Microfluidic Flow-focusing Device Research Articles

Bubble generation in a 3-D flow-focusing microchannel: From squeezing to jetting

Fine bubbles have been applied in many fields; however, the controllable preparation of monodispersed fine bubbles remains a challenge. In this study, a three-dimensional (3-D) flow-focusing microfluidic device was designed. Compared with bubble formation in one-dimensional (1-D) and two-dimensional (2-D) flow-focusing, the 3-D flow-focusing microchannel owns the features of making bubbles with much smaller sizes and wider operation range. Four different gas–liquid flow regimes (squeezing, squeezing-dripping transition, dripping, and jetting) and three different gas–liquid dispersion states (mono-dispersion, bi-dispersion, and poly-dispersion) were observed in the 3-D flow-focusing microchannels. Importantly, the bi-dispersion in the squeezing-dripping transition regime experimentally verified that the squeezing and viscous shearing mechanisms separately control bubble formation in the transition region based on the time sequence. The intrinsic reason for poly-dispersion in the squeezing-dripping transition and jetting regimes was revealed. Finally, a mathematical model is proposed to predict the bubble size in the flow-focusing microchannels.

Designing magnetic microcapsules for cultivation and differentiation of stem cell spheroids

Human pluripotent stem cells (hPSCs) represent an excellent cell source for regenerative medicine and tissue engineering applications. However, there remains a need for robust and scalable differentiation of stem cells into functional adult tissues. In this paper, we sought to address this challenge by developing magnetic microcapsules carrying hPSC spheroids. A co-axial flow-focusing microfluidic device was employed to encapsulate stem cells in core-shell microcapsules that also contained iron oxide magnetic nanoparticles (MNPs). These microcapsules exhibited excellent response to an external magnetic field and could be held at a specific location. As a demonstration of utility, magnetic microcapsules were used for differentiating hPSC spheroids as suspension cultures in a stirred bioreactor. Compared to standard suspension cultures, magnetic microcapsules allowed for more efficient media change and produced improved differentiation outcomes. In the future, magnetic microcapsules may enable better and more scalable differentiation of hPSCs into adult cell types and may offer benefits for cell transplantation.

Functionalized monodisperse microbubble production: microfluidic method for fast, controlled, and automated removal of excess coating material

Functionalized monodisperse microbubbles have the potential to boost the sensitivity and efficacy of molecular ultrasound imaging and targeted drug delivery using bubbles and ultrasound. Monodisperse bubbles can be produced in a microfluidic flow focusing device. However, their functionalization and sequential use require removal of the excess lipids from the bubble suspension to minimize the use of expensive ligands and to avoid competitive binding and blocking of the receptor molecules. To date, excess lipid removal is performed by centrifugation, which is labor intensive and challenging to automate. More importantly, as we show, the increased hydrostatic pressure during centrifugation can reduce bubble monodispersity. Here, we introduce a novel automated microfluidic ’washing’ method. First, bubbles are injected in a microfluidic chamber 1 mm in height where they are left to float against the top wall. Second, lipid-free medium is pumped through the chamber to remove excess lipids while the bubbles remain located at the top wall. Third, the washed bubbles are resuspended and removed from the device into a collection vial. We demonstrate that the present method can (i) reduce the excess lipid concentration by 4 orders of magnitude, (ii) be fully automated, and (iii) be performed in minutes while the size distribution, functionality, and acoustic response of the bubbles remain unaffected. Thus, the presented method is a gateway to the fully automated production of functionalized monodisperse microbubbles.

Multiresponsive Liquid Crystal Collagen Guides Mussel Byssus Formation.

