Droplet Sorting Research Articles

High‐Throughput Absorbance‐Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases

AbstractRecent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy‐intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet‐based microfluidic systems have emerged as a powerful high‐throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet‐based microfluidic systems, we present an absorbance‐activated droplet sorter that allows droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105‐member aldehyde dehydrogenase library towards D‐glyceraldehyde using a NADH mediated coupled assay that generates WST‐1 formazan as the colorimetric product. We successfully identify a variant with a 51 % improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

NOVAsort for error-free droplet microfluidics

High-throughput screening techniques are pivotal to unlocking the mysteries of biology. Yet, the promise of droplet microfluidics in enabling single-cell resolution, ultra-high-throughput screening remains largely unfulfilled. Droplet sorting errors caused by polydisperse droplet sizes that are often inevitable in multi-step assays have severely limited the effectiveness and utility of this technique, especially when screening large libraries. Even a relatively low 1% sorting error results in 10,000 false calls in a 1,000,000 droplet screen, imposing an unreasonably large burden on downstream validation. Here, we present NOVAsort (Next-generation Opto-Volume-based Accurate droplet sorter), a device capable of discerning droplets based on both size and fluorescence intensity. With a 1000- and 10,000-fold reduction in false positives and false negatives, respectively. NOVAsort addresses the challenges of conventional droplet sorting approaches and sets standards for accuracy and throughput in droplet microfluidic assays.

A cell-free bacteriophage synthesis system for directed evolution

Efficient phage production has always been an urgent need in fields such as drug discovery, disease treatment, and gene evolution. To meet this demand, we constructed a robust cell-free synthesis system for generating M13 phage by simplifying its genome, enabling a three-times faster efficiency compared with the traditional method in vivo. We further developed a cell-free directed evolution system in droplets, comprising a modified helper plasmid (ΔPS-ΔgIII-ΔgVI) and the simplified M13 genome-carrying gene mutation library. This system was greatly improved when coupled with fluorescence-activated droplet sorting (FADS). We successfully evolved the T7 RNA polymerase (RNAP), achieving a twofold higher activity to read through the T7 terminator. Moreover, we evolved the tryptophan tRNA into a suppressor tRNA with an eightfold increase in activity to read through the stop codon UAG.

Artificial intelligence-enabled multipurpose smart detection in active-matrix electrowetting-on-dielectric digital microfluidics

An active-matrix electrowetting-on-dielectric (AM-EWOD) system integrates hundreds of thousands of active electrodes for sample droplet manipulation, which can enable simultaneous, automatic, and parallel on-chip biochemical reactions. A smart detection system is essential for ensuring a fully automatic workflow and online programming for the subsequent experimental steps. In this work, we demonstrated an artificial intelligence (AI)-enabled multipurpose smart detection method in an AM-EWOD system for different tasks. We employed the U-Net model to quantitatively evaluate the uniformity of the applied droplet-splitting methods. We used the YOLOv8 model to monitor the droplet-splitting process online. A 97.76% splitting success rate was observed with 18 different AM-EWOD chips. A 99.982% model precision rate and a 99.980% model recall rate were manually verified. We employed an improved YOLOv8 model to detect single-cell samples in nanolitre droplets. Compared with manual verification, the model achieved 99.260% and 99.193% precision and recall rates, respectively. In addition, single-cell droplet sorting and routing experiments were demonstrated. With an AI-based smart detection system, AM-EWOD has shown great potential for use as a ubiquitous platform for implementing true lab-on-a-chip applications.

Droplet-based functional CRISPR screening of cell-cell interactions by SPEAC-seq.

