Droplet-based Microfluidic Systems Research Articles

High-Throughput Absorbance-Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases.

Recent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy-intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet-based microfluidic systems have emerged as a powerful high-throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet-based microfluidic systems, we present an absorbance-activated droplet sorter that allows droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105-member aldehyde dehydrogenase library towards D-glyceraldehyde using a NADH mediated coupled assay that generates WST-1 formazan as the colorimetric product. We successfully identify a variant with a 51 % improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Simultaneous effects of capillary number, viscosity ratio, and contraction ratio on droplet dynamics in contraction microchannel

Abstract In droplet-based microfluidic systems, fluid properties, flow conditions, and channel geometry play crucial roles in the movement and manipulation of droplets. This study employs a three-dimensional numerical simulation method to investigate droplet dynamics in a contraction microchannel under the simultaneous influence of different factors, such as capillary number (Ca), viscosity ratio (λ), and contraction ratio (C). Specifically, a theoretical model is introduced for predicting the transition of droplets from trap to squeeze, and it is observed to be 〖Ca〗_I = f(C,λ). Meanwhile, droplet dynamics are investigated for the transition from squeeze to breakup for finding critical capillary number value (CaII). In addition, the critical viscosity ratio for the process from squeeze to breakup has been figured out by numerical results for a wide range of contraction ratios. Eventually, droplet dynamics including deformation, velocity ratio, and breakup during contraction microchannel are also quantitatively studied under the factors above.

Thermo-controlled microfluidic generation of monodisperse alginate microspheres based on external gelation.

Droplet-based microfluidic systems have received much attention as promising tools for fabricating monodisperse microspheres of alginate solutions with high accuracy and reproducibility. The immediate and simple ionotropic gelation of alginate, its biocompatibility, and its tunability of mechanical properties make it a favorable hydrogel in the biomedical and tissue engineering fields. In these fields, micron-sized alginate hydrogel spheres have shown high potential as cell vehicles and drug delivery systems. Although on-chip microfluidic gelation of the produced alginate droplets is common, several challenges remain. Complicated chemical and microfabrication processes are required, and the risk of microchannel clogging is high. In the current study, we present an easy-to-use microfluidic external gelation process to produce highly spherical and monodisperse microspheres from very low-concentrated alginate-RGD solution [0.5% (w/v)]. To accomplish this, gelatin, a thermo-sensitive and inexpensive biomaterial, was incorporated into the alginate solution as a sacrificial biomaterial that mediates the off-chip external gelation of the alginate with Ca2+, and avoids droplet coalescence. Utilizing the methodology mentioned above, we successfully generated monodisperse alginate microspheres (AMs) with diameters ranging from 27 μm to 46 μm, with a coefficient of variation of 0.14, from a mixture of Arg-Gly-Asp (RGD)-modified very low viscosity alginate and gelatin. These RGD-AMs were used as microcarriers for human umbilical vein endothelial cells. The described easy-to-use and cost-effective microfluidic off-chip external gelation strategy exhibits comparable advantages to on-chip external gelation and demonstrates superiority over the latter since clogging is impossible.

Droplet Microfluidics for High-Throughput Screening and Directed Evolution of Biomolecules.

Directed evolution is a powerful technique for creating biomolecules such as proteins and nucleic acids with tailor-made properties for therapeutic and industrial applications by mimicking the natural evolution processes in the laboratory. Droplet microfluidics improved classical directed evolution by enabling time-consuming and laborious steps in this iterative process to be performed within monodispersed droplets in a highly controlled and automated manner. Droplet microfluidic chips can generate, manipulate, and sort individual droplets at kilohertz rates in a user-defined microchannel geometry, allowing new strategies for high-throughput screening and evolution of biomolecules. In this review, we discuss directed evolution studies in which droplet-based microfluidic systems were used to screen and improve the functional properties of biomolecules. We provide a systematic overview of basic on-chip fluidic operations, including reagent mixing by merging continuous fluid streams and droplet pairs, reagent addition by picoinjection, droplet generation, droplet incubation in delay lines, chambers and hydrodynamic traps, and droplet sorting techniques. Various microfluidic strategies for directed evolution using single and multiple emulsions and biomimetic materials (giant lipid vesicles, microgels, and microcapsules) are highlighted. Completely cell-free microfluidic-assisted in vitro compartmentalization methods that eliminate the need to clone DNA into cells after each round of mutagenesis are also presented.

