Droplet Array Research Articles

Binary-number-based digital electrochemical detection using a single working electrode with multiple sensors

While digital detection improves the sensitivity of electrochemical analysis, adapting it for array-based high-throughput assays has been challenging. The reason is that when integrating many electrochemical sensors in a small chip device, there may not be sufficient space for multiple lead connections to the external equipment. Here, we propose a new approach to prepare multiple sensors on one working electrode, and use the device for simultaneous digital electrochemical detection of droplet arrays. Based on the binary number system, sensor electrodes 4, 2, and 1mm2 in size are prepared on a single working electrode, and droplets with or without a target redox compound are placed on each sensor. The total signal from the working electrodes is converted to a three-digit binary number that specifies the presence or absence of the analyte at each position. Therefore, the droplets can be monitored simultaneously via a single line. This new concept allows us to prepare multiple electrochemical sensors on a small device for digital detection and digital imaging.

Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters

Drug assays with patient-derived cells such as circulating tumor cells requires manipulating small sample volumes without loss of rare disease-causing cells. Here, we report an effective technology for isolating and analyzing individual tumor cells and their clusters from minute sample volumes using an optimized microfluidic device integrated with pipettes. The method involves using hand pipetting to create an array of cell-laden nanoliter-sized droplets immobilized in a microfluidic device without loss of tumor cells during the pipetting process. Using this technology, we demonstrate single-cell analysis of tumor cell response to the chemotherapy drug doxorubicin. We find that even though individual tumor cells display diverse uptake profiles of the drug, the onset of apoptosis is determined by accumulation of a critical intracellular concentration of doxorubicin. Experiments with clusters of tumor cells compartmentalized in microfluidic drops reveal that cells within a cluster have higher viability than their single-cell counterparts when exposed to doxorubicin. This result suggests that circulating tumor cell clusters might be able to better survive chemotherapy drug treatment. Our technology is a promising tool for understanding tumor cell-drug interactions in patient-derived samples including rare cells.

High Throughput Analysis of Membrane Transport by using Arrayed Water-In-Oil Droplet Bilayers

The maintenance of an appropriate intracellular environment is a constant challenge for all living organisms, from prokaryotes to multicellular eukaryotes. Intracellular homeostasis is maintained by transporting various compounds such as ions, sugars, amino acids, and drugs across the cell membrane. Therefore, the analysis of transmembrane transport is crucial to understanding cell physiology as well as for exploring the bioavailability of drugs. In this study, we developed a novel micro-device in which a large number of droplet interface bilayers (> 500) are formed at a time by using femtoliter-sized droplet arrays immobilized on a hydrophobic/hydrophilic substrate.

Microfluidic platform for optimization of crystallization conditions

We describe a universal, high-throughput droplet-based microfluidic platform for crystallization. It is suitable for a multitude of applications, due to its flexibility, ease of use, compatibility with all solvents and low cost. The platform offers four modular functions: droplet formation, on-line characterization, incubation and observation. We use it to generate droplet arrays with a concentration gradient in continuous long tubing, without using surfactant. We control droplet properties (size, frequency and spacing) in long tubing by using hydrodynamic empirical relations. We measure droplet chemical composition using both an off-line and a real-time on-line method. Applying this platform to a complicated chemical environment, membrane proteins, we successfully handle crystallization, suggesting that the platform is likely to perform well in other circumstances. We validate the platform for fine-gradient screening and optimization of crystallization conditions. Additional on-line detection methods may well be integrated into this platform in the future, for instance, an on-line diffraction technique. We believe this method could find applications in fields such as fluid interaction engineering, live cell study and enzyme kinetics.

Large scale generation of micro-droplet array by vapor condensation on mesh screen piece

We developed a novel micro-droplet array system, which is based on the distinct three dimensional mesh screen structure and sintering and oxidation induced thermal-fluid performance. Mesh screen was sintered on a copper substrate by bonding the two components. Non-uniform residue stress is generated along weft wires, with larger stress on weft wire top location than elsewhere. Oxidation of the sintered package forms micro pits with few nanograsses on weft wire top location, due to the stress corrosion mechanism. Nanograsses grow elsewhere to show hydrophobic behavior. Thus, surface-energy-gradient weft wires are formed. Cooling the structure in a wet air environment nucleates water droplets on weft wire top location, which is more “hydrophilic” than elsewhere. Droplet size is well controlled by substrate temperature, air humidity and cooling time. Because warp wires do not contact copper substrate and there is a larger conductive thermal resistance between warp wire and weft wire, warp wires contribute less to condensation but function as supporting structure. The surface energy analysis of drops along weft wires explains why droplet array can be generated on the mesh screen piece. Because the commercial material is used, the droplet system is cost effective and can be used for large scale utilization.

