Droplet Array Research Articles

Picoliter droplet array based on bioinspired microholes for in situ single-cell analysis

The division of aqueous samples into microdroplet arrays has many applications in biochemical and medical analysis. Inspired by biological features, we propose a method to produce picoliter droplet arrays for single-cell analysis based on physical structure and interface. A 0.9 pL droplet array with an RSD (relative standard deviation) less than 6.3% and a density of 49,000 droplets/cm2 was successfully generated on a PDMS chip (polydimethylsiloxane) from a micromachined glass mold. The droplet generation principle of the wetting behavior in the microholes with splayed sidewalls on the PDMS chip by liquid smearing was exploited. The feasibility of the picoliter droplets for bacterial single-cell analysis was verified by the separation of mixed bacteria into single droplets and isolated in situ bacteria propagation.

Effect of VUV Radiation on Surface Modification of Polystyrene Exposed to Atmospheric Pressure Plasma Jet.

Precise tailoring of surface properties by gaseous plasma treatments remains a key scientific challenge, especially when adequate surface wettability should be laterally distributed, and sharp interfaces between hydrophobic and hydrophilic areas are desirable. The evolution of surface wettability and functional groups on polystyrene (PS) upon treatment with argon plasma jet was monitored by water contact angles and X-ray photoelectron spectroscopy (XPS). An array of water droplets was deposited on PS samples treated either directly by the plasma jet or only VUV radiation arising from the plasma. Rather sharp interfaces between the activated and not-affected regions were observed in both cases. The functionalization with highly-oxidized carbon functional groups, as determined by high-resolution C1s XPS spectra, was by far more efficient using the VUV radiation only. In contrast, the optimal wettability was achieved using direct plasma treatment. The results were explained by different mechanisms involved in the interaction of radiation and reactive plasma species with the polymer surface.

Rapid In Situ Photoimmobilization of a Planar Droplet Array for Digital PCR.

Digital PCR (dPCR) is a powerful technique capable of absolute quantification of nucleic acids with good accuracy. Droplet-based dPCR (ddPCR), among others, is one of the most important dPCR techniques. However, the surface tension-controlled droplets may suffer from fusion/fission due to the vigorous temperature change in PCR thermal cycling. Besides, the free movement of droplets makes them unsuitable for real-time fluorescence monitoring. In this paper, we first developed a photoimmobilized planar droplet array (PIPDA) by using a photocurable polyurethane as the continuous oil phase. It is found that uniform water-in-oil droplets of various sizes can be readily generated, and more importantly, the oil phase can be rapidly solidified in just a few seconds upon exposure to UV irradiation. This process will leave the droplets immobilized in the accommodation chamber as a stable planar array and, thus, effectively prevent the movement, coalescence, and breakup of droplets. In addition, a novel multilayered chip design has been proposed, which can thoroughly overcome the evaporation issue that commonly exists in polydimethylsiloxane (PDMS)-based dPCR chips. With these two innovations, the ddPCR experiment could be performed in a robust manner, and shows a promising potential in the development of real-time ddPCR technique. These features may therefore enable the wide application of PIPDA-based ddPCR in various fields.

Microfluidic Pneumatic Printed Sandwiched Microdroplet Array for High-Throughput Enzymatic Reaction and Screening

High-throughput enzyme screening for desired functionality is highly demanded. This paper utilizes a newly developed microfluidic pneumatic printing platform for high-throughput enzyme screening applications. The novel printing platform can achieve distinct features including a disposable cartridge, which avoids crosstalk; a flexible cartridge design, allowing for integration of multiple channels; and fast printing speed with submicroliter spot size. Moreover, a polydimethylsiloxane (PDMS)-based sandwich structure has been proposed and used during the printing and imaging, which can lead to better results, including reduced evaporation as well as a uniform light path during imaging. Using this microfluidic pneumatic printed PDMS sandwiched microdroplet array platform, we have demonstrated the capability of high-throughput generation of a combinatorial droplet array with concentration and volume gradients. Furthermore, the potential for enzymatic study has been validated by quantified cellulose reaction implemented with the printing platform.

Effect of additives on the growth of HKUST-1 crystals synthesized by microfluidic chips with concentration gradient.

