of droplets with high efficiency and minimum error. The platform combines two-photon photolithography-fabricated curved microstructures that allow high-efficiency (99.9%) re-flow of droplets and a unique droplet cleaving that automatically synchronizes paired droplets enabling high-efficiency (99.9%) downstream merging. We demonstrate that this method is applicable to a broad range of droplet sizes, including relatively large droplet sizes (hundreds of micrometers in diameter) that are typically more difficult to manipulate with high efficiency, yet are required in many cell assay applications requiring large organisms or multiple incubation steps. The utility of this highly efficient integrated droplet microfluidic platform was demonstrated by conducting a mock antibiotic screening assay against a bacterial pathogen. The approach and system presented here provides new avenues for the realization of ultra-high-efficiency multi-step droplet microfluidic systems with minimal error. [ABSTRACT FROM AUTHOR]

Droplet microfluidics has provided lab-on-a-chip platforms with the capability of bacteria encapsulation in biomaterials, controlled culture environments, and live monitoring of growth and proliferation. The droplets are mainly generated from biomaterials with temperature dependent gelation behavior which necessitates stable and size-controlled droplet formation within microfluidics. Here, the biomaterial is agar hydrogel with a non-Newtonian response at operating temperatures below 40 °C, the upper-temperature threshold for cells and pathogens. The size of the produced droplets and the formation regimes are examined when the agar is injected at a constant temperature of 37 °C with agar concentrations of 0.5%, 1%, and 2% and different flow rate ratios of the dispersed phase to the continuous phase (ϕ: 0.1 to 1). The numerical simulations show that ϕ and the capillary number (Ca) are the key parameters controlling the agar droplet size and formation regime, from dripping to jetting. Also, increasing the agar concentration produces smaller droplets. The simulation data were validated against experimental agar droplet generation and transport in microfluidics. This work helps to understand the physics of droplet generation in droplet microfluidic systems operating with non-Newtonian fluids. Pathogenic bacteria were successfully cultured and monitored in high resolution in agar droplets for further research in antibiotic susceptibility testing in bacteremia and urinary tract infection. [ABSTRACT FROM AUTHOR]

A microfluidic approach to seeded crystallization has been demonstrated using abacavir hemisulfate, a nucleoside analog reverse transcriptase inhibitor, in droplet reactors to control polymorphism and produce particles with a low particle size distribution. Two techniques are introduced: (1) the first technique involves an emulsion system consisting of a dispersed phase solvent and a continuous phase, which holds slight solubility of the dispersed phase solvent. The dispersed phase contains both a dissolved active pharmaceutical ingredient (API) and seeds of the desired polymorph. While the continuous phase enables solvent extraction, the negligible solubility of the API allows for growth of seeds inside droplets via extraction and subsequent API saturation. This technique demonstrates the ability to crystallize the API in spherical agglomerates via slow extraction of droplets. (2) The second technique utilizes a combined dispersed phase by joining in-flow a seed suspension stream with a supersaturated active pharmaceutical ingredient (API) stream. The combined dispersed phase is emulsified in a continuous phase for which the dispersed phase solvent and the API are both insoluble – droplets are incubated at temperatures below their saturation limit to induce crystal growth. Decreasing the concentration of seeds in its input stream resulted in a decreased number of crystals per droplet, increase in crystal size, and decrease in PSD. Temperature cycling was utilized as a proof of concept to demonstrate the ability to reduce the number of seeds per droplet where the optimal goal is to obtain a single seed per droplet for all droplets. Utilizing this approach in conjunction with the ability to produce monodispersed droplet reactors allows for enhanced control of particle size distribution (PSD) by precisely controlling the available mass for each individual seed crystal. The development of this technique as a proof-of-concept for crystallization can be expanded to manufacturing scales in a continuous manner using parallelized droplet generators and flow reactors to precisely control the temperature and crystal growth kinetics of individual droplets. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Our understanding of adipose tissue biology has steadily evolved. While structural and energy storage functionalities have been in the forefront, a key endocrine role for adipocytes was revealed only over the last few decades. In contrast to the wealth of information on dynamic function of other endocrine tissues, few studies have focused on dynamic adipose tissue function or on tool development toward that end. Here, we apply our unique droplet-based microfluidic devices to culture, perfuse, and sample secretions from primary murine epididymal white adipose tissue (eWAT), and from predifferentiated clusters of 3T3-L1 adipocytes. Through automated control, oil-segmented aqueous droplets (∼2.6 nL) were sampled from tissue or cells at 3.5 second temporal resolution (including sample and reference droplets), with integrated enzyme assays enabling real-time quantification of glycerol (down to 1.9 fmol per droplet). This high resolution revealed previously unreported oscillations in secreted glycerol at frequencies of 0.2 to 2.0 min−1 (∼30–300 s periods) present in the primary tissue but not in clustered cells. Low-level bursts (∼50 fmol) released in basal conditions were contrasted with larger bursts (∼300 fmol) during stimulation. Further, both fold changes and burst magnitudes were decreased in eWAT of aged and obese mice. These results, combined with immunostaining and photobleaching analyses, suggest that gap-junctional coupling or nerve cell innervation within the intact ex vivo tissue explants play important roles in this apparent tissue-level, lipolytic synchronization. High-resolution, quantitative sampling by droplet microfluidics thus permitted unique biological information to be observed, giving an analytical framework poised for future studies of dynamic oscillatory function of adipose and other tissues. [ABSTRACT FROM AUTHOR]

Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses. Cytokine capture assays that use engineered antibodies to anchor the secreted molecules to the secreting cells are widely used to characterize immune responses because they allow both sensitive identification and recovery of viable responding cells. However, if the cytokines diffuse away from the secreting cells, non-secreting cells will also be identified as responding cells. Here we encapsulate immune cells in microfluidic droplets and perform in-droplet cytokine capture assays to limit the diffusion of the secreted cytokines. We use microfluidic devices to rapidly encapsulate single natural killer NK-92 MI cells and their target K562 cells into microfluidic droplets. We perform in-droplet IFN-γ capture assays and demonstrate that NK-92 MI cells recognize target cells within droplets and become activated to secrete IFN-γ. Droplet encapsulation prevents diffusion of secreted products to neighboring cells and dramatically reduces both false positives and false negatives, relative to assays performed without droplets. In a sample containing 1% true positives, encapsulation reduces, from 94% to 2%, the number of true-positive cells appearing as negatives; in a sample containing 50% true positives, the number of non-stimulated cells appearing as positives is reduced from 98% to 1%. After cells are released from the droplets, secreted cytokine remains captured onto secreting immune cells, enabling FACS-isolation of populations highly enriched for activated effector immune cells. Droplet encapsulation can be used to reduce background and improve detection of any single-cell secretion assay. [ABSTRACT FROM AUTHOR]

Uncovering the heterogeneity of cellular populations and multicellular constructs is a long-standing goal in fields ranging from antimicrobial resistance to cancer research. Emerging technology platforms such as droplet microfluidics hold the promise to decipher such heterogeneities at ultra-high-throughput. However, there is a lack of methods able to rapidly identify and isolate single cells or 3D cell cultures. Here we demonstrate that deep neural networks can accurately classify single droplet images in real-time based on the presence and number of micro-objects including single mammalian cells and multicellular spheroids. This approach also enables the identification of specific objects within mixtures of objects of different types and sizes. The training sets for the neural networks consisted of a few hundred images manually picked and augmented to up to thousands of images per training class. Training required less than 10 minutes using a single GPU, and yielded accuracies of over 90% for single mammalian cell identification. Crucially, the same model could be used to classify different types of objects such as polystyrene spheres, polyacrylamide beads and MCF-7 cells. We applied the developed method for the selection of 3D cell cultures generated with Hek293FT cells encapsulated in agarose gel beads, highlighting the potential of the technology for the selection of objects with a high diversity of visual appearances. The real-time sorting of single droplets was in-line with droplet generation and occurred at rates up to 40 per second independently of image size up to 480 × 480 pixels. The presented microfluidic device also enabled storage of sorted droplets to allow for downstream analyses. [ABSTRACT FROM AUTHOR]