Cell-cell interactions are essential for the function and contextual regulation of biological tissues. We present a platform for high-throughput microfluidics-supported genetic screening of functional regulators of cell-cell interactions. Systematic perturbation of encapsulated associated cells followed by sequencing (SPEAC-seq) combines genome-wide CRISPR libraries, cell coculture in droplets and microfluidic droplet sorting based on functional read-outs determined by fluorescent reporter circuits to enable the unbiased discovery of interaction regulators. This technique overcomes limitations of traditional methods for characterization of cell-cell communication, which require a priori knowledge of cellular interactions, are highly engineered and lack functional read-outs. As an example of this technique, we describe the investigation of neuroinflammatory intercellular communication between microglia and astrocytes, using genome-wide CRISPR-Cas9 inactivation libraries and fluorescent reporters of NF-κB activation. This approach enabled the discovery of thousands of microglial regulators of astrocyte NF-κB activation important for the control of central nervous system inflammation. Importantly, SPEAC-seq can be adapted to different cell types, screening modalities, cell functions and physiological contexts, only limited by the ability to fluorescently report cell functions and by droplet cultivation conditions. Performing genome-wide screening takes less than 2 weeks and requires microfluidics capabilities. Thus, SPEAC-seq enables the large-scale investigation of cell-cell interactions.

High-Efficiency Interdigitated Electrode-Based Droplet Merger for Enabling Error-Free Droplet Microfluidic Systems.

Merging two droplets into a droplet to add and mix two contents is one of the common droplet microfluidic functions with droplet generation and sorting, performing broad ranges of biological and chemical assays in droplets. However, traditional droplet-merging techniques often encounter unsynchronized droplets, causing overmerging or mis-merging, and unwanted merging outside of the desired zone. This is more severe when the incoming droplets to be merged are polydisperse in their sizes, often observed in assays that require long-term incubation, elevated-temperature, and/or multiple droplet processing steps. Here, we developed an interdigitated electrode (IDE)-based droplet merger consisting of a droplet autosynchronizing channel and a merging channel. The autosynchronizing channel provides >95% merging efficiency even when 20% polydispersity in the droplet size exists. The highly localized and enhanced dielectrophoretic force generated by the IDEs on the channel bottom allows droplet merging at an extremely low voltage (4.5 V) and only locally at the IDE region. A systematic evaluation of how various design and operation parameters of the IDE merger, such as IDE finger dimensions, dielectric coating layer thickness, droplet size, and droplet flow speed impact the performance was conducted. The optimized device showed consistent performance even when operating for up to 100 h consecutively at high throughput (100 droplets/s). The presented technology has been integrated into a droplet microfluidics workflow to test the lytic activities of bacteriophage on bacterial host cells with 100% merging efficiency. We expect this function to be integrated into droplet microfluidic systems performing broad ranges of high-throughput chemical and biological assays.

Screening of polyurethane-degrading microbes using a quenching fluorescence probe by microfluidic droplet sorting

Excessive use of polyurethane (PU) polymers has led contributed to serious environmental pollution. The plastic recycling technology using microorganisms and enzymes as catalysts offers a promising green and low-carbon approach for managing plastic waste. However, current methods for screening PU-degrading strains suffer from drawbacks such as being time-consuming and inefficient. Herein, we present a novel approach for screening PU-degrading microorganisms using a quenching fluorescent probe along with the fluorescence-activated droplet sorting (FADS). The FPAP could specifically recognize the 4,4′-methylenedianiline (MDA) derivates released from PU degradation, with fluorescence quenching as a response. Based on the approach, we successfully screen two PU-degrading strains (Burkholderia sp. W38 and Bacillus sp. C1). After 20 d of cultivation, strain W38 and C1 could degrade 41.58% and 31.45% of polyester-PU film, respectively. Additionally, three metabolites were identified during the degradation of PU monomer (2,4-toluene diamine, 2,4-TDA) and a proposed degradation pathway was established. Consequently, the fluorescence probe integrated with microfluidic droplet systems, demonstrates potential for the development of innovative PU-biocatalysts. Furthermore, the identification of the 2,4-TDA degradation pathway provides valuable insights that can propel advancements in the field of PU biodegradation.