Marine mussels fabricate tough collagenous fibers known as byssal threads to anchor themselves. Threads are produced individually in minutes via secretion of liquid crystalline (LC) collagenous precursors (preCols); yet the physical and chemical parameters influencing thread formation remain unclear. Here, we characterized the structural anisotropy of native and artificially induced threads using quantitative polarized light microscopy and transmission electron microscopy to elucidate spontaneous vs regulated aspects of thread assembly, discovering that preCol LC phases form aligned domains of several hundred microns, but not the cm-level alignment of native threads. We then explored the hypothesized roles of mechanical shear, pH, and metal ions on thread formation through in vitro assembly studies employing a microfluidic flow focusing device using purified preCol secretory vesicles. Our results provide clear evidence for the role of all three parameters in modulating the structure and properties of the final product with relevance for fabrication of collagenous scaffolds for tissue engineering applications.

Synthesis of Gelatin Methacryloyl Analogs and Their Use in the Fabrication of pH-Responsive Microspheres.

pH-responsive hydrogels have numerous applications in tissue engineering, drug delivery systems, and diagnostics. Gelatin methacryloyl (GelMA) is a biocompatible, semi-synthetic polymer prepared from gelatin. When combined with aqueous solvents, GelMA forms hydrogels that have extensive applications in biomedical engineering. GelMA can be produced with different degrees of methacryloyl substitution; however, the synthesis of this polymer has not been tuned towards producing selectively modified materials for single-component pH-responsive hydrogels. In this work, we have explored two different synthetic routes targeting different gelatin functional groups (amine, hydroxyl, and/or carboxyl) to produce two GelMA analogs: gelatin A methacryloyl glycerylester (polymer A) and gelatin B methacrylamide (polymer B). Polymers A and B were used to fabricate pH-responsive hydrogel microspheres in a flow-focusing microfluidic device. At neutral pH, polymer A and B microspheres displayed an average diameter of ~40 µm. At pH 6, microspheres from polymer A showed a swelling ratio of 159.1 ± 11.5%, while at pH 10, a 288.6 ± 11.6% swelling ratio was recorded for polymer B particles.

Microfluidics single-cell encapsulation reveals that poly-l-lysine-mediated stem cell adhesion to alginate microgels is crucial for cell-cell crosstalk and its self-renewal

Microfluidic cell encapsulation has provided a platform for studying the behavior of individual cells and has become a turning point in single-cell analysis during the last decade. The engineered microenvironment, along with protecting the immune response, has led to increasingly presenting the results of practical and pre-clinical studies with the goals of disease treatment, tissue engineering, intelligent control of stem cell differentiation, and regenerative medicine. However, the significance of cell-substrate interaction versus cell-cell communications in the microgel is still unclear. In this study, monodisperse alginate microgels were generated using a flow-focusing microfluidic device to determine how the cell microenvironment can control human bone marrow-derived mesenchymal stem cells (hBMSCs) viability, proliferation, and biomechanical features in single-cell droplets versus multi-cell droplets. Collected results show insufficient cell proliferation (234 % and 329 %) in both single- and multi-cell alginate microgels. Alginate hydrogels supplemented with poly-l-lysine (PLL) showed a better proliferation rate (514 % and 780 %) in a comparison of free alginate hydrogels. Cell stiffness data illustrate that hBMSCs cultured in alginate hydrogels have higher membrane flexibility and migration potency (Young's modulus equal to 1.06 kPa), whereas PLL introduces more binding sites for cell attachment and causes lower flexibility and migration potency (Young's modulus equal to 1.83 kPa). Considering that cell adhesion is the most important parameter in tissue engineering, in which cells do not run away from a 3D substrate, PLL enhances cell stiffness and guarantees cell attachments. In conclusion, cell attachment to PLL-mediated alginate hydrogels is crucial for cell viability and proliferation. It suggests that cell-cell signaling is good enough for stem cell viability, but cell-PLL attachment alongside cell-cell signaling is crucial for stem cell proliferation and self-renewal.

Are monodisperse phospholipid-coated microbubbles “mono-acoustic?”

