Comparative Analysis of Droplet-Based Ultra-High-Throughput Single-Cell RNA-Seq Systems.

Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.

Immune quorum sensing dictates IFN-I response dynamics in human plasmacytoid dendritic cells.

Type I interferons (IFN-Is) are key in fighting viral infections, but also serve major roles beyond antiviral immunity. Crucial is the tight regulation of IFN-I responses, while excessive levels are harmful to the cells. In essence, immune responses are generated by single cells making their own decisions, which are based on the signals they perceive. Additionally, immune cells must anticipate the future state of their environment, thereby weighing the costs and benefits of each possible outcome, in the presence of other potentially competitive decision makers (i.e., IFN-I producing cells). A rather new cellular communication mechanism called quorum sensing describes the effect of cell density on cellular secretory behaviors, which fits well with matching the right amount of IFN-Is produced to fight an infection. More competitive decision makers must contribute relatively less and vice versa. Intrigued by this concept, we assessed the effects of immune quorum sensing in pDCs, specialized immune cells known for their ability to mass produce IFN-Is. Using conventional microwell assays and droplet-based microfluidics assays, we were able the characterize the effect of quorum sensing in human primary immune cells in vitro. These insights open new avenues to manipulate IFN-I response dynamics in pathological conditions affected by aberrant IFN-I signaling.

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.

Extending Droplet-Based Microfluidic Tools to Single-Atom Heterogeneous Catalysis.

From energy-related transformations to organic syntheses, single-atom heterogeneous catalysts (SACs) are offering new prospects to tackle sustainability challenges. However, scarce design guidelines and poor mechanistic understanding due to a lack of discovery and operando characterization tools impede theirbroader development. This perspective offers a glimpse into how droplet-based microfluidic technologies mayhelp solve both of these issues, and provides technical considerations for platform design to systematically fabricate SACs and study them under operational conditions during liquid-phase organic syntheses.

Droplet-Based Microfluidics Reveals Insights into Cross-Coupling Mechanisms over Single-Atom Heterogeneous Catalysts.

Single-atom heterogeneous catalysts (SACs) hold promise as sustainable alternatives to metal complexes in organic transformations. However, their working structure and dynamics remain poorly understood, hindering advances in their design. Exploiting the unique features of droplet-based microfluidics, we present the first in-situ assessment of a palladium SAC based on exfoliated carbon nitride in Suzuki-Miyaura cross-coupling using X-ray absorption spectroscopy. Our results confirm a surface-catalyzed mechanism, revealing the distinct electronic structure of active Pd centers compared to homogeneous systems, and providing insights into the stabilizing role of ligands and bases. This study establishes a valuable framework for advancing mechanistic understanding of organic syntheses catalyzed by SACs.

µIVC-Useq: a microfluidic-assisted high-throughput functionnal screening in tandem with next generation sequencing and artificial neural network to rapidly characterize RNA molecules.

The function of an RNA is intimately linked to its three-dimensional structure. X-ray crystallography or NMR allow the fine structural characterization of small RNA (e.g., aptamers) with a precision down to atomic resolution. Yet, these technics are time consuming, laborious and do not inform on mutational robustness and the extent to which a sequence can be modified without altering RNA function, an important set of information to assist RNA engineering. On another hand, thought powerful, in silico predictions still lack the required accuracy. These limitations can be overcome by using high-throughput microfluidic-assisted functional screening technologies, as they allow exploring large mutant libraries in a rapid and cost-effective manner. Among them, we recently introduced the microfluidic-assisted In Vitro Compartmentalization (µIVC), an efficient screening strategy in which reactions are performed in picoliter droplets at rates of several thousand per second. We later improved µIVC efficiency by using in tandem with high throughput sequencing, thought a laborious bioinformatic step was still required at the end of the process. In the present work, we strongly increased the automation level of the pipeline by implementing an artificial neural network enabling unsupervised bioinformatic analysis. We demonstrate the efficiency of this "µIVC-Useq" technology by rapidly identifying a set of sequences readily accepted by a key domain of the light-up RNA aptamer SRB-2. This work not only shed some new light on the way this aptamer can be engineered, but it also allowed us to easily identify new variants with an up-to 10-fold improved performance.

