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Acinetobacter baumannii is a remarkable microorganism known for its diversity of habitat and its multi-drug resistance, resulting in hard-to-treat infections. Thus, a sensitive method for the identification and detection of Acinetobacter baumannii is vital. However, current methods used for the detection of pathogens have not improved in the past decades and suffer from long process times and low detection limits. A cheap, quick, and easy detection mechanism is needed. In this work, we successfully prepared indium phosphide quantum dots with a zinc sulphide shell, conjugated to a targeting aptamer ligand, to specifically label Acinetobacter baumannii. The system retained both the photophysical properties of the quantum dots and the folded structure and molecular recognition function of the aptamer, therefore successfully targeting Acinetobacter baumannii. Confocal microscopy and transmission electron microscopy showed the fluorescent quantum dots surrounding the Acinetobacter baumannii cells confirming the specificity of the aptamer conjugated to indium phosphide quantum dots with a zinc sulphide shell. Controls were undertaken with a different bacteria species, showing no binding of the aptamer conjugated quantum dots. Our strategy offers a novel method to detect bacteria and engineer a scalable platform for fluorescence detection, therefore improving current methods and allowing for better treatment.
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Extracellular vesicles are spherical nanoparticles inherently released by almost all cell types. They acquire the cell's membrane and cytoplasmic characteristics offering abundant identical units that can be captured to recognize the cell of origin. The abundance of vital cell information and multifunctional roles in cellular processes has rendered them attention, particularly as promising biomarkers for disease diagnosis and use in potential drug delivery systems. This review provides insights into standard approaches towards cultivation and isolation of mammalian and bacterial extracellular vesicles. We assess gaps in conventional separation and detection technologies while also tracking developments in ongoing research. The review focuses on highlighting alternative state-of-the-art microfluidic devices that offer avenues for fast, cost-effective, precision-oriented capture and sensing of extracellular vesicles. Combining different detection technologies on an integrated "lab-on-a-chip" system has the prospective to provide customizable opportunities for clinical use of extracellular vesicles in disease diagnostics and therapeutic applications.
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Vesículas Extracelulares , Técnicas Analíticas Microfluídicas/métodos , Animais , Bactérias/química , Técnicas Biossensoriais/métodos , Humanos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
The physical characterisation, capture and detection of extracellular vesicles (EVs) and exosomes derived from breath condensate is reported. Breath-derived EVs were isolated from breath condensate and captured on a gold substrate using two complimentary methods. The characterised and isolated EVs were detected using surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS). EIS was done using aptamers as a targeting moiety and showed a larger change in resistance between dilute concentrations of EVs (less than 7 µg/mL).This is the first report of EVs and exosomes isolated and characterised from breath. In addition, EVs from a non-invasive and easily available source such as breath opens up further avenues in the detection of pulmonary diseases.
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Testes Respiratórios , Vesículas Extracelulares/metabolismo , Espectroscopia Dielétrica/métodos , Humanos , Pneumopatias/diagnóstico , Microscopia Eletrônica de Transmissão , Pontos Quânticos , Ressonância de Plasmônio de Superfície/métodosRESUMO
Redox active, photoluminescent silver nanoclusters templated with oligonucleotides were developed for glucose sensing. The silver nanoclusters had a photoluminescent emission at 610 nm that reversibly changed to 530 nm upon oxidation. The reversible emission change was measured with photoluminescent spectroscopy and used to detect H2O2, which is a by-product of the reaction of glucose with glucose oxidase. The ratio of the un-oxidised emission peak (610 nm) and the oxidised analogue (530 nm) was used to measure glucose concentrations up to 20 mM, well within glucose levels found in blood. Also, the reversibility of this system enables the silver nanoclusters to be reused.
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Indium phosphide quantum dots (QDs) passivated with zinc sulphide in a core/shell architecture (InP/ZnS) with different surface chemistries were introduced to RAW 264.7 murine "macrophage-like" cells to understand their potential toxicities. The InP/ZnS quantum dots were conjugated with an oligonucleotide, a carboxylic acid, or an amino-polyethylene glycol ligand, and cell viability and cell proliferation were investigated via a metabolic assay. Membrane integrity was measured through the production of lactate dehydrogenase. Fluorescence microscopy showed cellular uptake. All quantum dots exhibited cytotoxic behaviour less than that observed from cadmium- or lead-based quantum dots; however, this behaviour was sensitive to the ligands used. In particular, the amino-polyethylene glycol conjugated quantum dots proved to possess the highest cytotoxicity examined here. This provides quantitative evidence that aqueous InP/ZnS quantum dots can offer a safer alternative for bioimaging or in therapeutic applications.
