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Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles.
We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.
DNA origami protection and molecular interfacing through engineered sequence-defined peptoids.
DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson-Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1-9) with two types of architectures, termed as "brush" and "block," were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9), conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.
Combinatorial Low-Volume Synthesis of Well-Defined Polymers by Enzyme Degassing.
The synthesis of well-defined polymers in a low-volume, combinatorial fashion has long been a goal in polymer chemistry. Here, we report the preparation of a wide range of highly controlled homo and block co-polymers by Enz-RAFT (enzyme-assisted reversible addition-fragmentation chain transfer) polymerization in microtiter plates in the open atmosphere. The addition of 1 μm glucose oxidase (GOx) to water/solvent mixtures enables polymerization reactions to proceed in extremely low volumes (40 μL) and low radical concentrations. This procedure provides excellent control and high conversions across a range of monomer families and molecular weights, thus avoiding the need to purify for screening applications. This simple technique enables combinatorial polymer synthesis in microtiter plates on the benchtop without the need of highly specialized synthesizers and at much lower volumes than is currently possible by any other technique.
Multivalent nanoparticle networks enable point-of-care detection of human phospholipase-A2 in serum.
A rapid and highly sensitive point-of-care (PoC) lateral flow assay for phospholipase A2 (PLA2) is demonstrated in serum through the enzyme-triggered release of a new class of biotinylated multiarmed polymers from a liposome substrate. Signal from the enzyme activity is generated by the adhesion of polystreptavidin-coated gold nanoparticle networks to the lateral flow device, which leads to the appearance of a red test line due to the localized surface plasmon resonance effect of the gold. The use of a liposome as the enzyme substrate and multivalent linkers to link the nanoparticles leads to amplification of the signal, as the cleavage of a small amount of lipids is able to release a large amount of polymer linker and adhesion of an even larger amount of gold nanoparticles. By optimizing the molecular weight and multivalency of these biotinylated polymer linkers, the sensitivity of the device can be tuned to enable naked-eye detection of 1 nM human PLA2 in serum within 10 min. This high sensitivity enabled the correct diagnosis of pancreatitis in diseased clinical samples against a set of healthy controls using PLA2 activity in a point-of-care device for the first time.
Supramolecular replication of peptide and DNA patterned arrays
Here we present a novel approach to the parallel fabrication of protein and protein/DNA features on surfaces. © 2010 The Royal Society of Chemistry.
Correlated Heterospectral Lipidomics for Biomolecular Profiling of Remyelination in Multiple Sclerosis.
Analyzing lipid composition and distribution within the brain is important to study white matter pathologies that present focal demyelination lesions, such as multiple sclerosis. Some lesions can endogenously re-form myelin sheaths. Therapies aim to enhance this repair process in order to reduce neurodegeneration and disability progression in patients. In this context, a lipidomic analysis providing both precise molecular classification and well-defined localization is crucial to detect changes in myelin lipid content. Here we develop a correlated heterospectral lipidomic (HSL) approach based on coregistered Raman spectroscopy, desorption electrospray ionization mass spectrometry (DESI-MS), and immunofluorescence imaging. We employ HSL to study the structural and compositional lipid profile of demyelination and remyelination in an induced focal demyelination mouse model and in multiple sclerosis lesions from patients ex vivo. Pixelwise coregistration of Raman spectroscopy and DESI-MS imaging generated a heterospectral map used to interrelate biomolecular structure and composition of myelin. Multivariate regression analysis enabled Raman-based assessment of highly specific lipid subtypes in complex tissue for the first time. This method revealed the temporal dynamics of remyelination and provided the first indication that newly formed myelin has a different lipid composition compared to normal myelin. HSL enables detailed molecular myelin characterization that can substantially improve upon the current understanding of remyelination in multiple sclerosis and provides a strategy to assess remyelination treatments in animal models.
Re-Engineering Extracellular Vesicles as Smart Nanoscale Therapeutics.
