Ultrahigh Throughput Evolution of Tryptophan Synthase in Droplets via an Aptamer Sensor.

Tryptophan synthase catalyzes the synthesis of a wide array of noncanonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (up to 107 experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic readouts, which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a sensor to quantitatively report on tryptophan production in droplets. The utility of the sensor was validated by identifying five-fold improved tryptophan synthases from ∼100,000 protein variants. More generally, this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.

Droplet microfluidic system for high throughput and passive selection of bacteria producing biosurfactants.

Traditional methods for the enrichment of microorganisms rely on growth in a selective liquid medium or on an agar plate, followed by tedious characterization. Droplet microfluidic techniques have been recently used to cultivate microorganisms and preserve enriched bacterial taxonomic diversity. However, new methods are needed to select droplets comprising not only growing microorganisms but also those exhibiting specific properties, such as the production of value-added compounds. We describe here a droplet microfluidic screening technique for the functional selection of biosurfactant-producing microorganisms, which are of great interest in the bioremediation and biotechnology industries. Single bacterial cells are first encapsulated into picoliter droplets for clonal cultivation and then passively sorted at high throughput based on changes in interfacial tension in individual droplets. Our method expands droplet-based microbial enrichment with a novel approach that reduces the time and resources needed for the selection of surfactant-producing bacteria.

Versatile Product Detection via Coupled Assays for Ultrahigh-Throughput Screening of Carbohydrate-Active Enzymes in Microfluidic Droplets.

Enzyme discovery and directed evolution are the two major contemporary approaches for the improvement of industrial processes by biocatalysis in various fields. Customization of catalysts for improvement of single enzyme reactions or de novo reaction development is often complex and tedious. The success of screening campaigns relies on the fraction of sequence space that can be sampled, whether for evolving a particular enzyme or screening metagenomes. Ultrahigh-throughput screening (uHTS) based on in vitro compartmentalization in water-in-oil emulsion of picoliter droplets generated in microfluidic systems allows screening rates >1 kHz (or >107 per day). Screening for carbohydrate-active enzymes (CAZymes) catalyzing biotechnologically valuable reactions in this format presents an additional challenge because the released carbohydrates are difficult to monitor in high throughput. Activated substrates with large optically active hydrophobic leaving groups provide a generic optical readout, but the molecular recognition properties of sugars will be altered by the incorporation of such fluoro- or chromophores and their typically higher reactivity, as leaving groups with lowered pKa values compared to native substrates make the observation of promiscuous reactions more likely. To overcome these issues, we designed microdroplet assays in which optically inactive carbohydrate products are made visible by specific cascades: the primary reaction of an unlabeled substrate leads to an optical signal downstream. Successfully implementing such assays at the picoliter droplet scale allowed us to detect glucose, xylose, glucuronic acid, and arabinose as final products of complex oligosaccharide degradation by glycoside hydrolases by absorbance measurements. Enabling the use of uHTS for screening CAZyme reactions that have been thus far elusive will chart a route toward faster and easier development of specific and efficient biocatalysts for biovalorization, directing enzyme discovery by challenging catalysts for reaction with natural rather than model substrates.

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.

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.

Ultrahigh-Throughput Directed Evolution of a Metal-Free α/ß-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase.

Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91's activity by a factor of ≈360 (to a kcat/KM of ≈7 × 105 M-1 s-1) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.

Vector redesign and in-droplet cell-growth improves enrichment and recovery in live Escherichia coli.

