Droplet-based Microfluidics Research Articles

Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface.

Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG4 linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.

On-Chip Fabrication of Colloidal Suprastructures by Assembly and Supramolecular Interlinking of Microgels.

In this report, a versatile method is demonstrated to create colloidal suprastructures by assembly and supramolecular interlinking of microgels using droplet-based microfluidics. The behavior of the microgels is systematically investigated to evaluate the influence of their concentration on their distribution between the continuous, the droplet phase, and the interface. At low concentrations, microgels are mainly localized at the water-oil interface whereas an excess of microgels results, following the complete coverage of the water-oil interface, in their distribution in the continuous phase. To stabilize the colloidal suprastructure, on-chip gelation is introduced by adding natural polyphenol tannic acid (TA) in the water phase. TA forms interparticle linking between the poly(N-vinylcaprolactam) (PVCL) microgels by supramolecular interactions. The combination of supramolecular interlinking with the variation of the microgel concentration in microfluidic droplets enables on-chip fabrication of defined colloidal suprastructures with morphologies ranging from colloidosomes to colloidal supraballs. The obtained supracolloidal structures exhibit a pH-responsive behavior with a disintegration at alkaline conditions within a scale of seconds. The destabilization process results from the deprotonation of phenolic groups and destruction of hydrogen bonds with PVCL chains at higher pH.

3D free-assembly modular microfluidics inspired by movable type printing

Reconfigurable modular microfluidics presents an opportunity for flexibly constructing prototypes of advanced microfluidic systems. Nevertheless, the strategy of directly integrating modules cannot easily fulfill the requirements of common applications, e.g., the incorporation of materials with biochemical compatibility and optical transparency and the execution of small batch production of disposable chips for laboratory trials and initial tests. Here, we propose a manufacturing scheme inspired by the movable type printing technique to realize 3D free-assembly modular microfluidics. Double-layer 3D microfluidic structures can be produced by replicating the assembled molds. A library of modularized molds is presented for flow control, droplet generation and manipulation and cell trapping and coculture. In addition, a variety of modularized attachments, including valves, light sources and microscopic cameras, have been developed with the capability to be mounted onto chips on demand. Microfluidic systems, including those for concentration gradient generation, droplet-based microfluidics, cell trapping and drug screening, are demonstrated. This scheme enables rapid prototyping of microfluidic systems and construction of on-chip research platforms, with the intent of achieving high efficiency of proof-of-concept tests and small batch manufacturing.

Droplet-Based Microfluidics as a Platform to Design Food-Grade Delivery Systems Based on the Entrapped Compound Type.

Microfluidic technology has emerged as a powerful tool for several applications, including chemistry, physics, biology, and engineering. Due to the laminar regime, droplet-based microfluidics enable the development of diverse delivery systems based on food-grade emulsions, such as multiple emulsions, microgels, microcapsules, solid lipid microparticles, and giant liposomes. Additionally, by precisely manipulating fluids on the low-energy-demand micrometer scale, it becomes possible to control the size, shape, and dispersity of generated droplets, which makes microfluidic emulsification an excellent approach for tailoring delivery system properties based on the nature of the entrapped compounds. Thus, this review points out the most current advances in droplet-based microfluidic processes, which successfully use food-grade emulsions to develop simple and complex delivery systems. In this context, we summarized the principles of droplet-based microfluidics, introducing the most common microdevice geometries, the materials used in the manufacture, and the forces involved in the different droplet-generation processes into the microchannels. Subsequently, the encapsulated compound type, classified as lipophilic or hydrophilic functional compounds, was used as a starting point to present current advances in delivery systems using food-grade emulsions and their assembly using microfluidic technologies. Finally, we discuss the limitations and perspectives of scale-up in droplet-based microfluidic approaches, including the challenges that have limited the transition of microfluidic processes from the lab-scale to the industrial-scale.