High information content analysis, enrichment, and selection of rare events from a large population are of great importance in biological and biomedical research. The fluorescence lifetime of a fluorophore, a photophysical property which is independent of and complementary to fluorescence intensity, has been incorporated into various imaging and sensing techniques through microscopy, flow cytometry and droplet microfluidics. However, the throughput of fluorescence lifetime activated droplet sorting is orders of magnitude lower than that of fluorescence activated cell sorting, making it unattractive for applications such as directed evolution of enzymes, despite its highly effective compartmentalization of library members. We developed a microfluidic sorter capable of selecting fluorophores based on fluorescence lifetime and brightness at two excitation and emission colors at a maximum droplet rate of 2.5 kHz. We also present a novel selection strategy for efficiently analyzing and/or enriching rare fluorescent members from a large population which capitalizes on the Poisson distribution of analyte encapsulation into droplets. The effectiveness of the droplet sorter and the new selection strategy are demonstrated by enriching rare populations from a ∼108-member site-directed mutagenesis library of fluorescent proteins expressed in bacteria. This selection strategy can in principle be employed on many droplet sorting platforms, and thus can potentially impact broad areas of science where analysis and enrichment of rare events is needed. [ABSTRACT FROM AUTHOR]

Microfluidic droplet generators excel in generating monodisperse micrometer-sized droplets and particles. However, the low throughput of conventional droplet generators hinders their clinical and industrial translation. Current approaches to parallelize microdevices are challenged by the two-dimensional nature of the standard fabrication methods. Here, we report the facile production of three-dimensionally (3D) parallelized microfluidic droplet generators consisting of stacked and radially multiplexed channel designs. Computational fluid dynamics simulations form the design basis for a microflow distributor that ensures similar flow rates through all droplet generators. Stereolithography is the selected technique to fabricate microdevices, which enables the manufacturing of hollow channels with dimensions as small as 50 μm. The microdevices could be operated up to 4 bars without structural damage, including deformation of channels, or leakage of the on-chip printed Luer-Lok type connectors. The printed microdevices readily enable the production of water-in-oil emulsions, as well as polymer containing droplets that act as templates for both solid and core–shell hydrogel microparticles. The cytocompatibility of the 3D printed device is demonstrated by encapsulating mesenchymal stem cells in hydrogel microcapsules, which results in the controllable formation of stem cell spheroids that remain viable and metabolically active for at least 21 days. Thus, the unique features of stereolithography fabricated microfluidic devices allow for the parallelization of droplet generators in a simple yet effective manner by enabling the realization of (complex) 3D designs. [ABSTRACT FROM AUTHOR]

This study presented a flexible liquid metal-based microdroplet capacitive sensor that would simply and accurately measure the speed and length of droplets flowing in microchannels. A pair of coplanar U-shaped electrodes was used to form a capacitance through droplet microchannels. Liquid metal was injected into polydimethylsiloxane (PDMS) channels to form the U-shaped electrodes. The sensor would generate a multi-plateau capacitance waveform as a droplet passes through the sensing area, and each plateau period corresponds to the droplet position in the sensing area. The droplet speed and length would be directly calculated from the multi-plateau capacitance waveform. The errors for the capacitive result relative to the real value were <7.2% for length and <2.8% for speed. Moreover, the sensor still maintained excellent performance for droplet length and speed measurement even though the microfluidic chip was bent to 96°. We have demonstrated that the capacitive sensor would be used for sweat rate monitoring. [ABSTRACT FROM AUTHOR]