Directed evolution (DE) is a widely used method for improving the function of biomolecules via multiple rounds of mutation and selection. Microfluidic droplets have emerged as an important means to screen the large libraries needed for DE, but this approach was so far partially limited by the need to lyse cells, recover DNA, and retransform into cells for the next round, necessitating the use of a high-copy number plasmid or oversampling. The recently developed live cell recovery avoids some of these limitations by directly regrowing selected cells after sorting. However, repeated sorting cycles used to further enrich the most active variants ultimately resulted in unfavourable recovery of empty plasmid vector-containing cells over those expressing the protein of interest. In this study, we found that engineering of the original expression vector solved the problem of false positives (i.e. plasmids lacking an insert) cells containing empty vectors. Five approaches to measure activity of cell-displayed enzymes in microdroplets were compared. By comparing various cell treatment methods prior to droplet sorting two things were found. Substrate encapsulation from the start, that is prior to expression of enzyme, showed no disadvantage to post-induction substrate addition by pico-injection with respect to recovery of true positive variants. Furthermore in-droplet cell growth prior to induction of enzyme production improves the total amount of cells retrieved (recovery) and proportion of true positive variants (enrichment) after droplet sorting.

High-throughput total RNA sequencing in single cells using VASA-seq.

Most methods for single-cell transcriptome sequencing amplify the termini of polyadenylated transcripts, capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts and hinders alternative splicing analysis. We, therefore, developed VASA-seq to detect the total transcriptome in single cells, which is enabled by fragmenting and tailing all RNA molecules subsequent to cell lysis. The method is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to more than 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Analyzing the dynamics of the total single-cell transcriptome, we discovered cell type markers, many based on non-coding RNA, and performed in vivo cell cycle analysis via detection of non-polyadenylated histone genes. RNA velocity characterization was improved, accurately retracing blood maturation trajectories. Moreover, our VASA-seq data provide a comprehensive analysis of alternative splicing during mammalian development, which highlighted substantial rearrangements during blood development and heart morphogenesis.

Ultrahigh-Throughput Detection of Enzymatic Alcohol Dehydrogenase Activity in Microfluidic Droplets with a Direct Fluorogenic Assay.

The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh-throughput assays that use monodisperse water-in-oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8-hydroxy-1,3,6-pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for >6 weeks (i. e. 14-times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100 nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an >800-fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.

Ultrahigh throughput screening for enzyme function in droplets.

Water-in-oil droplets, made and handled in microfluidic devices, provide a new experimental format, in which ultrahigh throughput experiments can be conducted faster and with minimal reagent consumption. An increasing number of studies have emerged that applied this approach to directed evolution and metagenomic screening of enzyme catalysts. Here, we review the considerations necessary to implement robust workflows, based on choices of device design, detection modes, emulsion formulations and substrates, and scope out which enzyme classes have become amenable to droplet screening.

Gravity-driven microfluidic assay for digital enumeration of bacteria and for antibiotic susceptibility testing.

The alarming dynamics of antibiotic-resistant infections calls for the development of rapid and point-of-care (POC) antibiotic susceptibility testing (AST) methods. Here, we demonstrated the first completely stand-alone microfluidic system that allowed the execution of digital enumeration of bacteria and digital antibiograms without any specialized microfluidic instrumentation. A four-chamber gravity-driven step emulsification device generated ∼2000 monodisperse 2 nanoliter droplets with a coefficient of variation of 8.9% of volumes for 95% of droplets within less than 10 minutes. The manual workload required for droplet generation was limited to the sample preparation, the deposition into the sample inlet of the chip and subsequent orientation of the chip vertically without an additional pumping system. The use of shallow chambers imposing a 2D droplet arrangement provided superior stability of the droplets against coalescence and minimized the leakage of the reporter viability dye between adjacent droplets during long-term culture. By using resazurin as an indicator of the growth of bacteria, we were also able to reduce the assay time to ∼5 hours compared to 20 hours using the standard culture-based test.

Single-cell activity screening in microfluidic droplets.

Water-in-oil emulsion droplets can be used as microcompartments to contain single cells that can be subjected to activity assays in this format. Microfluidic devices produce droplets at > kHz rates and can be coupled to modules to, e.g., add reagents, incubate or measure analyte concentration optically (with sensitivities as low as 2nM). The range of optical assays includes fluorescence and absorbance detection and examples for the use of these technologies for ultrahigh-throughput sorting in directed evolution and functional metagenomics are described.

Microfluidic screening of antibiotic susceptibility at a single-cell level shows the inoculum effect of cefotaxime on E. coli.

