Microdroplet fabrication of silver–agarose nanocomposite beads for SERS optical accumulation

Microdroplets have been used as reactors for the fabrication of agarose beads with high uniformity in shape and size, and densely loaded with silver ions, which were subsequently reduced into nanoparticles using hydrazine. The resulting nanocomposite beads not only display a high plasmonic activity, but can also trap/concentrate analytes, which can be identified by means of surface-enhanced Raman scattering (SERS) spectroscopy. The size of the beads is such that it allows the detection of a single bead under a conventional optical microscope, which is very useful to reduce the amount of material required for SERS detectio

Hydrophilic PDMS microchannels for high-throughput formation of oil-in-water microdroplets and water-in-oil-in-water double emulsions

Here we present a novel surface modification method based on the sequential layer-by-layer deposition of polyelectrolytes yielding hydrophilic microchannels in PDMS-based microfluidic devices. The coatings are long-term stable and allow for the generation of monodisperse oil-in-water microdroplets even several months after the channel surface treatment. Due to the robustness of the polyelectrolyte multilayers ultra-high flow rates can be applied, making high-throughput droplet formation in the jetting mode possible. Furthermore, we successfully used our method to selectively modify the surface properties in certain areas of assembled microchannels. The resulting partially hydrophilic, partially hydrophobic microfluidic devices allow for the production of monodisperse water-in-oil-in-water double emulsions.<br/

Absorbance-activated-droplet sorting for directed enzyme evolution

The successful creation of custom-made enzymes by directed evolution relies in no small part on screening as many variants as possible. Massive scale-down of assay volumes by compartmentalization of library members in water-in-oil emulsion droplets has recently led to the development of ultrahigh-throughput screening platforms that use small volumes (typically picoliters) and allow sorting of more than 106 variants per hour 1,2. The key technical module to make this possible is a microfluidic droplet sorter that has so far relied exclusively on fluorescent readouts. To extend the range of assays amenable to this approach, we developed a highly efficient microfluidic absorbance-activated droplet sorter (AADS)3. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to more than a million droplets in 3 hours. To validate this device, we implemented a miniaturized coupled assay for an NAD+- dependent amino acid dehydrogenase. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts and sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ≈ 100 Hz. Furthermore, improved variants showing increased solubility (up to 60%) and thermostability (up to 12 °C) were identified after two rounds of directed evolution, thereby demonstrating the usefulness of this sorting module for enzyme engineering. This AADS makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows. We are currently expanding its range of applications towards the monitoring of cell growth for the development of survival assays and the detection of weak enzymatic reactions. 1. Colin P-Y, Kintses B, Gielen F, et al. Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics. Nat Commun. 2015; vol: 6, p:1-12. doi:10.1038/ncomms10008. 2. Colin PY, Zinchenko A, Hollfelder F. Enzyme engineering in biomimetic compartments. Curr Opin Struct Biol. 2015; vol: 33, p: 42-51. doi:10.1016/j.sbi.2015.06.001. 3. Gielen F, Hours R, Emond S, Fischlechner M, Schell U, Hollfelder F. Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS). Proc Natl Acad Sci U S A. 2016; vol: 113, p: E7383-E7389. doi:10.1073/pnas.160692711

One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution.

Directed evolution relies on iterative cycles of randomization and selection. The outcome of an artificial evolution experiment is crucially dependent on (i) the numbers of variants that can be screened and (ii) the quality of the assessment of each clone that forms the basis for selection. Compartmentalization of screening assays in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with good signal quality. Microfluidic systems have been developed to make and sort droplets, but the operator skill required precludes their ready implementation in nonspecialist settings. We now establish a protocol for the creation of monodisperse double-emulsion droplets in two steps in microfluidic devices with different surface characteristics (first hydrophobic, then hydrophilic). The resulting double-emulsion droplets are suitable for quantitative analysis and sorting in a commercial flow cytometer. The power of this approach is demonstrated in a series of enrichment experiments, culminating in the successful recovery of catalytically active clones from a sea of 1 000 000-fold as many low-activity variants. The modular workflow allows integration of additional steps: the encapsulated lysate assay reactions can be stopped by heat inactivation (enabling ready control of selection stringency), the droplet size can be contracted (to concentrate its contents), and storage (at -80 °C) is possible for discontinuous workflows. The control that can be thus exerted on screening conditions will facilitate exploitation of the potential of protein libraries compartmentalized in droplets in a straightforward protocol that can be readily implemented and used by protein engineers