Phospholipid-coated microbubbles with a uniform acoustic response are a promising avenue for functional ultrasound sensing. A uniform acoustic response requires both a monodisperse size distribution and uniform viscoelastic shell properties. Monodisperse microbubbles can be produced in a microfluidic flow focusing device. Here, we investigate whether such monodisperse microbubbles have uniform viscoelastic shell properties and thereby a uniform “mono-acoustic” response. To this end, we visualized phase separation of the DSPC and DPPE-PEG5000 lipid shell components and measured the resonance curves of nearly 2000 single and freely floating microbubbles using a high-frequency acoustic scattering technique. The results demonstrate inhomogeneous phase-separated shell microdomains across the monodisperse bubble population, which may explain the measured inhomogeneous viscoelastic shell properties. The shell viscosity varied over an order of magnitude and the resonance frequency by a factor of two indicating both a variation in shell elasticity and in initial surface tension despite the relatively narrow size distribution.

Microfluidic-based preparation of PLGA microspheres facilitating peptide sustained-release

It is challenging for the manufacturing of microspheres loaded with peptide due to the complicated preparation process and the issue of burst release. In this construct, a microfluidic flow-focusing device is developed to prepare and optimize poly (lactic-co-glycolic acid) (PLGA) Exenatide −loaded microspheres. Surfactants was added to greatly improve the stability of the emulsion, leading to increase drug loading capacity. By increasing the particle size of the drug-loaded microspheres to 30 μm, the burst release was significantly reduced. Additionally, multi-channel droplet microfluidic devices are prepared with the capability to achieve a 100-fold increase in throughput, which could achieve a production rate of 2 g per hour.

Effect of Mold Materials Used During Hot Embossing on Feature Fidelity for Microfabrication in Cyclic Olefin Polymer (COP) Substrate

During the transition from research to market, the fabrication of microfluidic devices in thermoplastic substrates is inevitable. For short production runs of several hundred products, hot embossing is the typical method before moving on to a typically more expensive injection molding process for higher production volumes. In this work, we investigated the effect of mold material used during hot embossing on feature fidelity for microfabrication in cyclic olefin polymer (COP) substrate. Specifically, we designed a simple flow-focusing microfluidic device and fabricated three different molds using silicon wafer by deep reactive ion etching (DRIE), aluminum filled high temperature epoxy by soft lithography and aluminum by CNC milling. We performed hot embossing experiments with 2mm thick COP substrates and these three different molds using automatic bench top Carver hot press. Finally, we characterized the hot embossed substrates by optical and scanning electron microscopy. Fabrication results demonstrate that the mold material plays a big role in feature fidelity. Among the mold materials used, silicon substrate performed the worst based on defects after demolding. Epoxy and aluminum molds were similar in terms of microfabricated feature defects in the substrate which could be mostly attributed to their coefficient of thermal expansion (CTE). A mold material with a CTE closer to the thermoplastic will result in much better feature fidelity.

One-Step Microfluidic Fabrication of Bioinspired Microfibers with a Spindle-Knot Structure for Fog Harvest.

Many biomimetic microfibers have been designed from spider silk to collect water efficiently from humid air as a result of its periodic spindle-knot structure, which enhances the direct movement and convergence of captured fog droplets. Here, a hydrodynamic flow-focusing microfluidic device with a theta-shaped tube is designed for the one-step fabrication of bioinspired microfibers with a spindle-knot structure for fog harvest. The morphology of the structured microfibers, including height, width, and spacing of spindle knots, can be adjusted readily by regulating the flow rate of specific phases. The production rate of these structured microfibers can reach 1100 cm/min. Moreover, the capture, transportation, and collection performance of fog droplets on various microfibers are investigated in a fog collection platform. It is demonstrated that our one-step microfluidic device presents a ready method for the fabrication of structured microfibers with spindle knots, which provide a significant facilitation on efficient fog capture and water collection.