Ultrahigh-throughput screening of Trichoderma reesei strains capable of carbon catabolite repression release and cellulase hyperproduction using a microfluidic droplet platform.

Trichoderma reesei is the most well-known cellulase producer in the biorefinery industry. Its cellulase biosynthesis is repressed by glucose via carbon catabolite repression (CCR), making CCR-releasing strains with cellulase hyperproduction desirable. Here, we employed a microfluidic droplet platform to culture and screen T. reesei mutants capable of CCR release and cellulase overproduction from extensive mutagenesis libraries. With 3 mutagenesis rounds, about 6.20 × 103 droplets were sorted from a population of 1.51 × 106 droplets in a period of 4.4 h; 76 recovery mutants were screened on flask fermentation, and 2 glucose uptake retarded mutants, MG-9-3 and MG-9-3-30, were eventually isolated. We also generated a hypercellulase producer, M-5, with CCR release via a single mutagenesis round. The hyphal morphology and molecular mechanisms in the mutants were analyzed. This versatile approach combined with a comprehensive understanding of CCR release mechanisms will provide innovative and effective strategies for low-cost cellulase production.

Metabolic cost of rapid adaptation of single yeast cells.

Cells can rapidly adapt to changing environments through nongenetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (1 in 1,000 cells adapt per hour) by simultaneously measuring metabolism and division of thousands of individual Saccharomyces cerevisiae cells using a droplet microfluidic system: droplets containing single cells are immobilized in a two-dimensional (2D) array, with osmotically induced changes in droplet volume being used to measure cell metabolism, while simultaneously imaging the cells to measure division. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated. Adaptation requires a characteristic amount of energy, indicating that it is an active process. The demonstration that metabolic trajectories predict a priori adaptation events provides evidence of tight energetic coupling between metabolism and regulatory reorganization in adaptation. This process allows S. cerevisiae to adapt on a physiological timescale, but related phenomena may also be important in other processes, such as cellular differentiation, cellular reprogramming, and the emergence of drug resistance in cancer.

2D MOF Nanosensor-Integrated Digital Droplet Microfluidic Flow Cytometry for In Situ Detection of Multiple miRNAs in Single CTC Cells.

Current circulating tumor cells (CTCs) detection strategies based on surface epithelial markers suffer from low specificity in distinguishing between CTCs and epithelial cells in hematopoietic cell population. Tumor-associated miRNAs within CTCs are emerging as new biomarkers due to their high correlation with tumor development and progress. However, in-situ simultaneous analysis of multiple miRNAs in single CTC cell is still challenging. To overcome this limitation, a digital droplet microfluidic flow cytometry based on biofunctionalized 2D metal-organic framework nanosensor (Nano-DMFC) is developed for in situ detection of dual miRNAs simultaneously in single living breast cancer cells. Here, 2D MOF-based fluorescent resonance energy transfer (FRET) nanosensors are established by conjugating dual-color fluorescence dye-labeled DNA probes on MOF nanosheet surface. In the Nano-DMFC, 2D MOF-based nanoprobes are precisely microinjected into each single-cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano-DMFC platform successfully detects dual miRNAs at single-cell resolution in 10 mixed positive MCF-7 cells out of 10 000 negative epithelial cells in serum biomimic samples. Moreover, this Nano-DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples, which demonstrate the high potential for CTC-based cancer early diagnosis and prognosis.

The prospects of tumor chemosensitivity testing at the single-cell level.