Droplet dynamics in asymmetric microfluidic junctions

Droplet-based microfluidics has received increasing interest over the last two decades, due to its extensive applications in drug delivery, cell encapsulation, protein crystallization, biochemical synthesis and high-throughput screening. As one of the key units in the microfluidic channels, asymmetric microfluidic junctions provide more opportunities to control and organize the droplets with different sizes, however, few studies have been reported to explore the effect of the junction angle on droplet behaviour in the asymmetric junctions. This work presents a numerical investigation of the droplet behaviour in asymmetric microfluidic junctions. The effect of the junction angle on the droplet breakup is explored in detail. Droplet breakup and non-breakup are found in asymmetric junctions. In the regime of the asymmetric breakup, the volume of the daughter droplet in the uphill branching channel is always larger than that in the downhill branching channel. Interestingly, in the regime of non-breakup, droplets are sorted into the uphill or downhill branching channels, suggesting that this asymmetric junction could be used for passive droplet sorting. The physical mechanisms behind droplet breakup and sorting are explored. Both the junction angle and the initial tip-to-tip length of the droplet play essential roles in the droplet behaviour in the asymmetric junctions. A phase diagram of the droplet behaviour is presented in this work. A scaling model is proposed to predict the transition between the breakup and non-breakup of the droplets in the asymmetric junctions.

On-demand delivery of therapeutic extracellular vesicles by encapsulating in monodispersed photodegradable hydrogel microparticles using a droplet microfluidic device

Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been studied for therapeutic applications due to easier endocytosis and fewer adverse effects than MSCs. However, EVs injected medicinally are diluted by body fluids and inefficiently delivered to the therapeutic target, limiting original therapeutic effects. Therefore, biocompatible carriers such as hydrogels are needed in EVs delivery systems to enhance EVs retention and release EVs sustainably at therapeutic targets. In addition to the advantages of delivering EVs using hydrogels, photodegradable hydrogels provide a controllable degradation process via ultraviolet (UV) irradiation. However, long-term UV irradiation to degrade the photodegradable hydrogel may potentially damage EVs and targets; therefore, a carrier that can be degraded with shorter UV irradiation is required. Herein, we generate photodegradable hydrogel microparticles (PHMPs) that can release encapsulated EVs sustainably within 2 min of UV irradiation by droplet-based microfluidics. By regulating the fluidic resistance of the channels, monodisperse PHMPs, which can avoid undesired variations in EVs release kinetics, were generated. EVs were successfully retained in PHMPs in the absence of external stimuli, and EVs were released from PHMPs and enhanced cell proliferation following UV irradiation. Therefore, PHMPs can overcome the limitations of conventional EVs delivery, including their inability to deliver EVs sustainably and on-demand, and low EVs retention.

Compartmentalized Polyampholyte Microgels by Depletion Flocculation and Coacervation of Nanogels in Emulsion Droplets.

In pH-responsive drug carriers, the distribution of charges has been proven to affect delivery efficiency but is difficult to control and verify. Herein, we fabricate polyampholyte nanogel-in-microgel colloids (NiM-C) and show that the arrangement of the nanogels (NG) can easily be manipulated by adapting synthesis conditions. Positively and negatively charged pH-responsive NG are synthesized by precipitation polymerization and labelled with different fluorescent dyes. The obtained NG are integrated into microgel (MG) networks by subsequent inverse emulsion polymerization in droplet-based microfluidics. By confocal laser scanning microscopy (CLSM), we verify that depending on NG concentration, pH value and ionic strength, NiM-C with different NG arrangements are obtained, including Janus-like phase-separation of NG, statistical distribution of NG, and core-shell arrangements. Our approach is a major step towards uptake and release of oppositely charged (drug) molecules.

Three-Dimensional Printing Enabled Droplet Microfluidic Device for Real-Time Monitoring of Single-Cell Viability and Blebbing Activity.

