Fluorescence-activated Droplet Sorting Research Articles

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.

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 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.

Detection methods for polyethylene terephthalate degrading enzymes: a review

Polyethylene terephthalate (PET) is one of the most widely used synthetic polyester. It poses serious threat to terrestrial, aquatic ecosystems and human health since it is difficult to be broken down and deposited in the environment. The biodegradation based on enzymatic catalysis offers a sustainable method for recycling PET. A number of PET hydrolases have been discovered in the last 20 years, and protein engineering has increased their degradation capabilities. However, no PET hydrolases that are practical for widespread industrial use have been identified. Screening of PET hydrolase using conventional detection techniques is laborious and inefficient process. Effective detection techniques are required to promote the commercialization of PET hydrolases. Using efficient detection techniques to screen potent industrial enzymes is essential for supporting the widespread industrial implementation of PET hydrolases. To define PET hydrolase, scientists have created a number of analytical techniques recently. The detection techniques that can be used to screen PET hydrolase, including high performance liquid chromatography, ultraviolet absorption spectrometric, and fluorescence activated droplet sorting method, are summarized in this study along with their potential applications.

SpinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

Establishment of picodroplet-based co-culture system to improve erythritol production in Yarrowia lipolytica

Fluorescence-activated droplet sorting (FADS) combined with transcription factor (TF)-based biosensors allows for the high-throughput isolation of clones that overproduce extracellular metabolites. Here, we developed a FADS-TF coculture pipeline for improving erythritol production in Yarrowia lipolytica. The pipeline comprises three droplet operation steps: the generation and incubation of droplets that encapsulate yeast mutants with single-cell resolution, the pico-injection of fluorescence-based erythritol-biosensing Escherichia coli, and the sorting of the highest fluorescence droplets with an approximately 1 ‰ survival ratio. With the picodroplet-based workflow, we initially carried out a two-stage temperature and pH control strategy on the droplets to separate the erythritol production and detection processes. We then eliminated the background expression of biosensors by utilizing pH-sensitive erythromycin, finally establishing a complete version of the coculture screening system. The optimal strain, Yarrowia lipolytica S4-9, contributed 231.2 g/L erythritol after a 114-h fermentation and was successfully screened from the fourth round of the mutant library. Compared to the parent, the erythritol yield and production rate of Yarrowia lipolytica S4-9 increased by 16.97 % and 26.09 %, respectively, in a 5-L bioreactor. Our results indicated that FADS-TFs have great potential for the high-throughput screening of extracellular products as additional biosensors become available.

Direct electrification of silicon microfluidics for electric field applications

Microfluidic systems are widely used in fundamental research and industrial applications due to their unique behavior, enhanced control, and manipulation opportunities of liquids in constrained geometries. In micrometer-sized channels, electric fields are efficient mechanisms for manipulating liquids, leading to deflection, injection, poration or electrochemical modification of cells and droplets. While PDMS-based microfluidic devices are used due to their inexpensive fabrication, they are limited in terms of electrode integration. Using silicon as the channel material, microfabrication techniques can be used to create nearby electrodes. Despite the advantages that silicon provides, its opacity has prevented its usage in most important microfluidic applications that need optical access. To overcome this barrier, silicon-on-insulator technology in microfluidics is introduced to create optical viewports and channel-interfacing electrodes. More specifically, the microfluidic channel walls are directly electrified via selective, nanoscale etching to introduce insulation segments inside the silicon device layer, thereby achieving the most homogeneous electric field distributions and lowest operation voltages feasible across microfluidic channels. These ideal electrostatic conditions enable a drastic energy reduction, as effectively shown via picoinjection and fluorescence-activated droplet sorting applications at voltages below 6 and 15 V, respectively, facilitating low-voltage electric field applications in next-generation microfluidics.

Modulating Sensitivity of an Erythromycin Biosensor for Precise High-Throughput Screening of Strains with Different Characteristics.

