Flow-focusing Droplet Generators Research Articles

A numerical analysis of particle encapsulation in a flow-focusing droplet generation device

In this paper, the process of encapsulating solid particle(s) into liquid droplets in a high-throughput flow-focusing microchannel is investigated numerically. Open source software is used, which computes fluid flow in an Eulerian framework and particle dynamics with a Lagrangian approach. Previous studies have demonstrated that if no action is taken, particles suspended in a liquid passing through a flow-focusing microchannel will be encapsulated at random. This is perhaps unsurprising, but in one such study, less than 35% of droplets were found to contain exactly one particle. The two aims of this study are (i) to explore the flow patterns arising in a microfluidic channel and (ii) to elucidate the effect of salient governing parameters on encapsulation efficiency (i.e., the fraction of droplets encapsulating one particle) by focusing on ordering the particles before reaching the droplet generation section. Following validation against experimental reference data, the capillary number is varied across the three droplet generation regimes: squeezing, dripping, and jetting. We demonstrate that under certain conditions, an encapsulation frequency of 100% can be achieved with ordered particles, but in most cases, this is significantly lower. We examine the flow field to help understand how this non-uniform distribution of particles occurs. Notably, we find the dripping to be the best option for particle encapsulation and in this case extend the study to explore the effect of junction angle, finding that an angle of 60° is the most favorable. Improved understanding of the encapsulation process derived from this study can help to improve design of high-throughput droplet generation microfluidic systems.

Computational simulation of the effects of interfacial tension in microfluidic flow focusing droplet generators

We present simulations of a square flow focusing droplet generator device exploring its performance characteristics over a range of interfacial surface tension values and varying neck width. Droplet generators have a wide range of applications from drug delivery to X-ray diffraction experiments. Matching the droplet frequency and volume to the experimental parameters is critical for maximising the data quality and minimising sample waste. Whilst varying the interfacial surface tension we observed that the lowest frequency of droplets is generated for surface tensions matching those typically reported for water-oil mixtures (around 40 mN/M). Decreasing or increasing the interfacial surface tension, for example by adding surfactant, results in an increase in droplet frequency. We also find that under the conditions simulated here, droplets are generated with much lower capillary numbers and higher Weber numbers than have typically been reported in the literature. The high ratio of flowrate-to-cross-section used here resulted in a velocity which was larger than has previously been reported for flow focusing devices and consequently we observe particularly large associated Reynolds numbers. However, in general, the simulated flow behaviour characteristics most closely match those typically observed for the jetting and tip-streaming regimes. The highest frequency of droplets achieved in our simulated devices was 36 kHz and 56 kHz corresponding to square neck channel widths of 12.5 and 25 µm respectively, an interfacial surface tension of 118.75 mN/m. We also examined the effect of varying neck width geometry for a fixed interfacial surface tension of 52 mN/m. We observed that the highest frequency droplet generation, 61 kHz, corresponded to a neck width of 37.5 µm with a corresponding droplet diameter of 22 µm. The high frequency, high monodispersity, and small droplet size predicted to occur through modification of the interfacial surface tension will have implications for the future design and optimisation of droplet-on-demand microfluidic devices.

Versatility and stability optimization of flow-focusing droplet generators via quality metric-driven design automation.

Droplet generation is a fundamental component of droplet microfluidics, compartmentalizing biological or chemical systems within a water-in-oil emulsion. As adoption of droplet microfluidics expands beyond expert labs or integrated devices, quality metrics are needed to contextualize the performance capabilities, improving the reproducibility and efficiency of operation. Here, we present two quality metrics for droplet generation: performance versatility, the operating range of a single device, and stability, the distance of a single operating point from a regime change. Both metrics were characterized in silico and validated experimentally using machine learning and rapid prototyping. These metrics were integrated into a design automation workflow, DAFD 2.0, which provides users with droplet generators of a desired performance that are versatile or flow stable. Versatile droplet generators with stable operating points accelerate the development of sophisticated devices by facilitating integration of other microfluidic components and improving the accuracy of design automation tools.