Droplet-based microfluidics with the characteristics of high throughput, low sample consumption, increasing reaction speed, and homogeneous volume control have been demonstrated as a useful platform for biomedical research and applications. The traditional fabrication methods of droplet microfluidics largely rely on expensive instruments, sophisticated operations, and even the requirement of an ultraclean room. In this manuscript, we present a 3D printing-based droplet microfluidic system with a specifically designed microstructure for droplet generation aimed at developing a more accessible and cost-effective method. The performance of droplet generation and the encapsulation capacity of the setup were examined. The device was further applied to measure the variation in cell viability over time and monitor the cell's blebbing activity to investigate its potential ability and feasibility for single-cell analysis. The result demonstrated that the produced droplets remained stable enough to enable the long-time detection of cell viability. Additionally, cell membrane protrusions featuring the life cycle of bleb initiation, expansion, and retraction can be well-observed. Three-dimensional printing-based droplet microfluidics benefit from the ease of manufacture, which is expected to simplify the fabrication of microfluidics and expand the application of the droplet approach in biomedical fields.

Design and Validation of a Droplet-based Microfluidic System To Study Non-Photochemical Laser-Induced Nucleation of Potassium Chloride Solutions.

Non-photochemical laser-induced nucleation (NPLIN) has emerged as a promising primary nucleation control technique offering spatiotemporal control over crystallization with potential for polymorph control. So far, NPLIN was mostly investigated in milliliter vials, through laborious manual counting of the crystallized vials by visual inspection. Microfluidics represents an alternative to acquiring automated and statistically reliable data. Thus we designed a droplet-based microfluidic platform capable of identifying the droplets with crystals emerging upon Nd:YAG laser irradiation using the deep learning method. In our experiments, we used supersaturated solutions of KCl in water, and the effect of laser intensity, wavelength (1064, 532, and 355 nm), solution supersaturation (S), solution filtration, and intentional doping with nanoparticles on the nucleation probability is quantified and compared to control cooling crystallization experiments. Ability of dielectric polarization and the nanoparticle heating mechanisms proposed for NPLIN to explain the acquired results is tested. Solutions with lower supersaturation (S = 1.05) exhibit significantly higher NPLIN probabilities than those in the control experiments for all laser wavelengths above a threshold intensity (50 MW/cm2). At higher supersaturation studied (S = 1.10), irradiation was already effective at lower laser intensities (10 MW/cm2). No significant wavelength effect was observed besides irradiation with 355 nm light at higher laser intensities (≥50 MW/cm2). Solution filtration and intentional doping experiments showed that nanoimpurities might play a significant role in explaining NPLIN phenomena.

Droplet microfluidic technologies for next-generation high-throughput screening

Droplet microfluidics has evolved into a promising platform for high-throughput screening (HTS), allowing for rapid and precise analysis of thousands of samples encapsulated within droplets. Droplet microfluidic platform offers versatility, high-throughput, and the ability to compartmentalize reactions for a wide range of applications including pharmaceutics, cell analysis, and combinatorial chemical analysis. While droplet-based microfluidics has made considerable advances in automating basic laboratory tasks, for instance, manipulation, storage, and analysis, there has been comparatively little advancement toward HTS applications. The complexity of the technology, the lack of standardization, and the challenges associated with screening large numbers of samples are all factors that have contributed to the limited adoption of droplet-based microfluidics in HTS. In this perspective, we provide a comprehensive overview of the progress of droplet microfluidics as a potential platform for next-generation HTS, specifically in the domain of droplet separation and library generation. We hope that this perspective will inspire further research in relevant academic fields and contribute to the development of innovative HTS strategies based on droplet microfluidic technologies.

Droplet-Based Microfluidics: Applications in Pharmaceuticals.

Droplet-based microfluidics offer great opportunities for applications in various fields, such as diagnostics, food sciences, and drug discovery. A droplet provides an isolated environment for performing a single reaction within a microscale-volume sample, allowing for a fast reaction with a high sensitivity, high throughput, and low risk of cross-contamination. Owing to several remarkable features, droplet-based microfluidic techniques have been intensively studied. In this review, we discuss the impact of droplet microfluidics, particularly focusing on drug screening and development. In addition, we surveyed various methods of device fabrication and droplet generation/manipulation. We further highlight some promising studies covering drug synthesis and delivery that were updated within the last 5 years. This review provides researchers with a quick guide that includes the most up-to-date and relevant information on the latest scientific findings on the development of droplet-based microfluidics in the pharmaceutical field.

