Droplet Array Research Articles

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis.

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 μm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.

Microfluidic Droplet Array as Optical Irises Actuated via Electrowetting

Initiated by a task in tunable microoptics, but not limited to this application, a microfluidic droplet array in an upright standing module with 3 × 3 subcells and droplet actuation via electrowetting is presented. Each subcell is filled with a single (of course transparent) water droplet, serving as a movable iris, surrounded by opaque blackened decane. Each subcell measures 1 × 1 mm2 and incorporates 2 × 2 quadratically arranged positions for the droplet. All 3 × 3 droplets are actuated synchronously by electrowetting on dielectric (EWOD). The droplet speed is up to 12 mm/s at 130 V (Vrms) with response times of about 40 ms. Minimum operating voltage is 30 V. Horizontal and vertical movement of the droplets is demonstrated. Furthermore, a minor modification of the subcells allows us to exploit the flattening of each droplet. Hence, the opaque decane fluid sample can cover each water droplet and render each subcell opaque, resulting in switchable irises of constant opening diameter. The concept does not require any mechanically moving parts or external pumps.

Single-Step Fabrication of High-Throughput Surface-Enhanced Raman Scattering Substrates.

The combination of surface-enhanced Raman scattering (SERS) with high-throughput screening (HTS) has significant importance for highly sensitive and massive workload assays. Although fabrication of HTS-SERS substrates can be achieved by several methods, the high cost as well as large-equipment dependence limit their applications. Here, we report a simple method to fabricate HTS-SERS substrates within one-step process. The HTS-SERS substrate is fabricated by simply UV-irradiating a fluoroalkylsilane (FAS)-modified liquid-repellent TiO2 surface in AgNO3 solution through a photomask. Owing to the photocatalytic nature of TiO2, the UV irradiation simultaneously triggers the degradation of the attached FAS and the generation of liquid-adhesive Ag nanoparticles (NPs) on exposed area. A HTS-SERS substrate could be directly obtained after UV irradiation. The deposited Ag NPs evidently enhance Raman signals, and the significant difference between the wettability of exposed area and masked area enables fast formation of high-throughput liquid droplet arrays through a simple dragging solution process. The fabrication method is applicable to various substrate materials, to introduce additional functionalities. The photocatalytic activity of TiO2 also allows us to photobleach the residual analyte and Ag NPs after detection to recycle substrate. This single-step method is a highly promising candidate for the fabrication of HTS-SERS substrates.

The WeIzmann Supercooled Droplets Observation on a Microarray (WISDOM) and application for ambient dust

Abstract. The WeIzmann Supercooled Droplets Observation on Microarray (WISDOM) is a new setup for studying ice nucleation in an array of monodisperse droplets for atmospheric implications. WISDOM combines microfluidics techniques for droplets production and a cryo-optic stage for observation and characterization of freezing events of individual droplets. This setup is designed to explore heterogeneous ice nucleation in the immersion freezing mode, down to the homogeneous freezing of water (235 K) in various cooling rates (typically 0.1–10 K min−1). It can also be used for studying homogeneous freezing of aqueous solutions in colder temperatures. Frozen fraction, ice nucleation active surface site densities and freezing kinetics can be obtained from WISDOM measurements for hundreds of individual droplets in a single freezing experiment. Calibration experiments using eutectic solutions and previously studied materials are described. WISDOM also allows repeatable cycles of cooling and heating for the same array of droplets. This paper describes the WISDOM setup, its temperature calibration, validation experiments and measurement uncertainties. Finally, application of WISDOM to study the ice nucleating particle (INP) properties of size-selected ambient Saharan dust particles is presented.

Flow-induced dissolution of femtoliter surface droplet arrays.