Genetically encoded biosensors are powerful tools for product-driven high-throughput screening in synthetic biology and metabolic engineering. However, most biosensors can only properly function in a limited concentration cutoff, and the incompatible performance characteristics of biosensors will lead to false positives or failure in screening. The transcription factor (TF)-based biosensors are usually organized in modular architecture and function in a regulator-depended manner, whose performance properties can be fine-tuned by modifying the expression level of the TF. In this study, we modulated the performance characteristics, including sensitivity and operating range, of an MphR-based erythromycin biosensor by fine-adjusting regulator expression levels via ribosome-binding site (RBS) engineering and obtained a panel of biosensors with varied sensitivities by iterative fluorescence-assisted cell sorting (FACS) in Escherichia coli to accommodate different screening purposes. To exemplify their application potential, two engineered biosensors with 10-fold different sensitivities were employed in the precise high-throughput screening by microfluidic-based fluorescence-activated droplet sorting (FADS) of Saccharopolyspora erythraea mutant libraries with different starting erythromycin productions, and mutants representing as high as 6.8 folds and over 100% of production improvements were obtained starting from the wild-type strain and the high-producing industrial strain, respectively. This work demonstrated a simple strategy to engineer biosensor performance properties, which was significant to stepwise strain engineering and production improvement.

ISort enables automated complex microfluidic droplet sorting in an effort to democratize technology

Fluorescence-activated droplet sorting (FADS) is a widely used microfluidic technique for high-throughput screening. However, it requires highly trained specialists to determine optimal sorting parameters, and this results in a large combinatorial space that is challenging to optimize systematically. Additionally, it is currently challenging to track every single droplet within a screen, leading to compromised sorting and "hidden" false-positive events. To overcome these limitations, we have developed a setup in which the droplet frequency, spacing, and trajectory at the sorting junction are monitored in real time using impedance analysis. The resulting data are used to continuously optimize all parameters automatically and to counteract perturbations, resulting in higher throughput, higher reproducibility, increased robustness, and a beginner-friendly character. We believe this provides a missing piece for the spreading of phenotypic single-cell analysis methods, similar to what we have seen for single-cell genomics platforms.

Label-free droplet-based bacterial growth phenotype screening by a mini integrated microfluidic platform

Rapid and accurate microbial screening, which is important in industrial breeding, enzyme directed evolution and synthetic biology, is currently time-consulting. Fluorescence-activated droplet sorting (FADS) system offers a promising alternative for microbial screening with high-throughput analysis. However, pre-labeling steps are required before use. Here, a low-cost, label-free droplet-based mini integrated microfluidic platform is developed for bacterial growth phenotype screening based on the difference in droplet autofluorescence properties. The platform integrates the main functions into a box, in which droplets are passed through the detection area and transmitted the signal of the light directly to the photomultiplier tubes (PMT) through the optical fiber inserted in a specially designed chip. After optimizing the chip structure and parameter, we firstly verify the capabilities of this platform for bacteria automatic counting. Then, label-free counting and sorting of the cultured bacteria in droplet have been well performed in this platform with the support of a voltage amplifier. We also employee this platform to determine the growth phenotype for microbial strain in droplets and screen the fast-growth bacteria in an automatic way. The label-free droplet-based platform provides an automated method for rapid bacteria growth phenotype screening, which can be further employed in high quality industrial strains screening, antibiotic resistance and directed evolution.

Droplet-based forward genetic screening of astrocyte-microglia cross-talk.

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Ultra-High-Throughput Absorbance-Activated Droplet Sorting for Enzyme Screening at Kilohertz Frequencies.

Droplet microfluidics is a valuable method to "beat the odds" in high throughput screening campaigns such as directed evolution, where valuable hits are infrequent and large library sizes are required. Absorbance-based sorting expands the range of enzyme families that can be subjected to droplet screening by expanding possible assays beyond fluorescence detection. However, absorbance-activated droplet sorting (AADS) is currently ∼10-fold slower than typical fluorescence-activated droplet sorting (FADS), meaning that, in comparison, a larger portion of sequence space is inaccessible due to throughput constraints. Here we improve AADS to reach kHz sorting speeds in an order of magnitude increase over previous designs, with close-to-ideal sorting accuracy. This is achieved by a combination of (i) the use of refractive index matching oil that improves signal quality by removal of side scattering (increasing the sensitivity of absorbance measurements); (ii) a sorting algorithm capable of sorting at this increased frequency with an Arduino Due; and (iii) a chip design that transmits product detection better into sorting decisions without false positives, namely a single-layered inlet to space droplets further apart and injections of "bias oil" providing a fluidic barrier preventing droplets from entering the incorrect sorting channel. The updated ultra-high-throughput absorbance-activated droplet sorter increases the effective sensitivity of absorbance measurements through better signal quality at a speed that matches the more established fluorescence-activated sorting devices.

Translating the Genome into Drugs.