Controllable domain morphology in coated poly(lactic acid) films for high-efficiency and high-precision transportation of water droplet arrays

Construction of superhydrophobic surfaces with tunable adhesion force has attracted considerable attention in past decades.

微小重力環境における燃料液滴列火炎燃え広がり速度に及ぼす雰囲気圧力の影響

微小重力環境における燃料液滴列火炎燃え広がり速度に及ぼす雰囲気圧力の影響

微小重力環境を用いた燃料液滴列火炎燃え広がりに及ぼす圧力の影響

微小重力環境を用いた燃料液滴列火炎燃え広がりに及ぼす圧力の影響

Pump- and Valve-Free Flow Injection Capillary Liquid Electrode Discharge Optical Emission Spectrometry Coupled to a Droplet Array Platform.

A miniature (2.5 cm length × 2.0 cm width × 1.0 cm height), low power (<10 W), and capillary liquid electrode microplasma optical emission spectrometer was developed for rapid determination of metallic species in aqueous solutions. The sample solution can be automatically introduced into the source without a pump owing to the inherent capillary attraction and the force arising from the solution vaporization induced by microplasma. A droplet array was used as a sampling platform to realize flow injection without using any valve and pump, significantly increasing throughput to 90 samples h-1. Sample volume is controlled through the sampling time and reduced to the nanoliter level. With a sampling time of 10 s (equal to 600 nL), detection limits of 30 μg L-1 (18 pg) and 75 μg L-1 (45 pg) were obtained for Cd and Hg, respectively, comparable to those reported for liquid electrode microplasma optical emission spectrometry. However, sample consumption is reduced more than 100-fold, making the proposed technique more suitable for the analysis of elements such as Cd, Hg, Li, Na, and K when sample volumes may be limited. The utility of this system was demonstrated by the determination of Cd and Hg in blood, real water samples, and Certified Reference Materials (rice powder, GBW07601a, and lobster hepatopancreas, TORT-3).

Droplet-Based Multivolume Digital Polymerase Chain Reaction by a Surface-Assisted Multifactor Fluid Segmentation Approach

Here we developed the surface-assisted multifactor fluid segmentation (SAMFS), an automated, fast, and flexible approach for generating a two-dimensional droplet array with tunable droplet volumes, for multivolume digital polymerase chain reaction (PCR). The SAMFS was developed based on the combination of robotic liquid handling and surface-assisted droplet generation techniques, where a continuous aqueous stream that flowed out from a capillary probe was segmented and immobilized on hydrophilic micropillars of a microchip into massive oil-covered droplets with the probe rapidly scanning over the microchip. We studied various factors affecting the droplet generation process, including micropillar top area, distance between adjacent micropillars, aqueous stream flow rate, and microchip moving speed, and demonstrated a high droplet generation throughput up to 50 droplets/s and a largest droplet volume adjusting range from 0.25 to 350 nL. The SAMFS approach was adopted to form an oil-covered array of 994 droplets with four different volumes (1.2, 6, 30, and 150 nL droplets) required for multivolume digital PCR within 8 min. The droplet array system was applied in absolute quantification of plasmid DNA under the multivolume digital PCR mode with a dynamic range spanning 4 orders of magnitude, as well as measurement of HER2 expression levels in different breast cancer cell lines. The results are consistent to those obtained by quantitative real-time PCR method, while the present one has higher precision.

Three-color crystal digital PCR

Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.

Water transport control on a patterned superhydrophobic surface via laser direct writing

A simple method for fabricating line patterns on a superhydrophobic surface is demonstrated using nanosecond laser direct writing. A laser-induced superhydrophobic surfaces exhibit the excellent superhydrophobicity with a contact angle of 158° and a sliding angle of less than 5°. By changing the superhydrophilic linewidth and line intervals, the authors investigated the anisotropic wetting and adhesive properties on the as-prepared line-patterned superhydrophobic surface. The experimental results showed that the linewidth and line intervals have an important influence in anisotropic wetting, including contact angles and sliding angles in both parallel and perpendicular directions. This data allow control of adhesion by having droplets slide off at designated tilting angles on the patterned superhydrophobic surface. The water droplet array can be easily separated into any configuration, such as an upper triangular array. The as-prepared patterned superhydrophobic surface can transfer different microliter-sized droplets from one superhydrophobic surface to another. This patterned superhydrophobic surface has some potential applications for the droplet manipulation, microfluidic devices, and cell culture.