Scalable Production of Size-Controlled Cholangiocyte and Cholangiocarcinoma Organoids within Liver Extracellular Matrix-Containing Microcapsules.

Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO (n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules (n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications.

Microfluidic formation of biodegradable PCLDA microparticles as sustainable sorbents for treatment of organic contaminants in wastewater

Polymeric microparticles (MPs) have attracted remarkable interest in environmental remediation owing to their economic benefits, simple manipulation and recyclability. However, most polymeric MPs have intrinsic issue for microplastic pollution, which can only be resolved for different materials using a biodegradable polymer. Here, we synthesize the photocurable polycaprolactone diacrylate (PCLDA) via an acrylation reaction for use as a sustainable sorbent in the treatment of organic contaminants. PCLDA can form chemically interconnected network, ensuring a solid state with the swelling phenomenon in their organic solvent. Using a flow-focusing droplet generator, we generate monodisperse oil-in-water (O/W) emulsions with the PCLDA solution as the oil dispersed phase and form MPs via in situ photopolymerization under UV light. Compared to the general solvent evaporation method, PCLDA MPs are found to have a fine morphology and narrow size distribution. We also evaluate the degree of swelling behavior of PCLDA MPs in various solvents based on their solubility parameter. Further we demonstrate that these PCLDA particles, as sustainable organic sorbents, exhibit high performance in the treatment of toluene oil droplets and solutes from water with good recyclability.

A Biodegradable Magnetic Microrobot Based on Gelatin Methacrylate for Precise Delivery of Stem Cells with Mass Production Capability.

A great deal of research has focused on small-scale robots for biomedical applications and minimally invasive delivery of therapeutics (e.g., cells, drugs, and genes) to a target area. Conventional fabrication methods, such as two-photon polymerization, can be used to build sophisticated micro- and nanorobots, but the long fabrication cycle for a single microrobot has limited its practical use. This study proposes a biodegradable spherical gelatin methacrylate (GelMA) microrobot for mass production in a microfluidic channel. The proposed microrobot is fabricated in a flow-focusing droplet generator by shearing a mixture of GelMA, photoinitiator, and superparamagnetic iron oxide nanoparticles (SPIONs) with a mixture of oil and surfactant. Human nasal turbinate stem cells (hNTSCs) are loaded on the GelMA microrobot, and the hNTSC-loaded microrobot shows precise rolling motion in response to an external rotating magnetic field. The microrobot is enzymatically degraded by collagenase, and released hNTSCs are proliferated and differentiated into neuronal cells. In addition, the feasibility of the GelMA microrobot as a cell therapeutic delivery system is investigated by measuring electrophysiological activity on a multielectrode array. Such a versatile and fully biodegradable microrobot has the potential for targeted stem cell delivery, proliferation, and differentiation for stem cell-based therapy.

Compact Empirical Model for Droplet Generation in a Lab-on-Chip Cytometry System

This study describes the construction of a compact empirical mathematical model for a flow-focusing microfluidic droplet generator. The application case is a portable, low-cost flow cytometry system for microbiological applications, with water droplet sizes of 50-70 micrometer range and droplet generation rates of 500-1500 per second. In this study, we demonstrate that for the design of reliable microfluidic systems, the availability of an empirical model of droplet generation is a mandatory precondition that cannot be achieved by time-consuming simulations based on detailed physical models. When introducing the concept of a compact empirical model, we refer to a mathematical model that considers general theoretical estimates and describes experimental behavioral trends with a minimal set of easily measurable parameters. By interpreting the experimental results for different water- and oil-phase flow rates, we constructed a minimal 3-parameter droplet generation rate model for every fixed water flow rate by relying on submodels of the water droplet diameter and effective ellipticity. As a result, we obtained a compact model with an estimated 5-10% accuracy for the planned typical application modes. The main novelties of this study are the demonstration of the applicability of the linear approximation model for droplet diameter suppression by the oil flow rate, introduction of an effective ellipticity parameter to describe the droplet form transformation from a bullet-like shape to a spherical shape, and introduction of a machine learning correction function that could be used to fine-tune the model during the real-time operation of the system.

Open-Source Pressure Controller Based on Compact Electro-Pneumatic Regulators for Droplet Microfluidics Applications

Microfluidics is a rapidly growing area that provides innovations in biotechnology, medical diagnostics, and life science. Along with the commercial solutions, there are open-source projects aimed to make such technologies more affordable and flexible for end users. In this article, we developed an open-source, low-cost, and easy-to-use microfluidic pressure controller with a modular design based on electropneumatic regulators to introduce liquids into microfluidic devices under constant pressures. For implementation of complex experimental protocols, the controller contains a vacuum pump, relay outputs, analog inputs, and digital inputs/outputs to connect external equipment. It can be operated using five displays and an encoder or from a personal computer in a manual or automatic regime through the custom open-source software. In this study, we applied this pressure controller for the generation of water-in-oil droplets in microfluidic flow-focusing droplet generators. Droplets&#x2019; diameters linearly depended on the dispersed and continuous phases&#x2019; ratio in the range 5&#x2013;<inline-formula> <tex-math notation="LaTeX">$65 \mu \text{m}$ </tex-math></inline-formula>. In all the cases, the generation regimes were stable for at least 4 h. Direct comparison of the droplet generation obtained with the developed pressure controller and precise syringe pumps showed that it will suit the needs of the microfluidic community for different flow-based lab-on-a-chip applications, as a more affordable and flexible solution for introducing liquids and controlling flow sensors, heaters, valves, light sources, and so on during experiments.

A 3D printed size-tunable flow-focusing droplet microdevice to produce cell-laden hydrogel microspheres

Flow-focusing droplet generators have been extensively employed for the generation of monodisperse droplets. However, the droplet device is usually designed with an application-specific performance that includes prescribed droplet size and generation frequency. To achieve an ideal device, cost- and time-inefficient iterations for the chip design and fabrication are usually needed. In this study, we take an advantage of 3D printing technology to rapidly prototype the droplet device that enables the facile control of the droplet size as well as the droplet generation frequency. Our device was designed with a screw-and-nut combination and the gap height (hg) between the dispersed phase outlet and the orifice could be easily and finely controlled by rotating the head of the threaded screw. The hg values can be precisely adjusted from 0 to 2000 μm supported by 20 designated control teeth on the screw head, which enable us to produce droplets in different sizes or in the same size with different generation frequencies. The proposed 3D printed device was employed to synthesize a variety of Ca-alginate microspheres containing A549 cells. The facile assembly of the screw-and-nut components allows us to prepare the droplet generator in a simple yet effective manner, and the size controllability of the droplets resulted in the production of various sizes of A549 cell-encapsulated microspheres, which can be used for drug screening.

Ultrahigh Throughput On‐Chip Synthesis of Microgels with Tunable Mechanical Properties

AbstractMicrogels, 1–100 µm sized hydrogel particles, have emerged as versatile materials for constructing injectable tissue scaffolds, with superb control over various properties. Microfluidic synthesis of microgels offers significant advantages over conventional batch synthesis such as high uniformity and precise control over emulsion size. Despite these positive attributes, the gelation of microfluidic droplets is typically performed off‐chip, compromising the particle homogeneity, and confounding the ability to control their mechanical properties. To address this, a multistep microgel synthesis line is developed, comprising of a flow focusing droplet generator followed by a UV‐curing stage that delivers a precise UV dosage to each droplet, allowing precise control of the mechanical properties of hydrogel microparticles. Furthermore, parallel operation of 4080 identical synthesis lines on a single chip is demonstrates, generating precisely defined microgels at a throughput appropriate for commercial and clinical translation. The system is validated by generating poly(ethylene glycol) diacrylate microgel particles with a diameter down to 40 µm at a throughput above 1 kg h−1 (106 particles s−1), with a coefficient of variation of 3%. It is demonstrated that the stiffness of the microgels can be precisely controlled from 103 to 104 Pa by varying the UV dosage.