Simultaneous influence of contact angle, capillary number, and contraction ratio on droplet dynamics in hydrophobic microchannel

In droplet-based microfluidic systems, droplet motion and behavior largely depend on fluid properties, flow conditions, and microchannel geometry. This study presents a model to predict droplet dynamics within a contraction microchannel, considering various factors, such as contact angle (θ), capillary number (Ca), and contraction ratio (C), within defined ranges, through the employment of a three-dimensional numerical simulation method. Droplets may undergo trap, squeeze, or breakup as they traverse the contraction microchannel, contingent upon the aforementioned factors. The transition from trap to squeeze is determined by the critical value of capillary number ( CaI ). Within the squeezed regime, the contact angle influences droplet deformation and velocity within the contraction microchannel. However, when capillary number ( CaII ) in contraction microchannel increases to a specific threshold corresponding to the contraction ratio, the droplet no longer in contact with the wall and becomes independent of the contact angle, a phenomenon referred to as detachment. Moreover, the contact angle’s impact on the transition of droplets from squeeze to breakup is contingent upon the contraction ratio. This study provides a comprehensive overview of droplet behavior within microfluidic systems, offering valuable insights for the optimization and design of microsystems.

Advances in Single-Cell Techniques for Linking Phenotypes to Genotypes.

Single-cell analysis has become an essential tool in modern biological research, providing unprecedented insights into cellular behavior and heterogeneity. By examining individual cells, this approach surpasses conventional population-based methods, revealing critical variations in cellular states, responses to environmental cues, and molecular signatures. In the context of cancer, with its diverse cell populations, single-cell analysis is critical for investigating tumor evolution, metastasis, and therapy resistance. Understanding the phenotype-genotype relationship at the single-cell level is crucial for deciphering the molecular mechanisms driving tumor development and progression. This review highlights innovative strategies for selective cell isolation based on desired phenotypes, including robotic aspiration, laser detachment, microraft arrays, optical traps, and droplet-based microfluidic systems. These advanced tools facilitate high-throughput single-cell phenotypic analysis and sorting, enabling the identification and characterization of specific cell subsets, thereby advancing therapeutic innovations in cancer and other diseases.

Investigating the effect of MnCl2 salt concentration on the droplet size and stability in a microfluidics droplets generation system

In the realm of droplet-based microfluidic systems, achieving optimal droplet size, shape, and distribution is crucial for efficient mass transfer and reaction performance. Traditionally, surfactants have been employed to modify interfacial properties and control droplet characteristics; however, they can impact reaction environments. A novel approach gaining traction is the creation of surfactant-free droplets, where additives are minimized to preserve the liquids' inherent properties. This study aims to investigate the creation of surfactant-free emulsions using divalent ions derived from Manganese(II) chloride (MnCl2) solutions. Emulsions play a vital role in various industries, but their stability often relies on surfactants. To address this, the research examines the influence of MnCl2 concentrations (ranging from 0.2 M to 1.0 M) on droplet dynamics within an oil phase using microfluidic analysis. Notably, a distinctive non-linear correlation between ion concentration and droplet size emerges, challenging conventional assumptions. This observation highlights the intricate and nuanced interfacial interactions governing surfactant-free emulsions, showcasing potential opportunities for designing stable emulsions without traditional surfactants. For instance, droplet sizes decreased from 171 µm to 138 µm as MnCl2 concentration increased from 0.2 M to 0.4 M, demonstrating this non-linear trend. However, further increases in MnCl2 concentration to 0.8 M and 1.0 M resulted in minimal changes in droplet size.