Measurement of antibiotic susceptibility at the level of single cells is important as it reveals the concentration of an antibiotic that leads to drug resistance in bacterial strains. To date, no solution for large-scale studies of antibiotic susceptibility at the single-cell level has been shown. Here, we present a method for production and separation of emulsions consisting of subnanoliter droplets that allows us to identify each emulsion by their spatial position in the train of emulsions without chemical barcoding. The emulsions of droplets are separated by a third immiscible phase, thus forming large compartments-tankers-each filled with an emulsion of droplet reactors. Each tanker in a train can be set under different reaction conditions for hundreds or thousands of replications of the same reaction. The tankers allow for long term incubation - needed to check for growth of bacteria under a screen of conditions. We use microfluidic tankers to analyze susceptibility to cefotaxime in ca. 1900 replications for each concentration of the antibiotic in one experiment. We test cefotaxime susceptibility for different initial concentrations of bacteria, showing the inoculum effect down to the level of single cells for more than a hundred single-cell events per tanker. Lastly, we use tankers to observe the formation of aggregates of bacteria in the presence of cefotaxime in the increasing concentration of the antibiotic. The microfluidic tankers allow for facile studies of the inoculum effect and antibiotic susceptibility, and constitute an attractive, label-free screening method for a variety of other experiments in chemistry and biology.

Droplet microfluidics for microbiology: techniques, applications and challenges.

Droplet microfluidics has rapidly emerged as one of the key technologies opening up new experimental possibilities in microbiology. The ability to generate, manipulate and monitor droplets carrying single cells or small populations of bacteria in a highly parallel and high throughput manner creates new approaches for solving problems in diagnostics and for research on bacterial evolution. This review presents applications of droplet microfluidics in various fields of microbiology: i) detection and identification of pathogens, ii) antibiotic susceptibility testing, iii) studies of microbial physiology and iv) biotechnological selection and improvement of strains. We also list the challenges in the dynamically developing field and new potential uses of droplets in microbiology.

Dodecylresorufin (C12R) Outperforms Resorufin in Microdroplet Bacterial Assays.

This paper proves that dodecylresorufin (C12R) outperforms resorufin (the conventional form of this dye) in droplet microfluidic bacterial assays. Resorufin is a marker dye that is widely used in different fields of microbiology and has increasingly been applied in droplet microfluidic assays and experiments. The main concern associated with resorufin in droplet-based systems is dye leakage into the oil phase and neighboring droplets. The leakage decreases the performance of assays because it causes averaging of the signal between the positive (bacteria-containing) and negative (empty) droplets. Here we show that C12R is a promising alternative to conventional resorufin because it maintains higher sensitivity, specificity, and signal-to-noise ratio over time. These characteristics make C12R a suitable reagent for droplet digital assays and for monitoring of microbial growth in droplets.

Antibiograms in five pipetting steps: precise dilution assays in sub-microliter volumes with a conventional pipette.

We demonstrate a standalone microfluidic chip that allows us to carry out commonly executed antibiotic susceptibility assays in an array of nanoliter droplets. We eliminated the need for automation in performing an exemplary complicated liquid handling assay on a chip. Operations on droplets are hard-wired into the microfluidic chip. The liquid handling protocol can be executed with a simple and commonly available source of flow such as an automatic manual pipette. The system passively prepares a series of dilutions of a chemical compound and mixes them with portions of the sample. The precision of metering, merging, mixing, and splitting of discrete portions of liquid samples is rooted in the passive capillary action in microfluidic traps and not in the precision of dosing with a pipette. We show an exemplary use of the device in the determination of the minimum inhibitory concentration (MIC) of ampicillin against E. coli ATCC 25922.

Rational design of digital assays.

Optimum algorithm for digital assays treats chemical compartments as bits of probabilistic information and arranges these bits in a fractional positional system. Maximization of information gain reduces, by orders of magnitude, the number of partitions required to achieve the requested dynamic range and precision of the assay. The method simplifies the execution of digital analytical methods providing for more accessible use of absolute quantization in research and in diagnostics.

A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings.

This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements.