Droplet-based System Research Articles

Enhancing the erythritol production of Yarrowia lipolytica by high-throughput screening based on highly sensitive artificial sensor and anchor protein cwp2.

Yarrowia lipolytica is widely used for the industrial production of the natural sweetener erythritol. Despite improvements in fermentation process control and metabolic pathway regulation, bottlenecks still exist in terms of yield and screening technology. Therefore, we constructed an artificial sensor system for effective erythritol detection, established a single-cell droplet-based high-throughput screening system based on fluorescence-activated cell sorting, and obtained Y. lipolytica with improved erythritol production through mutagenesis and high-throughput screening. We used a droplet generator to co-cultivate Y. lipolytica 5-14 with Escherichia coli and used the E. coli fluorescent signal to detect the concentration of erythritol synthesized by Y. lipolytica 5-14 for high-throughput screening. Strains were subjected to UV mutagenesis for 120 s. Under optimized fermentation conditions using Y. lipolytica mutants in 96-well plates, the screening efficiency reached 16.7%. Y. lipolytica 5-14-E6 showed a 21% increase in erythritol to 109.84 g/L. After fermentation at 30°C in a 100 m3 fermenter for 75 h, the mutant Y. lipolytica 5-14-E6 erythritol yield reached 178 g/L.

Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity. Existing droplet-based microfluidic methods produce many cell-free droplets, underutilizing bead barcodes and reagents. Combinatorial indexing on microplates is more efficient for barcoding but labor-intensive. Here we present Overloading And unpacKing (OAK), which uses a droplet-based barcoding system for initial compartmentalization followed by a second aliquoting round to achieve combinatorial indexing. We demonstrate OAK’s versatility with single-cell RNA sequencing as well as paired single-nucleus RNA sequencing and accessible chromatin profiling. We further showcase OAK’s performance on complex samples, including differentiated bronchial epithelial cells and primary retinal tissue. Finally, we examine transcriptomic responses of over 400,000 melanoma cells to a RAF inhibitor, belvarafenib, discovering a rare resistant cell population (0.12%). OAK’s ultra-high throughput, broad compatibility, high sensitivity, and simplified procedures make it a powerful tool for large-scale molecular analysis, even for rare cells.

Development of multi droplet-based electricity generator system for energy harvesting improvement from a single droplet

Abstract Due to high output performance, the droplet-based electricity generator (DEG) is garnering attention as a promising alternative power source for small electronic devices. Accordingly, to utilize the DEG as a power source, the efforts to boost the output have focused on methods to modify material modification and introduce surface structure. However, the behavior feature that the reconfigured droplet falls after the DEG operation leaves room for one more droplet energy harvesting from a single droplet. Here, a multi DEG system (MDEG) constructed with multiple DEG units is proposed to harvest more energy from a single droplet. The continuous movement of a water droplet is realized through the inclined stair structure of the MDEG, resulting in electrical energy generation from a single water droplet as many times as it falls. In particular, 2-step MDEG consisting of two DEG units can have 45% higher performance than a single DEG. Therefore, this study implies a contribution to the development of DEGs by considering the droplet dynamics, which has been overlooked in existing DEG studies.

Droplet-based mechanical transducers modulated by the symmetry of wettability patterns

Asymmetric mechanical transducers have important applications in energy harvesting, signal transmission, and micro-mechanics. To achieve asymmetric transformation of mechanical motion or energy, active robotic metamaterials, as well as materials with asymmetric microstructures or internal orientation, are usually employed. However, these strategies usually require continuous energy supplement and laborious fabrication, and limited transformation modes are achieved. Herein, utilizing wettability patterned surfaces for precise control of the droplet contact line and inner flow, we demonstrate a droplet-based mechanical transducer system, and achieve multimodal responses to specific vibrations. By virtue of the synergistic effect of surface tension and solid-liquid adhesion on the liquid dynamics, the droplet on the patterned substrate can exhibit symmetric/asymmetric vibration transformation when the substrate vibrates horizontally. Based on this, we construct arrayed patterns with distinct arrangements on the substrate, and employ the swarm effect of the arrayed droplets to achieve three-dimensional and multimodal actuation of the target plate under a fixed input vibration. Further, we demonstrate the utilization of the mechanical transducers for vibration management, object transport, and laser modulation. These findings provide a simple yet efficient strategy to realize a multimodal mechanical transducer, which shows significant potential for aseismic design, optical molding, as well as micro-electromechanical systems (MEMS).