Monodisperse microbubble-mediated drug delivery: Influence of microbubbles size on drug delivery outcome

As the microbubble's resonance frequency is size-dependent, polydisperse microbubbles induce varying drug delivery outcomes at one ultrasound frequency. This study aimed to investigate drug delivery outcome of monodisperse MBs (mMBs) with radii ranging from 1.5–2.9 μm insonified at 2 MHz. Phospholipid-coated mMBs were generated using the Horizon microfluidic flow-focusing device. In vitro experiments were conducted on single microbubble-endothelial cells (n = 68) using confocal microscopy and 10 Mfps ultra-high-speed imaging. At 220 kPa PNP for 10 cycles, the 1.5 μm mMBs exhibited the highest PI uptake in 81.3% of cases, followed by 77.3% for 2.2 μm, 36.8% for 2.7 μm and 13.3% for 2.9 μm mMBs. Conversely, the 1.5 μm mMBs had the second lowest excursion amplitude (R;max-R;0) of 0.8 ± 0.3 μm as this was 1.2 ± 0.3 μm for 2.2 μm mMBs, 1.0 ± 0.3 μm for 2.7 μm mMBs, and 0.7 ± 0.2 μm for 2.9 μm mMBs. Additionally, for the 1.5, 2.2, and 2.7 μm mMBs, PI uptake and tunnel formation occurred more often (1.6, 1.8, and 1.3 times, respectively) than PI uptake and a resealing membrane pore. During insonification, mMB pinch-off occurred more frequently in tunnel formation (70.8%) than resealing pore formation (53.3%). This research revealed the impact of mMBs size on drug delivery outcome.

Performance optimization of droplet formation and break up within a microfluidic device – Numerical and experimental evaluation

Droplet-based microfluidics, including the controlled manipulation of materials in the form of monodisperse droplets on the micrometer scale in microfluidic chips, has gained much attention due to their ability in biological assays. The capillary number of the continuous phase, as the ratio of viscous force and interfacial tension, is so important in the mechanism of droplet formation. This study aims to survey the type of continuous-phase fluid, the change in the parameters of the continuous phase, and the effect of the different flow rates ratios on the droplet formation dynamics, droplet size, distance between droplets and frequency of droplet formation. Three types of oil, including mineral, olive, and paraffin in a flow-focusing microfluidic device have been utilized numerically and experimentally. To characterize the parameters of the continuous phase affecting the droplet size, a microfluidic device has been designed to estimate of the droplet size for three different types of oil used in the assay. The results show that for a constant continuous phase velocity, the diameter of the droplets formed in olive oil has the lowest amount, followed by paraffin and mineral oil, respectively. Regarding the effects of oil type on the frequency of droplet formation and the distance between droplets, the rate of droplet formation in mineral oil, in the ratios of the variable flow rate and the constant continuous phase velocity, is the lowest and, the distance between the droplets has the highest level.

Large-Scale Preparation of Hair Follicle Germs Using a Microfluidic Device.

Hair follicle morphogenesis during embryonic development is driven by the formation of hair follicle germs (HFGs) via interactions between epithelial and mesenchymal cells. Bioengineered HFGs are potential tissue grafts for hair regenerative medicine because they can replicate interactions and hair follicle morphogenesis after transplantation. However, a mass preparation approach for HFGs is necessary for clinical applications, given that thousands of de novo hair follicles are required to improve the appearance of a single patient with alopecia. In this study, we developed a microfluidics-based approach for the large-scale preparation of HFGs. A simple flow-focusing microfluidic device allowed collagen solutions containing epithelial and mesenchymal cells to flow and generate collagen microbeads with distinct Janus structures. During the 3 days of culture, the collagen beads contracted owing to cellular traction forces, resulting in collagen- and cell-dense HFGs. The transplantation of HFGs into nude mice resulted in highly efficient de novo hair follicle regeneration. This method provides a scalable and robust tissue graft preparation approach for hair regeneration.

Effective colloidal emulsion droplet regulation in flow-focusing glass capillary microfluidic device via collection tube variation.