Tumor chemosensitivity testing plays a pivotal role in the optimal selection of chemotherapeutic regimens for cancer patients in a personalized manner. High-throughput drug screening approaches have been developed but they failed to take into account intratumor heterogeneity and therefore only provided limited predictive power of therapeutic response to individual cancer patients. Single cancer cell drug sensitivity testing (SCC-DST) has been recently developed to evaluate the variable sensitivity of single cells to different anti-tumor drugs. In this review, we discuss how SCC-DST overcomes the obstacles of traditional drug screening methodologies. We outline critical procedures of SCC-DST responsible for single-cell generation and sorting, cell-drug encapsulation on a microfluidic chip and detection of cell-drug interactions. In SCC-DST, droplet-based microfluidics is emerging as an important platform that integrated various assays and analyses for drug susceptibility tests for individual patients. With the advancement of technology, both fluorescence imaging and label-free analysis have been used for detecting single cell-drug interactions. We also discuss the feasibility of integrating SCC-DST with single-cell RNA sequencing to unravel the mechanisms leading to drug resistance, and utilizing artificial intelligence to facilitate the analysis of various omics data in the evaluation of drug susceptibility. SCC-DST is setting the stage for better drug selection for individual cancer patients in the era of precision medicine.

Droplet-Based Microfluidic Chip Design, Fabrication, and Use for Ultrahigh-Throughput DNA Analysis and Quantification.

DNA is widely used as a biomarker of contamination, infection, or disease, which has stimulated the development of a wide palette of detection and quantification methods. Even though several analytical approaches based on isothermal amplification have been proposed, DNA is still mainly detected and quantified by quantitative PCR (qPCR). However, for some analyses (e.g., in cancer research) qPCR may suffer from limitations arising from competitions between highly similar template DNAs, the presence of inhibitors, or suboptimal primer design. Nevertheless, digitalizing the analysis (i.e., individualizing DNA molecules into compartments prior to amplifying them in situ) allows to address most of these issues. By its capacity to generate and manipulate millions of highly similar picoliter volume water-in-oil droplets, microfluidics offers both the required miniaturization and parallelization capacity, and led to the introduction of digital droplet PCR (ddPCR). This chapter aims at introducing the reader to the basic principles behind ddPCR while also providing the key guidelines to fabricate, set up, and use his/her own ddPCR platform. We further provide procedures to detect and quantify DNA either purified in solution or directly from individualized cells. This approach not only gives access to DNA absolute concentration with unrivaled sensitivity, but it may also be the starting point of more complex in vitro analytical pipelines discussed at the end of the chapter.

Evolution and Single-Droplet Analysis of Fuel-Driven Compartments by Droplet-Based Microfluidics.

Active droplets are a great model for membraneless organelles. However, the analysis of these systems remains challenging and is often limited due to the short timescales of their kinetics. We used droplet-based microfluidics to encapsulate a fuel-driven cycle that drives phase separation into coacervate-based droplets to overcome this challenge. This approach enables the analysis of every coacervate-based droplet in the reaction container throughout its lifetime. We discovered that the fuel concentration dictates the formation of the coacervate-based droplets and their properties. We observed that coacervate-based droplets grow through fusion, decay simultaneously independent of their volume, and shrinkage rate scales with their initial volume. This method helps to further understand the regulation of membraneless organelles, and we believe the analysis of individual coacervate-based droplets enables future selection- or evolution-based studies.

Dynamic Actuation of DNA-Assembled Plasmonic Nanostructures in Microfluidic Cell-Sized Compartments.

Molecular motor proteins form the basis of cellular dynamics. Recently, notable efforts have led to the creation of their DNA-based mimics, which can carry out complex nanoscale motion. However, such functional analogues have not yet been integrated or operated inside synthetic cells toward the goal of realizing artificial biological systems entirely from the bottom-up. In this Letter, we encapsulate and actuate DNA-assembled dynamic nanostructures inside cell-sized microfluidic compartments. These encapsulated DNA nanostructures not only exhibit structural reconfigurability owing to their pH-sensitive molecular switches upon external stimuli but also possess optical feedback enabled by the integrated plasmonic probes. In particular, we demonstrate the power of microfluidic compartmentalization for achieving on-chip plasmonic enantiomer separation and substrate filtration. Our work exemplifies that the two unique tools, droplet-based microfluidics and DNA technology, offering high precision on the microscale and nanoscale, respectively, can be brought together to greatly enrich the complexity and diversity of functional synthetic systems.