The dissolution of liquid nanodroplets is a crucial step in many applied processes, such as separation and dispersion in the food industry, crystal formation of pharmaceutical products, concentrating and analysis in medical diagnosis, and drug delivery in aerosols. In this work, using both experiments and numerical simulations, we quantitatively study the dissolution dynamics of femtoliter surface droplets in a highly ordered array under a uniform flow. Our results show that the dissolution of femtoliter droplets strongly depends on their spatial positions relative to the flow direction, drop-to-drop spacing in the array, and the imposed flow rate. In some particular cases, the droplet at the edge of the array can dissolve about 30% faster than the ones located near the centre. The dissolution rate of the droplet increases by 60% as the inter-droplet spacing is increased from 2.5 μm to 20 μm. Moreover, the droplets close to the front of the flow commence to shrink earlier than those droplets in the center of the array. The average dissolution rate is faster for the faster flow. As a result, the dissolution time (Ti) decreases with the Reynolds number (Re) of the flow as Ti ∝ Re-3/4. The experimental results are in good agreement with the numerical simulations where the advection-diffusion equation for the concentration field is solved and the concentration gradient on the surface of the drop is computed. The findings suggest potential approaches to manipulate nanodroplet sizes in droplet arrays simply by dissolution controlled by an external flow. The obtained droplets with varying curvatures may serve as templates for generating multifocal microlenses in one array.

A Microfluidic Droplet Array System for Cell-Based Drug Combination Screening.

In the last few decades, drug combination therapy has been widely applied in oncology and in other complex diseases. Due to its potential advantage of lower drug toxicity and higher therapeutic efficacy, drug combination treatment has been more and more studied in fundamental labs and pharmacy companies. In this chapter, we report cell-based drug combination screening using a microfluidic droplet system based on a sequential operation droplet array (SODA) technique. In this system, an oil-covered two-dimensional droplet array chip was used as the platform for cell culture and analysis. This chip was fixed in an x-y-z translation stage under control of a computer program. A tapered capillary connected with a syringe pump was coupled with the droplet array chip to achieve multiple droplet manipulations including liquid metering, aspirating, depositing, mixing, and transferring. Complex multistep operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel using the present system. The drug consumption for each screening test was substantially decreased to 5ng-5μg, corresponding to 10- to 1000-fold reductions compared with traditional drug screening systems with 96- or 384-well plates.

Self-generation of two-dimensional droplet array using oil-water immiscibility and replacement.

Two-dimensional (2D) microdroplet arrays with indexed sample concentration gradients have been receiving considerable attention for high-throughput biological and medical analyses. However, the preparation of such an array by conventional methods mandates precise pipetting and/or pumping. In this paper, we introduce a method to spontaneously generate 2D-arrayed aqueous droplets using a well array, for which coarse pipetting is sufficient. The wells are connected in rows and columns via narrow channels. Aqueous solutions impregnated in the well array are split into droplets in every single well as a subsequently introduced immiscible solvent self-propagates and divides the solution at the channels. A concentration gradient of the samples can be formed across the connected solution in the well array; once droplets are generated, each droplet possesses a different sample concentration depending on its position in the array. We experimentally determined the optimal well dimensions and solvent species to obtain a high yield of droplet generation. We next demonstrated a 2D droplet array with a two-sample concentration gradient. Finally, the applicability of the system was demonstrated through a cell viability assay using a sample that induced apoptosis. We believe the proposed method contributes to simplification and miniaturization of the system to generate droplet arrays and thus is applicable to biological and medical analysis.

Droplet microarray: miniaturized platform for rapid formation and high-throughput screening of embryoid bodies.

Stem cells are influenced by various factors present in their in vivo microenvironment, such as interactions with neighboring cells, the extracellular matrix or soluble molecules. This demonstrates the high complexity of the in vivo microenvironment. Hence, many advances have been made in developing 3D screening models mimicking this complexity and the in vivo-like state in order to ensure more biomedically relevant investigations in drug discovery. In the field of stem cell research embryoid bodies are often used as relevant 3D systems. Embryoid bodies are embryonic stem cell aggregates that recapitulate the early embryonic development and that can differentiate into derivatives of the three germ layers. Embryoid bodies enable the investigation of processes underlying embryonic development, tissue generation and identification of drugs with developmental toxicity. The ability to perform high-throughput screenings using embryoid bodies could be extremely important to accelerate the progress in the field of stem cell research and embryonic development. To date, there are no simple methods to create high-density microarrays of embryoid bodies that further enable their high-throughput screening important for biomedical research. Here we demonstrate a new method that enables formation and high-throughput screening of embryoid bodies in arrays of defined, separated microdroplets. Using the superhydrophobic-hydrophilic micropattern of the droplet microarray, we demonstrate rapid and facile one-step formation of a dense array of multiple droplets containing homogeneous, single embryoid bodies. Thorough characterization of the influence of the initial cell number on embryoid body size, roundness and distribution was performed. We applied the embryoid body microarray to screen 774 FDA-approved compounds, identifying compounds with developmental toxicity such as mycophenolate mofetil or embryonic lethality such as eptifibatide. This work demonstrates the potential of the droplet microarray for the rapid formation of high-density microarrays of single embryoid bodies and their high-throughput drug screenings.