Experimental studies on droplet characteristics in a microfluidic flow focusing droplet generator: effect of continuous phase on droplet encapsulation.

The efficacy of droplet-based microfluidic assays depends on droplet size, pattern, generation rate, etc. The size of the droplet is affected by numerous variables as flow rate ratio, viscosity ratio, microchannel geometry, surfactants, nature of fluids and other dimensionless numbers. This work reports rigorous analysis and optimization of the behavior of droplets with change in flow rate ratio and viscosity ratio in a flow-focusing device. Droplets were produced for different flow rate ratios maintaining a constant aqueous phase and varying the continuous phase, to have capillary numbers ranging from 0.01 to 0.1. It was observed that the droplet size decreased with the increase in flow rate ratio, and vice versa. It was noted that as the viscosity ratio was increased, the dispersed phase elongated before the complete breakup and long droplets were formed in the microchannel. Smaller droplets were formed for lower viscosity ratios with a combination of higher flow rate ratios. An empirical relation has been developed to predict the droplet length in terms of capillary number and flow rate ratio for different viscosity ratios. In addition, microparticle encapsulation in individual droplets was attempted to realize the effect of flow rate of the continuous phase for various flow rate ratios on encapsulation efficiency.

Numerical and experimental investigation of a flow focusing droplet-based microfluidic device

Droplet-based microfluidic devices are widely used for drug delivery and cell transport. In most of these applications, flow characteristics and geometry strongly affect the droplet generation process. In this study, a flow focusing droplet generator was designed using numerical simulation. Among all the simulation methods, the 2D phase field method was used to predict the process in a newly designed microfluidic device before fabrication. To this end, the numerical method was verified according to previous research, including ten different cases, which demonstrated its applicability in this regard. The deviation of simulations from experiments was less than 5%. The device was then simulated over a wide range of flow rate ratios (φ) and fabricated based on these results. Following fabrication, experiments were conducted for different ratios. The experimental results were compared with the simulations qualitatively and quantitatively. The investigation revealed pre-estimation effectiveness, indicating that the deviation of simulations from experiments was at most 11%. A wide range of φ from 0.25 to 1.05 was applied to study the droplet generation characteristics during the tests. Furthermore, two fundamental modes were introduced, and the flow regime shift from squeezing to dripping was studied over a wide range of φ. Given the droplets’ shape, the transition procedure is divided into three stages: pre-transition, transition, and post-transition. This study’s results can be used to investigate the capabilities of a newly designed microdevice before fabrication and choose the best flow regime that simultaneously reduces shear stress on cells and increases droplet generation frequency and monodispersity. Finally, this passive design can transport cells inside droplets up to 280 samples per second.