Metal-organic frameworks (MOFs) have attracted considerable attention as novel nanoporous materials that combine the properties of organic and inorganic porous materials. HKUST-1 is one of the most well-developed and representative MOFs with wide applications in gas storage and separation, adsorption, and capture. In this study, we used microfluidics, an advanced technique of manipulation of small fluid volumes in microscale or even nanoscale channels, to investigate the effect of sodium dodecyl sulfate (SDS) on the growth of HKUST-1 crystals. We directly observed the morphological evolution of HKUST-1 crystals through droplet arrays with the SDS concentration gradient. The morphology of HKUST-1 evolved from cubic to cuboctahedron and finally to octahedron with increasing SDS concentration. The study results demonstrated the important role played by anions in solution in the structural regulation of HKUST-1.

Individual Control and Quantification of 3D Spheroids in a High-Density Microfluidic Droplet Array

SummaryAs three-dimensional cell culture formats gain in popularity, there emerges a need for tools that produce vast amounts of data on individual cells within the spheroids or organoids. Here, we present a microfluidic platform that provides access to such data by parallelizing the manipulation of individual spheroids within anchored droplets. Different conditions can be applied in a single device by triggering the merging of new droplets with the spheroid-containing drops. This allows cell-cell interactions to be initiated for building microtissues, studying stem cells’ self-organization, or observing antagonistic interactions. It also allows the spheroids’ physical or chemical environment to be modulated, as we show by applying a drug over a large range of concentrations in a single parallelized experiment. This convergence of microfluidics and image acquisition leads to a data-driven approach that allows the heterogeneity of 3D culture behavior to be addressed across the scales, bridging single-cell measurements with population measurements.

A Droplet Microfluidic System to Fabricate Hybrid Capsules Enabling Stem Cell Organoid Engineering.

Organoids derived from self‐organizing stem cells represent a major technological breakthrough with the potential to revolutionize biomedical research. However, building high‐fidelity organoids in a reproducible and high‐throughput manner remains challenging. Here, a droplet microfluidic system is developed for controllable fabrication of hybrid hydrogel capsules, which allows for massive 3D culture and formation of functional and uniform islet organoids derived from human‐induced pluripotent stem cells (hiPSCs). In this all‐in‐water microfluidic system, an array of droplets is utilized as templates for one‐step fabrication of binary capsules relying on interfacial complexation of oppositely charged Na‐alginate (NaA) and chitosan (CS). The produced hybrid capsules exhibit high uniformity, and are biocompatible, stable, and permeable. The established system enables capsule production, 3D culture, and self‐organizing formation of human islet organoids in a continuous process by encapsulating pancreatic endocrine cells from hiPSCs. The generated islet organoids contain islet‐specific α‐ and β‐like cells with high expression of pancreatic hormone specific genes and proteins. Moreover, they exhibit sensitive glucose‐stimulated insulin secretion function, demonstrating the capability of these binary capsules to engineer human organoids from hiPSCs. The proposed system is scalable, easy‐to‐operate, and stable, which can offer a robust platform for advancing human organoids research and translational applications.

Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling droplet array-based capillary electrophoresis and PCR-RFLP.

The efficacy of targeted therapy is associated with multi-gene mutation status. Carrying out effective multi-genotyping analysis in combination has been a challenge in clinical settings. We therefore developed a droplet-based capillary electrophoresis (CE) system coupled with PCR-restriction fragment length polymorphism (PCR-RFLP) technology to detect multi-gene mutations from a small volume of samples. A 16 × 16 200-nL droplet array for sample encapsulation was constructed on a glass chip. The electrophoresis system consisted of a tapered vertical capillary filled with polyvinylpyrrolidone, a laser-induced fluorescence detector, and a high voltage power supply. Notably, a droplet-based electrokinetic sample introduction method and a "∩" shape capillary were developed to facilitate consecutive droplet sampling using a home-made automatic control module. The DL2000 DNA marker was consecutively separated, achieving high migration time and plate number reproducibility. The system was further applied to detect PCR-RFLP products. For colorectal cancer (CRC) cell lines, KRAS, BRAF, and PIK3CA were genotyped with a sensitivity of 0.25%. For CRC patient specimens, 30 samples were consecutively and automatically multi-genotyped without inter-sample contamination, with a lowest mutation frequency of 0.37%. For the first time, we developed a droplet-based CE system for consecutive DNA analysis with low sample consumption. This automated CE system could be further developed to integrate the full process of gene mutation detection, serving as a more effective platform for individualised therapy.