An on-demand droplet injection method for controlled delivery of nanolitre-volume liquid samples to scientific instruments for subsequent analysis is presented. We employ pulsed focussed surface acoustic waves (SAW) to eject droplets from an enclosed microfluidic channel into an open environment. The 3D position of individual droplets and their time of arrival can be precisely controlled to within 61 μs in a 500 μm square target region 40 μm wide. The continuous ejection rate of 16 000 droplets per second can be tuned to produce pulsed trains of droplets from 0 up to 357 Hz. The main benefit of this technique is its ease of integration with complex microfluidic processing steps, such as droplet merging, sorting, and encapsulation, prior to sample delivery. With its ability to precisely deliver a small quantity of fluid to a pre-defined location this technology is applicable in X-ray based molecular studies, including the rapidly expanding field of X-ray free electron lasers. Fabrication procedures for this device, the underlying forcing mechanism, the role of nozzle design, and demonstration of the performance in both continuous and on-demand modes are reported. [ABSTRACT FROM AUTHOR]

The detection of toxic gases is becoming an important element in tackling increased air pollution. This has led to the development of gas sensors based on porous solid materials, which are produced using sol–gel chemistry and functionalized to change their optical qualities when in contact with the gas. In this context it is interesting to explore how microfluidics can be used to miniaturize these sensors, to improve their sensitivity and dynamic range, or to multiplex many gas measurements on a single chip. In this article we show how the sol–gel process can be implemented using anchored droplet microfluidics. The sensor material is partitioned into droplets while in the sol phase and maintained using capillary anchors. The ability to hold the droplets in place first allows us to study the sol–gel process. We use an original rheology method, which consists of observing the flows within stationary droplets that are submitted to an external flow, to measure the gelation time of the droplets. These measurements show a gelation time that decreases from 50 minutes to below 10 minutes as the temperature increases from 20 to 50 °C. We also measure the shrinkage of individual gel beads after gelation and find that this syneresis process is nearly finished after about 12 hours, leading to a final bead size that is 50% smaller than the initial droplet. Finally, we show that the beads can be functionalized and used to detect the presence of formaldehyde. These results first provide a new way to observe the physics of the sol–gel process in a well-controlled and quantitative fashion. Moreover they highlight how the coupling of microfluidics and sol–gel chemistry can be used to detect toxic gases, in view of answering the challenges surrounding gas detection in real-world settings. [ABSTRACT FROM AUTHOR]

The alarming dynamics of antibiotic-resistant infections calls for the development of rapid and point-of-care (POC) antibiotic susceptibility testing (AST) methods. Here, we demonstrated the first completely stand-alone microfluidic system that allowed the execution of digital enumeration of bacteria and digital antibiograms without any specialized microfluidic instrumentation. A four-chamber gravity-driven step emulsification device generated ∼2000 monodisperse 2 nanoliter droplets with a coefficient of variation of 8.9% of volumes for 95% of droplets within less than 10 minutes. The manual workload required for droplet generation was limited to the sample preparation, the deposition into the sample inlet of the chip and subsequent orientation of the chip vertically without an additional pumping system. The use of shallow chambers imposing a 2D droplet arrangement provided superior stability of the droplets against coalescence and minimized the leakage of the reporter viability dye between adjacent droplets during long-term culture. By using resazurin as an indicator of the growth of bacteria, we were also able to reduce the assay time to ∼5 hours compared to 20 hours using the standard culture-based test. [ABSTRACT FROM AUTHOR]

