Passive Droplet Microfluidic Platform for High-Throughput Screening of Microbial Proteolytic Activity.

Traditional bacterial isolation methods are often costly, have limited throughput, and may not accurately reflect the true microbial community composition. Consequently, identifying rare or slow-growing taxa becomes challenging. Over the past decade, a new approach has been proposed to replace traditional flasks or multiwell plates with ultrahigh-throughput droplet microfluidic screening assays.

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 10 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.

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.

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.

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.

Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Automated generation of libraries of nL droplets.

We demonstrate an integrated system for rapid and automated generation of multiple, chemically distinct populations of ~10(3)-10(4) sub-nanoliter droplets. Generation of these 'libraries of droplets' proceeds in the following automated steps: i) generation of a sequence of micro-liter droplets of individually predetermined composition, ii) injection of these 'parental' droplets onto a chip, iii) transition from a mm- to a μm-scale of the channels and splitting each of the parental drops with a flow-focusing module into thousands of tightly monodisperse daughter drops and iv) separation of such formed homogeneous populations with plugs of a third immiscible fluid. This method is compatible both with aspiration of microliter portions of liquid from a 96-well plate with a robotic station and with automated microfluidic systems that generate (~μL) droplets of preprogrammed compositions.

Characterization of Caulobacter crescentus FtsZ protein using dynamic light scattering.

The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering (DLS) spectroscopy as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP, and FtsZ monomers.

Rapid screening of antibiotic toxicity in an automated microdroplet system.

We report an automated microfluidic platform for 'digitally' screening the composition space of droplets containing cocktails of small molecules and demonstrate the features of this system by studying epistatic interactions between antibiotics and Escherichia coli ATCC 25922. This system has several key characteristics: (i) it uses small (<100 μL) samples of liquids and suspensions of bacteria that are introduced directly into the chip; (ii) it generates a sequence of droplets with compositions, including reagents and bacterial cell suspensions that are programmed by the user; (iii) it exports the sequence of droplets to an external segment of tubing that is subsequently disconnected for incubation and storage; and (iv) after incubation of bacteria in droplets, the droplets are injected into a second device equipped with an in-line fiber optic spectrophotometer that measures cell growth. The system generates and fuses droplets with precise (<1% in standard deviation) control of liquid volumes and of the concentrations of input substrates.