Droplet Microfluidics Research Articles

High‐Throughput Single‐Cell, Single‐Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics

AbstractThere is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single‐cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single‐cell genomic, RNA, and protein analysis, their application to sub‐cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose‐based droplet microfluidic approach for single‐cell, single‐mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi‐step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single‐mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

Optimization of a tunable process for rapid production of calcium phosphate microparticles using a droplet-based microfluidic platform.

Calcium phosphate (CaP) biomaterials are amongst the most widely used synthetic bone graft substitutes, owing to their chemical similarities to the mineral part of bone matrix and off-the-shelf availability. However, their ability to regenerate bone in critical-sized bone defects has remained inferior to the gold standard autologous bone. Hence, there is a need for methods that can be employed to efficiently produce CaPs with different properties, enabling the screening and consequent fine-tuning of the properties of CaPs towards effective bone regeneration. To this end, we propose the use of droplet microfluidics for rapid production of a variety of CaP microparticles. Particularly, this study aims to optimize the steps of a droplet microfluidic-based production process, including droplet generation, in-droplet CaP synthesis, purification and sintering, in order to obtain a library of CaP microparticles with fine-tuned properties. The results showed that size-controlled, monodisperse water-in-oil microdroplets containing calcium- and phosphate-rich solutions can be produced using a flow-focusing droplet-generator microfluidic chip. We optimized synthesis protocols based on in-droplet mineralization to obtain a range of CaP microparticles without and with inorganic additives. This was achieved by adjusting synthesis parameters, such as precursor concentration, pH value, and aging time, and applying heat treatment. In addition, our results indicated that the synthesis and fabrication parameters of CaPs in this method can alter the microstructure and the degradation behavior of CaPs. Overall, the results highlight the potential of the droplet microfluidic platform for engineering CaP microparticle biomaterials with fine-tuned properties.

Deep Learning-Based Inkjet Droplet Detection for Jetting Characterizations and Multijet Synchronization.

Inkjet printing is a powerful direct material writing process. It can be used to deposit microfluidic droplets in designated patterns at submicrometer resolution, which reduces materials usage. Nonetheless, predicting jetting characterizations is not easy because of the intrinsic complexity of the ink-nozzle-air interactions. Thus, inkjet processes are monitored by skilled engineers to ensure process reliability. This is a bottleneck in industry, resulting in high labor costs for multiple nozzles. To address this, we present a deep learning-based method for jetting characterizations. Inkjet printing is recorded by an in situ CCD camera and each droplet is detected by YOLOv5, a 1-stage detector using a convolutional neural network (CNN). The precision, recall, and mean average precision (mAP) at a 0.5 intersection over the union (IoU) threshold of the trained model were 0.86, 0.89, and 0.90, respectively. Each regression result for a detected droplet is accumulated in chronological order for each class of droplet and nozzle. The quantified information includes velocity, diameter, length, and translation, which can be used to synchronize multinozzle jetting and, eventually, the printed patterns. This demonstrates the feasibility of autonomous real-time process testing for large-scale electronics manufacturing, such as the high-resolution patterning of biosensor electrodes and QD display pixels while exploiting big data obtained from jetting characterizations.

Abstract 3976: Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach

Abstract Single-cell immune receptor profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000-5,000 cells) using microwell arrays or droplet microfluidics technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput when compared to bulk immune receptor profiling methods. Here, we describe a low-throughput, single-cell immune profiling strategy using sorted cells in 96-well plates. The plate is pre-aliquoted with either T-cell receptor (TCR) ɑ/β or TCR γ/δ primers along with 30 crucial T-cell markers. We perform multiplex RT-PCR amplification and sequencing of the CDR3 regions. The resulting data provides the abundant clonotype counts along with the chain pairing information for these ɑ/β and γ/δ chains, together with the T-cell subtype information using gene-expression profiles. By analyzing the TCR gene rearrangement at the single-cell level, researchers can better understand T cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. This method provides a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay make it a valuable tool for unraveling the immune dynamics in various diseases. Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Lester Kobzik, Khadija Ghias, Paul Diehl. Single-cell TCRɑ/β and TCRγ/δ immune receptor profiling and immunophenotyping using a 96-well plate sorted-cell approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3976.