In the past decade, extracellular vesicles (EVs) have emerged as a key cell-free strategy for the treatment of a range of pathologies, including cancer, myocardial infarction, and inflammatory diseases. Indeed, the field is rapidly transitioning from promising in vitro reports toward in vivo animal models and early clinical studies. These investigations exploit the high physicochemical stability and biocompatibility of EVs as well as their innate capacity to communicate with cells via signal transduction and membrane fusion. This review focuses on methods in which EVs can be chemically or biologically modified to broaden, alter, or enhance their therapeutic capability. We examine two broad strategies, which have been used to introduce a wide range of nanoparticles, reporter systems, targeting peptides, pharmaceutics, and functional RNA molecules. First, we explore how EVs can be modified by manipulating their parent cells, either through genetic or metabolic engineering or by introducing exogenous material that is subsequently incorporated into secreted EVs. Second, we consider how EVs can be directly functionalized using strategies such as hydrophobic insertion, covalent surface chemistry, and membrane permeabilization. We discuss the historical context of each specific technology, present prominent examples, and evaluate the complexities, potential pitfalls, and opportunities presented by different re-engineering strategies.
Multiplex sensing of protease and kinase enzyme activity via orthogonal coupling of quantum dot-peptide conjugates.
Nanoparticle-based labels are emerging as simpler and more sensitive alternatives to traditional fluorescent small molecules and radioactive reporters in biomarker assays. The determination of biomarker levels is a recommended clinical practice for the assessment of many diseases, and detection of multiple analytes in a single assay, known as multiplexing, can increase predictive accuracy. While multiplexed detection can also simplify assay procedures and reduce systematic variability, combining multiple assays into a single procedure can lead to complications such as substrate cross-reactivity, signal overlap, and loss of sensitivity. By combining the specificity of biomolecular interactions with the tunability of quantum dot optical properties, we have developed a detection system capable of simultaneous evaluation of the activity of two critical enzyme classes, proteases and kinases. We avoid cross-reactivity and signal overlap by synthesizing enzyme-specific peptide sequences with orthogonal terminal functionalization for attachment to quantum dots with distinct emission spectra. Enzyme activity is reported via binding of either gold nanoparticle-peptide conjugates or FRET acceptor dye-labeled antibodies, which mediate changes in quantum dot emission spectra. To the best of our knowledge, this is the first demonstration of the multiplexed sensing of the activity of two different classes of enzymes via a nanoparticle-based activity assay. Using the quantum dot-based assay described herein, we were able to detect the protease activity of urokinase-type plasminogen activator at concentrations ≥ 50 ng/mL and the kinase activity of human epidermal growth factor receptor 2 at concentrations ≥ 7.5 nM, levels that are clinically relevant for determination of breast cancer prognosis. The modular nature of this assay design allows for the detection of different classes of enzymes simultaneously and represents a generic platform for high-throughput enzyme screening in rapid disease diagnosis and drug discovery.
Highly controlled open vessel RAFT polymerizations by enzyme degassing
Intolerance to oxygen is a key limitation in many reactions and particularly in controlled radical polymerizations. Here we introduce the use of enzymes such as glucose oxidase (GOx) to deoxygenate reversible addition-fragmentation chain transfer polymerizations (Enz-RAFT), facilitating the preparation of highly controlled polymers in vessels open to ambient oxygen. Because the removal of oxygen is so efficient, very low concentrations of GOx and initiator can be used, enabling excellent control which is demonstrated by pseudoliving polymerization kinetics and the preparation of multiblock copolymers with narrow molecular weight distributions (Mw/Mn < 1.15). GOx retains sufficient activity to facilitate polymerization not only in aqueous solutions but also in a range of water/organic solvent mixtures, and we demonstrate the use of this technique to perform open vessel Enz-RAFT polymerizations in various methanol and dioxane/water mixtures.
Single Particle Automated Raman Trapping Analysis of Breast Cancer Cell-Derived Extracellular Vesicles as Cancer Biomarkers.