Bottom-Up Assembly of Functional Intracellular Synthetic Organelles by Droplet-Based Microfluidics.

Bottom-up synthetic biology has directed most efforts toward the construction of artificial compartmentalized systems that recreate living cell functions in their mechanical, morphological, or metabolic characteristics. However, bottom-up synthetic biology also offers great potential to study subcellular structures like organelles. Because of their intricate and complex structure, these key elements of eukaryotic life forms remain poorly understood. Here, the controlled assembly of lipid enclosed, organelle-like architectures is explored by droplet-based microfluidics. Three types of giant unilamellar vesicles (GUVs)-based synthetic organelles (SOs) functioning within natural living cells are procedured: (A) synthetic peroxisomes supporting cellular stress-management, mimicking an organelle innate to the host cell by using analogous enzymatic modules; (B) synthetic endoplasmic reticulum (ER) as intracellular light-responsive calcium stores involved in intercellular calcium signalling, mimicking an organelle innate to the host cell but utilizing a fundamentally different mechanism; and (C) synthetic magnetosomes providing eukaryotic cells with a magnetotactic sense, mimicking an organelle that is not natural to the host cell but transplanting its functionality from other branches of the phylogenetic tree. Microfluidic assembly of functional SOs paves the way for high-throughput generation of versatile intracellular structures implantable into living cells. This in-droplet SO design may support or expand cellular functionalities in translational nanomedicine.

Droplet-based optofluidic systems for measuring enzyme kinetics.

The study of enzyme kinetics is of high significance in understanding metabolic networks in living cells and using enzymes in industrial applications. To gain insight into the catalytic mechanisms of enzymes, it is necessary to screen an enormous number of reaction conditions, a process that is typically laborious, time-consuming, and costly when using conventional measurement techniques. In recent times, droplet-based microfluidic systems have proved themselves to be of great utility in large-scale biological experimentation, since they consume a minimal sample, operate at high analytical throughput, are characterized by efficient mass and heat transfer, and offer high levels of integration and automation. The primary goal of this review is the introduction of novel microfluidic tools and detection methods for use in high-throughput and sensitive analysis of enzyme kinetics. The first part of this review focuses on introducing basic concepts of enzyme kinetics and describing most common microfluidic approaches, with a particular focus on segmented flow. Herein, the key advantages include accurate control over the flow behavior, efficient mass and heat transfer, multiplexing, and high-level integration with detection modalities. The second part describes the current state-of-the-art platforms for high-throughput and sensitive analysis of enzyme kinetics. In addition to our categorization of recent advances in measuring enzyme kinetics, we have endeavored to critically assess the limitations of each of these detection approaches and propose strategies to improve measurements in droplet-based microfluidics. Graphical abstract.

Microfluidic Production of Autofluorescent BSA Hydrogel Microspheres and Their Sequential Trapping for Fluorescence-Based On-Chip Permanganate Sensing.