This paper presents a novel microfluidic method for the controllable generation of uniform gas-in-oil-in-water double emulsions with ultra-thin liquid films (2–12 μm) covering microbubbles (116–180 μm). This method combines one dispersion stage and a mass transfer-induced phase separation process in a simple co-flowing microchannel, instead of a complicated microfluidic device fabrication and a multistage dispersion process. In our experiments, CO2–alkane gas mixtures (dispersed phase) and NaOH aqueous solutions (continuous phase) were utilized as working systems. The main factors affecting the diameter of the inner bubble and the volume of the oil film are discussed. Through our method, the monodispersed gas-in-oil-in-water emulsions can be prepared in a simple and precise way. [ABSTRACT FROM AUTHOR]

Droplet-based microfluidic systems require a precise control of droplet physical properties; hence, measuring the morphological properties of droplets is critical to obtain high sensitivity analysis. The ability to perform such measurements in real-time is another demand which has not been addressed yet. In this study, we used coplanar electrodes configured in the differential measurement mode for impedimetric measurement of size and velocity. To obtain the size of the droplets, detailed 3D finite element simulations of the system were performed. The interaction of the non-uniform electric field and the droplet was investigated. Electrode geometry optimization steps were described and design guideline rules were laid out. User-friendly software was developed for real-time observation of droplet length and velocity together with in situ statistical analysis results. A comparison between impedimetric and optical measurement tools is given. Finally, to illustrate the benefit of having real-time analysis, iDM was used to synthesize particles with a predefined monodispersity limit and to study the response times of syringe pump and pressure pump driven droplet generation devices. This analysis allows one to evaluate the 'warm-up' time for a droplet generator system, after which droplets reach the desired steady-state size required by the application of interest. [ABSTRACT FROM AUTHOR]

We have developed a multistep microfluidic device that expands the current SlipChip capabilities by enabling multiple steps of droplet merging and multiplexing. Harnessing the interfacial energy between carrier and sample phases, this manually operated device accurately meters nanoliter volumes of reagents and transfers them into on-device reaction wells. Judiciously shaped microfeatures and surface-energy traps merge droplets in a parallel fashion. Wells can be tuned for different volumetric capacities and reagent types, including for pre-spotted reagents that allow for unique identification of original well contents even after their contents are pooled. We demonstrate the functionality of the multistep SlipChip by performing RNA transcript barcoding on-device for synthetic spiked-in standards and for biologically derived samples. This technology is a good candidate for a wide range of biological applications that require multiplexing of multistep reactions in nanoliter volumes, including single-cell analyses. [ABSTRACT FROM AUTHOR]

We report spatial separation of extracellular vesicle (EVs) populations based on particle size by using an approach that exploits Marangoni flow and the coffee-ring effect in microdroplets. Sequential transfer of a drying droplet progressively increases the mean size of EVs in the sample by repeated subsampling of a droplet during coffee-ring formation. This method allows size-based sorting, separation, and eventual retrieval of EVs for RNA and protein analysis. To demonstrate the biomedical relevance of this method, EVs from prostate cancer patients were analyzed; results revealed that the expression of cancer-associated genes and proteins was higher in small EVs than in large EVs. This ability to sort EVs using a combination of coffee ring with Marangoni flow and sequential droplet-transfer allows analysis of subpopulations of EVs, and will facilitate further studies of EVs. [ABSTRACT FROM AUTHOR]

Recent advances in transcriptomic analysis at single-cell resolution reveal cell-to-cell heterogeneity in a biological sample with unprecedented resolution. Partitioning single cells in individual micro-droplets and harvesting each cell's mRNA molecules for next-generation sequencing has proven to be an effective method for profiling transcriptomes from a large number of cells at high throughput. However, the assays to recover the full transcriptomes are time-consuming in sample preparation and require expensive reagents and sequencing cost. Many biomedical applications, such as pathogen detection, prefer highly sensitive, reliable and low-cost detection of selected genes. Here, we present a droplet-based microfluidic platform that permits seamless on-chip droplet sorting and merging, which enables completing multi-step reaction assays within a short time. By sequentially adding lysis buffers and reactant mixtures to micro-droplet reactors, we developed a novel workflow of single-cell reverse transcription loop-mediated-isothermal amplification (scRT-LAMP) to quantify specific mRNA expression levels in different cell types within one hour. Including single cell encapsulation, sorting, lysing, reactant addition, and quantitative mRNA detection, the fully on-chip workflow provides a rapid, robust, and high-throughput experimental approach for a wide variety of biomedical studies. [ABSTRACT FROM AUTHOR]