Cooperative evaporation in two-dimensional droplet arrays

The evaporation of a sessile drop in a gaseous environment may be critical to many practical applications. Evaporation dynamics of interacting sessile droplets is strongly influenced by the proximity of adjacent droplets. We study the effects of droplet-droplet vapor-mediated interactions on the evaporation lifetime of two-dimensional arrays of sessile water droplets. We observe that the presence of neighboring droplets acts as a mode of vapor accumulation which slows down the evaporation process. By considering an arbitrarily configured two-dimensional array of droplets, here we provide a simple generalized theoretical limit to their lifetime in an evaporating state. Using a scaling analysis, we put forward that the sessile droplet lifetime in a two-dimensional array is a linear function of the extent of confinement for various surface wettability and droplet geometric parameters (contact angle and contact radius). Notwithstanding the geometrical and physical complexity of the effective confinement generated due to their cooperative interactions, we show that the consequent evaporation characteristics may be remarkably insensitive to the topographical details of the overall droplet organization for a wide range of droplet-substrate combinations. With subsequent deployment of particle-laden droplets, however, our results lead to the discovery of a unique pathway towards tailoring the internal flows within the collective system by harnessing an exclusive topologically driven symmetry-breaking phenomenon, yielding a strategy of patterning particulate matters around the droplet array.

Parallel Droplet Deposition via a Superhydrophobic Plate with Integrated Heater and Temperature Sensors.

A simple setup, which is suitable for parallel deposition of homogenous liquids with a precise volume (dosage), is presented. First, liquid is dispensed as an array of droplets onto a superhydrophobic dosage plate, featuring a 3 × 3 array of holes. The droplets rest on these holes and evaporate with time until they are small enough to pass through them to be used on the final target, where a precise amount of liquid is required. The system can be fabricated easily and operates in a highly parallel manner. The design of the superhydrophobic dosage plate can be adjusted, in terms of the hole positions and sizes, in order to meet different specifications. This makes the proposed system extremely flexible. The initial dispensed droplet mass is not significant, as the dosing takes place during the evaporation process, where the dosage is determined by the hole diameter. In order to speed up the evaporation process, microheaters are screen printed on the back side of the dosage plate. To characterize the temperature distribution caused by the microheaters, temperature sensors are screen printed on the top side of the dosage plate as well. Experimental data regarding the temperature sensors, the microheaters, and the performance of the setup are presented, and the improvement due to the heating of the dosage plate is assessed. A significant reduction of the total evaporation time due to the microheaters was observed. The improvement caused by the temperature increase was found to follow a power law. At a substrate temperature of 80 °C, the total evaporation time was reduced by about 79%.

Collective nucleation dynamics in two-dimensional emulsions with hexagonal packing.

We report a mechanism for nucleation in a monolayer of hexagonally packed monodisperse droplet arrays. Upon cooling, we observe solidified droplets to nucleate their supercooled neighbors giving rise to an autocatalyticlike mechanism for accelerated crystallization. This collective mode of nucleation depends on the strength and nature of droplet contacts. Intriguingly, the statistical distribution of the solidified droplet clusters is found to be independent of emulsion characteristics except surfactant. In contrast to classical nucleation theory, our work highlights the need to consider collective effects of nucleation in supercooled concentrated emulsions where droplet crowding is inevitable.

Membrane Structure Drives Synchronization Patterns in Arrays of Diffusively Coupled Self-Oscillating Droplets.

Networks of diffusively coupled inorganic oscillators, confined in nano- and microcompartments, are effective for predicting and understanding the global dynamics of those systems where the diffusion of activatory or inhibitory signals regulates the communication among different individuals. By taking advantage of a microfluidic device, we study the dynamics of arrays of diffusively coupled Belousov-Zhabotinsky (BZ) oscillators encapsulated in water-in-oil single emulsions. New synchronization patterns are induced and controlled by modulating the structural and chemical properties of the phospholipid-based biomimetic membranes via the introduction of specific dopants. Doping molecules do not alter the membrane basic backbone, but modify the lamellarity (and, in turn, the permeability) or interact chemically with the reaction intermediates. A transition from two-period clusters showing 1:2 period-locking to one-period antiphase synchronization is observed by decreasing the membrane lamellarity. An unsynchronized scenario is found when the dopant is able to interfere with chemical communication by reacting with the chemical messengers.