Digital enzyme assay using attoliter droplet array.

Single-molecule digital enzyme assay using micron-sized droplet array is a promising analysis method to quantify biomolecules at extremely low concentrations. However, multiplex digital enzyme assays are still difficult to access because the best buffer conditions can vary largely among enzymes. In addition, the best conditions for flurogenic compounds to retain high quantum efficiency and to avoid leakage into the oil phase can be also very different. In this study, digital enzyme assay was performed using an array of nanometer-sized droplets of 200 aL volume, termed 'nanocell'. Due to the small reaction volume, nanocell enhanced the accumulation rate of fluorescent products by a factor of 100 when compared with micron-sized reactors. Nanocell also enabled oil-free sealing of reactors: when flushed with an air flow, nanocell displayed water droplets under air, allowing enzymes to catalyze the reaction at the same rate as in oil-sealed reactors. Dual digital enzyme assay was also demonstrated using β-galactosidase and alkaline phosphatase (ALP) at pH 7.4, which is far from the optimum condition for ALP. Even under such a non-optimum condition, ALP molecules were successfully detected. Nanocell could largely expand the applicability of digital bioassay for enzymes under non-optimum conditions or enzymes of low turnover rate. The sealing of the reactor with air would also expand the applicability, allowing the use of fluorescent dyes that leak into oil.

Influence of forced convection on the evaporation and internal dynamics inside of an array of salt solution droplets

The effects of a gentle forced air convection on the internal dynamics of an array of multiple pinned sessile salt solution droplets are investigated via fully-coupled transient ALE finite element analysis. Results highlight the competition between the shear-induced circulation within the droplets and the gravity-driven flow in the droplets arising from increasing liquid density in regions of high water evaporation. At low air speeds, gravity effects dominate, resulting in a non-uniform concentration distribution. However, at higher speeds the shear-induced circulation within the droplets becomes sufficient to mix the liquid within the droplets via a 3D flow pattern, resulting in greater concentration uniformity. In addition, the shielding effect of leading droplets on downstream droplets is explored for various air speeds, with results showing differences in average concentration levels.

Geometric Replication Integrity of Micro Features Fabricated Using Variotherm Assisted Micro Injection Moulding

The geometric surface integrity of micro patterns is critical for functionality of polymeric micro devices, e.g. replication accuracy for micro channels and individual lens for the element of a compound eye lens. Micro injection moulding has been used widely for mass production of these devices. However, achieving high replication integrity is a critical challenge. In the present work, we developed a variotherm assisted micro injection moulding process to replicate a micro droplet and ellipse feature arrays in order to study the deformation behaviour of sickled blood cells. The effects of warm circuit temperature and cold circuit temperature on replication integrity, including filling depth, profile and morphology, were characterized based on moulding process monitoring, measurement and imaging. We have found that the warm circuit temperature is critical for feature filling due to its influence on the polymer-mould interface temperature, and that too high a warm circuit temperature can cause significant adhesion of polymer to a micro cavity. The cold circuit temperature controls shrinkage of polymer melts and the mould, and subsequently influences demoulding. Both temperatures need to be optimized to achieve the best replication of micro features.

A microfluidic chip capable of generating and trapping emulsion droplets for digital loop-mediated isothermal amplification analysis.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that rapidly amplifies specific DNA molecules at high yield. In this study, a microfluidic droplet array chip was designed to execute the digital LAMP process. The novel device was capable of 1) creating emulsion droplets, 2) sorting them into a 30 × 8 droplet array, and 3) executing LAMP across the 240 trapped and separated droplets (with a volume of 0.22 nL) after only 40 min of reaction at 56 °C. Nucleic acids were accurately quantified across a dynamic range of 50 to 2.5 × 103 DNA copies per μL, and the limit of detection was a single DNA molecule. This is the first time that an arrayed emulsion droplet microfluidic device has been used for digital LAMP analysis. When compared to microwell digital nucleic acid amplification assays, this droplet array-based digital LAMP assay eliminates the constraint on the size of the digitized target, which was determined by the dimension of the microwells for its counterparts. Moreover, the capacity for hydrodynamic droplet trapping allows the chip to operate in a one-droplet-to-one-trap manner. This microfluidic chip may therefore become a promising device for digital LAMP-based diagnostics in the near future.

