High-throughput bacterial co-encapsulation in microfluidic gel beads for discovery of antibiotic-producing strains.

Bacteria with antagonistic activity inhibit the growth of other bacteria through different mechanisms, including the production of antibiotics. As a result, these microorganisms are a prolific source of such compounds. However, searching for antibiotic-producing strains requires high-throughput techniques due to the vast diversity of microorganisms. Here, we screened and isolated bacteria with antagonistic activity against Escherichia coli expressing the green fluorescent protein (E. coli-GFP). We used microfluidics to co-encapsulate and co-culture single cells from different strains within picoliter gel beads and analyzed them using fluorescence-activated cell sorting (FACS). To test the methodology, we used three bacterial isolates obtained from Mexican maize, which exhibit high, moderate, or no antagonistic activity against E. coli-GFP, as determined previously using agar plate assays. Single cells from each strain were separately co-incubated into gel beads with E. coli-GFP. We monitored the development of the maize bacteria microcolonies and tracked the growth or inhibition of E. coli-GFP using bright-field and fluorescent microscopy. We correlated these images with distinctive light scatter and fluorescence signatures of each incubated bead type using FACS. This analysis enabled us to sort gel beads filled with an antagonistic strain, starting from a mixture of the three different types of maize bacteria and E. coli-GFP. Likewise, culturing the FACS-sorted beads on agar plates confirmed the isolation and recovery of the two antagonistic strains. In addition, enrichment assays demonstrated the methodology's effectiveness in isolating rare antibiotic-producer strains (0.01% abundance) present in a mixture of microorganisms. These results show that associating light side scatter and fluorescent flow cytometry signals with microscopy images provides valuable controls to establish successful high-throughput methods for sorting beads in which microbial interaction assays are performed.

Prebiotic Chemistry Experiments Using Microfluidic Devices.

Microfluidic devices are small tools mostly consisting of one or more channels, with dimensions between one and hundreds of microns, where small volumes of fluids are manipulated. They have extensive use in the biomedical and chemical fields; however, in prebiotic chemistry, they only have been employed recently. In prebiotic chemistry, just three types of microfluidic devices have been used: the first ones are Y-form devices with laminar co-flow, used to study the precipitation of minerals in hydrothermal vents systems; the second ones are microdroplet devices that can form small droplets capable of mimic cellular compartmentalization; and the last ones are devices with microchambers that recreate the microenvironment inside rock pores under hydrothermal conditions. In this review, we summarized the experiments in the field of prebiotic chemistry that employed microfluidic devices. The main idea is to incentivize their use and discuss their potential to perform novel experiments that could contribute to unraveling some prebiotic chemistry questions.

Ultrasound-assisted production and optimization of mini-emulsions in a microfluidic chip in continuous-flow.

The use of ultrasound to generate mini-emulsions (50 nm to 1 µm in diameter) and nanoemulsions (mean droplet diameter < 200 nm) is of great relevance in drug delivery, particle synthesis and cosmetic and food industries. Therefore, it is desirable to develop new strategies to obtain new formulations faster and with less reagent consumption. Here, we present a polydimethylsiloxane (PDMS)-based microfluidic device that generates oil-in-water or water-in-oil mini-emulsions in continuous flow employing ultrasound as the driving force. A Langevin piezoelectric attached to the same glass slide as the microdevice provides enough power to create mini-emulsions in a single cycle and without reagents pre-homogenization. By introducing independently four different fluids into the microfluidic platform, it is possible to gradually modify the composition of oil, water and two different surfactants, to determine the most favorable formulation for minimizing droplet diameter and polydispersity, employing less than 500 µL of reagents. It was found that cavitation bubbles are the most important mechanism underlying emulsions formation in the microchannels and that degassing of the aqueous phase before its introduction to the device can be an important factor for reduction of droplet polydispersity. This idea is demonstrated by synthetizing solid polymeric particles with a narrow size distribution starting from a mini-emulsion produced by the device.

Droplet-Based Microfluidics Methods for Detecting Enzyme Inhibitors.

Sub-nanoliter droplets produced in microfluidic devices have gained an enormous importance for performing all kinds of biochemical assays. One of the main reasons is that the amounts of reagents employed can be reduced in approximately five orders of magnitude compared to conventional microplate assays. In this chapter, we describe how to carry out the design, fabrication, and operation of a microfluidic device that allows performing enzyme kinetics and enzyme inhibition assays in droplets. This procedure can be used effectively to screen a small size library of compounds. Then, we describe how to use this droplet microfluidic setup to screen for potential inhibitor compounds eluted from a coupled high-performance liquid chromatography (HPLC) system that separates crude natural extracts.

Detection of Enzyme Inhibitors in Crude Natural Extracts Using Droplet-Based Microfluidics Coupled to HPLC.