Effect of entry geometry on droplet dynamics in contraction microchannel

In droplet-based microfluidic systems, the movement and control of liquid droplets are primarily governed by microchannel geometry. The aim of this study is to examine droplet dynamics in contraction microchannels by using three-dimensional simulation and theoretical modeling. This work specifically describes three regimes of the droplet dynamics, including the trap, squeeze, and breakup regimes, and investigates the effects of capillary number (Ca), entry angle (α), and contraction channel ratio (C). Additionally, a theoretical model is proposed to describe the transition between squeeze and trap regimes, which depends on the entry angle. The critical value of capillary number (Ca1c) for this transition is observed to be Ca1c=a(CM−b/α), where a and b are fitted parameters. Meanwhile, the entry angle is found to have no influence on the transition from squeeze to breakup regime. The droplet deformations, retraction, and/or breakup position are quantitatively investigated for a wide range of capillary number and entry angle. The aforementioned findings would provide valuable recommendations for designing contraction micro channels.

Microfluidic platform using focused ultrasound passing through hydrophobic meshes towards automatic biological experiment

Laboratory automation is critical in improving productivity and data quality. Droplet-based microfluidic systems offer a solution that enables parallel handling of small samples with high reconfigurability and scalability. The most common technique is electrowetting-on-dielectric (EWOD), which manipulates droplets by exploiting the imbalance of wetting. However, this method is often limited by biofouling. In our previous study, we proposed a novel microfluidic platform using focused ultrasound passing through a hydrophobic mesh, which reduces the contact area and thus the hydrophobic interactions and electrostatic attractions of biomolecules. Our platform demonstrates the manipulation of protein-rich droplets at concentrations up to 1 mg/ml without the need for any additives. This is a significant improvement over existing EWOD methods which are limited to handling samples with protein concentrations as low as 0.005 mg/ml without additives. Here, we further investigate the influence of protein concentration, pH, and buffers using fluorescence microscopy to determine the effect on the system. Our platform also provides basic functions such as moving, merging, and splitting, as well as superior jumping capabilities. With reduced biofouling and the ability to directly transfer droplets to adjacent devices and multi-step processes, our platform opens up new possibilities for the automation of biology.

Textured surfaces for oil droplet transport

Droplet-based microfluidic systems bring the advantage of handling samples in discrete forms and these systems can be built as a surface-based platform, eliminating the need of a carrier fluid. Droplet transport on surfaces can be achieved by creating local energy gradients by using electrowetting, thermocapillary forces, chemical gradients and surface texture. These methods are developed for aqueous droplets; while controlled transportation of oil droplets remains as a challenge due to their low surface tension. One of the major challenges in achieving controlled oil droplet manipulation is to obtain oil repellent -oleophobic- surfaces. In this work, pillar arrays with mushroom shaped profiles were designed, fabricated, and tested to study oil wetting on textured surfaces. Contact angles higher than 170° were achieved for hexadecane droplets in Fakir state. Based on these findings, surface texture ratchet tracks that are composed of an array of arc shaped pillars were built to generate local energy gradients to manipulate oil droplets in a continuous fashion. Motion of hexadecane droplets at a speed of 7 mm/s was achieved. The results of this study are crucial for applications of oil droplet transport such as in biochemistry, smart surface development, wearable device design as well as microsystem packaging.

Multiparametric, Label-Free Analysis of Microfluidic Droplets via a Dynamic Phase Grating Approach.

Droplet-based microfluidic systems possess many fundamental advantages as a platform for the analysis of chemical and biological species. However, whereas on-chip operations have rapidly developed over the past decades, approaches for analyzing target molecules within droplets have largely remained limited to methods requiring bulky and expensive instrumentation. In this work, we describe a droplet analysis approach whereby the droplet train itself is the sensing construct. Specifically, the droplet train is interrogated as a transmission phase grating, allowing high-throughput, label-free, solution-phase, and multi-parametric analysis of droplet contents. Importantly, three distinct properties of generated droplets can be simultaneously extracted using this conceptually simple and experimentally straightforward measurement approach. Under constant droplet generation conditions, measurement of droplet viscosity is achieved by monitoring changes in zero order to first order peak separation in the far-field diffraction pattern, with a sensitivity of 2.28 × 10-4 cSt per μm change in peak separation. In parallel, measurement of droplet refractive index (RI) is achieved by measuring changes in the ratio of the zero order to first order peak intensity, with a sensitivity of 2.14 × 10-4 RI units per unit change in a diffracted peak intensity ratio. Finally, droplet generation frequency is determined from the time-varying oscillation of the peak height ratio, yielding comparable results to an expensive high-speed camera commonly used for droplet imaging. Importantly, the experimental strategy for this approach is straightforward and does not require expensive instrumentation; therefore, it may find utility in affordable and portable analysis approaches applied to diverse droplet microfluidic assays.