Abstract 533: Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models

Abstract Triple Negative breast cancer (TNBC) accounts for 10-15% of all breast cancers where patients encounter poor clinical outcomes. Treatment modalities for TNBC are conventional cytotoxic chemotherapy and radiation leads to 35%-40% relapse within 5 years of diagnosis. There is a growing need for more effective targeted therapeutics to inhibit TNBC growth. The role of EZH2 in maintaining tumor growth and metastasis in TNBC is well documented. EZH2 promotes breast tumor-initiating cell expansion (BITC) and cancer progression by impairing the DNA damage repair process favoring activation of oncogenes. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity, resistance to these small molecules limits their use in solid tumors. The neurotransmitter dopamine via its D1 receptor activation in TNBC cell lines induces apoptosis and autophagy, as well as inhibits the invasion and regress in mammary tumors in vivo. Here we hypothesize that the presence of dopamine D1 receptor agonist (A77636) enhances the efficacy of EZH2 inhibitors (GSK126) to inhibit in vitro TNBC tumor growth and metastasis. To test the efficacy of the combination we employed a 3D culture system of MDA-MB-231 cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. We also employed a 3D organ-on-chip-based microphysiological (MPS) platform (SynTumor) to study the effect of the drug combination on metastasis in vitro. The SynTumor MPS devices replicate the pathophysiological architecture of native vascularized breast tumors. The combination treatment enhanced a significant reduction of tumor spheroid diameter compared to the vehicle and indicated spheroid regression over treatment. Furthermore, the combination therapy induced necrosis and caused complete inhibition of EZH2 expression. The knockdown of EZH2 inhibited tumor spheroid formation and displayed cytoskeleton de-arrangement. In the microfluidic SynTumor model, circulating tumor cell numbers were reduced by half at 96 hrs after EZH2 combination treatment. Our data indicate that the combinatorial effect of DRD1 agonist and EZH2 inhibitor efficiently attenuates the EZH2-mediated in vitro tumor growth and metastasis (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: xilal Rima, Gautam Sarathy, Chunyu Hu, Divya S. Patel, Deborah Ramsey, Lara Rizotto, Dario Palmeri, Gwen Fewell, Bhuvaneswari Ramaswamy, Eduardo Reátegu, Eswar Shankar. Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 533.

Monitoring Aqueous Sucrose Solutions Using Droplet Microfluidics: Ice Nucleation, Growth, Glass Transition, and Melting.

Freezing and freeze-drying processes are commonly used to extend the shelf life of drug products and to ensure their safety and efficacy upon use. When designing a freezing process, it is beneficial to characterize multiple physicochemical properties of the formulation, such as nucleation rate, crystal growth rate, temperature and concentration of the maximally freeze-concentrated solution, and melting point. Differential scanning calorimetry has predominantly been used in this context but does have practical limitations and is unable to quantify the kinetics of crystal growth and nucleation. In this work, we introduce a microfluidic technique capable of quantifying the properties of interest and use it to investigate aqueous sucrose solutions of varying concentration. Three freeze-thaw cycles were performed on droplets with 75-μm diameters at cooling and warming rates of 1 °C/min. During each cycle, the visual appearance of the droplets was optically monitored as they experienced nucleation, crystal growth, formation of the maximally freeze-concentrated solution, and melting. Nucleation and crystal growth manifested as increases in droplet brightness during the cooling phase. Heating was associated with a further increase as the temperature associated with the maximally freeze-concentrated solution was approached. Heating beyond the melting point corresponded to a decrease in brightness. Comparison with the literature confirmed the accuracy of the new technique while offering new visual data on the maximally freeze-concentrated solution. Thus, the microfluidic technique presented here may serve as a complement to differential scanning calorimetry in the context of freezing and freeze-drying. In the future, it could be applied to a plethora of mixtures that undergo such processing, whether in pharmaceutics, food production, or beyond.