Electrowetting-driven digital (droplet-based) microfluidics has a tremendous impact on lab-on-a-chip applications. However, the biofouling problem impedes the real applications of such digital microfluidics. Here we report antifouling digital microfluidics by introducing lubricant infused porous film to electrowetting (more exactly, electrowetting on dielectric or EWOD). Such film minimizes direct contact between droplets and the solid surface but provides liquid–liquid contact between droplets and the lubricant liquid, which thus prevents unspecific adsorption of biomolecules to the solid surface. We demonstrate the compatibility of the lubricant infused film with EWOD to transport bio droplets. This configuration shows robust and high performance even for long cyclic operations without fouling in a wide range of concentrations of protein solutions. In addition, a variety of conductive droplets, including deionized (DI) water, saline, protein solution, DNA solution, sheep blood, milk, ionic liquid and honey, are examined, similarly showing high performance in cyclic transportations. In addition, using the same electrode patterns used in EWOD, transportations of dielectric (non-conductive) droplets including light crude oil, propylene carbonate and alcohol are also achieved. Such capability of droplet handling without fouling will certainly benefit the practical applications of digital microfluidics in droplet handling, sampling, reaction, diagnosis in clinic medicine, biotechnology and chemistry fields. [ABSTRACT FROM AUTHOR]

The high-throughput selection of individual droplets is an essential function in droplet-based microfluidics. Fluorescence-activated droplet sorting is achieved using electric fields triggered at rates up to 30 kHz, providing the ultra-high throughput relevant in applications where large libraries of compounds or cells must be analyzed. To achieve such sorting frequencies, electrodes have to create an electric field distribution that generates maximal actuating forces on the droplet while limiting the induced droplet deformation and avoid disintegration. We propose a metric characterizing the performance of an electrode design relative to the theoretical optimum and analyze existing devices using full 3D simulations of the electric fields. By combining parameter optimization with numerical simulation we derive rational design guidelines and propose optimized electrode configurations. When tested experimentally, the optimized design show significantly better performance than the standard designs. [ABSTRACT FROM AUTHOR]

A novel method for crystallization utilizing solvent/antisolvent extraction in microfluidic droplet liquid reactors has been developed for rapid and low-cost screening of crystal polymorphism (i.e. molecular crystallographic arrangement or internal structure) and habit (i.e. crystallographic shape or external structure). The method involves a ternary solvent system consisting of a dispersed phase of two miscible fluids, one in which the active pharmaceutical ingredient (API) is soluble (solvent) and one in which the API is insoluble (antisolvent). The solvent/antisolvent dispersed phase is immiscible with a third continuous phase. Crystallization of an API, GSK1, was controlled within droplets by altering the rate of solvent extraction from droplets into the continuous phase, thereby decreasing API solubility. Crystal size, habit, and population per droplet were directly impacted by the solvent's rate of extraction. Single crystals were grown in individual droplets by slow extraction of solvent into the surrounding continuous phase, which occurs when crystal growth gradually reduces API concentration such that it is maintained within the metastable zone throughout extraction. Rapid extraction of solvent from droplets results in API concentration significantly exceeding its metastable limit, producing a greater number of crystal nuclei compared to slow extraction conditions. When holding constant solubilized API mass per droplet, crystal sizes were larger for slow extraction rates (l = 96.3, w = 16.6 μm) compared to fast extraction rates (l = 48.8, w = 9.5 μm) as a result of crystal growth occurring on fewer crystal nuclei per droplet. Crystal habit can be controlled by adjusting the solvent extraction rate and consequently the saturation, where minimal saturation resulted in a rhombohedral habit and comparatively higher saturation resulted in an acicular habit with a higher aspect ratio. Antisolvents were tested using two hydrophobic APIs demonstrating the method's capability for rapidly identifying favorable crystal morphologies for downstream manufacturability using miniscule amounts of API. [ABSTRACT FROM AUTHOR]