Extracellular vesicles (EVs) secreted by cancer cells provide an important insight into cancer biology and could be leveraged to enhance diagnostics and disease monitoring. This paper details a high-throughput label-free extracellular vesicle analysis approach to study fundamental EV biology, toward diagnosis and monitoring of cancer in a minimally invasive manner and with the elimination of interpreter bias. We present the next generation of our single particle automated Raman trapping analysis─SPARTA─system through the development of a dedicated standalone device optimized for single particle analysis of EVs. Our visualization approach, dubbed dimensional reduction analysis (DRA), presents a convenient and comprehensive method of comparing multiple EV spectra. We demonstrate that the dedicated SPARTA system can differentiate between cancer and noncancer EVs with a high degree of sensitivity and specificity (>95% for both). We further show that the predictive ability of our approach is consistent across multiple EV isolations from the same cell types. Detailed modeling reveals accurate classification between EVs derived from various closely related breast cancer subtypes, further supporting the utility of our SPARTA-based approach for detailed EV profiling.
Silica-gelatin hybrids with tailorable degradation and mechanical properties for tissue regeneration
Nature has evolved mechanisms to create a diversity of specialized materials through nanoscale organization. Inspired by nature, hybrid materials are designed with highly tailorable properties, which are achieved through careful control of their nanoscale interactions. These novel materials, based on a silica-gelatin hybrid system, have the potential to serve as a platform technology for human tissue regeneration. Covalent interactions between the inorganic and organic constituents of the hybrid are essential to enable the precise control of mechanical and dissolution properties. Furthermore, hybrid scaffold porosity is found to highly influence mechanical properties, to the extent where scaffolds of particular strength could be specified based on their porosity. The hybrids also demonstrate a non-cytotoxic effect when mesenchymal stem cells are cultured on the material. Cytoskeletal proteins of the cells are imaged using actin and vimentin staining. It is envisaged these hybrid materials will find a diverse application in both hard and soft tissue regenerating scaffolds. Utilizing the enhanced properties of hybrid materials, novel silica-gelatin hybrids are synthesized and formed into macroporous scaffolds (imaged here with X-ray micro-computed tomography). These scaffolds demonstrate great promise in the field of tissue regeneration as the mechanical and dissolution properties can be carefully tailored for the intended application and studies with mesenchymal stem cells show a favorable response to the hybrids. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Assessing the impact of silicon nanowires on bacterial transformation and viability of Escherichia coli.
We investigated the biomaterial interface between the bacteria Escherichia coli DH5α and silicon nanowire patterned surfaces. We optimised the engineering of silicon nanowire coated surfaces using metal-assisted chemical etching. Using a combination of focussed ion beam scanning electron microscopy, and cell viability and transformation assays, we found that with increasing interfacing force, cell viability decreases, as a result of increasing cell rupture. However, despite this aggressive interfacing regime, a proportion of the bacterial cell population remains viable. We found that the silicon nanowires neither resulted in complete loss of cell viability nor partial membrane disruption and corresponding DNA plasmid transformation. Critically, assay choice was observed to be important, as a reduction-based metabolic reagent was found to yield false-positive results on the silicon nanowire substrate. We discuss the implications of these results for the future design and assessment of bacteria-nanostructure interfacing experiments.
Insights into the AIEE of 1,8-naphthalimides (NPIs): inverse effects of intermolecular interactions in solution and aggregates.
Systematic structural perturbation has been used to fine-tune and understand the luminescence properties of three new 1,8-naphthalimides (NPIs) in solution and aggregates. The NPIs show blue emission in the solution state and their fluorescence quantum yields are dependent upon their molecular rigidity. In concentrated solutions of the NPIs, intermolecular interactions were found to quench the fluorescence due to the formation of excimers. In contrast, upon aggregation (in THF/H2O mixtures), the NPIs show aggregation-induced emission enhancement (AIEE). The NPIs also show moderately high solid-state emission quantum yields (ca. 10-12.7 %). The AIEE behaviour of the NPIs depends on their molecular rigidity and the nature of their intermolecular interactions. The NPIs 1-3 show different extents of intermolecular (π-π and C-H⋅⋅⋅O) interactions in their solid-state crystal structures depending on their substituents. Detailed photophysical, computational and structural investigations suggest that an optimal balance of structural flexibility and intermolecular communication is necessary for achieving AIEE characteristics in these NPIs.