An intelligent droplet sorter using optimized liquid-metal electrodes for droplet sorting under a low voltage

The sorting of target droplets is an essential function in droplet microfluidics. Previous dielectrophoresis-based droplet sorters were often designed based on experience and required ultrahigh driving voltages of more than 1 kV. In this study, an intelligent droplet sorter (IDS) using optimized liquid-metal electrodes was developed for droplet sorting at a low voltage of 80 V. To explore the optimal structure, we carried out numerical simulations to optimize the dimensions, shapes, and layouts of the electrodes. The optimized IDS with a square active electrode and two pairs of shielding triangular electrodes was obtained based on the normalized dielectrophoresis force ξ and comprehensive efficiency η. Subsequently, the optimized electrodes made of liquid metal were used to deflect droplets under a low driving voltage of 80 V. Finally, we integrated our IDS with an intelligent droplet detection system and successfully applied the system for the sorting of target-encapsulated and empty droplets with encapsulation and collection efficiencies of over 90 %. Our IDS offers the advantages of low voltage, easy fabrication, and excellent performance and thus shows significant potential in sorting target droplets.

High-Throughput Absorbance-Activated Droplet Sorting for Engineering Aldehyde Dehydrogenases.

Recent decades have seen a dramatic increase in the commercial use of biocatalysts, transitioning from energy-intensive traditional chemistries to more sustainable methods. Current enzyme engineering techniques, such as directed evolution, require the generation and testing of large mutant libraries to identify optimized variants. Unfortunately, conventional screening methods are unable to screen such large libraries in a robust and timely manner. Droplet-based microfluidic systems have emerged as a powerful high-throughput tool for library screening at kilohertz rates. Unfortunately, almost all reported systems are based on fluorescence detection, restricting their use to a limited number of enzyme types that naturally convert fluorogenic substrates or require the use of surrogate substrates. To expand the range of enzymes amenable to evolution using droplet-based microfluidic systems, we present an absorbance-activated droplet sorter that allows of droplet sorting at kilohertz rates without the need for optical monitoring of the microfluidic system. To demonstrate the utility of the sorter, we rapidly screen a 105-member aldehyde dehydrogenase library towards D-glyceraldehyde using a NADH mediated coupled assay that generates WST-1 formazan as the colorimetric product. We successfully identify a variant with a 51% improvement in catalytic efficiency and a significant increase in overall activity across a broad substrate spectrum.

Detachable acoustofluidic droplet-sorter

BackgroundCell sorting is crucial in isolating specific cell populations. It enables detailed analysis of their functions and characteristics and plays a vital role in disease diagnosis, drug discovery, and regenerative medicine. Fluorescence-activated cell sorting (FACS) is considered the gold standard for high-speed single-cell sorting. However, its high cost, complex instrumentation, and lack of portability are significant limitations. Additionally, the high pressure and electric fields used in FACS can harm cell integrity. In this work, an acoustofluidic device was developed in combination with surface acoustic wave (SAW) and droplet microfluidics to isolate single-cell droplets with high purity while maintaining high cell viability. ResultHuman embryonic kidney cells, transfected with fluorescent reporter plasmids, were used to demonstrate the targeted droplet sorting containing single cells. The acoustofluidic sorter achieved a recovery rate of 81 % and an accuracy rate higher than 97 %. The device maintained a cell viability rate of 95 % and demonstrated repeatability over 20 consecutive trials without compromising efficiency, thus underscoring its reliability. Thermal image analysis revealed that the temperature of the interdigital transducer (IDT) during SAW operation remained within the permissible range for maintaining cell viability. SignificanceThe findings highlighted the sensitivity and effectiveness of the developed acoustofluidic device as a tool for single-cell sorting. The detachable microfluidic chip design enables the reusability of the expensive IDT, making it cost-effective and reducing the risk of cross-contamination between different biological samples. The results underscore its capability to accurately isolate individual cells on the basis of specific criteria, showcasing its potential to advance research and clinical applications requiring precise cell sorting methodologies.