Simple and flexible production of controllable emulsion droplets from open-type co-flow microfluidics

An open-type co-flow microfluidic technique that allows simple microflow manipulation with a broad operation range of flow rates for controllably producing uniform droplets with widely adjustable sizes is developed. The microfluidic device employs a co-flow geometry with relatively open shearing space to largely reduce the pressure drop in the microchannel to facilitate the flow of liquids. Such a device significantly extends the operation window of the dripping regime and eliminates the back flow, to benefit easy microflow manipulation with broadly adjustable flow rates for producing monodisperse droplets with widely tunable sizes. Typically, at Wed = 0.0028, the operation window of the dripping regime and adjustable range of flow rates for this device are both at least ∼3 times wider than those of traditional co-flow devices with confined shearing space for droplet generation. This work provides a facile microfluidic platform with simple flow manipulation and a wide operation range for the wide-scale impact of droplet microfluidics.

Multiplexed fluorescence and scatter detection with single cell resolution using on-chip fiber optics for droplet microfluidic applications

Droplet microfluidics has emerged as a critical component of several high-throughput single-cell analysis techniques in biomedical research and diagnostics. Despite significant progress in the development of individual assays, multiparametric optical sensing of droplets and their encapsulated contents has been challenging. The current approaches, most commonly involving microscopy-based high-speed imaging of droplets, are technically complex and require expensive instrumentation, limiting their widespread adoption. To address these limitations, we developed the OptiDrop platform; this platform is a novel optofluidic setup that leverages the principles of flow cytometry. Our platform enables on-chip detection of the scatter and multiple fluorescence signals from the microfluidic droplets and their contents using optical fibers. The highly customizable on-chip optical fiber-based signal detection system enables simplified, miniaturized, low-cost, multiparametric sensing of optical signals with high sensitivity and single-cell resolution within each droplet. To demonstrate the ability of the OptiDrop platform, we conducted a differential expression analysis of the major histocompatibility complex (MHC) protein in response to IFNγ stimulation. Our results showed the platform’s ability to sensitively detect cell surface biomarkers using fluorescently labeled antibodies. Thus, the OptiDrop platform combines the versatility of flow cytometry with the power of droplet microfluidics to provide wide-ranging, scalable optical sensing solutions for research and diagnostics.

A Modular and Cost-Effective Droplet Microfluidic Device for Controlled Emulsion Production.

The droplet microfluidic device has become a widely used tool in fields such as physics, chemistry, and biology, but its complexity has limited its widespread application. This report introduces a modular and cost-effective droplet microfluidic device for the controlled production of complex emulsions, including oil and aqueous single emulsions, and double emulsions with varying numbers of encapsulated droplets. The droplet sizes can be precisely controlled by easily replacing flat needles and adjusting the needle position within an axially accelerated co-flow field. This modular device not only allows for easy repair and maintenance in case of device clogging or damage but can also be readily expanded to produce complex emulsions. The low-cost and user-friendly nature of the device greatly facilitates the widespread adoption and utilization of droplet microfluidics.

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells.

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

Self-Metallized Whole Cell Vaccines Prepared by Microfluidics for Bioorthogonally Catalyzed Antitumor Immunotherapy.

Tumor whole cell, carrying a complete set of tumor-associated antigens and tumor-specific antigens, has shown great potential in the construction of tumor vaccines but is hindered by the complex engineering means and limited efficacy to cause immunity. Herein, we provided a strategy for the self-mineralization of autologous tumor cells with palladium ions in microfluidic droplets, which endowed the engineered cells with both immune and catalytic functions, to establish a bioorthogonally catalytic tumor whole-cell vaccine. This vaccine showed strong inhibition both in the occurrence and recurrence of tumor by invoking the immediate antitumor immunity and building a long-term immunity.

An Automated Single-Cell Droplet-Digital Microfluidic Platform for Monoclonal Antibody Discovery.

Monoclonal antibody (mAb) discovery plays a prominent role in diagnostic and therapeutic applications. Droplet microfluidics has become a standard technology for high-throughput screening of antibody-producing cells due to high droplet single-cell confinement frequency and rapid analysis and sorting of the cells of interest with their secreted mAbs. In this work, a new method is described for on-demand co-encapsulation of cells that eliminates the difficulties associated with washing in between consecutive steps inside the droplets and enables the washing and addition of fresh media. The new platform identifies hybridoma cells that are expressing antibodies of interest using antibody-characterization assays to find the best-performing or rare-cell antibody candidates.