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Quantum dots are attractive alternatives to organic fluorophores for the purposes of fluorescent labeling and the detection of biomarkers. They can also be made to specifically target a protein of interest by conjugating biomolecules, such as antibodies. However, the majority of the fluorescent labeling using quantum dots is done using toxic materials such as cadmium or lead due to the well-established synthetic processes for these quantum dots. Here, we demonstrate the use of indium phosphide quantum dots with a zinc sulfide shell for the purposes of labeling and the detection of exosomes derived from the THP-1 cell line (monocyte cell line). Exosomes are nano-sized vesicles that have the potential to be used as biomarkers due to their involvement in complex cell processes. However, the lack of standardized methodology around the detection and analysis of exosomes has made it difficult to detect these membrane-containing vesicles. We targeted a protein that is known to exist on the surface of the exosomes (CD63) using a CD63 antibody. The antibody was conjugated to the quantum dots that were first made water-soluble using a ligand-exchange method. The conjugation was done using carbodiimide coupling, and was confirmed using a range of different methods such as dynamic light scattering, surface plasmon resonance, fluorescent microscopy, and Fourier transform infrared spectroscopy. The conjugation of the quantum dot antibody to the exosomes was further confirmed using similar methods. This demonstrates the potential for the use of a non-toxic conjugate to target nano-sized biomarkers that could be further used for the detection of different diseases.
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Cádmio , Exossomos/química , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Pontos Quânticos , Carbodi-Imidas/química , Linhagem Celular , Exossomos/imunologia , Exossomos/metabolismo , Humanos , Índio , Fosfinas , Sulfetos , Tetraspanina 30/imunologia , Compostos de ZincoRESUMO
Exosomes are biomolecular nanostructures released from cells. They carry specific biomolecular information and are mainly researched for their exquisite properties as a biomarker source and delivery system. We introduce exosomes in the context of other extracellular vesicles, describe their biophysical isolation and characterisation and discuss their biochemical profiling. Motivated by our interest in early-life nutrition and health, and corresponding studies enrolling lactating mothers and their infants, we zoom into exosomes derived from human breast milk. We argue that these should be more extensively studied at proteomic and micronutrient profiling level, because breast milk exosomes provide a more specific window into breast milk quality from an immunological (proteomics) and nutritional (micronutrient) perspective. Such enhanced breast milk exosome profiling would thereby complement and enrich the more classical whole breast milk analysis and is expected to deliver more functional insights than the rather descriptive analysis of human milk, or larger fractions thereof, such as milk fat globule membrane. We substantiate our arguments by a bioinformatic analysis of two published proteomic data sets of human breast milk exosomes.
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The authors describe the synthesis of water-soluble and fluorescent graphene oxide quantum dots via acid exfoliation of graphite nanoparticles. The resultant graphene oxide quantum dots (GoQDs) were then modified with folic acid. Folic acid receptors are overexpressed in cancer cells and hence can bind to functionalized graphene oxide quantum dots. On excitation at 305 nm, the GoQDs display green fluorescence with a peak wavelength at ~520 nm. The modified GoQDs are non-toxic to macrophage cells even after prolonged exposure and high concentrations. Fluorescence lifetime imaging and multiphoton microscopy was used (in combination) to image HeCaT cells exposed to GoQDs, resulting in a superior method for bioimaging. Graphical abstract Schematic representation of graphene oxide quantum dots, folic acid modified graphene oxide quantum dots (red), and the use of fluorescence lifetime to discriminate against green auto-fluorescence of HeCaT cells.
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Ácido Fólico/química , Grafite/química , Neoplasias/diagnóstico por imagem , Imagem Óptica/métodos , Pontos Quânticos/química , Linhagem Celular Tumoral , Receptores de Folato com Âncoras de GPI/análise , Receptores de Folato com Âncoras de GPI/metabolismo , Ácido Fólico/metabolismo , Humanos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias/patologiaRESUMO
Since their advent in the early 1990s, nanomaterials hold promise to constitute improved technologies in the biomedical area. In particular, graphene quantum dots (GQDs) were conjectured to produce new or improve current methods used for bioimaging, drug delivery, and biomarker sensors for early detection of diseases. This review article critically compares and discusses current state-of-the-art use of GQDs in biology and health sciences. It shows the ability of GQDs to be easily functionalised for use as a targeted multimodal treatment and imaging platform. The in vitro and in vivo toxicity of GQDs are explored showing low toxicity for many types of GQDs.