Comparative analysis of 10X Chromium vs. BD Rhapsody whole transcriptome single-cell sequencing technologies in complex human tissues

The development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromium vs. microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy.Although high technical consistency was observed in unraveling the whole transcriptome, the relative abundance of cell populations differed. Cells with low mRNA content such as T cells were underrepresented in the droplet-based system, at least partly due to lower RNA capture rates. In contrast, microwell-based scRNA-seq recovered less cells of epithelial origin. Moreover, we discovered platform-dependent variabilities in mRNA quantification and cell-type marker annotation. Overall, our study provides important information for selection of the appropriate scRNA-seq platform and for the interpretation of published results.

Comprehensive Parameter Space Mapping of Cell Cycle Dynamics under Network Perturbations.

Studies of quantitative systems and synthetic biology have extensively utilized models to interpret data, make predictions, and guide experimental designs. However, models often simplify complex biological systems and lack experimentally validated parameters, making their reliability in perturbed systems unclear. Here, we developed a droplet-based synthetic cell system to continuously tune parameters at the single-cell level in multiple dimensions with full dynamic ranges, providing an experimental framework for global parameter space scans. We systematically perturbed a cell-cycle oscillator centered on cyclin-dependent kinase (Cdk1), enabling comprehensive mapping of period landscapes in response to network perturbations. The data allowed us to challenge existing models and refine a new model that matches the observed response. Our analysis demonstrated that Cdk1 positive feedback inhibition restricts the cell cycle frequency range, confirming model predictions; furthermore, it revealed new cellular responses to the inhibition of the Cdk1-counteracting phosphatase PP2A: monomodal or bimodal distributions across varying inhibition levels, underscoring the complex nature of cell cycle regulation that can be explained by our model. This comprehensive perturbation platform may be generalizable to exploring other complex dynamic systems.

Continuous Production of Monodisperse Silver Nanoparticles Suitable for Catalysis in a Droplet-Based Microreactor System

Silver nanoparticles (Ag NPs) with sizes below 50 nm have high value in catalysis, especially in electrocatalysis. However, due to the low efficiency of mass and heat transfer, conventional batch synthesis is unable to produce high-quality Ag NPs repeatedly at a large scale. To tackle this obstacle, a droplet-based microreactor system was designed in this work. Continuous and controllable synthesis of Ag NPs with sizes ranging from 7 to 30 nm, polydispersity indexes less than 10%, and high stability which could be stored at room temperature for 2.5 months were achieved by regulating residence time and introducing seed-mediated growth. Based on the Lifshitz–Slyozov–Wagner model and experimental data, growth kinetics were analyzed and revealed it as a diffusion-limited reaction, providing more powerful theoretical support for the synthesis of metal nanomaterials by the microreactor system.

Demonstration of doxorubicin's cardiotoxicity and screening using a 3D bioprinted spheroidal droplet-based system.

Doxorubicin (DOX) is a highly effective anthracycline chemotherapy agent effective in treating a broad range of life-threatening malignancies but it causes cardiotoxicity in many subjects. While the mechanism of its cardiotoxic effects remains elusive, DOX-related cardiotoxicity can lead to heart failure in patients. In this study, we investigated the effects of DOX-induced cardiotoxicity on human cardiomyocytes (CMs) using a three-dimensional (3D) bioprinted cardiac spheroidal droplet based-system in comparison with the traditional two-dimensional cell (2D) culture model. The effects of DOX were alleviated with the addition of N-acetylcysteine (NAC) and Tiron. Caspase-3 activity was quantified, and reactive oxygen species (ROS) production was measured using dihydroethidium (DHE) staining. Application of varying concentrations of DOX (0.4 μM-1 μM) to CMs revealed a dose-specific response, with 1 μM concentration imposing maximum cytotoxicity and 0.22 ± 0.11% of viable cells in 3D samples versus 1.02 ± 0.28% viable cells in 2D cultures, after 5 days of culture. Moreover, a flow cytometric analysis study was conducted to study CMs proliferation in the presence of DOX and antioxidants. Our data support the use of a 3D bioprinted cardiac spheroidal droplet as a robust and high-throughput screening model for drug toxicity. In the future, this 3D spheroidal droplet model can be adopted as a human-derived tissue-engineered equivalent to address challenges in other various aspects of biomedical pre-clinical research.

CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion

Genome-scale target identification promises to guide microbial cell factory engineering for higher-titer production of biomolecules such as recombinant proteins (r-protein), but challenges remain due to the need not only for comprehensive genotypic perturbation but also in conjunction with high-throughput phenotypic screening strategies. Here, we developed a CRISPRi-microfluidics screening platform to systematically identify crucial gene targets that can be engineered to enhance r-protein secretion in Corynebacterium glutamicum. We created a CRISPR interference (CRISPRi) library containing 46,549 single-guide RNAs, where we aimed to unbiasedly target all genes for repression. Meanwhile, we developed a highly efficient droplet-based microfluidics system integrating the FlAsH-tetracysteine assay that enables screening of millions of strains to identify potential knockdowns conducive to nanobody VHH secretion. Among our highest-ranking candidates are a slew of previously unknown targets involved in transmembrane transport, amino-acid metabolism and redox regulation. Guided by these findings, we eventually constructed a hyperproducer for multiple proteins via combinatorial engineering of redox-response transcription factors. As the near-universal applicability of CRISPRi technology and the FlAsH-based screening platform, this procedure might be expanded to include a varied variety of microbial species and recombinant proteins.

Single cell RNA sequencing reveals hemocyte heterogeneity in Biomphalaria glabrata: Plasticity over diversity.

The freshwater snail Biomphalaria glabrata is an intermediate host of Schistosoma mansoni, the agent of human intestinal schistosomiasis. However, much is to be discovered about its innate immune system that appears as a complex black box, in which the immune cells (called hemocytes) play a major role in both cellular and humoral response towards pathogens. Until now, hemocyte classification has been based exclusively on cell morphology and ultrastructural description and depending on the authors considered from 2 to 5 hemocyte populations have been described. In this study, we proposed to evaluate the hemocyte heterogeneity at the transcriptomic level. To accomplish this objective, we used single cell RNA sequencing (scRNAseq) technology coupled to a droplet-based system to separate hemocytes and analyze their transcriptome at a unique cell level in naive Biomphalaria glabrata snails. We were able to demonstrate the presence of 7 hemocyte transcriptomic populations defined by the expression of specific marker genes. As a result, scRNAseq approach showed a high heterogeneity within hemocytes, but provides a detailed description of the different hemocyte transcriptomic populations in B. glabrata supported by distinct cellular functions and lineage trajectory. As a main result, scRNAseq revealed the 3 main population as a super-group of hemocyte diversity but, on the contrary, a great hemocytes plasticity with a probable capacity of hemocytes to engage to different activation pathways. This work opens a new field of research to understand the role of hemocytes particularly in response to pathogens, and towards S. mansoni parasites.

Self-Organization Emerging from Marangoni and Elastocapillary Effects Directed by Amphiphile Filament Connections.

Self-organization of meso- and macroscale structures is a highly active research field that exploits a wide variety of physicochemical phenomena, including surface tension, Marangoni flow, and (elasto)capillary effects. The release of surface-active compounds generates Marangoni flows that cause repulsion, whereas capillary forces attract floating particles via the Cheerios effect. Typically, the interactions resulting from these effects are nonselective because the gradients involved are uniform. In this work, we unravel the mechanisms involved in the self-organization of amphiphile filaments that connect and attract droplets floating at the air–water interface, and we demonstrate their potential for directional gradient formation and thereby selective interaction. We simulate Marangoni flow patterns resulting from the release and depletion of amphiphile molecules by source and drain droplets, respectively, and we predict that these flow patterns direct the growth of filaments from the source droplets toward specific drain droplets, based on their amphiphile depletion rate. The interaction between such droplets is then investigated experimentally by charting the flow patterns in their surroundings, while the role of filaments in source–drain attraction is studied using microscopy. Based on these observations, we attribute attraction of drain droplets and even solid objects toward the source to elastocapillary effects. Finally, the insights from our simulations and experiments are combined to construct a droplet-based system in which the composition of drain droplets regulates their ability to attract filaments and as a consequence be attracted toward the source. Thereby, we provide a novel method through which directional attraction can be established in synthetic self-organizing systems and advance our understanding of how complexity arises from simple building blocks.

Digital polymerase chain reaction duplexing method in a single fluorescence channel

The digital polymerase chain reaction (dPCR) technique can quantify specific sequences of deoxyribonucleic acid using either a droplet-based or chip-based system. dPCR duplexing methods in a single fluorescence channel are typically based on the difference in fluorescence amplitude (F) between two targets. The different targets are distinguished from each other by the F-value variation using non-equal probe concentrations or different target lengths. In the present study, we propose a single fluorescence channel-based dPCR duplexing method that combines a specific probe and intercalating dye to increase the difference in F values between the two targets. We selected two sequences, one from chromosome 18 (Chr18) detected only by the intercalating dye EvaGreen and the other from chromosome 21 (Chr21) detected by a combination of a 6-carboxyfluorescein (FAM) probe and EvaGreen. We performed the dPCR protocol and imaged the dPCR chip at room temperature to verify the proposed duplexing method. The result revealed that the difference in F values between Chr18 and Chr21 increased from ≈5% to 20% when using the FAM probe for Chr21 compared with the detection of both amplicons using EvaGreen only. The added FAM probe enabled two-target discrimination using a single-color fluorescent channel. We further determined the difference in F values at different temperatures using artificial dPCR images. This proposed method represents a simple option for single fluorescence channel dPCR duplexing, making it suitable for simplified dPCR systems used for point-of-care applications.

Design of a simple nanoscale hydrophilic-hydrophobic heterojunction system with under-liquid dual superlyophobicity for application in controllable droplet-based microreactor system and oil/water emulsions separation

Recently, many superwetting materials have emerged to deal with environmental problems. Among them, the under-liquid dual superlyophobic superwetting surface has attracted widespread attention due to the possessing of underwater superoleophobicity and underoil superhydrophobicity at the same time, and without the requirement of any energy stimulation when compared to conventional or responsive superwetting surfaces. However, A rational design of this deliciated surface state still lacking. Herein, a novel dual superlyophobic surface was achieved by simply modulating the relative proportion of a nanoscale hydrophilic-hydrophobic heterojunction system - cellulose nanofibrils (CNFs) and graphited multiwalled carbon nanotubes (gMWCNTs), and without any additional chemical modification. Besides, the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory or extended-DLVO (EDLVO) theory was introduced first to help understand the wettability variation of such a heterogeneous surface, which is mainly attributed to the changing stability of oil or water film covering the membrane surface. Additionally, this novel nanoscale hydrophilic-hydrophobic heterojunction system with dual superlyophobicity and excellent flexibility can not only separate both oil-in-water emulsion and water-in-oil emulsion simultaneously, but also achieve the lossless transfer in the droplet-based microreactor system under both water and oil environments. This may bring novel applications to the field of dual superlyophobicity. Moreover, this nanoscale hydrophilic-hydrophobic heterojunction system offers new and universal theoretical guidance for facile preparation of dual superlyophobic surfaces, owing to the richness in the functionality of low dimensional nanomaterials, it is foreseeable that more potential applications in various fields can be coupled with such an adaptable platform.

Large-Scale Production of Ligand-Engineered Robust Lead Halide Perovskite Nanocrystals by a Droplet-Based Microreactor System.

Cesium lead halide perovskite nanocrystals (CLHP NCs) have a wide range of potential applications benefited from the properties of high photoluminescence quantum yield (PLQY), wide luminous gamut, and narrow half peak width. However, due to the ionic nature and sensitivity to moisture, oxygen, or heat, perovskite nanocrystals are too fragile to maintain their crystal structure and optical properties. This work proposes solutions to two key issues in the development of CLHP NCs. First, a productive droplet-based microreactor system is designed to accomplish the scale-up production of CLHP NCs, obtaining sub-gram high-purity nanocrystal powders in a single production process. Second, CLHP NCs which are stable in polar solvents, air environment, and high temperature by using 3-aminopropyl triethoxysilane (APTES) as basic ligand are obtained. Wrapped with Si-O-Si generated by APTES, the CLHP NCs exhibit a longer fluorescence lifetime and higher quantum yield. Especially, the PLQY of CsPbBr3 @APTES can be stable at higher than 90% for more than 10 days. The Si-O-Si protective layer can also suppress the anion exchange between CsPbBr3 and CsPbI3 , maintaining the monochromaticity of nanocrystal luminescence. Eventually, full-spectrum quantum light-emitting diode (QLED) beads with robust nanocrystals are fabricated. The gamut of CsPbX3 @APTES encompasses 140% of the NTSC color gamutstandard.

Droplet-based bioprinting enables the fabrication of cell–hydrogel–microfibre composite tissue precursors

Composites offer the option of coupling the individual benefits of their constituents to achieve unique material properties, which can be of extra value in many tissue engineering applications. Strategies combining hydrogels with fibre-based scaffolds can create tissue constructs with enhanced biological and structural functionality. However, developing efficient and scalable approaches to manufacture such composites is challenging. Here, we use a droplet-based bioprinting system called reactive jet impingement (ReJI) to integrate a cell-laden hydrogel with a microfibrous mesh. This system uses microvalves connected to different bioink reservoirs and directed to continuously jet bioink droplets at one another in mid-air, where the droplets react and form a hydrogel that lands on a microfibrous mesh. Cell–hydrogel–fibre composites are produced by embedding human dermal fibroblasts at two different concentrations (5 × 106 and 30 × 106 cells/mL) in a collagen–alginate–fibrin hydrogel matrix and bioprinted onto a fibre-based substrate. Our results show that both types of cell–hydrogel–microfibre composite maintain high cell viability and promote cell–cell and cell–biomaterial interactions. The lower fibroblast density triggers cell proliferation, whereas the higher fibroblast density facilitates faster cellular organisation and infiltration into the microfibres. Additionally, the fibrous component of the composite is characterised by high swelling properties and the quick release of calcium ions. The data indicate that the created composite constructs offer an efficient way to create highly functional tissue precursors for laminar tissue engineering, particularly for wound healing and skin tissue engineering applications.Graphic abstract

Single cell transfection of human-induced pluripotent stem cells using a droplet-based microfluidic system.

Microfluidic tools have recently made possible many advances in biological and biomedical research. Research in fields such as physics, engineering, chemistry and biology have combined to produce innovation in microfluidics which has positively impacted diverse areas such as nucleotide sequencing, functional genomics, single-cell studies, single molecules assays and biomedical diagnostics. Among these areas, regenerative medicine and stem cells have benefited from microfluidics since these tools have had a profound impact on their applications. In this study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We will demonstrate that this system has great efficiency in single cells and captured droplets, like other microfluidic methods but with lower cost. Moreover, this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations and cell therapy, among other potential applications.

Droplet-Based Evaporative System for the Estimation of Protein Crystallization Kinetics.

Crystallization is a potential cost-effective alternative to chromatography for the purification of biotherapeutic proteins. Crystallization kinetics are required for the design and control of such processes, but only a limited quantity of proteins is available during the initial stage of process development. This article describes the design of a droplet-based evaporative system for the evaluation of candidate crystallization conditions and the estimation of kinetics using only a droplet (on the order of μL) of protein solution. The temperature and humidity of air fed to a flow cell containing the droplet are controlled for evaporation and rehydration of the droplet, which are used for manipulating supersaturation. Dual-angle images of the droplet are taken and analyzed on-line to obtain the droplet volume and crystal sizes. Crystallization kinetics are estimated based on a first-principles process model and experimental data. Tight control of temperature and humidity of the air, fast and accurate image analysis, and accurate estimation of crystallization kinetics are experimentally demonstrated for a model protein lysozyme. The estimated kinetics are suitable for the model-based design and control of protein crystallization processes.