Highly Sensitive Blocker Displacement Amplification and Droplet Digital PCR Reveal Low-Level Parental FOXF1 Somatic Mosaicism in Families with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins

Detection of low-level somatic mosaicism [alternate allele fraction (AAF) ≤ 10%] in parents of affected individuals with the apparent de novo pathogenic variants enables more accurate estimate of recurrence risk. To date, only a few systematic analyses of low-level parental somatic mosaicism have been performed. Herein, highly sensitive blocker displacement amplification, droplet digital PCR, quantitative PCR, long-range PCR, and array comparative genomic hybridization were applied in families with alveolar capillary dysplasia with misalignment of pulmonary veins. We screened 18 unrelated families with the FOXF1 variant previously determined to be apparent de novo (n=14), of unknown parental origin (n=1), or inherited from a parent suspected to be somatic and/or germline mosaic (n=3). We identified four (22%) families with FOXF1 parental somatic mosaic single-nucleotide variants (n=3) and copy number variant deletion (n=1) detected in parental blood samples and an AAF ranging between 0.03% and 19%. In one family, mosaic allele ratio in tissues originating from three germ layers ranged between <0.03% and 0.65%. Because the ratio of parental somatic mosaicism have significant implications for the recurrence risk, this study further implies the importance of a systematic screening of parental samples for low-level and very-low-level (AAF ≤ 1%) somatic mosaicism using methods that are more sensitive than those routinely applied in diagnostics.

Activity-expression profiling of glucose-6-phosphate dehydrogenase in tissues of normal and diabetic mice

Glucose-6-phosphate dehydrogenase (G6PD) plays a principal role in the regulation of oxidative stress by modulating the nicotinamide adenine dinucleotide phosphate pool and is expected to be associated with metabolic diseases such as diabetes mellitus (DM). However, it is unclear whether hyperglycemia increases G6PD activity levels in DM because suitable assays for quantifying the activity in a high-throughput manner are lacking. Using liquid droplet arrays tailored to analyze tissue lysates, we performed G6PD activity profiling in eight tissues of normal and diabetic mice: brain, heart, kidney, liver, lung, muscle, spleen, and thyroid. Diabetic mice exhibited significantly higher G6PD activities in the kidney, liver, spleen, and thyroid than normal mice; no significant difference was found in the brain, heart, lung, or muscle. We also performed G6PD expression profiling in the eight tissues using Western blot analysis. Diabetic mice showed significantly elevated G6PD expression levels in the kidney, lung, spleen, and thyroid compared with normal mice; no significant difference was found in the brain, heart, liver, or muscle. An analysis of G6PD activity-expression profiles demonstrated tissue-specific changes in response to hyperglycemia. Thus, our approach would be helpful for understanding the role of G6PD in tissue-based pathogenesis of diabetic complications.

Parallel Sampling of Nanoliter Droplet Arrays for Noninvasive Protein Analysis in Discrete Yeast Cultivations by MALDI-MS.

Miniaturization of cell-based assays enables the analysis of secreted compounds from low cell numbers down to a single cell. Droplet microfluidics is a well-established tool for high-throughput single-cell analysis. Nevertheless, the integration of label-free bioanalytical techniques like mass spectrometry is still ongoing. For example, without additional separation steps, droplet-enclosed cells do not survive the analysis. Cell separation techniques for droplets have been reported, but could not yet be coupled to mass spectrometric analysis. Here, we present a simple approach for high-throughput cell separation in parallel in nanoliter droplets and demonstrate that it can be used for qualitative analysis of protein secretion by the yeast Komagataella phaffii. Using a custom-made droplet spotter, we generated an array of 200 droplets of nanoliter volumes on a glass plate, each containing approximately 500 cells. After cultivation for 24 h, a second plate was placed above the droplet array and brought in contact with the droplets. All droplets were sampled in parallel by plate-based droplet splitting. The nanoliter samples of the supernatant could be interfaced with mass spectrometry and we were able to detect the protein brazzein (his-tagged, 7445 Da) in all but two droplets. Additionally, we show that the cells were viable after the cell separation and a sample from one spot could be transferred to a cultivation tube. An advantage of our protocol is that each cell suspension is directly linked to the analysis result by its position. Furthermore, we demonstrate that our method is capable of splitting around 6000 droplets in a few seconds. In the future, additional processing steps on a small scale, such as desalting and protein digestion, could be developed and will enable structural proteomics in nanoliter volumes.

Nanomolar Synthesis in Droplet Microarrays with UV-Triggered On-Chip Cell Screening.