Regulation of droplet size and flow regime by geometrical confinement in a microfluidic flow-focusing device

We have developed a coupled level set and volume of fluid-based computational fluid dynamics model to analyze the droplet formation mechanism in a square flow-focusing microchannel. We demonstrate a flexible manipulation of droplet formation and flow regime based on the modified flow-focusing microchannel with a constricted orifice. Furthermore, we have systematically studied the influence of geometrical confinement, flow rate, and interfacial tension on the droplet formation regime, length, volume, velocity, and shape. Three different flow regimes, namely squeezing, dripping, and jetting, are observed, and the flow regime maps are formulated based on the Reynolds and capillary numbers. After an extensive numerical investigation, we described the boundaries between the different regimes. Droplet shape is also quantified based on the deformation index value. Plug-shaped droplets are observed in the squeezing regime, and near spherical droplets are found in the dripping and jetting regimes. Our study provides insights into the transition of a regime under various geometrical confinement and fluid properties. The results reveal that the modified flow-focusing microchannel can substantially enhance dripping while decreasing the squeezing regime, which is of paramount importance from the standpoint of producing high throughput stable and monodisperse microdroplets. Eventually, this work emphasizes the importance of geometrical confinement, fluid properties, and flow conditions on the droplet formation process in a flow-focusing microchannel that can effectively provide helpful guidelines on the design and operations of such droplet-based microfluidic systems.

Droplet digital microfluidic system for screening filamentous fungi based on enzymatic activity

Fungal cell-wall-degrading enzymes have great utility in the agricultural and food industries. These cell-wall-degrading enzymes are known to have functions that can help defend against pathogenic organisms. The existing methods used to discover these enzymes are not well adapted to fungi culture and morphology, which prevents the proper evaluation of these enzymes. We report the first droplet-based microfluidic method capable of long-term incubation and low-voltage conditions to sort filamentous fungi inside nanoliter-sized droplets. The new method was characterized and validated in solid-phase media based on colloidal chitin such that the incubation of single spores in droplets was possible over multiple days (2–4 days) and could be sorted without droplet breakage. With long-term culture, we examined the activity of cell-wall-degrading enzymes produced by fungi during solid-state droplet fermentation using three highly sensitive fluorescein-based substrates. We also used the low-voltage droplet sorter to select clones with highly active cell-wall-degrading enzymes, such as chitinases, β-glucanases, and β-N-acetylgalactosaminidases, from a filamentous fungi droplet library that had been incubated for >4 days. The new system is portable, affordable for any laboratory, and user-friendly compared to classical droplet-based microfluidic systems. We propose that this system will be useful for the growing number of scientists interested in fungal microbiology who are seeking high-throughput methods to incubate and sort a large library of fungal cells.

Single-cell sorting using integrated pneumatic valve droplet microfluidic chip

Single-cell analysis and sorting have been increasingly prevalent in scientific study and industry in recent years. Droplet-based microfluidic systems are significant tools for single cell separation due to the advantages of high-throughput screening flux and relatively simple chip manufacturing. Herein, this work integrated single-cell droplet generation, incubation, and sorting functions into a single microfluidic chip using pneumatic microvalve for single-cell screening of hybridoma cells. The chip realized the generation, storage, incubation, and assay sorting of droplets in order by adjusting the switch of the microvalve. Numerical methods were used to simulate the effects of membrane size and pressure on valve deformation. Incubation results showed that fluorescent signals were generated in the droplets containing both hybridoma cells and microspheres under the control of pneumatic microvalves. In addition, the purity of target droplets was increased from 4.87% before sorting to 89.70% after sorting using fluorescence-activated droplet sorting platform, demonstrating the feasibility of the integrated chip with a pneumatic valve for single-cell screening. The integrated chip is proved to be operated efficiently with minimum cell loss and human labor through reducing the risk of droplet fusion and facilitating the development of automated devices. So, the present study provides an integrated design for microdroplet-based single-cell sorting with various applications, such as single-cell sequencing, antibody screening, and directed evolutionary studies.