Laser-induced superhydrophobic grid patterns on PDMS for droplet arrays formation

We demonstrate a facile single step laser treatment process to render a polydimethylsiloxane (PDMS) surface superhydrophobic. By synchronizing a pulsed nanosecond laser source with a motorized stage, superhydrophobic grid patterns were written on the surface of PDMS. Hierarchical micro and nanostructures were formed in the irradiated areas while non-irradiated areas were covered by nanostructures due to deposition of ablated particles. Arrays of droplets form spontaneously on the laser-patterned PDMS with superhydrophobic grid pattern when the PDMS sample is simply immersed in and withdrawn from water due to different wetting properties of the irradiated and non-irradiated areas. The effects of withdrawal speed and pitch size of superhydrophobic grid on the size of formed droplets were investigated experimentally. The droplet size increases initially with increasing the withdrawal speed and then does not change significantly beyond certain points. Moreover, larger droplets are formed by increasing the pitch size of the superhydrophobic grid. The droplet arrays formed on the laser-patterned PDMS with wettability contrast can be used potentially for patterning of particles, chemicals, and bio-molecules and also for cell screening applications.

3D Lattice Structure Control of Ordered Macroporous Material by Self‐Assembly of Liquid Droplets

Microfluidic devices, which can continuously fabricate single emulsion with monodispersed droplets having a pore diameter of more than 100 μm in large numbers, can be applied to manufacture ordered macroporous films. 3D ordered macroporous films with a diameter of more than 100 μm can be fabricated using ordered arrays of the monodispersed droplets as templates of the macropores, which are self-assembled in the space between two parallel flat glass plates. As the gap between the glass plates increases, the number of the layer increases. Furthermore, in the case with two or more layers, the lattice structure of the macroporous films also changes due to the confinement effects.

Spontaneous assembly of chemically encoded two-dimensional coacervate droplet arrays by acoustic wave patterning.

The spontaneous assembly of chemically encoded, molecularly crowded, water-rich micro-droplets into periodic defect-free two-dimensional arrays is achieved in aqueous media by a combination of an acoustic standing wave pressure field and in situ complex coacervation. Acoustically mediated coalescence of primary droplets generates single-droplet per node micro-arrays that exhibit variable surface-attachment properties, spontaneously uptake dyes, enzymes and particles, and display spatial and time-dependent fluorescence outputs when exposed to a reactant diffusion gradient. In addition, coacervate droplet arrays exhibiting dynamical behaviour and exchange of matter are prepared by inhibiting coalescence to produce acoustically trapped lattices of droplet clusters that display fast and reversible changes in shape and spatial configuration in direct response to modulations in the acoustic frequencies and fields. Our results offer a novel route to the design and construction of ‘water-in-water' micro-droplet arrays with controllable spatial organization, programmable signalling pathways and higher order collective behaviour.

Femtosecond laser-induced ultra-fine nanostructures on silicon surface

A new type of periodic nanostructuring, including melting droplet arrays, ablated grid arrays, and ultra-fine periodic surface structures, was produced during the interaction between femtosecond laser pulses and silicon. The co-existence of these nano-patterns as well as sharp transitions between them were observed along the scan tracks. Physical processes such as ultrafast melting, material ablation, and thermal melting are associated with these nanostructures. Coulomb explosion, interference, and local field arrangement are proposed as the possible mechanisms underlying this new nano-patterning process.

Insights into Vapor-Mediated Interactions in a Nanocolloidal Droplet System: Evaporation Dynamics and Affects on Self-Assembly Topologies on Macro- to Microscales.

Particle-laden droplet-based systems ranging from micro- to nanoscale have become increasingly popular in applications such as inkjet printing, pharmaceutics, nanoelectronics, and surface patterning. All such applications involve multidroplet arrays in which vapor-mediated interactions can significantly affect the evaporation dynamics and morphological topology of precipitates. A fundamental study was conducted on nanocolloidal paired droplets (droplets kept adjacent to each other as in an array) to understand the physics related to the evaporation dynamics, internal flow pattern, particle transport, and nanoparticle self-assembly, primarily using optical diagnostic techniques [such as micro-particle image velocimetry (μPIV)]. Paired droplets exhibit contact angle asymmetry, inhomogeneous contact line slip, and unique single-toroid microscale flow, which are unobserved in single droplets. Furthermore, nanoparticles self-assemble (at the nanoscale) to form a unique variable-thickness (microscale) tilted dome-shaped structure that eventually ruptures at an angle because of evaporation at a nanopore scale to form cavities (miniscale). The geometry and morphology of the dome can be further fine-tuned at a macro- to microscale by varying the initial particle concentration and substrate properties. This concept has been extended to a linear array of droplets to showcase how to custom design two-dimensional drop arrangements to create controlled surface patterns at multiple length scales.

