RESUMO
Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 µM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at â¼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module 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.
Assuntos
Evolução Molecular Direcionada/métodos , Microfluídica/instrumentação , Aminoácido Oxirredutases/metabolismo , Cinética , Miniaturização , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Tradeoffs provide a rationale for the outcome of natural selection. A prominent example is the negative correlation between the growth rate and the biomass yield in unicellular organisms. This tradeoff leads to a dilemma, where the optimization of growth rate is advantageous for an individual, whereas the optimization of the biomass yield would be advantageous for a population. High-rate strategies are observed in a broad variety of organisms such as Escherichia coli, yeast, and cancer cells. Growth in suspension cultures favors fast-growing organisms, whereas spatial structure is of importance for the evolution of high-yield strategies. Despite this realization, experimental methods to directly select for increased yield are lacking. We here show that the serial propagation of a microbial population in a water-in-oil emulsion allows selection of strains with increased biomass yield. The propagation in emulsion creates a spatially structured environment where the growth-limiting substrate is privatized for populations founded by individual cells. Experimental evolution of several isogenic Lactococcus lactis strains demonstrated the existence of a tradeoff between growth rate and biomass yield as an apparent Pareto front. The underlying mutations altered glucose transport and led to major shifts between homofermentative and heterofermentative metabolism, accounting for the changes in metabolic efficiency. The results demonstrated the impact of privatizing a public good on the evolutionary outcome between competing metabolic strategies. The presented approach allows the investigation of fundamental questions in biology such as the evolution of cooperation, cell-cell interactions, and the relationships between environmental and metabolic constraints.
Assuntos
Lactococcus lactis , Evolução Biológica , Lactococcus lactis/crescimento & desenvolvimento , Lactococcus lactis/metabolismo , Seleção GenéticaRESUMO
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.
Assuntos
Emulsões , Citometria de Fluxo/métodos , MicrofluídicaRESUMO
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 ß-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.
Assuntos
Nanotecnologia , beta-Glucosidase/antagonistas & inibidores , beta-Glucosidase/metabolismo , Cinética , Nanotecnologia/instrumentação , Tamanho da Partícula , Prunus/enzimologia , Fatores de TempoRESUMO
The wide use of 3D-organotypic cell models is imperative for advancing our understanding of basic cell biological mechanisms. For this purpose, easy-to-use enabling technology is required, which should optimally link standardized assessment methods to those used for the formation, cultivation, and evaluation of cell aggregates or primordial tissue. We thus conceived, manufactured, and tested devices which provide the means for cell aggregation and online monitoring within a hanging drop. We then established a workflow for spheroid manipulation and immune phenotyping. This described workflow conserves media and reagent, facilitates the uninterrupted tracking of spheroid formation under various conditions, and enables 3D-marker analysis by means of 3D epifluorescence deconvolution microscopy. We provide a full description of the low-cost manufacturing process for the fluidic devices and microscopic assessment tools, and the detailed blueprints and building instructions are disclosed. Conclusively, the presented compilation of methods and techniques promotes a quick and barrier-free entry into 3D cell biology.
RESUMO
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.
RESUMO
Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.
Assuntos
Microfluídica/instrumentação , Técnicas Eletroquímicas , Géis/química , Espectrometria de Massas , Microfluídica/métodos , Reação em Cadeia da Polimerase , Polímeros/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Espectrometria de Fluorescência , Análise Espectral Raman , Tensoativos/químicaRESUMO
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.
Assuntos
FermentaçãoRESUMO
Surface-enhanced Raman spectroscopy (SERS) is well known for its high sensitivity that emerges due to the plasmonic enhancement of electric fields typically on gold and silver nanostructures. However, difficulties associated with the preparation of nanostructured substrates with uniform and reproducible features limit reliability and quantitation using SERS measurements. In this work we use layer-by-layer (LbL) self-assembly to incorporate multiple functional building blocks of collaborative assemblies of nanoparticles on colloidal spheres to fabricate SERS sensors. Gold nanoparticles (AuNPs) are packaged in discrete layers, effectively 'freezing nano-gaps', on spherical colloidal cores to achieve multifunctionality and reproducible sensing. Coupling between layers tunes the plasmon resonance for optimum SERS signal generation to achieve a 10 nM limit of detection. Significantly, using the layer-by-layer construction, SERS-active AuNP layers are spaced out and thus optically isolated. This uniquely allows the creation of an internal standard within each colloidal sensor to enable highly reproducible self-calibrated sensing. By using 4-mercaptobenzoic acid (4-MBA) as the internal standard adenine concentrations are quantified to an accuracy of 92.6-99.5%. Our versatile approach paves the way for rationally designed yet quantitative colloidal SERS sensors and their use in a variety of sensing applications.