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Diagnóstico por Imagem/métodos , Grafite/administração & dosagem , Pontos Quânticos/administração & dosagem , Nanomedicina Teranóstica/métodos , Animais , Diagnóstico por Imagem/tendências , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/tendências , Grafite/metabolismo , Humanos , Pontos Quânticos/metabolismo , Nanomedicina Teranóstica/tendênciasRESUMO
Silver nanoparticles are well-known for their antibacterial properties. However, the detailed mechanism describing the interaction between the nanoparticles and a cell membrane is not fully understood, which can impede the use of the particles in biomedical applications. Here, a tethered bilayer lipid membrane has been used as a model system to mimic a natural membrane and to study the effect of exposure to small silver nanoparticles with diameters of about 2 nm. The solid supported membrane architecture allowed for the application of surface analytical techniques such as electrochemical impedance spectroscopy and atomic force microscopy. Exposure of the membrane to solutions of the silver nanoparticles led to a small but completely reversible perturbation of the lipid bilayer.
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Bicamadas Lipídicas/química , Nanopartículas Metálicas/química , Prata/química , Espectroscopia Dielétrica , Microscopia de Força AtômicaRESUMO
Advanced approaches to direct the differentiation of embryonic stem cells are highly sought after. The surface-bound chemical gradient format is a powerful screening approach that can be deployed to study changes in stem cell behavior as a function of subtle changes in surface chemistry. Here, we investigate the spontaneous differentiation of cells derived from differentiating mouse embryoid body (mEB) cells into endoderm, mesoderm, and ectoderm following culture on surface-bound gradients of chemical functional groups in the absence of differentiation-biasing bioactive factors. Gradients were created using a diffusion-controlled plasma polymerization technique. The generated coating ranged from hydrophobic 1,7-octadiene (OD) plasma polymer at one end of the gradient to a more hydrophilic allylamine (AA) plasma polymer on the opposite end. The gradient surface was divided into seven equal regions of progressively increasing AA plasma polymer content and mEB cell response within these regions was compared. Cells adhered preferentially to the central regions of the gradient; however, cell proliferation increased toward AA-plasma-polymer-rich end of the gradient. Variation in the expression of germ layer markers was noted across the gradient surface. High AA:OD plasma polymer ratios triggered cell differentiation toward both mesoderm and ectoderm. Expression of tissue-specific markers, in particular, KRT18, AFP, and TNNT2, was strikingly responsive to subtle changes in surface chemistry, exhibiting vastly different expression levels between adjacent regions. Our results suggest that the surface-bound gradient platform is well suited to screening surface chemistries for use in the field of stem cell technologies and regenerative medicine.
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Alilamina/farmacologia , Diferenciação Celular , Corpos Embrioides/citologia , Alcenos/farmacologia , Animais , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Corpos Embrioides/efeitos dos fármacos , Imunofluorescência , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camundongos , Espectroscopia Fotoeletrônica , Propriedades de SuperfícieRESUMO
Nanoporous alumina (PA) arrays produced by self-ordering growth, using electrochemical anodization, have been extensively explored for potential applications based upon the unique thermal, mechanical and structural properties, and high surface-to-volume ratio of these materials. However, the potential applications and functionality of these materials may be further extended by molecular-level engineering of the surface of the pore rims. In this paper we present a method for the generation of chemical gradients on the surface of PA arrays based upon plasma co-polymerization of two monomers. We further extend these chemical gradients, which are also gradients of surface charge, to those of bound ligands and number density gradients of nanoparticles. The latter represent a highly exotic new class of materials, comprising aligned PA, capped by gold nanoparticles around the rim of the pores. Gradients of chemistry, ligands and nanoparticles generated by our method retain the porous structure of the substrate, which is important in applications that take advantage of the inherent properties of these materials. This method can be readily extended to other porous materials.
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This paper presents a novel and facile method for the generation of efficient antibacterial coatings which can be applied to practically any type of substrate. Silver nanoparticles were stabilized with an adsorbed surface layer of polyvinyl sulphonate (PVS). This steric layer provided excellent colloidal stability, preventing aggregation over periods of months. PVS-coated silver nanoparticles were bound onto amine-containing surfaces, here produced by deposition of an allylamine plasma polymer thin film onto various substrates. SEM imaging showed no aggregation upon surface binding of the nanoparticles; they were well dispersed on amine surfaces. Such nanoparticle-coated surfaces were found to be effective in preventing attachment of Staphylococcus epidermidis bacteria and also in preventing biofilm formation. Combined with the ability of plasma polymerization to apply the thin polymeric binding layer onto a wide range of materials, this method appears promising for the fabrication of a wide range of infection-resistant biomedical devices.