To address current limitations in adapting solid phase sample capture and washing techniques to continuously flowing droplet microfluidics, we have developed the “Coalesce-Attract-Resegment Wash” (CAR-Wash) approach. This module provides efficient, high-throughput magnetic washing by electrocoalescing magnetic bead-laden input droplets with a washing buffer flow and magnetophoretically transporting beads through the buffer into a secondary droplet formation streamline. In this work, we first characterized the technology in terms of throughput, sample retention, and flow-based exclusion of waste volume, demonstrating ＞500 Hz droplet processing with ＞98% bead retention and ＞100-fold dilution in final droplets. Next, we showed that the technique can be adapted to alternative commercially available magnetic beads with lower magnetite content per particle. Then, we demonstrated the CAR-Wash module's effectiveness in washing away a small molecule competitive inhibitor to restore the activity of magnetic bead-immobilized β-galactosidase. Finally, we applied the system to immunomagnetically enrich a green fluorescent protein–histone H2B fusion protein from cell lysate while washing away mCherry and other lysate components. We believe this approach will bridge the gap between powerful biochemical and bioanalytical techniques and current droplet microfluidic capabilities, and we envision future application in droplet-based immunoassays, solid phase extraction, and other complex, multi-step operations. [ABSTRACT FROM AUTHOR]

Automated control of individual droplets in microfluidic channels offers tremendous potential for applications requiring high accuracy and minimal user involvement. The feasibility of active droplet control has been previously demonstrated with pressure-driven flow control and visual feedback, but the manual operation required to perform droplet manipulations limited the accuracy, repeatability, and throughput. The present study improves upon the aforementioned challenges with a higher-level algorithm capturing the dynamics of droplet motion for a semi-automated control system. With a simple T junction geometry, droplets can now be automatically and precisely controlled on-demand. Specifically, there is ±10% accuracy for droplet generation, ±1.3% monodispersity for 500 μm long droplets and ±4% accuracy for splitting ratios. On-demand merging, mixing, and sorting are also demonstrated as well as the application of a drug screening assay related to neurodegenerative disorders. Overall, this system serves as a foundation for a fully automated system that does not require valves, embedded electrodes, or complex multi-layer fabrication. [ABSTRACT FROM AUTHOR]

Selection of live cells from a population is critical in many biological studies and biotechnologies. We present here a novel droplet microfluidic approach that allows for label-free and passive selection of live cells using the glycolytic activity of individual cells. It was observed that with the use of a specific surfactant utilized to stabilize droplet formation, the interfacial tension of droplets was very sensitive to pH. After incubation, cellular lactate release results in droplets containing a live cell to attain a lower pH than other droplets. This enables the sorting of droplets containing live cells when confined droplets flow over a microfabricated trench oriented diagonally with respect to the direction of flow. The technique is demonstrated with human U87 glioblastoma cells for the selection of only droplets containing a live cell while excluding either empty droplets or droplets containing a dead cell. This label-free sorting method, dubbed sorting by interfacial tension (SIFT) presents a new strategy to sort diverse cell types based on metabolic activity. [ABSTRACT FROM AUTHOR]