Droplet Microfluidics for High-Throughput Screening and Directed Evolution of Biomolecules.

Directed evolution is a powerful technique for creating biomolecules such as proteins and nucleic acids with tailor-made properties for therapeutic and industrial applications by mimicking the natural evolution processes in the laboratory. Droplet microfluidics improved classical directed evolution by enabling time-consuming and laborious steps in this iterative process to be performed within monodispersed droplets in a highly controlled and automated manner. Droplet microfluidic chips can generate, manipulate, and sort individual droplets at kilohertz rates in a user-defined microchannel geometry, allowing new strategies for high-throughput screening and evolution of biomolecules. In this review, we discuss directed evolution studies in which droplet-based microfluidic systems were used to screen and improve the functional properties of biomolecules. We provide a systematic overview of basic on-chip fluidic operations, including reagent mixing by merging continuous fluid streams and droplet pairs, reagent addition by picoinjection, droplet generation, droplet incubation in delay lines, chambers and hydrodynamic traps, and droplet sorting techniques. Various microfluidic strategies for directed evolution using single and multiple emulsions and biomimetic materials (giant lipid vesicles, microgels, and microcapsules) are highlighted. Completely cell-free microfluidic-assisted in vitro compartmentalization methods that eliminate the need to clone DNA into cells after each round of mutagenesis are also presented.

Spontaneous charging-induced droplets directional steering

Droplet sorting is crucial for biochemical applications involving biomedicine, chemical reactions, and material synthesis. Owing to its advantages of high precision and low sample consumption, electrical sorting technology has been widely used. Despite this, existing electrical controls rely heavily on high-voltage sources and pose the risk of cross-contamination. Consequently, we developed a self-powered, non-electrode contacted droplet-based microfluidic sorting technique, drawing inspiration from triboelectric nanogenerators. This methodology unveils an unforeseen phenomenon, wherein sliding droplets spontaneously steered on overlapping conductor area. Our investigation discerns that this droplet comportment emanates from the triboelectric charge heterogeneity of the substrate, evoking asymmetric drag and attraction forces that govern the directional steering of the droplets. Utilizing this principle, a spontaneous triboelectric induced droplet sorting method was constructed for the screening of various solutions and multi-channel sorting. This work not only introduces an innovative approach to microfluidic droplet sorting, but also augments the fundamental comprehension of droplet dynamics.

Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

High-precision screening and sorting of double emulsion droplets.

Mounting evidence suggests that cell populations are extremely heterogeneous, with individual cells fulfilling different roles within the population. Flow cytometry (FC) is a high-throughput tool for single-cell analysis that works at high optical resolution. Sub-populations with unique properties can be screened, isolated and sorted through fluorescence-activated cell sorting (FACS), using intracellular fluorescent products or surface-tagged fluorescent products of interest. However, traditional FC and FACS methods cannot identify or isolate cells that secrete extracellular products of interest. Double emulsion (DE) droplets are an innovative approach to retaining these extracellular products so cells producing them can be identified and isolated with FC and FACS. The water-in-oil-in-water structure makes DE droplets compatible with the sheath flow of flow cytometry. Single cells can be encapsulated with other reagents into DEs, which act as pico-reactors. These droplets allow biological activities to take place while allowing for cell cultivation monitoring, rare mutant identification, and cellular events characterization. However, using DEs in FACS presents technical challenges, including rupture of DEs, poor accuracy and low sorting efficiency. This study presents high-performance sorting using fluorescent beads (as simulants for cells). This study aims to guide researchers in the use of DE-based flow cytometry, offering insights into how to resolve the technical difficulties associated with DE-based screening and sorting using FC.

Directed Evolution of Escherichia coli Nissle 1917 to Utilize Allulose as Sole Carbon Source.