Miniaturization and parallelization of combinatorial organic synthesis is important to accelerate the process of drug discovery while reducing the consumption of reagents and solvents. This work presents a miniaturized platform for on-chip solid-phase combinatorial library synthesis with UV-triggered on-chip cell screening. The platform is based on a nanoporous polymer coating on a glass slide, which is modified via photolithography to yield arrays of hydrophilic (HL) spots surrounded by superhydrophobic (SH) surface. The combination of HL spots and SH background enables confinement of nanoliter droplets, functioning as miniaturized reactors for the solid-phase synthesis. The polymer serves as support for nanomolar solid-phase synthesis, while a photocleavable linker enables the release of the synthesized compounds into the droplets containing live cells. A 588 compound library of bisamides is synthesized via a four-component Ugi reaction on the chip and products are detected via stamping of the droplet array onto a conductive substrate and subsequent matrix-assisted laser desorption ionization mass spectrometry. The light-induced cleavage shows high flexibility in screening conditions by spatial, temporal, and quantitative control.

Highly ordered water droplet arrays on a volatile solvent surface

The condensation of water vapor on cold surfaces is termed as breath figures. It is fascinating that the highly ordered breath figure arrays can be formed by simple evaporation of a volatile polymer solution. It has been speculated that the ordered breath figure arrays originate from ordered water droplet arrays induced by the evaporation of volatile solvents. However, it is unknown what the real formation process of ordered water droplet arrays on volatile surfaces of pure solvents is. Herein, we report the real-time observation of the growth process of water droplets on the volatile surface of the pure solvent of carbon disulfide by using an optical microscope. We observed the highly ordered water droplet arrays on the volatile surface. We found six distinct stages during the evaporation process. The growth mechanism of water droplets obeys individual growth before coalescing. In addition, a few defects of large water droplets, vacancies and irregular boundaries were observed. The rapid movement of water droplets indicated the strong convective current in the solvent, promoting the rearrangement of water droplets to form more ordered arrays. The highly ordered water droplet arrays only need a moist environment, which is very convenient for ordered structures. Our findings not only provide direct evidence for ordered water droplet arrays but also quantitatively clarify their dynamic process, which helps to understand the mechanism of ordered breath figure arrays.

Self-Assembled Microlens Array with Controllable Focal Length Formed on a Selective Wetting Surface.

We report a microlens array with a controllable focal length that is based on a selective wetting surface. A substrate modified with a hydrophobic layer that has an array of microholes exhibits different wettability for inside holes (hydrophilic) versus outside holes (hydrophobic). When liquid flows over the surface, a small amount of liquid is adhered to hydrophilic holes and forms a lens-shaped droplet array that self-assembles because of surface tension. A large-sized plano-convex lens array that has good uniformity is obtained via the blade coating method, and controlling the amount of liquid enables control of the focal length. Our self-assembled microlens array has the merits of high optical performance, a simple fabrication procedure, and good mechanical stability, and thus, it has potential applications in imaging processing, light extraction, protein detection, light-emitting diodes, sensors, and displays.

A programmable microfluidic platform for multisample injection, discretization, and droplet manipulation.

A programmable microfluidic platform enabling on-demand sampling, compartmentalization, and manipulation of multiple aqueous volumes is presented. The system provides random-access actuation of a microtrap array supporting selective discretization of picoliter volumes from multiple sample inputs. The platform comprises two interconnected chips, with parallel T-junctions and multiplexed microvalves within one chip enabling programmable injection of aqueous sample plugs, and nanoliter volumes transferred to a second microtrap array chip in which the plugs are actively discretized into picoliter droplets within a static array of membrane displacement actuators. The system employs two different multiplexer designs that reduce the number of input signals required for both sample injection and discretization. This versatile droplet-based technology offers flexible sample workflows and functionalities for the formation and manipulation of heterogeneous picoliter droplets, with particular utility for applications in biochemical synthesis and cell-based assays requiring flexible and programmable operation of parallel and multistep droplet processes. The platform is used here for the selective encapsulation of differentially labeled cells within a discrete droplet array.

Smearing Observation of Picoliter Droplets Pinning on Bio-Inspired Negative Lotus Leaf Replicas

Separating aqueous samples into large numbers of independent micro-droplets enables efficient study of single cells and detection of single molecules. However, as the droplet size becomes smaller, the reinforced handling process is possibly required to ensure droplets operability, resulting in increased experimental complexity. Herein, we propose a method of imitating the bio-structure of lotus leave reversal to generate a picoliter droplet array effectively. This bio-inspired structure is made of a single material PDMS (polydimethylsiloxane) without any additional chemical surface treatments. Only smearing is required for embedding liquid samples in such microwells with splayed walls. In the present experiments, we have obtained a regular array of 2 pL droplets with a density of 62,500 droplets/cm2, which equivalents to 5.4 million droplets on the surface a 96 well plate. We believe that such a method contributes to the development of the efficient micro-droplet generator with a pure physical interface and thus it is applicable to biological and medical analysis.