Microfabrication technologies have extensively advanced over the past decades, realizing a variety of well-designed compact devices for material synthesis, separation, analysis, monitoring, sensing, and so on. The performance of such devices has been undoubtedly improved, while it is still challenging to build up a platform by rationally combining multiple processes toward practical demands which become more diverse and complicated. Here, we present a simple and effective microfluidic system to produce and immobilize a well-defined functional material for on-chip permanganate (MnO4-) sensing. A droplet-based microfluidic approach that can continuously produce monodispersed droplets in a water-in-oil system is employed to prepare highly uniform microspheres (average size: 102 µm, coefficient of variation: 3.7%) composed of bovine serum albumin (BSA) hydrogel with autofluorescence properties in the presence of glutaraldehyde (GA). Each BSA hydrogel microsphere is subsequently immobilized in a microchannel with a hydrodynamic trapping structure to serve as an independent fluorescence unit. Various anions such as Cl-, NO3-, PO43-, Br-, BrO3-, ClO4-, SCN-, HCO3-, and MnO4- are individually flowed into the microchannel, resulting in significant fluorescence quenching only in the case of MnO4-. Linear correlation is confirmed at an MnO4- concentration from 20 to 80 µM, and a limit of detection is estimated to be 1.7 µM. Furthermore, we demonstrate the simultaneous immobilization of two kinds of different microspheres in parallel microchannels, pure BSA hydrogel microspheres and BSA hydrogel microspheres containing rhodamine B molecules, making it possible to acquire two fluorescence signals (green and yellow). The present microfluidics-based combined approach will be useful to record a fingerprint of complicated samples for sensing/identification purposes by flexibly designing the size and composition of the BSA hydrogel microspheres, immobilizing them in a desired manner and obtaining a specific pattern.

A Quartz Crystal Microbalance, Which Tracks Four Overtones in Parallel with a Time Resolution of 10 Milliseconds: Application to Inkjet Printing.

A quartz crystal microbalance (QCM) is described, which simultaneously determines resonance frequency and bandwidth on four different overtones. The time resolution is 10 milliseconds. This fast, multi-overtone QCM is based on multi-frequency lockin amplification. Synchronous interrogation of overtones is needed, when the sample changes quickly and when information on the sample is to be extracted from the comparison between overtones. The application example is thermal inkjet-printing. At impact, the resonance frequencies change over a time shorter than 10 milliseconds. There is a further increase in the contact area, evidenced by an increasing common prefactor to the shifts in frequency, Δf, and half-bandwidth, ΔΓ. The ratio ΔΓ/(-Δf), which quantifies the energy dissipated per time and unit area, decreases with time. Often, there is a fast initial decrease, lasting for about 100 milliseconds, followed by a slower decrease, persisting over the entire drying time (a few seconds). Fitting the overtone dependence of Δf(n) and ΔΓ(n) with power laws, one finds power-law exponents of about 1/2, characteristic of semi-infinite Newtonian liquids. The power-law exponents corresponding to Δf(n) slightly increase with time. The decrease of ΔΓ/(-Δf) and the increase of the exponents are explained by evaporation and formation of a solid film at the resonator surface.

High-throughput droplet-based microfluidics for directed evolution of enzymes.

Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user-defined goal) coupled with droplet-based microfluidics allows the detailed analysis of millions of enzyme variants on ultra-short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet-based microfluidics, made in this context towards the high-throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell-based assays, as in the case of directed evolution experimentations.

An integrated chip-mass spectrometry and epifluorescence approach for online monitoring of bioactive metabolites from incubated Actinobacteria in picoliter droplets.

We present a lab-on-a-chip approach for the analysis of secondary metabolites produced in microfluidic droplets by simultaneous epifluorescence microscopy and electrospray ionization mass spectrometry (ESI-MS). The approach includes encapsulation and long-term off-chip incubation of microbes in surfactant-stabilized droplets followed by a transfer of droplets into a microfluidic chip for subsequent analysis. Before the reinjected droplets are spaced and electrosprayed from an integrated emitter into a mass spectrometer, the presence of fluorescent marker molecules is monitored nearly simultaneously with a custom-made portable epifluorescence microscope. This combined fluorescence and MS-detection setup allows the analysis of metabolites and fluorescent labels in a complex biological matrix at a single droplet level. Using hyphae of Streptomyces griseus, encapsulated in microfluidic droplets of ~ 200 picoliter as a model system, we show the detection of in situ produced streptomycin by ESI-MS and the feasibility of detecting fluorophores inside droplets shortly before they are electrosprayed. The presented method expands the analytical toolbox for the discovery of bioactive metabolites such as novel antibiotics, produced by microorganisms.

Dissecting enzyme function with microfluidic-based deep mutational scanning.

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.