Patient-Derived Organoids as Therapy Screening Platforms in Cancer Patients.

Patient-derived organoids (PDOs) developed ex vivo and in vitro are increasingly used for therapeutic screening. They provide a more physiologically relevant model for drug discovery and development compared to traditional cell lines. However, several challenges remain to be addressed to fully realize the potential of PDOs in therapeutic screening. This paper summarizes recent advancements in PDO development and the enhancement of PDO culture models. This is achieved by leveraging materials engineering and microfabrication technologies, including organs-on-a-chip and droplet microfluidics. Additionally, this work discusses the application of PDOs in therapy screening to meet diverse requirements and overcome bottlenecks in cancer treatment. Furthermore, this work introduces tools for data processing and analysis of organoids, along with their microenvironment. These tools aim to achieve enhanced readouts. Finally, this work explores the challenges and future perspectives of using PDOs in drug development and personalized screening for cancer patients.

Envisioning the Future of High-Throughput Biomedical Assays through the Convergence of AI and Droplet Microfluidics

The advent of droplet microfluidics has revolutionized the landscape of biomedical research, offering a platform for the precise control and manipulation of fluids at the microscale [1,2].

Self-Generating Gold Nanocatalysts in Autologous Tumor Cells for Targeted Catalytic Immunotherapy.

Employing tumor whole cells for tumor immunotherapy is a promising tumor therapy proposed in the early stage, but its therapeutic efficacy is weakened by the methods of eliminating pathogenicity and the mass ratio of the effective antigen carried by itself. Here, by adding gold ion to live cancer cells in the microfluidic droplets, this work obtains dead tumor whole cells with NIR-controlled catalytic ability whose pathogenicity is removed while plenary tumor antigens, major structure, and homing ability are reserved. The engineered tumor cell (Cell-Au) with the addition of prodrug provides 1O2 in an O2-free Russell mechanism, which serves better in a hypoxic tumor microenvironment. This tumor whole-cell catalytic vaccine (TWCV) promotes the activation of dendritic cells and the transformation of macrophages into tumor suppressor phenotype. In 4T1 tumor-bearing mice, the Cell-Au-based vaccine supports the polarization of cytotoxicity T cells, resulting in tumor eradication and long-term animal survival. Compared with antigen vaccines or adoptive cell therapy which takes months to obtain, this TWCV can be prepared in just a few days with satisfactory immune activation and tumor therapeutic efficacy, which provides an alternative way for the preparation of personalized tumor vaccines across tumor types and gives immunotherapy a new path.

Droplet microfluidics for time-resolved serial crystallography.

Serial crystallography requires large numbers of microcrystals and robust strategies to rapidly apply substrates to initiate reactions in time-resolved studies. Here, we report the use of droplet miniaturization for the controlled production of uniform crystals, providing an avenue for controlled substrate addition and synchronous reaction initiation. The approach was evaluated using two enzymatic systems, yielding 3 µm crystals of lysozyme and 2 µm crystals of Pdx1, an Arabidopsis enzyme involved in vitamin B6 biosynthesis. A seeding strategy was used to overcome the improbability of Pdx1 nucleation occurring with diminishing droplet volumes. Convection within droplets was exploited for rapid crystal mixing with ligands. Mixing times of <2 ms were achieved. Droplet microfluidics for crystal size engineering and rapid micromixing can be utilized to advance time-resolved serial crystallography.

Quantitative study of droplet generation by pressure-driven microfluidic flows in a flow-focusing microdroplet generator

To precisely control the size of droplets is of great importance for the applications of the droplet microfluidics. In a flow-focusing microdroplet generator, the pressure-driven microfluidic device is designed to control the flow rates of the fluids. For a specific geometry of the flow-focusing microchannel, a mathematical model of droplet formation is established, and the nonlinear relation between the droplet length and the driven-pressure ratio can be described by our model. For pressure-driven microfluidic flows, the nonlinear relation between the droplet length and the driving-pressure ratio is measured experimentally in the flow-focusing microchannel. Particularly, by using the closed-loop control method of droplet generation, good agreements are shown between the measured size of droplets and the predicted size of the droplets. As a result, the control precision of the droplet size can be increased drastically by the closed-loop control method of droplet generation. Consequently, monodisperse droplets of extremely small size can be produced in the flow-focusing microdroplet generator.