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From the viewpoint of a materials scientist, viruses can be regarded as organic nanoparticles. They are composed of a small number of different (bio)polymers: proteins and nucleic acids. Many viruses are enveloped in a lipid membrane and all viruses do not have a metabolism of their own, but rather use the metabolic machinery of a living cell for their replication. Their surface carries specific tools designed to cross the barriers of their host cells. The size and shape of viruses, and the number and nature of the functional groups on their surface, is precisely defined. As such, viruses are commonly used in materials science as scaffolds for covalently linked surface modifications. A particular quality of viruses is that they can be tailored by directed evolution by taking advantage of their inbuilt colocalization of geno- and phenotypes. The powerful techniques developed by life sciences are becoming the basis of engineering approaches towards nanomaterials, opening a wide range of applications far beyond biology and medicine.
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Materiais Biomiméticos/química , Nanoestruturas/química , Nanotecnologia , Tensoativos/química , Vírus/química , Autoanticorpos/imunologia , Permeabilidade da Membrana Celular , Humanos , Masculino , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Ácidos Nucleicos/química , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/imunologiaRESUMO
We have developed a disposable microfluidic chip with integrated cavity mirrors comprised of two pieces of 3M Vikuiti™ enhanced specular reflector II (ESRII) film, for performing cavity-enhanced absorption spectroscopy with a white light-emitting diode (LED). Compared to measurements made with a chip without cavity mirrors, the absorption path length is enhanced by a maximum factor of 28 at 544 nm, and the sensitivity is enhanced by approximately 5 times, enabling micromolar range detection limits to be achieved in an optical path length of only 50 µm.
Assuntos
Luz , Polímeros/química , Análise Espectral/instrumentação , Desenho de Equipamento , Dispositivos Lab-On-A-ChipRESUMO
Natural evolution relies on the improvement of biological entities by rounds of diversification and selection. In the laboratory, directed evolution has emerged as a powerful tool for the development of new and improved biomolecules, but it is limited by the enormous workload and cost of screening sufficiently large combinatorial libraries. Here we describe the production of gel-shell beads (GSBs) with the help of a microfluidic device. These hydrogel beads are surrounded with a polyelectrolyte shell that encloses an enzyme, its encoding DNA and the fluorescent reaction product. Active clones in these man-made compartments can be identified readily by fluorescence-activated sorting at rates >10(7) GSBs per hour. We use this system to perform the directed evolution of a phosphotriesterase (a bioremediation catalyst) caged in GSBs and isolate a 20-fold faster mutant in less than one hour. We thus establish a practically undemanding method for ultrahigh-throughput screening that results in functional hybrid composites endowed with evolvable protein components.
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Biomimética , Diester Fosfórico Hidrolases/química , Catálise , Géis , Técnicas Analíticas MicrofluídicasRESUMO
Extreme miniaturization of biological and chemical reactions in pico- to nanoliter microdroplets is emerging as an experimental paradigm that enables more experiments to be carried out with much lower sample consumption, paving the way for high-throughput experiments. This review provides the protein scientist with an experimental framework for (a) formation of polydisperse droplets by emulsification or, alternatively, of monodisperse droplets using microfluidic devices; (b) construction of experimental rigs and microfluidic chips for this purpose; and (c) handling and analysis of droplets.
Assuntos
Proteínas/química , Evolução Molecular Direcionada , Emulsões , Hexoses/química , Técnicas Analíticas Microfluídicas , Óleo Mineral/química , Nanotecnologia , Tamanho da Partícula , Distribuição de Poisson , Polissorbatos/química , Biossíntese de Proteínas , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Tensoativos/químicaRESUMO
We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 × 10(7) monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viable.