Microgels as Platforms for Antibody-Mediated Cytokine Scavenging.

Therapeutic antibodies are the key treatment option for various cytokine-mediated diseases, such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. However, systemic injection of these antibodies can cause side effects and suppress the immune system. Moreover, clearance of therapeutic antibodies from the blood is limiting their efficacy. Here, water-swollen microgels are produced with a size of 25µm using droplet-based microfluidics. The microgels are functionalized with TNFα antibodies to locally scavenge the pro-inflammatory cytokine TNFα. Homogeneous distribution of TNFα-antibodies is shown throughout the microgel network and demonstrates specific antibody-antigen binding using confocal microscopy and FLIM-FRET measurements. Due to the large internal accessibility of the microgel network, its capacity to bind TNFα is extremely high. At a TNFα concentration of 2.5µgmL-1 , the microgels are able to scavenge 88% of the cytokine. Cell culture experiments reveal the therapeutic potential of these microgels by protecting HT29 colorectal adenocarcinoma cells from TNFα toxicity and resulting in a significant reduction of COX II and IL8 production of the cells. When the microgels are incubated with stimulated human macrophages, to mimic the in vivo situation of inflammatory bowel disease, the microgels scavenge almost all TNFα that is produced by the cells.

Refractory colorectal cancer patient derived MicroOrganoSpheres (MOS) to enable correlation of targeted therapy combination response with clinical outcomes.

e15574 Background: Metastatic colorectal cancer (CRC) patients who have become resistant to standard-of-care (SOC) treatments are often put on targeted therapies, including experimental drugs still in clinical trials. However, patients with targeted mutations still often do not respond to the targeted therapies alone, hence it remains a critically unmet need to explore potential combinatorial regimens that will enhance the efficacy. Methods: We performed a high-throughput screen using MicroOrganoSpheres (MOS) derived from metastatic refractory patients to correlate with clinical outcomes and explore alternative combinations that might benefit the patients. Biopsies from metastatic CRC patients who became resistant to SOC and were about to receive targeted therapies in clinical trials were molecularly profiled and implanted into immunodeficient mice to generate patient-derived xenografts (PDX). Results: MOS were derived and established from PDX using droplet-based microfluidics for high-throughput 3D screening. These MOS-based refractory patient avatars contain either targetable mutations including KRAS G12C and BRAF 600E or no such mutations. Response of the MOS to dose-titrated single and combination drug treatments including the available targeted therapies were measured by a fully automated robotic dispensing and imaging pipeline and quantified by AI-based live imaging analytical algorithm to generate robust drug response curves, which were used to derive IC50 and EC50. We tested both FDA-approved and experimental targeted therapies as single agent or in combination in 5-dose titrations. Longitudinal tracking of each MOS with Live/dead fluorescence dye signals was obtained for 5 days. MOS drug response curves and IC50s show correlation with clinical outcomes. This comprehensive dataset enabled us to assess the potential benefit of different combinations, help deconvolute the efficacy and synergy of individual drugs, and provide insight into patients who fail to response to targeted therapies despite possessing the targeted mutations. As the MOS diagnostic assay can be completed from a patient biopsy within 10 days and is currently in a registered, multi-site clinical trial (ClinicalTrials.gov # NCT05189171), this study provides proof-of-principle for a potential clinical MOS-based assay to guide refractory patients to the optimal combination regimen containing targeted therapy. Conclusions: Refractory patients may benefit from a MOS-based high-throughput screen assay to select the optimal combination regimen containing targeted therapy.