The Human Genome Project ultimately aimed to translate DNA sequence into drugs. With the draft in hand, the Molecular Libraries Program set out to prosecute all genome-encoded proteins for drug discovery with automated high-throughput screening (HTS). This ambitious vision remains unfulfilled, even while innovations in sequencing technology have fully democratized access to genome-scale sequencing. Why? While the central dogma of biology allows us to chart the entirety of cellular metabolism through sequencing, there is no direct coding for chemistry. The rules of base pairing that relate DNA gene to RNA transcript and amino acid sequence do not exist for relating small-molecule structure with macromolecular binding partners and subsequently cellular function. Obtaining such relationships genome-wide is unapproachable via state-of-the-art HTS, akin to attempting genome-wide association studies using turn-of-the-millennium Sanger DNA sequencing.Our laboratory has been engaged in a multipronged technology development campaign to revolutionize molecular screening through miniaturization in pursuit of genome-scale drug discovery capabilities. The compound library was ripe for miniaturization: it clearly needed to become a consumable. We employed DNA-encoded library (DEL) synthesis principles in the development of solid-phase DELs prepared on microscopic beads, each harboring 100 fmol of a single library member and a DNA tag whose sequence describes the structure of the library member. Loading these DEL beads into 100 pL microfluidic droplets followed by online photocleavage, incubation, fluorescence-activated droplet sorting, and DNA sequencing of the sorted DEL beads reveals the chemical structures of bioactive compounds. This scalable library synthesis and screening platform has proven useful in several proof-of-concept projects involving current clinical targets.Moving forward, we face the problem of druggability and proteome-scale assay development. Developing biochemical or cellular assays for all genome-encoded targets is not scalable and likely impossible as most proteins have ill-defined or unknown activity and may not function outside of their native contexts. These are the dark undruggable expanses, and charting them will require advanced synthesis and analytical technologies that can generalize probe discovery, irrespective of mature protein function, to fulfill the Genome Project's vision of proteome-wide control of cellular pharmacology.

Single-cell sorting using integrated pneumatic valve droplet microfluidic chip

Single-cell analysis and sorting have been increasingly prevalent in scientific study and industry in recent years. Droplet-based microfluidic systems are significant tools for single cell separation due to the advantages of high-throughput screening flux and relatively simple chip manufacturing. Herein, this work integrated single-cell droplet generation, incubation, and sorting functions into a single microfluidic chip using pneumatic microvalve for single-cell screening of hybridoma cells. The chip realized the generation, storage, incubation, and assay sorting of droplets in order by adjusting the switch of the microvalve. Numerical methods were used to simulate the effects of membrane size and pressure on valve deformation. Incubation results showed that fluorescent signals were generated in the droplets containing both hybridoma cells and microspheres under the control of pneumatic microvalves. In addition, the purity of target droplets was increased from 4.87% before sorting to 89.70% after sorting using fluorescence-activated droplet sorting platform, demonstrating the feasibility of the integrated chip with a pneumatic valve for single-cell screening. The integrated chip is proved to be operated efficiently with minimum cell loss and human labor through reducing the risk of droplet fusion and facilitating the development of automated devices. So, the present study provides an integrated design for microdroplet-based single-cell sorting with various applications, such as single-cell sequencing, antibody screening, and directed evolutionary studies.

Combining genetically encoded biosensors with droplet microfluidic system for enhanced glutaminase production by Bacillus amyloliquefaciens

A high-throughput screening technology for single-cell by fluorescence-activated droplet sorting (FADS) has been applied to improve industrial enzymes and production strains, which usually require fluorophore-modified substrates to measure enzyme activity in microfluidic droplets. However, the difficulty of developing fluorophore-modified substrates limits their further application. Here, we employed the intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) within microfluidic droplet sorting for screening Bacillus amyloliquefaciens variants with high glutaminase production. A rapid microplate assay based on the biosensor was developed to quantify glutaminase activity over 700-fold faster than high-performance liquid chromatography (HPLC). The novel FADS workflow was established subsequently for high-throughput screening of glutaminase-producing strains and achieved an enrichment ratio of 56 times. Furthermore, a 105 whole-genome mutant library was sorted, obtaining a clone that over 47 % increase in glutaminase production. Compared with the microtiter plate screening system, the biosensor-based microfluidic platform can give a 5 × 102 screening rate enhancement and decrease reagent consumption by 1.8 × 104 times. This strategy based on genetically encoded fluorescent biosensors will accelerate the application of FADS to the directed evolution of industrial enzymes and cell factories.

Application of a novel fluorogenic polyurethane analogue probe in polyester-degrading microorganisms screening by microfluidic droplet.

