1,720,976 research outputs found
Viruses as building blocks for materials and devices
No full textFrom 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 medicin
Droplet Kitchen
No full textOpen Instrumentation and Howtos for Microfluidics: Flexible and affordable droplet microfluidics with digital fabrication, open hardware and open source software.</span
Tuning plasmons layer-by-layer for quantitative colloidal sensing with surface-enhanced Raman spectroscopy
No full textSurface-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
Engineering virus functionalities on colloidal polyelectrolyte lipid composites
No full textPolyelectrolyte-multilayered colloids were prepared employing the layer-by-layer technology. A lipid bilayer was then added, and finally rubella-like particles were fused. Immunofluorescence studies demonstrate that the colloidal biocomposites have a virus-like surface. Indeed, they were taken up into cells, while particles with an outer lipid layer were rarely found inside cell
Fusion of enveloped virus nanoparticles with polyelectrolyte-supported lipid membranes for the design of bio/nonbio interfaces
No full textFusion 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 materia
Characterization of lipid bilayers adsorbed on spherical LbL-support
Full text linkStructural and dynamic properties of membranes composed of phosphatidylcholine (PC) and phosphatidylserine (PS) on layer-by-layer (LbL) polyelectrolyte coated particles were investigated using solid-state nuclear magnetic resonance (NMR) and fluorescence methods. These spherically supported membranes showed structural, dynamic, and elastic properties similar to free-standing membranes as proved by 31P and 2H NMR. Small differences between behaviour of PC and PS on LbL support due to interaction with the polyelectrolyte were observed. Fluorescence lifetime imaging microscopy (FLIM) using 7-nitro-2-1,3-benzoxadiazol (NBD) labeled PC and PS showed a stronger impact of the outermost polyelectrolyte (PAH) on the fluorescence lifetimes of NBD-PS compared to NBD-PC. Although small defects in nm range allowing passage of Mn2+ to both layers of the membrane coat were present, a rather homogeneous coating observed by fluorescence microscopy, complete fluorescence recovery after photobleaching, and NMR results reveal that somewhat continuous lipid bilayers were formed around the LbL particles<br/
Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)
Full text linkUltrahigh-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 ?M 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 ?100 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
Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution
No full textWe 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 × 107 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 viabl
Virus-coated layer-by-layer colloids as a multiplex suspension array for the detection and quantification of virus-specific antibodies
Full text linkBackground: 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 ?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 human
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