Colloidal emulsion droplets, created using glass capillary microfluidic devices, have been found in a myriad of applications, serving as subtle microcarriers, delicate templates, etc. To meet the objective requirements under varying circumstances, it is crucial to efficiently control the morphology and dimensions of the droplets on demand. The glass capillary collection tube is a crucial component of the flow-focusing microfluidic system due to its close association with the geometrical confinement of the multiphasic flow. However, there are currently no guidelines for the design of the morphology and dimensions of the glass capillary collection tube, which shall result in a delay in assessing serviceability until after the microfluidic device is prepared, thereby causing a loss of time and effort. Herein, an experimental study was conducted to investigate the effect of the geometrical characteristics of glass capillary collection tubes on the production of colloidal emulsion droplets. After characterizing the generated colloidal emulsion droplets, it was found that the geometrical variations of the glass capillary collection tube resulted in numerical disparities of droplets due to different degrees of flow-focusing effects. The stronger flow-focusing effect produced smaller droplets at a higher frequency, and the dimensional variation of colloidal emulsion droplets was more responsive to varying flow rates. Furthermore, the transformation from colloidal single-core double-emulsion droplets to multi-core double-emulsion droplets also changed with the flow rate due to the glass capillary collection tube morphology-determined varying flow-focusing effect. These experimental findings can offer qualitative guidance for the design of glass capillary microfluidic devices in the preliminary stage, thus facilitating the smooth production of desired colloidal emulsion droplets.

A Versatile Photocrosslinkable Silicone Composite for 3D Printing Applications.

Embedded printing has emerged as a valuable tool for fabricating complex structures and microfluidic devices. Currently, an ample of amount of research is going on to develop new materials to advance its capabilities and increase its potential applications. Here, we demonstrate a novel, transparent, printable, photocrosslinkable, and tuneable silicone composite that can be utilized as a support bath or an extrudable ink for embedded printing. Its properties can be tuned to achieve ideal rheological properties, such as optimal self-recovery and yield stress, for use in 3D printing. When used as a support bath, it facilitated the generation microfluidic devices with circular channels of diameter up to 30 μm. To demonstrate its utility, flow focusing microfluidic devices were fabricated for generation of Janus microrods, which can be easily modified for multitude of applications. When used as an extrudable ink, 3D printing of complex-shaped constructs were achieved with integrated electronics, which greatly extends its potential applications towards soft robotics. Further, its biocompatibility was tested with multiple cell types to validate its applicability for tissue engineering. Altogether, this material offers a myriad of potential applications (i.e., soft robotics, microfluidics, bioprinting) by providing a facile approach to develop complicated 3D structures and interconnected channels.

Parameter control, characterization and stability of soy protein emulsion prepared by microfluidic technology.

A flow-focusing microfluidic device driven by pressure was employed in soy protein emulsions with uniform droplet size and good morphology. The results suggested that pressure was an essential factor for droplet formation. The optimum parameter was at a continuous phase pressure of 140mbar and dispersed phase pressure of 80mbar. Under this condition, the droplet formation time was shortened to 0.20s, with average sizes of 39-43μm and coefficient of variation of about 2%. Emulsion stability was improved with increasing soy protein isolate (SPI) concentrations. At SPI concentrations higher than 20mg/mL, the emulsions exhibited improved stability against changes in temperature, pH and salt concentration. Emulsions prepared in this manner exhibited superior oxidative stability than those prepared by conventional methods utilizing homogenizers. This study showed that microfluidic technology can be applied to soy protein emulsions as an effective tool for preparing droplets with uniform size and enhanced stability.

Simplified 3D hydrodynamic flow focusing for lab-on-chip single particle study

Accurately control of the position of a fluid and particle within lab-on-a-chip platform is a critical prerequisite for many downstream analysis processes, such as detection, trapping and separation, moving the sensing at the single-particle level. With the development of microfluidic fabrication technology, particle/cell focusing has shifted from two to three dimensions. 3D hydrodynamic focusing, which sorts and aligns the incoming cloud of particles so that they pass through the interrogation area one by one, enables new possibilities and breakthroughs in the single-cell analysis system. Despite the excellent results shown in literature, there is still a lack of a device that can simultaneously fulfilling the requirements of high throughput, compactness, high integrability, and ease of use operation to become a widely accepted work center for biomedical research and clinical applications. Here, we proposed a unique 3D flow focusing microfluidic device buried in fused silica substrate that potentially combines all this advantages. By designing a sample channel suspended inside a larger buffer channel, manufactured by exploiting the laser-assisted micromachine technique, a not size-dependent focusing capability is shown. A spatially and temporally stable central flow of a mixture of 15 μm and 6 μm PS particles to a 1 μm PS microsphere solution has been obtained with high accuracy. Finally, to test the achievable focusing resolution, the chip was tested for the detection of Escherichia Coli bacteria in water solution as proof of concept of biological application.