Application of polyester-degrading microorganisms or enzymes should be considered as an eco-friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. However, current impranil DLN-based screening of polyester-degrading microorganisms is time-consuming, labour-intensive and unable to distinguish polyesterases from other protease- or amidase-like enzymes. Herein, we present an approach that combined a novel synthetic fluorescent polyurethane analogue probe (FPAP), along with the droplet-based microfluidics to screen polyurethane-degrading microorganisms through fluorescence-activated droplet sorting (FADS) pipeline. The fluorescent probe FPAP exhibited a fluorescence enhancement effect once hydrolysed by polyesterases, along with a strong specificity in discriminating polyesterases from other non-active enzymes. Application of FPAP in a microfluidic droplet system demonstrated that this probe exhibited high sensitivity and efficiency in selecting positive droplets containing leaf-branch compost cutinase (LCC) enzymes. This novel fluorogenic probe, FPAP, combined with the droplet microfluidic system has the potential to be used in the exploitation of novel PUR-biocatalysts for biotechnological and environmental applications.

Transcription-Associated Fluorescence-Activated Droplet Sorting for Di-rhamnolipid Hyperproducers.

Rhamnolipids (RLs) are biosurfactants with great economic significance that have been used extensively in multiple industries. Pseudomonas aeruginosa is a promising microorganism for sustainable RL production. However, current CTAB-MB based screening of RL-producing strains is time-consuming, labor-intensive, and unable to distinguish mono- and di-RL. In this study, we developed a novel transcription-associated fluorescence-activated droplet sorting (FADS) method to specifically target the di-RL hyperproducers. We first investigated critical factors associated with this method, including the specificity and sensitivity for discriminating di-RL overproducers from other communities. Validation of genotype-phenotype linkage between the GFP intensity, rhlC transcription, and di-RL production showed that rhlC transcription is closely correlated with di-RL production, and the GFP intensity is responsive to rhlC transcription, respectively. Using this platform, we screened out ten higher di-RL producing microorganisms, which produced 54-208% more di-RL than the model P.aeruginosa PAO1. In summary, the droplet-based microfluidic platform not only facilitates a more specific, reliable, and rapid screening of P.aeruginosa colonies with desired phenotypes, but also shows that intracellular transcription-associated GFP intensity can be used to measure the yield of di-RL between populations of droplets containing different environmental colonies. This method also can be integrated with transposon mutation libraries to target P.aeruginosa mutants.

Ultrahigh-throughput screening of industrial enzyme-producing strains by droplet-based microfluidic system.

Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultrahigh throughput, but its widespread application remains limited by the accessibility of a droplet microfluidic high-throughput screening (HTS) platform, especially to common laboratories having no background in microfluidics. Here, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; and (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed, and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing Bacillus licheniformis strains from a mutant library generated by atmospheric and room temperature plasma mutagenesis, and clones displaying over 50% improvement in α-amylase productivity were isolated. This droplet screening system could be applied to the engineering of other industrially valuable strains.

High-throughput screening of cell-free riboswitches by fluorescence-activated droplet sorting

Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets.

7-Aminocoumarin-4-acetic Acid as a Fluorescent Probe for Detecting Bacterial Dipeptidyl Peptidase Activities in Water-in-Oil Droplets and in Bulk.

Droplet-based microfluidic systems are a powerful tool for biological assays with high throughput. Water-in-oil droplets (WODLs) are typically used in droplet-based microfluidic systems to culture microorganisms and perform enzyme assays. However, because of the oil surrounding the nanoliter and picoliter volumes of WODLs, availability of suitable substrates is limited. For instance, although 7-amino-4-methylcoumarin (AMC) is commonly used as a fluorescent probe of the substrate to detect peptidase activity, AMC leaks from WODLs to the oil phase due to its high hydrophobicity. Thus, AMC substrates cannot be used in droplet-based microfluidic systems with WODLs. In this study, we developed a peptidase substrate consisting of a dipeptide and 7-aminocoumarin-4-acetic acid (ACA), an AMC-derived fluorogenic compound. ACA was retained in the WODL for more than 7 days, and the dipeptidyl ACA substrate detected dipeptidyl peptidase (DPP) activity in the WODL. Compared to AMC substrates, the substrate specificity constants of DPPs for ACA substrates increased up to 4.7-fold. Fluorescence-activated droplet sorting made high-throughput screening of microorganisms based on DPP activity using the dipeptidyl ACA substrate possible. Since ACA could be applied to various substrates as a fluorescent probe, detectable microbial enzyme activities for droplet-based microfluidic systems can be largely expanded.