Microdroplet screening and selection for improved microbial production of extracellular compounds

Microorganisms produce extracellular compounds that affect the final product quality in fermentation processes. Selection of overproducing mutants requires coupling of the extracellular product to the producer genotype, which can be achieved by single-cell compartmentalization. Emulsions contain up to billions of microdroplets/mL which significantly increases the screening throughput compared to microtiter-plate-based selections. Factors affecting the success of screening in microdroplets include the nature of the producing organism (robust, no invasive growth), the product (not soluble in oil) and the product assay (preferably fluorescence based). Together these factors determine the required microdroplet production technique and sorting set-up. Because microdroplets allow relatively inexpensive ultrahigh-throughput screening, they are likely to become a standard tool in the strain selection toolbox of the fermentation industry.(VLID)460219

Lipid layers on polyelectrolyte multilayer supports

The mechanism of formation of supported lipid layers from phosphatidylcholine and phosphatidylserine vesicles in solution on polyelectrolyte multilayers was studied by a variety of experimental techniques. The interaction of zwitterionic and acidic lipid vesicles, as well as their mixtures, with polyelectrolyte supports was followed in real time by micro-gravimetry. The fabricated lipid–polyelectrolyte composite structures on top of multilayer coated colloidal particles were characterized by flow cytometry and imaging techniques. Lipid diffusion over the macroscopic scale was quantified by fluorescence recovery after photobleaching, and the diffusion was related to layer connectivity. The phospholipid–polyelectrolyte binding mechanism was investigated by infrared spectroscopy. A strong interaction of polyelectrolyte primary amino groups with phosphate and carboxyl groups of the phospholipids, leading to dehydration, was observed. Long-range electrostatic attraction was proven to be essential for vesicle spreading and rupture. Fusion of lipid patches into a homogeneous bilayer required lateral mobility of the lipids on the polyelectrolyte support. The binding of amino groups to the phosphate group of the zwitterionic lipids was too weak to induce vesicle spreading, but sufficient for strong adsorption. Only the mixture of phosphatidylcholine and phosphatidylserine resulted in the spontaneous formation of bilayers on polyelectrolyte multilayers. The adsorption of phospholipids onto multilayers displaying quarternary ammonium polymers produced a novel 3D lipid polyelectrolyte structure on colloidal particles.<br/

A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition.

The ability to miniaturize biochemical assays in water-in-oil emulsion droplets allows a massive scale-down of reaction volumes, so that high-throughput experimentation can be performed more economically and more efficiently. Generating such droplets in compartment-on-demand (COD) platforms is the basis for rapid, automated screening of chemical and biological libraries with minimal volume consumption. Herein, we describe the implementation of such a COD platform to perform high precision nanoliter assays. The coupling of a COD platform to a droplet absorbance detection set-up results in a fully automated analytical system. Michaelis-Menten parameters of 4-nitrophenyl glucopyranoside hydrolysis by sweet almond β-glucosidase can be generated based on 24 time-courses taken at different substrate concentrations with a total volume consumption of only 1.4 μL. Importantly, kinetic parameters can be derived in a fully unsupervised manner within 20 min: droplet production (5 min), initial reading of the droplet sequence (5 min), and droplet fusion to initiate the reaction and read-out over time (10 min). Similarly, the inhibition of the enzymatic reaction by conduritol B epoxide and 1-deoxynojirimycin was measured, and Ki values were determined. In both cases, the kinetic parameters obtained in droplets were identical within error to values obtained in titer plates, despite a >10(4)-fold volume reduction, from micro- to nanoliters

Resolving Structure and Mechanical Properties at the Nanoscale of Viruses with Frequency Modulation Atomic Force Microscopy

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25–50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called “dissipation channel” in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used

Droplet Kitchen

Open Instrumentation and Howtos for Microfluidics: Flexible and affordable droplet microfluidics with digital fabrication, open hardware and open source software.</span