Artificial neural network-based predictions of surface electrocoalescence of water droplets in hydrocarbon media

Electrowetting (EW) is a widely-studied microfluidic technique to promote coalescence of droplets and also to generate droplets. However, analytical or computational modeling of the intricate phenomena associated with electrocoalescence of water droplets in hydrocarbon media is very challenging. Presently, EW-assisted surface electrocoalescence experiments are used to train machine learning (ML) algorithms. These comprise an artificial neural network (ANN), eXtreme gradient boosting (XGBoost) and polynomial regression. These models are then used to predict the influence of parameters such as applied voltage, frequency, electrode spacing, concentration and initial droplet density (normalized with uncovered area ratio (δi/αi)), to predict nine targets: uncovered area ratio (αf), final droplet density normalized with uncovered area (δf/αf), and seven droplet density distribution (radius) bins. ANN was the most accurate predictive tool among the three ML models with R2 of 0.89. ANN accurately predicted the droplet distribution bins for three distinct cases of good electrocoalescence, poor electrocoalescence and satellite droplet ejection (droplet generation). SHAP (Shapley Additive exPlanations) dependence plots were used to quantify the parametric influence of various parameters on each output. For δf/αf, frequency and electrode spacing were the most and least influential, respectively. Interestingly, the feature influence on droplet density distributions was observed to reverse (magnitude and direction) with droplet radius. The key novelty of this study is the coupling of data from a multifunctional microfluidic device to an ANN model to accurately predict complex stochastic droplet-related phenomena (coalescence, generation). As such, the framework developed here can be utilized for other data-rich droplet-based microfluidic systems. δf/αf

Flexible on-chip droplet generation, switching and splitting via controllable hydrodynamics

Flexible droplet preparation and manipulation are significant for lots applications such as immunoreaction and monocellular culture. Herein, we present a novel method for effective on-chip droplet generation, splitting and switching via controllable hydrodynamics. The microchannel of the designed chip has 6 inlets and 3 three outlets. The water solution is injected from a specific inlet (inlet d), and the other 5 inlets are used to inject oil fluids. Under the shearing effect of immiscible oils, the water phase breaks into dispersed droplets first, and the generated droplets can be further split into daughter drops or switched into side outlets from the middle outlet. To investigate the hydrodynamic droplet manipulation behaviors, a two-dimensional simulation model based on phase-field method is established. Utilizing the computational model, we systematically analyze the influences of the flow rates of continuous and dispersed fluids and the manipulation modes on droplet generation, splitting and switching. The numerical results indicate that the droplets can be generated with controlled sizes. For instance, at Qd = 5 μL/min and Qc1,2 = 5 μL/min, the droplet diameter decreases from 89.2 μm to 49.2 μm as Qs1,2 gradually rises from 15 μL/min to 40 μL/min. Moreover, the prepared droplets can realize on-demand splitting and switching. When Qd, Qc1,2, and Qs1,2 are fixed at 5 μL/min, 5 μL/min and 25 μL/min, respectively, the generated droplet is split into different proportional daughter drops with the rising of Qs3 (or Qs4) at first, and finally it is switched into the side-outlets when Qs3 (or Qs4) is higher than 80 μL/min. Therefore, this proposed droplet manipulation approach will be promising for various applications, and the numerical simulations can provide useful guidelines on the design and operation of droplet-based microfluidic systems.