Machine learning enhanced droplet microfluidics

Machine learning has recently been introduced in the context of droplet microfluidics to simplify the process of droplet formation, which is usually controlled by a variety of parameters. However, the studies introduced so far have mainly focused on droplet size control using water and mineral oil in microfluidic devices fabricated using soft lithography or rapid prototyping. This approach negated the applicability of machine learning results to other types of fluids more relevant to biomedical applications, while also preventing users that do not have access to microfluidic fabrication facilities to take advantage of previous findings. There are a number of different algorithms that could be used as part of a data driven approach, and no clear comparison has been previously offered among multiple machine learning architectures with respect to the predictions of flow rate values and generation rate. We here employed machine learning to predict the experimental parameters required for droplet generation in three commercialized microfluidic flow-focusing devices using phosphate buffer saline and biocompatible fluorinated oil as dispersed and continuous liquid phases, respectively. We compared three different machine learning architectures and established the one leading to more accurate predictions. We also compared the predictions with a new set of experiments performed at a different day to account for experimental variability. Finally, we provided a proof of concept related to algae encapsulation and designed a simple app that can be used to generate accurate predictions for a given droplet size and generation rate across the three commercial devices.

Deep reinforcement learning-based digital twin for droplet microfluidics control

This study applied deep reinforcement learning (DRL) with the Proximal Policy Optimization (PPO) algorithm within a two-dimensional computational fluid dynamics (CFD) model to achieve closed-loop control in microfluidics. The objective was to achieve the desired droplet size with minimal variability in a microfluidic capillary flow-focusing device. An artificial neural network was utilized to map sensing signals (flow pressure and droplet size) to control actions (continuous phase inlet pressure). To validate the numerical model, simulation results were compared with experimental data, which demonstrated a good agreement with errors below 11%. The PPO algorithm effectively controlled droplet size across various targets (50, 60, 70, and 80 μm) with different levels of precision. The optimized DRL + CFD framework successfully achieved droplet size control within a coefficient of variation (CV%) below 5% for all targets, outperforming the case without control. Furthermore, the adaptability of the PPO agent to external disturbances was extensively evaluated. By subjecting the system to sinusoidal mechanical vibrations with frequencies ranging from 10 Hz to 10 KHz and amplitudes between 50 and 500 Pa, the PPO algorithm demonstrated efficacy in handling disturbances within limits, highlighting its robustness. Overall, this study showcased the implementation of the DRL+CFD framework for designing and investigating novel control algorithms, advancing the field of droplet microfluidics control research.

Modular assembly of microswimmers with liquid compartments.

Artificial microswimmers, i.e. colloidal scale objects capable of self-propulsion, have garnered significant attention due to their central role as models for out of equilibrium systems. Moreover, their potential applications in diverse fields such as biomedicine, environmental remediation, and materials science have long been hypothesized, often in conjunction with their ability to deliver cargoes to overcome mass transport limitations. A very efficient way to load molecular cargoes is to disperse them in a liquid compartment, however, fabricating microswimmers with multiple liquid compartments remains a significant challenge. To address this challenge, we present a modular fabrication platform that combines microfluidic synthesis and sequential capillarity-assisted particle assembly (sCAPA) for microswimmers with various liquid compartments. We demonstrate the synthesis of monodisperse, small polymer-based microcapsules (Ø = 3-6μm) with different liquid cargoes using a flow-focusing microfluidic device. By employing the sCAPA technique, we assemble multiple microcapsules into microswimmers with high precision, resulting in versatile microswimmers with multiple liquid compartments and programmable functionalities. Our work provides a flexible approach for the fabrication of modular microswimmers, which could potentially actively transport cargoes and release them on demand in the future.