Bioinspired Wire-on-Pillar Magneto-Responsive Superhydrophobic Arrays

Versatile surfaces demonstrating multiple interfacial functionalities are highly demanded as a surface typically serves various duties and faces multiple challenges in real practice. However, such versatile surfaces are rarely reported mainly due to the challenges in integrating multiple structural characteristics. Here, by mimicking lotus leaves, butterfly wing, and respiratory cilia, we develop a surface termed wire-on-pillar magneto-responsive superhydrophobic arrays (WP-MRSA), which possess interfacial properties of structural superhydrophobicity, anisotropicity, stimuli responsiveness, and flexibility. By combining soft lithography and self-alignment of iron-laden aerosols under a magnetic field, iron-laden wires are planted atop prefabricated pillar arrays, resulting in well-ordered, sparse, high-aspect-ratio, flexible, and superhydrophobic wires, which largely deflect in response to a magnetic field. This unique integration of structural properties and configurations enables various functionalities, such as on-demand control of droplet impact dynamics, real-time regulation of surface lateral adhesion force, fast removal and sorting of objects, and precise manipulation of droplets for selective reactions. Those functionalities benefit various applications especially droplet-based microfluidics and active self-cleaning surfaces.

Hundred-micron droplet ejection by focused ultrasound for genomic applications

Droplet-based microfluidic (DMF) technology is widely used in genetic analysis. Among multiple droplet generation techniques, acoustic droplet ejection (ADE) is a non-contact, high-precision method that utilizes the energy of focused ultrasound. The ejection of a single droplet of controlled size, the performance of multi-droplet ejections and the effects of the ultrasound energy on nucleic acid molecules in the droplets, are the main research interests in droplet-based genomics. In this work, a focused ultrasonic transducer with a frequency of 20 MHz was used, and we achieved droplet ejection successfully. Various experiments were carried out to study ADE and the beneficial results were obtained, including the characterization of ADE conditions, the relationship between droplet volume and parameters (including power, duration and distance), the energy required for ADE, and the effects of ultrasound energy on DNA strands, etc. It was first noticed that the superior limit of the droplet size could be increased by adjusting the height of the transducer. In addition, multi-droplet ejections at the optimal delay time were realized with good linearity. Above all, we demonstrated that the nucleic acid molecules remained their integrity when the exciting power was the maximum and the exciting duration was the longest in our system. In summary, ADE was proved to have an important role and a vast prospect in DMF technology for genomics-related applications.

Application of Droplet-Based Microfluidics in Microbial Research

Droplet-based microfluidics is a technology that generates and manipulates highly uniform droplets, ranging from picoliter to nanoliter droplets, in microchannels under precise control. In biological research, each droplet can be used to encapsulate a small group of cells or even a single cell, and then serve as an individual container for biochemical reaction, which is well suited for high-throughput and high-resolution biochemical analysis. In the field of microbial research, from cultivation and identification of microbes to the investigation of the spatiotemporal dynamics of microbial communities, from precise quantitation of microbiota to systematic study of microbial interactions, and from the isolation of rare and unculturable microbes to the development of genetically engineered strains, droplet microfluidic technology has played an important promotional role in all these aspects. Droplet microfluidics shows potential for becoming a basic tool for exploring single-cell microbes in microbiological research. In this review, we gave a brief overview of the technical basis of droplet microfluidics. Then, we presented its latest applications in microbial research and had some discussions, aiming to provide a reference for relevant research on microorganisms.

Role and Effect of Meso-Structuring Surfactants on Properties and Formation Mechanism of Microfluidic-Enabled Mesoporous Silica Microspheres.