Microfluidic protein detection and quantification using droplet morphology

Sensitive, inline detection of proteins is required for post-chromatographic analyses in proteomics, cell-based assays, and drug discovery workflows. Among the common inline methods, post-column derivatization requires chemical labels, while label-free methods are either expensive (mass spectrometry) or have limited sensitivity at small length scales (UV–Vis). This paper presents a label-free detection technique based on the concept that dissolved proteins can function as surfactants and decrease the dynamic interfacial tension (IFT) of an immiscible (water–oil) interface. Existing methods for measuring IFT, such as axisymmetric drop shape analysis (ADSA), operate in batch mode and are not suitable for continuous detection. Here we show that a microfluidic flow-focusing droplet generator operating at a frequency of > 100 Hz can track IFT changes continuously, with high temporal resolution and small detection volumes. Variations in protein concentration alter the size and shape of the drops/plugs formed, and these changes can be quantified in time using a high-speed camera and in-house image processing software. Moreover, the continuously refreshing interface alleviates issues related to surface aging. Two applications are demonstrated: (1) direct injection of a single protein into a microfluidic chip. (2) post-column detection of protein mixtures separated by high performance size exclusion chromatography (SEC HPLC). Of interest, the dynamic range of protein (bovine serum albumin, BSA) was 50 -104 μg/ml without using HPLC unit. The lowest limit of detection without HPLC unit was ~ 1 μg/ml of thyroglobulin protein in a 1 nl droplet, which equates to 1 fg of total protein. When used as a detector, the aforementioned detection method offered a sensitivity of six orders of magnitude higher than conventional UV–VIS detectors.

An asymmetric flow-focusing droplet generator promotes rapid mixing of reagents

Nowadays droplet microfluidics is widely used to perform high throughput assays and for the synthesis of micro- and nanoparticles. These applications usually require packaging several reagents into droplets and their mixing to start a biochemical reaction. For rapid mixing microfluidic devices usually require additional functional elements that make their designs more complex. Here we perform a series of 2D numerical simulations, followed by experimental studies, and introduce a novel asymmetric flow-focusing droplet generator, which enhances mixing during droplet formation due to a 2D or 3D asymmetric vortex, located in the droplet formation area of the microfluidic device. Our results suggest that 2D numerical simulations can be used for qualitative analysis of two-phase flows and droplet generation process in quasi-two-dimensional devices, while the relative simplicity of such simulations allows them to be easily applied to fairly complicated microfluidic geometries. Mixing inside droplets formed in the asymmetric generator occurs up to six times faster than in a conventional symmetric one. The best mixing efficiency is achieved in a specific range of droplet volumes, which can be changed by scaling the geometry of the device. Thus, the droplet generator suggested here can significantly simplify designs of microfluidic devices because it enables both the droplet formation and fast mixing of the reagents within droplets. Moreover, it can be used to precisely estimate reaction kinetics.

Shear-dependent microvortices in liquid–liquid flow-focusing geometry: A theoretical, numerical, and experimental study

In this work, we describe the mechanism of particle trapping and release at the flow-focusing microfluidic droplet generation junction, utilizing the hydrodynamic microvortices generated in the dispersed phase. This technique is based solely on our unique flow-focusing geometry and the flow control of the two immiscible phases and, thus, does not require any on-chip active components. The effectiveness of this technique to be used for particle trapping and the subsequent size selective release into the droplets depends on the fundamental understanding of the nature of the vortex streamlines. Here, we utilized theoretical, computational, and experimental fluid dynamics to study in detail these microvortices and parameters affecting their formation, trajectory, and magnitude.

Machine learning enables design automation of microfluidic flow-focusing droplet generation

Droplet-based microfluidic devices hold immense potential in becoming inexpensive alternatives to existing screening platforms across life science applications, such as enzyme discovery and early cancer detection. However, the lack of a predictive understanding of droplet generation makes engineering a droplet-based platform an iterative and resource-intensive process. We present a web-based tool, DAFD, that predicts the performance and enables design automation of flow-focusing droplet generators. We capitalize on machine learning algorithms to predict the droplet diameter and rate with a mean absolute error of less than 10 μm and 20 Hz. This tool delivers a user-specified performance within 4.2% and 11.5% of the desired diameter and rate. We demonstrate that DAFD can be extended by the community to support additional fluid combinations, without requiring extensive machine learning knowledge or large-scale data-sets. This tool will reduce the need for microfluidic expertise and design iterations and facilitate adoption of microfluidics in life sciences.