A flexible microfluidic system for single-cell transcriptome profiling elucidates phased transcriptional regulators of cell cycle

Single cell transcriptome profiling has emerged as a breakthrough technology for the high-resolution understanding of complex cellular systems. Here we report a flexible, cost-effective and user-friendly droplet-based microfluidics system, called the Nadia Instrument, that can allow 3′ mRNA capture of ~ 50,000 single cells or individual nuclei in a single run. The precise pressure-based system demonstrates highly reproducible droplet size, low doublet rates and high mRNA capture efficiencies that compare favorably in the field. Moreover, when combined with the Nadia Innovate, the system can be transformed into an adaptable setup that enables use of different buffers and barcoded bead configurations to facilitate diverse applications. Finally, by 3′ mRNA profiling asynchronous human and mouse cells at different phases of the cell cycle, we demonstrate the system's ability to readily distinguish distinct cell populations and infer underlying transcriptional regulatory networks. Notably this provided supportive evidence for multiple transcription factors that had little or no known link to the cell cycle (e.g. DRAP1, ZKSCAN1 and CEBPZ). In summary, the Nadia platform represents a promising and flexible technology for future transcriptomic studies, and other related applications, at cell resolution.

Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities.

Antibiotics from few culturable microorganisms have saved millions of lives since the 20th century. But with resistance formation, these compounds become increasingly ineffective, while the majority of microbial and with that chemical compound diversity remains inaccessible for cultivation and exploration. Culturing recalcitrant bacteria is a stochastic process. But conventional methods are limited to low throughput. By increasing (i) throughput and (ii) sensitivity by miniaturization, we innovate microbiological cultivation to comply with biological stochasticity. Here, we introduce a droplet-based microscale cultivation system, which is directly coupled to a high-throughput screening for antimicrobial activity prior to strain isolation. We demonstrate that highly parallelized in-droplet cultivation starting from single cells results in the cultivation of yet uncultured species and a significantly higher bacterial diversity than standard agar plate cultivation. Strains able to inhibit intact reporter strains were isolated from the system. A variety of antimicrobial compounds were detected for a selected potent antibiotic producer.

Droplet-Based Bioprinting Enables the Fabrication of Cell-Hydrogel-Microfibre Composite Tissue Precursors

Composites offer an opportunity to couple particular benefits of their constituents to achieve unique material properties that can be of extra value in many tissue engineering applications. Strategies combining hydrogels with fibre-based scaffolds can create more biologically and structurally functional tissue constructs. However, developing efficient and scalable approaches to manufacture such composites is challenging. Here, we use a droplet-based bioprinting system called Reactive Jet Impingement (ReJI) to integrate a cell-laden hydrogel with a microfibrous mesh. The ReJI system uses microvalves that are connected to different bio-ink reservoirs and directed to continuously jet bio-ink droplets at one another in mid-air where the droplets react and form a hydrogel that lands on a microfibrous mesh. We produce cell-hydrogel-fibre composites by embedding human dermal fibroblasts at two different concentrations (5 x 106 and 30 x 106 cells/ml respectively) in a collagen-alginate-fibrin hydrogel matrix and bioprint it onto a fibre-based substrate. Our results show that both types of cell-hydrogel-microfibre composites maintain high cell viability and promote cell-cell and cell-biomaterial interactions. The lower fibroblast density thereby triggers cell proliferation whereas the higher fibroblast density facilitates faster cellular organisation and infiltration into the microfibres. Additionally, the fibrous component of the composite is characterised by its high swelling properties and quick calcium ions release. The data indicate that the composite constructs we create offer an efficient way to create highly functional tissue precursors for laminar tissue engineering in particular wound healing and skin tissue engineering applications.