We have shown in a previous work that the combination of the emulsion solvent evaporation technique and droplet-based microfluidics allows for the synthesis of well-defined monodisperse mesoporous silica microcapsules (hollow microspheres), whose size, shape and composition may be finely and easily controlled. In this study, we focus on the crucial role played by the popular Pluronic® P123 surfactant, used for controlling the mesoporosity of synthesised silica microparticles. We show in particular, that although both types of initial precursor droplets, prepared with and without P123 meso-structuring agent, namely P123+ and P123- droplets, have a similar diameter (≃30 μm) and a similar TEOS silica precursor concentration (0.34 M), the resulting microparticles exhibit two noticeably different sizes and mass densities. Namely, 10 μm and 0.55 g/cm3 for P123+ microparticles, and 5.2 μm and 1.4 g/cm3 for P123- microparticles. To explain such differences, we used optical and scanning electron microscopies, small-angle X-ray diffraction and BET measurements to analyse structural properties of both types of microparticles and show that in the absence of Pluronic molecules, P123- microdroplets divide during their condensation process, on average, into three smaller droplets before condensing into silica solid microspheres with a smaller size and a higher mass density than those obtained in the presence of P123 surfactant molecules. Based on these results and on condensation kinetics analysis, we also propose an original mechanism for the formation of silica microspheres in the presence and in the absence of the meso-structuring and pore-forming P123 molecules.

Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics Screening.

Bacillus subtilis is one of the commonly used industrial strains for riboflavin production. High-throughput screening is useful in biotechnology, but there are still an insufficient number of articles focusing on improving the riboflavin production of B. subtilis by this powerful tool. With droplet-based microfluidics technology, single cells can be encapsulated in droplets. The screening can be carried out by detecting the fluorescence intensity of secreted riboflavin. Thus, an efficient and high-throughput screening method suitable for riboflavin production strain improvement could be established. In this study, droplet-based microfluidics screening was applied, and a more competitive riboflavin producer U3 was selected from the random mutation library of strain S1. The riboflavin production and biomass of U3 were higher than that of S1 in flask fermentation. In addition, the results of fed-batch fermentation showed that the riboflavin production of U3 was 24.3 g/L, an 18% increase compared with the parent strain S1 (20.6 g/L), and the yield (g riboflavin/100 g glucose) increased by 19%, from 7.3 (S1) to 8.7 (U3). Two mutations of U3 (sinRG89R and icdD28E) were identified through whole genome sequencing and comparison. Then they were introduced into BS168DR (parent of S1) for further analysis, which also caused riboflavin production to increase. This paper provides protocols for screening riboflavin-producing B. subtilis with droplet-based microfluidics technology and reveals mutations in riboflavin overproduction strains.

Miniaturizing chemistry and biology using droplets in open systems.

Open droplet microfluidic systems manipulate droplets on the picolitre-to-microlitre scale in an open environment. They combine the compartmentalization and control offered by traditional droplet-based microfluidics with the accessibility and ease-of-use of open microfluidics, bringing unique advantages to applications such as combinatorial reactions, droplet analysis and cell culture. Open systems provide direct access to droplets and allow on-demand droplet manipulation within the system without needing pumps or tubes, which makes the systems accessible to biologists without sophisticated setups. Furthermore, these systems can be produced with simple manufacturing and assembly steps that allow for manufacturing at scale and the translation of the method into clinical research. This Review introduces the different types of open droplet microfluidic system, presents the physical concepts leveraged by these systems and highlights key applications.

Combinatorial drug screening on 3D Ewing sarcoma spheroids using droplet-based microfluidics

Culturing and screening cells in microfluidics, particularly in three-dimensional formats, has the potential to impact diverse areas from fundamental biology to cancer precision medicine. Here, we use a platform based on anchored droplets for drug screening. The response of spheroids of Ewing sarcoma (EwS) A673 cells to simultaneous or sequential combinations of etoposide and cisplatin was evaluated. This was done by culturing spheroids of EwS cells inside 500nL droplets then merging them with secondary droplets containing fluorescent-barcoded drugs at different concentrations. Differences in EwS spheroid growth and viability were measured by microscopy. After drug exposure such measurements enabled estimation of their IC50 values, which were in agreement with values obtained in standard multiwell plates. Then, synergistic drug combination was evaluated. Sequential combination treatment of EwS with etoposide applied 24h before cisplatin resulted in amplified synergistic effect. As such, droplet-based microfluidics offers the modularity required for evaluation of drug combinations.