Sugar substitutes are popular due to their akin taste and low calories. However, excessive use of aspartame and erythritol can have varying effects. While D-allulose is presently deemed a secure alternative to sugar, its excessive consumption is not devoid of cellular stress implications. In this study, the evolution of Escherichia coli Nissle 1917 (EcN) is directed to utilize allulose as sole carbon source through a combination of adaptive laboratory evolution (ALE) and fluorescence-activated droplet sorting (FADS) techniques. Employing whole genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats interference (CRISPRi) in conjunction with compensatory expression displayed those genetic mutations in sugar and amino acid metabolic pathways, including glnP, glpF, gmpA, nagE, pgmB, ybaN, etc., increased allulose assimilation. Enzyme-substrate dynamics simulations and deep learning predict enhanced substrate specificity and catalytic efficiency in nagE A247E and pgmB G12R mutants. The findings evince that these mutations hold considerable promise in enhancing allulose uptake and facilitating its conversion into glycolysis, thus signifying the emergence of a novel metabolic pathway for allulose utilization. These revelations bear immense potential for the sustainable utilization of D-allulose in promoting health and well-being.

An integrated microfluidic platform for on-demand single droplet dispenser with high accuracy by electrohydrodynamic (EHD) printing technique

Droplet microfluidics has revolutionized single-cell analysis, allowing for ultra-high-throughput sorting of droplets to meet the demands of single-cell sorting. However, accurately and efficiently dispensing target droplets into specific containers for downstream analysis has remained a challenge. Here, we present an integrated microfluidic droplet dispenser based on the electrohydrodynamic (EHD) principle, capable of on-demand single droplet dispensing with high accuracy. Our system uses optical signal activation and EHD actuation to isolate and dispense droplets. The microfluidic droplet auto dispenser consists of four main modules: a pump-drive chip module, an optical automatic signal recognition module, a high voltage control module, and an automated X-Y translation stage module. By optimizing the chip structure and voltage parameter, this platform successfully isolates single Escherichia coli (E. coli) cells onto a Petri dish for downstream cultivation and heterogeneity analysis. In addition, the dispenser microfluidic system dispenses the sorted droplets in a "one droplet, one well" manner, providing an automated method for droplet interface from the micro- to macro- platform. This dispenser system provides a platform for phenotypic screening of single colony pick and single cell analysis

SeParate: multiway fluorescence-activated droplet sorting based on integration of serial and parallel triaging concepts.

Fluorescence-activated droplet sorting (FADS) has emerged as a versatile high-throughput sorting tool that is, unlike most fluorescence-activated cell sorting (FACS) platforms, capable of sorting droplet-compartmentalized cells, cell secretions, entire enzymatic reactions and more. Recently, multiplex FADS platforms have been developed for the sorting of multi-fluorophore populations towards different outlets in addition to the standard, more commonly used, 2-way FADS platform. These multiplex FADS platforms consist of either multiple 2-way junctions one after the other (i.e. serial sorters) or of one junction sorting droplets in more than 2 outlets (i.e. parallel sorters). In this work, we present SeParate, a novel platform based on integrating s̲e̲rial and p̲a̲r̲allel sorting principles for accura̲t̲e̲ multiplex droplet sorting that is able to mitigate limitations of current multiplex sorters. We show the SeParate platform and its capability in highly accurate 4-way sorting of a multi-fluorophore population into four subpopulations with the potential to expand to more. More specifically, the SeParate platform was thoroughly validated using mixed populations of fluorescent beads and picoinjected droplets, yielding sorting accuracies up to 100% and 99.9%, respectively. Finally, transfected HEK-293T cells were sorted employing two different optical setups, resulting in an accuracy up to 99.5%. SeParate's high accuracy for a diverse set of samples, including highly variable biological specimens, together with its scalability beyond the demonstrated 4-way sorting, warrants a broad applicability for multi-fluorophore studies in life sciences, environmental sciences and others.

A centrifugal-driven spiral microchannel microfiltration chip for emulsion and deformable particle sorting.