Natural product screening for new bioactive compounds can greatly benefit from low reagents consumption and high throughput capacity of droplet-based microfluidic systems. However, the creation of large droplet libraries in which each droplet carries a different compound is a challenging task. A possible solution is to use an HPLC coupled to a droplet generating microfluidic device to sequentially encapsulate the eluting compounds. In this work we demonstrate the feasibility of carrying out enzyme inhibiting assays inside nanoliter droplets with the different components of a natural crude extract after being separated by a coupled HPLC column. In the droplet formation zone, the eluted components are mixed with an enzyme and a fluorogenic substrate that permits to follow the enzymatic reaction in the presence of each chromatographic peak and identify those inhibiting the enzyme activity. Using a fractal shape channel design and automated image analysis, we were able to identify inhibitors of Clostridium perfringens neuraminidase present in a root extract of the Pelargonium sidoides plant. This work demonstrates the feasibility of bioprofiling a natural crude extract after being separated in HPLC using microfluidic droplets online and represents an advance in the miniaturization of natural products screening.

Isothermal titration calorimetry determination of individual rate constants of trypsin catalytic activity.

Determination of individual rate constants for enzyme-catalyzed reactions is central to the understanding of their mechanism of action and is commonly obtained by stopped-flow kinetic experiments. However, most natural substrates either do not fluoresce/absorb or lack a significant change in their spectra while reacting and, therefore, are frequently chemically modified to render adequate molecules for their spectroscopic detection. Here, isothermal titration calorimetry (ITC) was used to obtain Michaelis-Menten plots for the trypsin-catalyzed hydrolysis of several substrates at different temperatures (278-318K): four spectrophotometrically blind lysine and arginine N-free esters, one N-substituted arginine ester, and one amide. A global fitting of these data provided the individual rate constants and activation energies for the acylation and deacylation reactions, and the ratio of the formation and dissociation rates of the enzyme-substrate complex, leading also to the corresponding free energies of activation. The results indicate that for lysine and arginine N-free esters deacylation is the rate-limiting step, but for the N-substituted ester and the amide acylation is the slowest step. It is shown that ITC is able to produce quality kinetic data and is particularly well suited for those enzymatic reactions that cannot be measured by absorption or fluorescence spectroscopy.

Choline sulfatase from Ensifer (Sinorhizobium) meliloti: Characterization of the unmodified enzyme.

Ensifer (Sinorhizobium) meliloti is a nitrogen-fixing α-proteobacterium able to biosynthesize the osmoprotectant glycine betaine from choline sulfate through a metabolic pathway that starts with the enzyme choline-O-sulfatase. This protein seems to be widely distributed in microorganisms and thought to play an important role in their sulfur metabolism. However, only crude extracts with choline sulfatase activity have been studied. In this work, Ensifer (Sinorhizobium) meliloti choline-O-sulfatase was obtained in a high degree of purity after expression in Escherichia coli. Gel filtration and dynamic light scattering experiments showed that the recombinant enzyme exists as a dimer in solution. Using calorimetry, its catalytic activity against its natural substrate, choline-O-sulfate, gave a k cat=2.7×10-1 s-1 and a K M=11.1 mM. For the synthetic substrates p-nitrophenyl sulfate and methylumbelliferyl sulfate, the k cat values were 3.5×10-2 s-1 and 4.3×10-2 s-1, with K M values of 75.8 and 11.8 mM respectively. The low catalytic activity of the recombinant sulfatase was due to the absence of the formylglycine post-translational modification in its active-site cysteine 54. Nevertheless, unmodified Ensifer (Sinorhizobium) meliloti choline-O-sulfatase is a multiple-turnover enzyme with remarkable catalytic efficiency.

Effect of velocity on microdroplet fluorescence quantified by laser-induced fluorescence.

Microdroplets generated inside microfluidic devices have been widely used as miniaturized chemical and biological reactors allowing important reductions in experimental fluid volumes and making it possible to carry out high-throughput assays. Laser-induced fluorescence (LIF) is commonly used to detect and quantify the product, marker or cell content inside each individual droplet. In this work, we employed this technique to characterize the response of in-flow microdroplets filled with fluorescein dye at different laser powers and flow velocities. Using two parallel laser beams closely focused inside a microchannel we determined the microdroplet velocities and showed that the droplet fluorescence intensity decreases exponentially with reducing velocities because of the effects of photobleaching. In contrast, the fluorescence intensity increases linearly with laser power in the 4-10 mW range. When LIF is used for microdroplet measurements it is important to consider not just the fluorophore concentration but also the droplet velocity and laser power in the development of quantitative assays.

Simultaneous determination of gene expression and enzymatic activity in individual bacterial cells in microdroplet compartments.

A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes has been fabricated in PDMS. Once captured in droplets that remain static in the device, bacteria express both a red fluorescent protein (mRFP1) and the enzyme, alkaline phosphatase (AP), from a biscistronic construct. By measuring the fluorescence intensity of both the mRFP1 inside the cells and a fluorescent product formed as a result of the enzymatic activity outside the cells, gene expression and enzymatic activity can be simultaneously and continuously monitored. By collecting data from many individual cells, the distribution of activities in a cell is quantified and the difference in activity between two AP mutants is measured.

