High-throughput droplet-based analysis of influenza A virus genetic reassortment by single-virus RNA sequencing.

The segmented RNA genome of influenza A viruses (IAVs) enables viral evolution through genetic reassortment after multiple IAVs coinfect the same cell, leading to viruses harboring combinations of eight genomic segments from distinct parental viruses. Existing data indicate that reassortant genotypes are not equiprobable; however, the low throughput of available virology techniques does not allow quantitative analysis. Here, we have developed a high-throughput single-cell droplet microfluidic system allowing encapsulation of IAV-infected cells, each cell being infected by a single progeny virion resulting from a coinfection process. Customized barcoded primers for targeted viral RNA sequencing enabled the analysis of 18,422 viral genotypes resulting from coinfection with two circulating human H1N1pdm09 and H3N2 IAVs. Results were highly reproducible, confirmed that genetic reassortment is far from random, and allowed accurate quantification of reassortants including rare events. In total, 159 out of the 254 possible reassortant genotypes were observed but with widely varied prevalence (from 0.038 to 8.45%). In cells where eight segments were detected, all 112 possible pairwise combinations of segments were observed. The inclusion of data from single cells where less than eight segments were detected allowed analysis of pairwise cosegregation between segments with very high confidence. Direct coupling analysis accurately predicted the fraction of pairwise segments and full genotypes. Overall, our results indicate that a large proportion of reassortant genotypes can emerge upon coinfection and be detected over a wide range of frequencies, highlighting the power of our tool for systematic and exhaustive monitoring of the reassortment potential of IAVs.

Advancing Point-of-Care Applications with Droplet Microfluidics: From Single-Cell to Multicellular Analysis.

Recent advances in single-cell and multicellular microfluidics technology have provided powerful tools for studying cancer biology and immunology. The ability to create controlled microenvironments, perform high-throughput screenings, and monitor cellular interactions at the single-cell level has significantly advanced our understanding of tumor biology and immune responses. We discuss cutting-edge multicellular and single-cell microfluidic technologies and methodologies utilized to investigate cancer-immune cell interactions and assess the effectiveness of immunotherapies. We explore the advantages and limitations of the wide range of 3D spheroid and single-cell microfluidic models recently developed, highlighting the various approaches in device generation and applications in immunotherapy screening for potential opportunities for point-of-care approaches.

Characterizing cell interactions at scale with made-to-order droplet ensembles (MODEs).

Cell-cell interactions are important to numerous biological systems, including tissue microenvironments, the immune system, and cancer. However, current methods for studying cell combinations and interactions are limited in scalability, allowing just hundreds to thousands of multicell assays per experiment; this limited throughput makes it difficult to characterize interactions at biologically relevant scales. Here, we describe a paradigm in cell interaction profiling that allows accurate grouping of cells and characterization of their interactions for tens to hundreds of thousands of combinations. Our approach leverages high-throughput droplet microfluidics to construct multicellular combinations in a deterministic process that allows inclusion of programmed reagent mixtures and beads. The combination droplets are compatible with common manipulation and measurement techniques, including imaging, barcode-based genomics, and sorting. We demonstrate the approach by using it to enrich for chimeric antigen receptor (CAR)-T cells that activate upon incubation with target cells, a bottleneck in the therapeutic T cell engineering pipeline. The speed and control of our approach should enable valuable cell interaction studies.

Uniform sized cancer spheroids production using hydrogel-based droplet microfluidics: a review.

Three-dimensional (3D) cell culture models have been extensively utilized in various mechanistic studies as well as for drug development studies as superior in vitro platforms than conventional two-dimensional (2D) cell culture models. This is especially the case in cancer biology, where 3D cancer models, such as spheroids or organoids, have been utilized extensively to understand the mechanisms of cancer development. Recently, many sophisticated 3D models such as organ-on-a-chip models are emerging as advanced in vitro models that can more accurately mimic the in vivo tissue functions. Despite such advancements, spheroids are still considered as a powerful 3D cancer model due to the relatively simple structure and compatibility with existing laboratory instruments, and also can provide orders of magnitude higher throughput than complex in vitro models, an extremely important aspects for drug development. However, creating well-defined spheroids remain challenging, both in terms of throughputs in generation as well as reproducibility in size and shape that can make it challenging for drug testing applications. In the past decades, droplet microfluidics utilizing hydrogels have been highlighted due to their potentials. Importantly, core-shell structured gel droplets can avoid spheroid-to-spheroid adhesion that can cause large variations in assays while also enabling long-term cultivation of spheroids with higher uniformity by protecting the core organoid area from external environment while the outer porous gel layer still allows nutrient exchange. Hence, core-shell gel droplet-based spheroid formation can improve the predictivity and reproducibility of drug screening assays. This review paper will focus on droplet microfluidics-based technologies for cancer spheroid production using various gel materials and structures. In addition, we will discuss emerging technologies that have the potential to advance the production of spheroids, prospects of such technologies, and remaining challenges.