Study of droplet formation regimes in a pressure control mode in microfluidic chip for screening cell libraries

To create a high throughput system for screening single cells in “water-in-oil” droplets, a key role is played by the method of controlling fluid flows in a microfluidic droplet generator. This is due to the fact that it is required to obtain monodisperse drops-microcapsules, place cells in them and be able to manipulate them. For these purposes, not only systems based on syringe pumps are suitable, but also pressure control units may be used. Unfortunately, pressure control instruments on the market for researchers are represented by only a few companies and are quite expensive. In this work, we develop a homemade microfluidic pressure controller based on SMC ITV 0010 pneumatic regulators and show its operability. Also, we investigated the conditions for reproducible generation of water-in-oil emulsions in a microfluidic flow focusing droplet generator by pressure driven fluid flows.

An Interface-Particle Interaction Approach for Evaluation of the Co-Encapsulation Efficiency of Cells in a Flow-Focusing Droplet Generator.

Droplet-based microfluidics offers significant advantages, such as high throughput and scalability, making platforms based on this technology ideal candidates for point-of-care (POC) testing and clinical diagnosis. However, the efficiency of co-encapsulation in droplets is suboptimal, limiting the applicability of such platforms for the biosensing applications. The homogeneity of the bioanalytes in the droplets is an unsolved problem. While there is extensive literature on the experimental setups and active methods used to increase the efficiency of such platforms, passive techniques have received less attention, and their fundamentals have not been fully explored. Here, we develop a novel passive technique for investigating cell encapsulation using the finite element method (FEM). The level set method was used to track the interfaces of forming droplets. The effects of walls and the droplet interfaces on relatively large cells were calculated to track them more accurately during encapsulation. The static surface tension force was used to account for the effects of the interfaces on cells. The results revealed that the pairing efficiency is highly sensitive to the standard deviation (SD) of the distance between the cells in the entrance channel. The pairing efficiency prediction error of our model differed by less than 5% from previous experiments. The proposed model can be used to evaluate the performance of droplet-based microfluidic devices to ensure higher precision for co-encapsulation of cells.

Smartphone-Based Droplet Digital LAMP Device with Rapid Nucleic Acid Isolation for Highly Sensitive Point-of-Care Detection.

While advances in microfluidics have enabled rapid and highly integrated detection of nucleic acid targets, the detection sensitivity is still unsatisfactory in the current POC (point-of-care) detection systems, especially for low abundance samples. In this study, a chip that integrates rapid nucleic acid extraction based on IFAST (immiscible phase filtration assisted by surface tension) and digital isothermal detection was developed to achieve highly sensitive POC detection within 60 min. Based on the interface theory, the factors influencing the interface stability of the IFAST process were studied, and the IFAST nucleic acid extraction conditions were optimized to increase the nucleic acid extraction recovery rate to 75%. Spiral mixing channel and flow-focusing droplet generation structure were designed to achieve the mixing and sample partitioning by applying negative pressure. A portable microdroplet fluorescence detection device was developed based on smartphone imaging. Validation tests were carried out for quantification of low-abundance cfDNA and detection of mutations.

On-chip integration of normal phase high-performance liquid chromatography and droplet microfluidics introducing ethylene glycol as polar continuous phase for the compartmentalization of n-heptane eluents

We introduce an integrated chip-approach for a postcolumn segmentation of normal phase liquid chromatography. This is achieved by the seamless integration of a chiral NP-chip-HPLC column, a flow-focusing droplet generator, and a segmented flow channel in a single microfluidic glass chip. This allows a continuous segmentation of the eluent into droplets which are picked up and transported via an immiscible continuous phase. The combination of NP-chip-HPLC and droplet microfluidics enables to fractionate and conserve chromatographic runs for further downstream processes at picoliter scale. An essential aspect is the proper choice of the continuous phase concerning polarity, wetting properties and viscosity. For this purpose, ethylene glycol is introduced which facilitates this first combination of normal phase chromatography and droplet microfluidics. By adjusting the flow rates and varying the generator geometry, the size and frequency of the droplets could be precisely controlled.