Simultaneous measurement of reactions in microdroplets filled by concentration gradients.

This work describes a technology for performing and monitoring simultaneously several reactions confined in strings of microdroplets having identical volumes but different composition, and travelling with the same speed in parallel channels of a microfluidic device. This technology, called parallel microdroplets technology (PmicroD), uses an inverted optical microscope and a charge-coupled device (CCD) camera to collect images and analyze them so as to report on the reactions occurring in these microdroplets. A concentration gradient of one reactant is created in the microfluidic device. In each channel, a different concentration of this reactant is mixed with a fixed amount of a second reactant. Using planar flow-focusing methodology, these mixtures are confined in microdroplets of pL size which travel in oil as continuous medium, avoiding laminar dispersion. By analyzing the images of parallel strings of microdroplets, the time courses of several reactions with different reagent compositions are investigated simultaneously. In order to design the microfluidic device that consists in a complex network of channels having well-defined geometries and restricted positions, the theoretical concept of equivalent channels (i.e. channels having identical hydraulic resistance) is exploited and developed. As a demonstration of the PmicroD technology, an enzyme activity assay was carried out and the steady-state kinetic constants were determined.

Efficient catalytic promiscuity for chemically distinct reactions.

High catalytic proficiencies observed for the native and promiscuous reaction of the Pseudomonas aeruginosa arylsulfatase (PAS; the picture shows transition states of the two substrates with corresponding binding constants K(tx)) suggest that the trade-off between high activity and tight specificity can be substantially relaxed.

Controlling the retention of small molecules in emulsion microdroplets for use in cell-based assays.

Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carried out over periods up to 18 h, without substantial leaking (<5%). We demonstrate the utility of this additive by following the enzymatic activity of alkaline phosphatase expressed by Escherichia coli cells. The ability to reliably compartmentalize genotype (cell) and phenotype (reaction product) is the basis for using microdroplets in directed evolution studies, and the approaches described herein provide a test system for assessing emulsion formulations for such purposes.

Kinetic analysis of beta-phosphoglucomutase and its inhibition by magnesium fluoride.

The isomerization of beta-glucose-1-phosphate (betaG1P) to beta-glucose-6-phosphate (G6P) catalyzed by beta-phosphoglucomutase (betaPGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of beta-glucose-1,6-bisphosphate (betaG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (kcat = 65 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.7 microM, Ki = 122 microM). If alphaG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (kcat = 74 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.8 microM, Ki = 122 microM, K(alphaG16BP) = 91 microM for productive binding, K(alphaG16BP) = 21 microM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140,000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the betaPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a betaG1P x MgF3(-) x betaPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 x 10(-16) M(5) and suggests an affinity of the MgF3(-) moiety to this transition-state analogue (TSA) of < or = 70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of betaPGM.

Development of quantitative cell-based enzyme assays in microdroplets.

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

An integrated device for monitoring time-dependent in vitro expression from single genes in picolitre droplets.

Microdroplets have great potential for high-throughput biochemical screening. We report the design of an integrated microfluidic device for droplet formation, incubation and screening. Picolitre water-in-oil droplets can be stored in a reservoir that contains approximately 10(6) droplets. In this reservoir droplets are stable for at least 6 h, which gives an extended timescale for biochemical experiments. We demonstrate the utility of the system by following the in vitro expression of green fluorescent protein. The high efficiency allows protein expression from a single molecule of DNA template, creating "monoclonal droplets" in which genotype and phenotype are combined in one emulsion compartment.

Efficient catalytic promiscuity in an enzyme superfamily: an arylsulfatase shows a rate acceleration of 10(13) for phosphate monoester hydrolysis.

We report a second catalytic activity of Pseudomonas aeruginosa arylsulfatase (PAS). Besides hydrolyzing sulfate monoesters, this enzyme catalyzes the hydrolysis of phosphate monoesters with multiple turnovers (>90), a k(cat) value of 0.023 s(-1), a K(M) value of 29 microM, and a kcat/K(M) ratio of 790 M(-1) s(-1) at pH 8.0. This corresponds to a remarkably high rate acceleration of 10(13) relative to the nonenzymatic hydrolysis [(k(cat)/K(M))/k(w)] and a transition-state binding constant (K(tx)) of 3.4 pM. Promiscuous phosphatase and original sulfatase activities only differ by a factor of 620 (measured by k(cat)), so the enzyme provides high accelerations for both reactions. The magnitudes and relative similarity of the kinetic parameters suggest that a functional switch from sulfatase to phosphatase activities is feasible, either by gene duplication or by direct evolution via an intermediate enzyme with dual specificity.

A Trojan horse transition state analogue generated by MgF3- formation in an enzyme active site.

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. beta-Phosphoglucomutase catalyses the isomerization of beta-glucose-1-phosphate to beta-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using (19)F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF(3)(-) mimics the transferring PO(3)(-) moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of beta-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.