High-throughput G protein-coupled receptor-based autocrine screening for secondary metabolite production in yeast.

Biosensors are valuable tools in accelerating the test phase of the design-build-test-learn cycle of cell factory development, as well as in bioprocess monitoring and control. G protein-coupled receptor (GPCR)-based biosensors enable cells to sense a wide array of molecules and environmental conditions in a specific manner. Due to the extracellular nature of their sensing, GPCR-based biosensors require compartmentalization of distinct genotypes when screening production levels of a strain library to ensure that detected levels originate exclusively from the strain under assessment. Here, we explore the integration of production and sensing modalities into a single Saccharomyces cerevisiae strain and compartmentalization using three different methods: (1) cultivation in microtiter plates, (2) spatial separation on agar plates, and (3) encapsulation in water-in-oil-in-water double emulsion droplets, combined with analysis and sorting via a fluorescence-activated cell sorting machine. Employing tryptamine and serotonin as proof-of-concept target molecules, we optimize biosensing conditions and demonstrate the ability of the autocrine screening method to enrich for high producers, showing the enrichment of a serotonin-producing strain over a nonproducing strain. These findings illustrate a workflow that can be adapted to screening for a wide range of complex chemistry at high throughput using commercially available microfluidic systems.

Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Droplet Microfluidic-Based In Situ Analyzer for Monitoring Free Nitrate in Soil.

Monitoring nutrients in the soil can provide valuable information for understanding their spatiotemporal variability and informing precise soil management. Here, we describe an autonomous in situ analyzer for the real-time monitoring of nitrate in soil. The analyzer can sample soil nitrate using either microdialysis or ultrafiltration probes placed within the soil and quantify soil nitrate using droplet microfluidics and colorimetric measurement. Compared with traditional manual sampling and lab analysis, the analyzer features low reagent consumption (96 µL per measurement), low maintenance requirement (monthly), and high measurement frequency (2 or 4 measurements per day), providing nondrifting lab-quality data with errors of less than 10% using a microdialysis probe and 2-3% for ultrafiltration. The analyzer was deployed at both the campus garden and forest for different periods of time, being able to capture changes in free nitrate levels in response to manual perturbation by the addition of nitrate standard solutions and natural perturbation by rainfall events.

Fabrication of Crescent Shaped Microparticles for Particle Templated Droplet Formation.

Crescent-shaped hydrogel microparticles are shown to template uniform volume aqueous droplets upon simple mixing with aqueous and oil media for various bioassays. This emerging "lab on a particle" technique requires hydrogel particles with tunable material properties and dimensions. The crescent shape of the particles is attained by aqueous two-phase separation of polymers followed by photopolymerization of the curable precursor. In this work, the phase separation of poly(ethylene glycol) diacrylate (PEGDA, Mw 700) and dextran (Mw 40 000) for tunable manufacturing of crescent-shaped particles is investigated. The particles' morphology is precisely tuned by following a phase diagram, varying the UV intensity, and adjusting the flow rates of various streams. The fabricated particles with variable dimensions encapsulate uniform aqueous droplets upon mixing with an oil phase. The particles are fluorescently labeled with red and blue emitting dyes at variable concentrations to produce six color-coded particles. The blue fluorescent dye shows a moderate response to the pH change. The fluorescently labeled particles are able to tolerate an extremely acidic solution (pH 1) but disintegrate within an extremely basic solution (pH 14). The particle-templated droplets are able to effectively retain the disintegrating particle and the fluorescent signal at pH 14.

Protein design-scapes generated by microfluidic DNA assembly elucidate domain coupling in the bacterial histidine kinase CpxA.

The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence-function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant "S helix" located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.

Droplet digital molecular beacon-LAMP assay via pico-injection for ultrasensitive detection of pathogens.

A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/µL in a model plasmid containing the malB gene and 3 CFU/µL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.

Tailored Fluorosurfactants through Controlled/Living Radical Polymerization for Highly Stable Microfluidic Droplet Generation.

Droplet-based microfluidics represents a disruptive technology in the field of chemistry and biology through the generation and manipulation of sub-microlitre droplets. To avoid droplet coalescence, fluoropolymer-based surfactants are commonly used to reduce the interfacial tension between two immiscible phases to stabilize droplet interfaces. However, the conventional preparation of fluorosurfactants involves multiple steps of conjugation reactions between fluorinated and hydrophilic segments to form multiple-block copolymers. In addition, synthesis of customized surfactants with tailored properties is challenging due to the complex synthesis process. Here, we report a highly efficient synthetic method that utilizes living radical polymerization (LRP) to produce fluorosurfactants with tailored functionalities. Compared to the commercialized surfactant, our surfactants outperform in thermal cycling for polymerase chain reaction (PCR) testing, and exhibit exceptional biocompatibility for cell and yeast culturing in a double-emulsion system. This breakthrough synthetic approach has the potential to revolutionize the field of droplet-based microfluidics by enabling the development of novel designs that generate droplets with superior stability and functionality for a wide range of applications.

High-Throughput Host-Microbe Single-Cell RNA Sequencing Reveals Ferroptosis-Associated Heterogeneity during Acinetobacter baumannii Infection.

Interactions between host and bacterial cells are integral to human physiology. The complexity of host-microbe interactions extends to different cell types, spatial aspects, and phenotypic heterogeneity, requiring high-resolution approaches to capture their full complexity. The latest breakthroughs in single-cell RNA sequencing (scRNA-seq) have opened up a new era of studies in host-pathogen interactions. Here, we first report a high-throughput cross-species dual scRNA-seq technology by using random primers to simultaneously capture both eukaryotic and bacterial RNAs (scRandom-seq). Using reference cells, scRandom-seq can detect individual eukaryotic and bacterial cells with high throughput and high specificity. Acinetobacter baumannii (A.b) is a highly opportunistic and nosocomial pathogen that displays resistance to many antibiotics, posing a significant threat to human health, calling for discoveries and treatment. In the A.b infection model, scRandom-seq witnessed polarization of THP-1 derived-macrophages and the intracellular A.b-induced ferroptosis-stress in host cells. The inhibition of ferroptosis by Ferrostatin-1 (Fer-1) resulted in the improvement of cell vitality and resistance to A.b infection, indicating the potential to resist related infections. scRandom-seq provides a high-throughput cross-species dual single-cell RNA profiling tool that will facilitate future discoveries in unraveling the complex interactions of host-microbe interactions in infection systems and tumor micro-environments.

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

There 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.

FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride.

Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions.

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed. Different genotype variants induced by mutagens are encapsulated in microemulsions and cultured to secrete proteins inside the emulsions. The secreted protein of interest is detected as a fluorescence signal by the fluorescent immunosensor quenchbody (Q-body), and a cell sorter is used to select emulsions containing improved protein-secreting strains based on the fluorescence intensity. The concept of the screening method is demonstrated by culturing Corynebacterium glutamicum in emulsions and detecting the secreted proteins. Finally, productive strains of fibroblast growth factor 9 (FGF9) are screened, and the FGF9 secretion increased threefold compared to that of parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag. This is a highly simple process that requires only the addition of a Q-body to the medium and does not require the addition of any substrates or chemical treatments. Furthermore, this method shortens the development period of industrial strains for biopharmaceutical protein production.

Photothermal Responsive Digital Polymerase Chain Reaction Resolving Exosomal microRNAs Expression in Liver Cancer.