Formation and Parallel Manipulation of Gradient Droplets on a Self-Partitioning SlipChip for Phenotypic Antimicrobial Susceptibility Testing

Flexible, robust, and user-friendly screening systems with a large dynamic range are highly desired in scientific research, industrial development, and clinical diagnostics. Droplet-based microfluidic systems with gradient concentrations of chemicals have been demonstrated as promising tools to provide confined microenvironments for screening tests with small reaction volumes. However, the generation and manipulation of gradient droplets, such as droplet merging, generally require sophisticated fluidic manipulation systems, potentially limiting their application in decentralized settings. We present a gradient-droplet SlipChip (gd-SlipChip) microfluidic device that enables instrument-free gradient droplet formation and parallel manipulation. The device can establish a gradient profile by free interfacial diffusion in a continuous fluidic channel. With a simple slipping step, gradient droplets can be generated by a surface tension-driven self-partitioning process. Additional reagents can be introduced in parallel to these gradient droplets with further slipping operations to initiate screening tests of the droplets over a large concentration range. To profile the concentration in the gradient droplets, we establish a numerical simulation model and verify it with hydrogen chloride (HCl) diffusion, as tested with a dual-color pH indicator (methyl orange and aniline blue). As a proof of concept, we tested this system with a gradient concentration of nitrofurantoin for the phenotypic antimicrobial susceptibility testing (AST) of Escherichia coli. The results of our gd-SlipChip-based AST on both reference and clinical strains of E. coli can be indicated by the bacterial growth profile within 3 h and are consistent with the clinical culture-based AST.

Generation and Screening of Antigen-Specific Nanobodies from Mammalian Cells Expressing the BCR Repertoire Library Using Droplet-Based Microfluidics.

Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.

7-Aminocoumarin-4-acetic Acid as a Fluorescent Probe for Detecting Bacterial Dipeptidyl Peptidase Activities in Water-in-Oil Droplets and in Bulk.

Droplet-based microfluidic systems are a powerful tool for biological assays with high throughput. Water-in-oil droplets (WODLs) are typically used in droplet-based microfluidic systems to culture microorganisms and perform enzyme assays. However, because of the oil surrounding the nanoliter and picoliter volumes of WODLs, availability of suitable substrates is limited. For instance, although 7-amino-4-methylcoumarin (AMC) is commonly used as a fluorescent probe of the substrate to detect peptidase activity, AMC leaks from WODLs to the oil phase due to its high hydrophobicity. Thus, AMC substrates cannot be used in droplet-based microfluidic systems with WODLs. In this study, we developed a peptidase substrate consisting of a dipeptide and 7-aminocoumarin-4-acetic acid (ACA), an AMC-derived fluorogenic compound. ACA was retained in the WODL for more than 7 days, and the dipeptidyl ACA substrate detected dipeptidyl peptidase (DPP) activity in the WODL. Compared to AMC substrates, the substrate specificity constants of DPPs for ACA substrates increased up to 4.7-fold. Fluorescence-activated droplet sorting made high-throughput screening of microorganisms based on DPP activity using the dipeptidyl ACA substrate possible. Since ACA could be applied to various substrates as a fluorescent probe, detectable microbial enzyme activities for droplet-based microfluidic systems can be largely expanded.

A numerical study of droplet splitting in branched T-shaped microchannel using the two-phase level-set method

Droplet splitting as a significant feature of droplet-based microfluidic systems has been widely employed in biotechnology, biomedical engineering, tissue engineering, and it has been preferred over continuous flow systems. In the present paper, two-dimensional numerical simulations have been done to examine the asymmetrical droplet splitting process. The two-phase level set method (LSM) has been predicted to analyze the mechanism of droplet formation and droplet splitting in immiscible liquid/liquid two-phase flow in the branched T-junction microchannel. Governing equations on flow field have been discretized and solved using finite element-based COMSOL Multiphysics software (version 5.3a). Obtained numerical results were validated by experimental data reported in the literature which show acceptable agreement. The model was developed to simulate the mechanism of droplet splitting at the branched T-junction microchannel. This study provides a passive technique to asymmetrically split up microdroplets at the downstream T-junctions. The results show that outlet branches’ pressure gradient affects the droplet splitting. Specifically, it has been shown that the splitting ratio increases by increasing the length ratio, and equal droplet splitting can be achieved where the ratio is LL/ Lu = 1. We have used two outlet branches having the same width but different lengths to create the required pressure gradient. As the length ratio of the outlet branches increases, the diameter ratio increases as well.