Droplet sorting and enrichment, as a prominent field within microfluidic technology, represent a pivotal stage in the manipulation of droplets and particles. In recent times, droplet sorting methods based on lab-on-disk (LOD) have garnered significant interest among researchers for their inherent merits, including high throughput, ease of operation, seamless device integration, and independence from supplementary driving forces. This study introduces a centrifugal force-driven microfluidic chip comprising spiral microchannels. The chip incorporates microhole arrays along the sidewall of the spiral channels, enabling size-based sorting and enrichment of microdroplets under the influence of multiple forces. Firstly, a comparative analysis was performed to assess the influence of the separation port structure and rotational speed on efficiency, and a mechanical modeling approach was employed to conduct kinetic analyses of droplet behavior during instantaneous separation. Those findings demonstrated a good agreement with the experimental results at ω < 100 rpm. Subsequently, sorting experiments on homogeneous droplets indicated that repetitive sorting could increase the recovery ratios, RT(α), of high-concentration droplets (20.7%) from 35.3% to over 80%. We also conducted a sorting experiment on three-component homogeneous-phase emulsions using a serially connected chip array, and the sorting throughput was 0.58 mL min-1. As a result, the RT(α) for 60 and 160 μm droplets were 99.4% and 88.9%, respectively. Lastly, we conducted elution experiments and dual-sample sorting on a single chip, and the fluorescence results demonstrated that this study provided an efficient and non-cross-contaminating sorting method for non-homogenous phase multi-sample microreactor units.

On-chip droplet analysis and cell spheroid screening by capillary wrapping enabled shape-adaptive ferrofluid transporters.

Non-invasive droplet manipulation with no physical damage to the sample is important for the practical value of manipulation tools in multidisciplinary applications from biochemical analysis and diagnostics to cell engineering. It is a challenge to achieve this for most existing photothermal, electric stimuli, and magnetic field-based technologies. Herein, we present a droplet handling toolbox, the ferrofluid transporter, for non-invasive droplet manipulation in an oil environment. It involves the transport of droplets with high robustness and efficiency owing to low interfacial friction. This capability caters to various scenarios including droplets with varying components and solid cargo. Moreover, we fabricated a droplet array by transporter positioning and achieved droplet gating and sorting for complex manipulation in the droplet array. Benefiting from the ease of scale-up and high biocompatibility, the transporter-based droplet array can serve as a digital microfluidic platform for on-chip droplet-based bioanalysis, cell spheroid culture, and downstream drug screening tests.

A Functional Hydrogel Bead-Based High-Throughput Screening System for Mammalian Cells with Enhanced Secretion of Therapeutic Antibodies.

Droplet-based high-throughput screening systems are an emerging technology that provides a quick test to screen millions of cells with distinctive characteristics. Biopharmaceuticals, specifically therapeutic proteins, are produced by culturing cells that secrete heterologous recombinant proteins with different populations and expression levels; therefore, a technology to discriminate cells that produce more target proteins is needed. Here, we present a droplet-based microfluidic strategy for encapsulating, screening, and selecting target cells with redox-responsive hydrogel beads (HBs). As a proof-of-concept study, we demonstrate the enrichment of hybridoma cells with enhanced capability of antibody secretion using horseradish peroxidase (HRP)-catalyzed hydrogelation of tetra-thiolate poly(ethylene glycol); hybridoma cells were encapsulated in disulfide-bonded HBs. Recombinant protein G or protein M with a C-terminal cysteine residue was installed in the HBs via disulfide bonding to capture antibodies secreted from the cells. HBs were fluorescently stained by adding the protein L-HRP conjugate using a tyramide signal amplification system. HBs were then separated by fluorescence-activated droplet sorting and degraded by reducing the disulfide bonds to recover the target cells. Finally, we succeeded in the selection of hybridoma cells with enhanced antibody secretion, indicating the potential of this system in the therapeutic protein production.