Exosomal microRNAs have been studied as a good source of noninvasive biomarkers due to their functions in genetic exchange between cells and have been already well documented in many biological activities; however, inaccuracy remains a key challenge for liver cancer surveillance. Herein, a versatile duplex photothermal digital polymerase chain reaction (PCR) strategy combined with a lipid nanoparticle-based exosome capture approach is proposed to profile microRNAs expression through a 3-h easy-to-operate process. The microfluidically-generated molybdenum disulfide-nanocomposite-doped gelatin microcarriers display attractive properties as a 2-4 °C s-1 ramping-up rate triggered by near-infrared and reversible sol-gel transforming in step with PCR activation. To achieve PCR thermocycling, the corresponding irradiation coordinating with fan cooling are automatically performed via a homemade control module with programs. Thus, taking the multiplexing capability of dual-color labeling, 19-31 folds higher in exosomal microRNA-200b-3p and microRNA-21-5p, and tenfold lower in microRNA-22-3p expressions relative to the control microRNA-26a-5p are quantified in two liver cancer cells (Huh7 and HepG2) than in those from the healthy cells. It is believed that this exosomal microRNA genotyping method would be highly applicable for liver cancer diagnostics.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

Quantitative flow cytometry leveraging droplet-based constriction microchannels with high reliability and high sensitivity.

This study presented a quantitative flow cytometry leveraging droplet-based constriction microchannels with high reliability and high sensitivity. Droplets encapsulating single cells and even distribution of fluorescein labeled antibodies removed from targeted cells deformed through the constriction microchannel where the excited fluorescent signals were sampled and interpreted into numbers of proteins based on volume equivalence in measurement of droplets and calibration of fluorescence. To improve the detection reliability, a comprehensive analysis and comparison of multiple stripping agents such as proteinase K, guanidine hydrochloride, and urea was conducted. To improve the detection sensitivity, light modulation was used to address electrical noises and quartz microchannels were fabricated to address optical noises. As a demonstration, based on this quantitative flow cytometry of droplet microfluidics, (1) mutant p53 expressions of single cells were quantified as 1.95 ± 0.60 × 105 (ncell  = 2918 of A431) and 1.30 ± 0.70 × 105 (ncell  = 3954 of T47D); (2) single-cell expressions of Ras, c-Myc, and ß-tubulin were quantified as 1.90 ± 0.59 × 105 , 4.39 ± 1.44 × 105 , and 2.97 ± 0.81 × 105 (ncell  = 3298 of CAL 27), 1.83 ± 0.58 × 105 , 2.08 ± 0.13 × 106 , and 1.96 ± 0.74 × 105 (ncell  = 5459 of WSU-HN6). As a microfluidic tool capable of quantitatively estimating single-cell protein expressions, this methodology may provide a new quantitative perspective for the field of flow cytometry.

Hybrid Digital-Droplet Microfluidic Chip for Applications in Droplet Digital Nucleic Acid Amplification: Design, Fabrication and Characterization.

Microfluidic-based platforms have become a hallmark for chemical and biological assays, empowering micro- and nano-reaction vessels. The fusion of microfluidic technologies (digital microfluidics, continuous-flow microfluidics, and droplet microfluidics, just to name a few) presents great potential for overcoming the inherent limitations of each approach, while also elevating their respective strengths. This work exploits the combination of digital microfluidics (DMF) and droplet microfluidics (DrMF) on a single substrate, where DMF enables droplet mixing and further acts as a controlled liquid supplier for a high-throughput nano-liter droplet generator. Droplet generation is performed at a flow-focusing region, operating on dual pressure: negative pressure applied to the aqueous phase and positive pressure applied to the oil phase. We evaluate the droplets produced with our hybrid DMF-DrMF devices in terms of droplet volume, speed, and production frequency and further compare them with standalone DrMF devices. Both types of devices enable customizable droplet production (various volumes and circulation speeds), yet hybrid DMF-DrMF devices yield more controlled droplet production while achieving throughputs that are similar to standalone DrMF devices. These hybrid devices enable the production of up to four droplets per second, which reach a maximum circulation speed close to 1540 µm/s and volumes as low as 0.5 nL.

Shaking Device for Homogeneous Dispersion of Magnetic Beads in Droplet Microfluidics.

Magnetic beads (or particles) having a size between 1 and 5 µm are largely used in many biochemical assays devoted to both purification and quantification of cells, nucleic acids, or proteins. Unfortunately, the use of these beads within microfluidic devices suffers from natural precipitation because of their size and density. The strategies applied thus far to cells or polymeric particles cannot be extended to magnetic beads, mainly due to their magnetization and their higher densities. We report an effective shaking device capable of preventing the sedimentation of beads that are stored in a custom PCR tube. After the characterization of the operating principle, the device is validated for magnetic beads in droplets, leading to an equal distribution between the droplets, barely affecting their generation.