High-Throughput Peptide Derivatization toward Supramolecular Diversification in Microtiter Plates.
The evolution of life on earth eventually leads to the emergence of species with increased complexity and diversity. Similarly, evolutionary chemical space exploration in the laboratory is a key step to pursue the structural and functional diversity of supramolecular systems. Here, we present a powerful tool that enables rapid peptide diversification and employ it to expand the chemical space for supramolecular functions. Central to this strategy is the exploitation of palladium-catalyzed Suzuki-Miyaura cross-coupling reactions to direct combinatorial synthesis of peptide arrays in microtiter plates under an open atmosphere. Taking advantage of this in situ library design, our results unambiguously deliver a fertile platform for creating a set of intriguing peptide functions including green fluorescent protein-like peptide emitters with chemically encoded emission colors, hierarchical self-assembly into nano-objects, and macroscopic hydrogels. This work also offers opportunities for quickly surveying the diversified peptide arrays and thereby identifying the structural factors that modulate peptide properties.
Changes in embryonic stem cell colony morphology and early differentiation markers driven by colloidal crystal topographical cues.
The use of materials properties to guide cell behaviour is an attractive option for regenerative medicine, where controlling stem cell behaviour is important for the establishment of a functioning cell population. A wide range of materials properties have been shown to influence many types of cells but little is known about the effects of topography on embryonic stem cells (ESCs). In order to advance this knowledge, we synthesised and characterised substrates formed of silica colloidal crystal (SCC) microspheres to present highly ordered and reproducible topographical features from 120-600 nm in diameter. We found that, compared to cells cultured on flat glass, cells cultured on the SCC substrates retained transcription of stem cell (Dppa5a, Nanog, and Pou5f1) and endoderm (Afp, Gata4, Sox17, and Foxa2) markers more similar to undifferentiated ESCs, suggesting the substrates are restricting differentiation, particularly towards the endoderm lineage. Additionally, five days after seeding, we observed strikingly different colony morphology, with cells on the SCC substrates growing in spherical colonies approximately ten cells thick, while cells on glass were growing in flat monolayers. Colonies on the SCC substrates developed a central pit, which was never observed in cells cultured on glass, and expressed proteins related to epithelialisation. Together, these data demonstrate the potential of using topographical cues to control stem cell behaviour in vitro.
A Serological Point-of-Care Test for the Detection of IgG Antibodies against Ebola Virus in Human Survivors.
Ebola virus disease causes widespread and highly fatal epidemics in human populations. Today, there is still great need for point-of-care tests for diagnosis, patient management and surveillance, both during and post outbreaks. We present a point-of-care test comprising an immunochromatographic strip and a smartphone reader, which detects and semiquantifies Ebola-specific antibodies in human survivors. We developed a Sudan virus glycoprotein monoplex platform and validated it using sera from 90 human survivors and 31 local noninfected controls. The performance of the glycoprotein monoplex was 100% sensitivity and 98% specificity compared to standard whole antigen enzyme-linked immunosorbent assay (ELISA), and it was validated with freshly collected patient samples in Uganda. Moreover, we constructed a multiplex test for simultaneous detection of antibodies against three recombinant Sudan virus proteins. A pilot study comprising 15 survivors and 5 noninfected controls demonstrated sensitivity and specificity of 100% compared to standard ELISA. Finally, we developed a second multiplex subtype assay for the identification of exposure to three related EVD species: Sudan virus, Bundibugyo virus and Ebola virus (formerly Zaire) using recombinant viral glycoprotein. This multiplex test could distinguish between the host's immunity to specific viral species and identify cross-reactive immunity. These developed serological platforms consisted of capture ligands with high specificity and sensitivity, in-house developed strips and a compatible smartphone application. These platforms enabled rapid and portable testing, data storage and sharing as well as geographical tagging of the tested individuals in Uganda. This platform holds great potential as a field tool for diagnosis, vaccine development, and therapeutic evaluation.