Drop manipulation on hydrophobic surfaces is of importance in lab-on-a-chip applications. Recently, superhydrophobic surface-assisted lab-on-a-chips have attracted significant attention from researchers due to their advantages of contamination resistance and low adhesion between the drop and the surface during manipulation. However, control over both static and dynamic interactions between a drop and a superhydrophobic surface has been rarely achieved. In this study, we designed an electric-field-dependent liquid-dielectrophoresis force to manipulate a drop on a superhydrophobic surface. This type of control has been found to be fast in response, bio-friendly, convenient, repeatable, and energy efficient. Moreover, the adhesion force and rebounding for both the static and the dynamic interactions between the drop and the surface under an electric field have been explored. It was found that the adhesion force could be reversibly tuned three-fold without breaking the Cassie–Baxter state. Rebounding experiments showed a close to linear relation between energy dissipation and the applied voltage. This relation was used to tune the on-demand behaviors of a drop on a surface in a proof-of-concept experiment for drop sorting. This electric-field-dependent drop manipulation may have potential applications in digital microfluidics, micro-reactors and advanced lab-on-a-drop platforms. [ABSTRACT FROM AUTHOR]

Microfluidic step emulsification passively produces highly monodisperse droplets and can be easily parallelized for high throughput emulsion production. The two main techniques used for step emulsification are: i) edge-based droplet generation (EDGE), where droplets are formed in a single, very wide and shallow nozzle, and ii) microchannel emulsification (MCE), where droplets are formed in many separated narrow nozzles. These techniques differ in modes of droplet formation that influence the throughput and monodispersity of produced emulsions. Here we report a systematic study of novel grooved step emulsifying geometries, a hybrid of MCE and EDGE architectures. We introduce partitions of different heights to a wide (EDGE-like) slit to establish optimal geometries for high-throughput droplet production. We demonstrate that the volume and monodispersity of the produced emulsion can be tuned solely by changing the height of these partitions. We show that the spacing of the partitions influences the size of the produced droplets, but not the population monodispersity. We also determine the moment of transition between two distinct droplet generation modes as a function of the geometrical parameters of the nozzle. The optimized grooved geometry appears to combine the advantages of both MCE and EDGE, i.e. spatial localization of droplet forming units (DFUs), high-throughput formation of tightly monodisperse droplets from parallel DFUs, and low sensitivity to variation in the flow rate of the dispersed phase. As a proof-of-concept we show grooved devices that for a 260-fold increase of flow rate produce droplets with volume increased by just 75%, as compared to 91% increase in volume over a 180-fold increase of flow rate of the dispersed phase in MCE devices. We also present the optimum microfluidic device geometry that almost doubles the throughput of an MCE device in the generation of nanoliter droplets. [ABSTRACT FROM AUTHOR]

Current cancer detection systems lack the required sensitivity to reliably detect minimal residual disease (MRD) and recurrence at the earliest stages when treatment would be most effective. To address this issue, we present a novel liquid biopsy approach that utilizes an integrated comprehensive droplet digital detection (IC3D) digital PCR system which combines microfluidic droplet partitioning, fluorescent multiplex PCR chemistry, and our rapid 3D, large-volume droplet counting technology. The IC3D ddPCR assay can detect cancer-specific, ultra-rare genomic targets due to large sample input and high degree of partitioning. We first demonstrate our droplet digital PCR assay can robustly detect common cancer mutants including KRAS G12D spiked in wild-type genomic background or isolated from patient samples with 100% specificity. We then demonstrate that the IC3D ddPCR system can detect oncogenic KRAS G12D mutant alleles against a background of wild-type genomes at a sensitivity of 0.00125–0.005% with a false positive rate of 0% which is 50 to 1000× more sensitive than existing commercial liquid biopsy ddPCR and qPCR platforms, respectively. In addition, our technology can uniquely enable detection of circulating tumor cells using their genetic markers without a pre-enrichment step, and analysis of total tumor DNA isolated from blood samples, which will increase clinical sensitivity and specificity, and minimize inter-assay variability. Therefore, our technology holds the potential to provide clinicians with a powerful decision-making tool to monitor and treat MRD with unprecedented sensitivity for earlier stage intervention. [ABSTRACT FROM AUTHOR]