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Evolução Molecular Direcionada , Técnicas Analíticas Microfluídicas , Sulfatases/metabolismo , Escherichia coli/metabolismo , Fluoresceína/síntese química , Fluoresceína/química , Fluoresceína/metabolismo , Hidrólise , Cinética , Técnicas Analíticas Microfluídicas/instrumentação , Miniaturização , Óleos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfatases/genética , Água/químicaRESUMO
Fusion of lipid-enveloped viruses with endosomal membranes triggered by low pH in the endosome is a key step in the course of viral infection. This ubiquitous mechanism can be used to integrate functional nanoparticles of viral origin into composite materials consisting of a polyelectrolyte multilayer with an adsorbed lipid membrane in a natural and biomimetic way. Polyelectrolyte multilayers as the support for the lipid membrane are a versatile means to combine the biological functions of the viral surface with the multiplicity of polyelectrolyte borne functions into a novel bio/nonbio composite material.
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Materiais Biocompatíveis/química , Eletrólitos/química , Membranas/química , Nanopartículas/química , Nanotecnologia/métodos , Proteínas do Envelope Viral/química , Coloides/química , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Lipídeos/química , Microscopia de Força Atômica , Propriedades de Superfície , Fatores de Tempo , Vírus/metabolismoRESUMO
The heme protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes, a cell species found in increasing amounts in the male and female genital tract of patients with genital tract inflammations. Myeloperoxidase binds only to a fraction of freshly prepared human spermatozoa. The number of spermatozoa able to bind myeloperoxidase raised considerably in samples containing pre-damaged cells or in acrosome-reacted samples. In addition, myeloperoxidase released from zymosan-stimulated polymorphonuclear leukocytes was also able to bind to pre-damaged spermatozoa. The ability of spermatozoa to bind myeloperoxidase coincided with the binding of annexin V to externalized phosphatidylserine epitopes indicating the loss of plasma membrane integrity and with the incorporation of ethidium homodimer I. Myeloperoxidase did not interact with intact spermatozoa. Annexin V and myeloperoxidase bind to the same binding sites as verified by double fluorescence techniques, flowcytometry analyses as well as competition experiments. We demonstrated also that myeloperoxidase is eluted together with pure phosphatidylserine liposomes or liposomes composed of phosphatidylserine and phosphatidylcholine in gel filtration, but not with pure phosphatidylcholine liposomes. In conclusion, myeloperoxidase interacts with apoptotic spermatozoa via binding to externalized phosphatidylserine indicating a yet unknown role of this protein in recognition and removal of apoptotic cells during inflammation.
Assuntos
Apoptose , Peroxidase/metabolismo , Fosfatidilserinas/metabolismo , Espermatozoides/fisiologia , Animais , Epitopos , Feminino , Humanos , Inflamação/metabolismo , Masculino , Neutrófilos/citologia , Neutrófilos/metabolismo , Ligação Proteica , Espermatozoides/citologia , Espermatozoides/patologia , Sistema Urogenital/metabolismo , Sistema Urogenital/patologia , Zimosan/metabolismoRESUMO
BACKGROUND: Suspension array technology has surpassed ELISA for automated, simultaneous detection and quantification of soluble biomarkers such as virus-specific antibodies. We describe assays in which antigens are attached to a lipid bilayer surrounding color-coded particles. METHODS: We used layer-by-layer technology to establish a multiplex suspension array with distinguishable microbeads coated with authentic viral surfaces to catch and quantify virus-specific antibodies in a flow cytometric analysis. Antigenic surfaces were generated by chimeric and wild-type baculoviruses plus 2 different influenza A virus subtypes fused to a lipid bilayer surrounding distinctly colored particles. Specificity of binding of chosen antibodies and sera was detected by immunofluorescence. Results of multiplex analysis were compared with results of ELISA. RESULTS: Titrations of virus-specific antibodies in the multiplex suspension array demonstrated specific binding to the viral surface proteins. The multiplex suspension array gave positive results for up to log 5-diluted primary antibodies with an approximately 5- to 10-fold reduced dynamic range compared with the respective ELISA. CONCLUSIONS: The bead-based multiplex suspension array is customizable and easy to establish. By displaying native influenza A virus surfaces and recombinant HIV-1 epitopes, the new assay provides a tool for the detection of major viral infections in humans.