Understanding the voltage-induced electrowetting and microfluidic droplet movement phenomena on a Teflon-on-flexible (TOF) substrate

The current work focuses on the basic principle of voltage-induced electrowetting and relevant movement of the microfluidic droplets. The prototype of microfluidic devices are fabricated on the Teflon-on-flexible substrate. Three different liquid droplets, namely, the de-ionized (DI) water, sucrose (aq.) solution, and eugenol, have been studied for such purpose within the voltage range of 1–16 V. Electrowetting and subsequent changes in contact angle are extensively investigated with the modification of “work of adhesion” and “work of cohesion” upon application of external voltage. The liquid droplet is positioned on the dielectric-hydrophobic layer which also separates it from the metal electrodes. Eugenol exhibits more susceptibility to electrowetting compared to sucrose solution and DI water. Consequently, sucrose (aq.) solution and DI water show comparatively more droplet displacement. The “work of spreading” for the liquids under test on Teflon surface is obtained. The spreading of eugenol starts at relatively low voltages than sucrose (aq.) solution and DI water. Eugenol follows the Young–Lippmann equation, i.e., linear relation between {cos(θv) − cos(θ0)} with voltage2 (V2); however, sucrose (aq.) solution and DI water deviate from such nature. Here, θ0 and θv are the initial and voltage modified contact angles, respectively. Thus, the current study provides an accurate approach to analyze the interaction of solid–liquid surfaces and its consequent effect upon application of external voltages.

Droplet Microfluidics for Current Cancer Research: From Single-Cell Analysis to 3D Cell Culture.

Cancer is the second leading cause of death worldwide. Differences in drug resistance and treatment response caused by the heterogeneity of cancer cells are the primary reasons for poor cancer therapy outcomes in patients. In addition, current in vitro anticancer drug-screening methods rely on two-dimensional monolayer-cultured cancer cells, which cannot accurately predict drug behavior in vivo. Therefore, a powerful tool to study the heterogeneity of cancer cells and produce effective in vitro tumor models is warranted to leverage cancer research. Droplet microfluidics has become a powerful platform for the single-cell analysis of cancer cells and three-dimensional cell culture of in vitro tumor spheroids. In this review, we discuss the use of droplet microfluidics in cancer research. Droplet microfluidic technologies, including single- or double-emulsion droplet generation and passive- or active-droplet manipulation, are concisely discussed. Recent advances in droplet microfluidics for single-cell analysis of cancer cells, circulating tumor cells, and scaffold-free/based 3D cell culture of tumor spheroids have been systematically introduced. Finally, the challenges that must be overcome for the further application of droplet microfluidics in cancer research are discussed.

Microbially Inspired Calcium Carbonate Precipitation Pathway Integrated Polyelectrolyte Capsules (MICPC) for Biomolecules Release.

Complexation between oppositely charged polyelectrolytes offers a facile single-step strategy for assembling functional micro-nano carriers for efficient drug and vaccine delivery. However, the stability of the delivery system within the physiological environment is compromised due to the swelling of the polyelectrolyte complex, driven by the charge shielding effect, and consequently leads to uncontrollable burst release, thereby limiting its potential applications. In a pioneering approach, cellular pathway-inspired calcium carbonate precipitation pathways are developed that are integrated into polyelectrolyte capsules (MICPC). These innovative capsules are fabricated at the interface of all-aqueous microfluidic droplets, resulting in a precisely controllable and sustained release profile in physiological conditions. Unlike single-step polyelectrolyte assembly capsules which always perform rapid burst release, the MICPC exhibits a sustainable and tunable release pattern, releasing biomolecules at an average rate of 3-10% per day. Remarkably, the degree of control over MICPC's release kinetics can be finely tuned by adjusting the quantity of synthesized calcium carbonate particles within the polyelectrolyte complex. This groundbreaking work not only deepens the insights into polyelectrolyte complexation but also significantly enhances the overall stability of these complexes, opening up new avenues for expanding the range of applications involving polyelectrolyte complex-related materials.