Static droplet array for culturing single live adherent cells in an isolated chemical microenvironment.

We present here a new method to easily and reliably generate an array of hundreds of dispersed nanoliter-volume semi-droplets for single-cells culture and analysis. The liquid segmentation step occurs directly in indexed traps by a tweezer-like mechanism and is stabilized by spatial confinement. Unlike common droplet-based techniques, the semi-droplet wets its surrounding trap walls thus supporting the culturing of both adherent and non-adherent cells. To eliminate cross-droplet cell migration and chemical cross-talk each semi-droplet is separated from a nearby trap by an ∼80 pL air plug. The overall setup and injection procedure takes less than 10 minutes, is insensitive to fabrication defects and supports cell recovery for downstream analysis. The method offers a new approach to easily capture, image and culture single cells in a chemically isolated microenvironment as a preliminary step towards high-throughput single-cell assays.

Trapping a moving droplet train by bubble guidance in microfluidic networks.

Trapping a train of moving droplets into preset positions within a microfluidic device facilitates the long-term observation of biochemical reactions inside the droplets. In this paper, a new bubble-guided trapping method, which can remarkably improve the limited narrow two-phase flow rate range of uniform trapping, was proposed by taking advantage of the unique physical property that bubbles do not coalescence with two-phase fluids and the hydrodynamic characteristic of large flow resistance of bubbles. The flow behaviors of bubble-free and bubble-guided droplet trains were compared and analyzed under the same two-phase flow rates. The experimental results show that the droplets trapped by bubble-free guided trapping exhibit the four trapping modes of sequentially uniform trapping, non-uniform trapping induced by break-up and collision, and failed trapping due to squeezing through, and the droplets exhibit the desired uniform trapping in a relatively small two-phase flow rate range. Compared with bubble-free guided droplets, bubble-guided droplets also show four trapping modes. However, the two-phase flow rate range in which uniform trapping occurs is increased significantly and the uniformity of the trapped droplet array is improved. This investigation is beneficial to enhance the applicability of microfluidic chips for storing droplets in a passive way.

Active droplet-array (ADA) microfluidics enables multiplexed complex bioassays for point of care testing.

We introduce a novel and versatile microfluidic technology that allows parallel and multi-step bioanalytical procedures to be simply implemented by switching reagent-containing droplet arrays among alternative interaction zones for intended mass or energy transport in a programmable manner. This enables multiplexed complex bioassays for point-of-care testing.

System-level analysis of a novel air-cooled condenser using spray freezing of phase change materials

A comprehensive system-level analysis is performed for a novel air-cooled condenser based on spray freezing of phase change materials (PCMs). This novel air-cooled condenser uses PCMs to decouple the process of steam condensation and heat rejection to air in order to significantly improve air-side heat transfer and reduce steam condensation temperature as compared to conventional air-cooled condensers (ACCs). Melting of solid PCM particles in a two-phase PCM slurry flow anchors the steam condensation temperature close to the PCM melting point regardless of the change in ambient air temperature. Spray freezing of millimeter-sized liquid PCM droplets increases the air-side heat transfer coefficient by five times compared to the finned-tubed ACCs. A multiscale model, which directly captures the melting and settling of PCM particles at the microscopic level and accounts for phase change through energy source terms at the macroscopic level, has been developed to simulate the PCM slurry flow over heated tube bundles. Using this multiscale model, the effects of particle volume fraction, Reynolds number, and particle to steam tube diameter ratio on the averaged wall Nusselt number of the steam tubes are investigated. It is found that the averaged wall Nusselt number for a PCM slurry flow of 20% solid fraction achieves a 38% enhancement over the PCM single-phase flow of same Reynolds number. On the air side, the freezing/melting of PCM droplet/particle is approximated based on a 1-D transient heat conduction model and the air-side pressure drop across the PCM droplet array is determined using a 3-D k-ε turbulence model. The performance of this spray-freezing PCM ACC is compared against a baseline ACC of a 500 MWe power plant. It is found that, for comparable footprint area and ambient conditions, the spray-freezing PCM ACC reduces the initial temperature difference to as low as 16.8 °C and provides up to 10.8 MW net power production gain compared to the baseline ACC.