Fabrication of Micro-Needle Electrodes for Bio-Signal Recording by a Magnetization-Induced Self-Assembly Method.

Micro-needle electrodes (MEs) have attracted more and more attention for monitoring physiological electrical signals, including electrode-skin interface impedance (EII), electromyography (EMG) and electrocardiography (ECG) recording. A magnetization-induced self-assembling method (MSM) was developed to fabricate a microneedle array (MA). A MA coated with Ti/Au film was assembled as a ME. The fracture and insertion properties of ME were tested by experiments. The bio-signal recording performance of the ME was measured and compared with a typical commercial wet electrode (Ag/AgCl electrode). The results show that the MA self-assembled from the magnetic droplet array under the sum of gravitational surface tension and magnetic potential energies. The ME had good toughness and could easily pierce rabbit skin without being broken or buckling. When the compression force applied on the ME was larger than 2 N, ME could stably record EII, which was a lower value than that measured by Ag/AgCl electrodes. EMG signals collected by ME varied along with the contraction of biceps brachii muscle. ME could record static ECG signals with a larger amplitude and dynamic ECG signals with more distinguishable features in comparison with a Ag/AgCl electrode, therefore, ME is an alternative electrode for bio-signal monitoring in some specific situations.

Dynamic analysis of immune and cancer cell interactions at single cell level in microfluidic droplets.

Cell-cell communication mediates immune responses to physiological stimuli at local and systemic levels. Intercellular communication occurs via a direct contact between cells as well as by secretory contact-independent mechanisms. However, there are few existing methods that allow quantitative resolution of contact-dependent and independent cellular processes in a rapid, precisely controlled, and dynamic format. This study utilizes a high-throughput microfluidic droplet array platform to analyze cell-cell interaction and effector functions at single cell level. Controlled encapsulation of distinct heterotypic cell pairs was achieved in a single-step cell loading process. Dynamic analysis of dendritic cell (DC)-T cell interactions demonstrated marked heterogeneity in the type of contact and duration. Non-stimulated DCs and T cells interacted less frequently and more transiently while antigen and chemokine-loaded DCs and T cells depicted highly stable interactions in addition to transient and sequential contact. The effector function of CD8+ T cells was assessed via cytolysis of multiple myeloma cell line. Variable cell conjugation periods and killing time were detected irrespective of the activation of T cells, although activated T cells delivered significantly higher cytotoxicity. T cell alloreactivity against the target cells was partially mediated by secretion of interferon gamma, which was abrogated by the addition of a neutralizing antibody. These results suggest that the droplet array-based microfluidic platform is a powerful technique for dynamic phenotypic screening and potentially applicable for evaluation of novel cell-based immunotherapeutic agents.

Ionic Liquid-Assisted Electropolymerization for Lithographical Perfluorocarbon Deposition and Hydrophobic Patterning

We developed a novel approach for hydrophobic patterning: combining the photolithography technique with ionic-liquid (IL)-based electropolymerization to fabricate a hydrophobic pattern. Perfluoro-functionalized 3,4-ethylenedioxythiophene (EDOT-F) dispersed in ILs was directly electropolymerized on substrates, which were patterned in advance with positive photoresists. The positive photoresists did not dissolve in ionic liquids during the electropolymerization process, and the poly(EDOT-F) film created hydrophobic domains, which resulted in hydrophobic patterning. This approach provides desired patterns with a lateral resolution consistent with the mask for photolithography. Two kinds of modified indium-tin-oxide-coated glass (ITO-glass) substrates were used to demonstrate the feasibility of process for creating a hydrophobic pattern: ITO-glass substrates coated with nanostructured PEDOT, and the same substrates coated with Au nanoparticles. By confining water droplets on these two patterned substrates to form droplet arrays, we demonstrated two potential applications: multiple droplet-type electrochemical cells and surface-enhanced Raman scattering platforms. In addition, we also applied this approach to create hydrophobic patterning on ITO-coated polyethylene terephthalate (ITO-PET) substrates. The droplet arrays remained well-organized on the ITO-PET substrates even when the substrates were bent. Our work successfully introduced ILs into the photolithography process, implying great potential for these green solvents.