Large-Scale Femtoliter Droplet Array for Single Cell Efflux Assay of Bacteria.

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

Fully resolved DNS of droplet array combustion in turbulent convective flows and modelling for mixing fields in inter-droplet space

The combustion of droplets in turbulent convective flows is simulated using direct numerical simulations (DNS). The liquid–vapour interface between kerosene droplets and the surrounding air is fully resolved with realistic boundary conditions for mass conservation, heat conduction and species diffusion. The study focuses on the characterisation of the mixing process between the evaporating fuel and the surrounding gas in regions that are dominated by small turbulent scales. For inertial droplets, these regions are characterised by Kolmogorov time scales and the mean relative velocity between droplets and the surrounding gas phase. Scaling laws for quantities of interest that require sub-grid modelling for LES, such as mixture fraction, its conditional scalar dissipation and probability density functions (PDF), are presented and assessed by comparisons with the DNS data. The scaling laws provide satisfactory estimates and are validated for different inflow Reynolds numbers, turbulence intensities and integral length scales, droplet diameters, inter-droplet distances, droplet combustion regimes and various instants of the transient evaporation process. Suitable modelling parameters are extracted from the DNS and functional dependencies of the parameters are suggested. This study demonstrates that the scaling laws are suitable to serve as sub-grid scale models for mixture fraction based approaches such as flamelet, conditional moment closure (CMC) or multiple mapping conditioning (MMC) methods.

Behavior of a Weak Polyelectrolyte at Oil-water Interfaces under Different Environmental Conditions

Understanding the behavior of macromolecules adsorbed to a surface or interface under different environmental conditions is very important in stabilization of colloidal formulations, coating, drug delivery and lubrications. However, the experimental understanding on polyelectrolyte behavior at interfaces under different environmental conditions is lacking. Here, we probe the conformational changes of a weak polyelectrolyte (Polyacrylic acid, PAA) adsorbed at an oil-water interface, under different physiochemical condition such as pH and salt of various concentrations and valences, using a simple spectroscopic approach. The conformational change of PAA adsorbed at magnetically polarizable emulsions droplets is manifested from the Bragg spectral shift or interdroplet spacing change from an array of droplets. In addition, the intermolecular repulsive interaction between the polyelectrolyte covered droplets is studied in details. In the pH range of 3–11.8, at zero salt concentration, the interdroplet spacing increased from 40 to 173nm, indicating a large extended conformation of adsorbed PAA, which undergoes a sharp collapse in the presence of multi valence salt. The decay length obtained from the force measurement results, at zero salt concentration and at low pH, suggest that the adsorbed PAA exhibits a compact structure at an oil-water interface whereas it adopts an extended structure at high pH. Interestingly, the scaling exponents observed from the force decay length during swelling and collapse of the adsorbed PAA at different pH and salts are in good agreement with mean field theory and counter-ion fluctuation theory, though the interface and anchoring behavior of polyelectrolyte are quite different. Our results provide new experimental insights into macromolecular conformation at oil-water interfaces under different practical situations and offer new possibilities in probing macromolecular behavior at interfaces, for creating stimuli responsive nanostructures (pH, temperature, ion etc), lubrication and better stabilization of colloids.

Fabrication and Actuation of an Electrowetting Droplet Array on a Flexible Substrate.

Electrowetting-on-dielectric (EWOD) is a fast, well-established actuation method for a variety of applications, from microfluidics to electrowetting displays to electrowetting lenses. We therefore seek to develop a robust, scalable fabrication method for the realization of EWOD on a flexible polydimethylsiloxane (PDMS) substrate in order to increase the range of possible applications. We fabricated a 5 × 5 array of individually controlled electrowetting cells to manipulate silicone oil droplets via EWOD. The fabrication process utilized exclusively flexible materials to improve the robustness of the overall device, and processing methods were adapted to accommodate the particular challenges posed by flexible materials. Simulation of the EWOD devices was conducted using ANSYS Fluent and showed the change in contact angle in response to voltage applied. Fabricated devices were also tested, with actuation of the oil droplet observed with up to 100 V (RMS) AC applied across underlying electrodes. We demonstrated fabrication of a fully flexible array and verified actuation to center droplets over the electrodes. This work may be expanded to address more specific flexible applications for EWOD.