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1.
Talanta ; 243: 123355, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1778463

ABSTRACT

Mass testing for the diagnosis of COVID-19 has been hampered in many countries owing to the high cost of genetic material detection. This study reports on a low-cost immunoassay for detecting SARS-CoV-2 within 30 min using dynamic light scattering (DLS). The immunosensor comprises 50-nm gold nanoparticles (AuNPs) functionalized with antibodies against SARS-CoV-2 spike glycoprotein, whose bioconjugation was confirmed using transmission electron microscopy (TEM), UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and surface-enhanced Raman scattering spectroscopy (SERS). The specific binding of the bioconjugates to the spike protein led to an increase in bioconjugate size, with a limit of detection (LOD) 5.29 × 103 TCID50/mL (Tissue Culture Infectious Dose). The immunosensor was also proven to be selective upon interaction with influenza viruses once no increase in size was observed after DLS measurement. The strategy proposed here aimed to use antibodies conjugated to AuNPs as a generic platform that can be extended to other detection principles, enabling technologies for low-cost mass testing for COVID-19.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Biosensing Techniques/methods , COVID-19/diagnosis , COVID-19 Testing , Dynamic Light Scattering , Gold/chemistry , Humans , Immunoassay/methods , Metal Nanoparticles/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Viral Proteins
2.
Blood ; 138(22): 2256-2268, 2021 12 02.
Article in English | MEDLINE | ID: covidwho-1443788

ABSTRACT

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.


Subject(s)
Antigen-Antibody Complex/immunology , Autoantibodies/immunology , COVID-19/prevention & control , Capsid Proteins/adverse effects , Drug Contamination , Genetic Vectors/adverse effects , HEK293 Cells/immunology , Immunoglobulin G/immunology , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/etiology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/adverse effects , Adenoviridae/immunology , Animals , Antigen-Antibody Complex/ultrastructure , Autoantibodies/biosynthesis , Capillary Leak Syndrome/etiology , Capsid Proteins/immunology , Cell Line, Transformed , /immunology , Dynamic Light Scattering , Epitopes/chemistry , Epitopes/immunology , Extracellular Traps/immunology , Extravasation of Diagnostic and Therapeutic Materials/etiology , Genetic Vectors/immunology , HEK293 Cells/chemistry , Humans , Imaging, Three-Dimensional , Immunoglobulin G/biosynthesis , Inflammation , Mice , Microscopy/methods , Platelet Activation , Proteomics , Purpura, Thrombocytopenic, Idiopathic/blood , Purpura, Thrombocytopenic, Idiopathic/immunology , Sinus Thrombosis, Intracranial/diagnostic imaging , Sinus Thrombosis, Intracranial/immunology , Spike Glycoprotein, Coronavirus/immunology , Virus Cultivation
3.
J Phys Chem Lett ; 12(39): 9557-9563, 2021 Oct 07.
Article in English | MEDLINE | ID: covidwho-1439482

ABSTRACT

Lipid-enveloped viruses, such as Ebola, influenza, or coronaviruses, are a major threat to human health. Ethanol is an efficient disinfectant that is widely used to inactivate these viruses and prevent their transmission. However, the interactions between ethanol and enveloped viruses leading to their inactivation are not yet fully understood. This study demonstrates the link between ethanol-induced viral inactivation and the nanostructural and chemical transformations of the model virus Phi6, an 85 nm diameter lipid-enveloped bacterial virus that is commonly used as surrogate for human pathogenic viruses. The virus morphology was investigated using small-angle X-ray scattering and dynamic light scattering and was related to its infectivity. The Phi6's surface chemistry was characterized by cryogenic X-ray photoelectron spectroscopy, and the modifications in protein structure were assessed by circular dichroism and fluorescence spectroscopy. Ethanol-triggered structural modifications were found in the lipid envelope, detaching from the protein capsid and forming coexisting nanostructures.


Subject(s)
Bacteriophage phi 6/chemistry , Ethanol/pharmacology , Virus Inactivation/drug effects , Bacteriophage phi 6/drug effects , Bacteriophage phi 6/ultrastructure , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Circular Dichroism , Dynamic Light Scattering , Ethanol/chemistry , Microscopy, Electron, Transmission , Photoelectron Spectroscopy , Scattering, Small Angle , X-Ray Diffraction
4.
Mol Biotechnol ; 64(1): 1-8, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1366408

ABSTRACT

Because of the essential roles of SARS-CoV-2 papain-like protease (PLpro) in the viral polyprotein processing and suppression of host immune responses, it is a crucial target for drug discovery against COVID-19. To develop robust biochemical methodologies for inhibitor screening against PLpro, extensive characterization of recombinant protein is important. Here we report cloning, expression, and purification of the recombinant SARS-CoV-2 PLpro, and explore various parameters affecting its stability and the catalytic activity. We also report the optimum conditions which should be used for high-throughput inhibitor screening using a fluorogenic tetrapeptide substrate.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , High-Throughput Screening Assays/methods , Antiviral Agents/pharmacology , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/isolation & purification , Coumarins/chemistry , Coumarins/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Dimethyl Sulfoxide/chemistry , Dynamic Light Scattering , Edetic Acid/chemistry , Enzyme Stability , Fluorometry/methods , Hydrogen-Ion Concentration , Osmolar Concentration , Peptides/chemistry , Peptides/metabolism , Temperature
5.
Chem Commun (Camb) ; 57(57): 6979-6982, 2021 Jul 15.
Article in English | MEDLINE | ID: covidwho-1287828

ABSTRACT

The infection of coronavirus initiates with the binding between its spike protein receptor binding domain (RBD) and a human cellular receptor called angiotensin-converting enzyme 2 (ACE2). Here, we construct truncated ACE2 peptide-conjugated gold nanoparticles as antiviral scaffolds and study their binding with the SARS-CoV-2 RBD using dynamic light scattering (DLS). Systematic DLS analysis identifies the effective peptide-nanoparticle conjugation and its efficient, specific, and long-lasting multivalent binding towards the RBD with a binding affinity of 41 nM, indicating the potential of this antiviral platform to compete with natural ACE2-RBD interactions for viral blocking and showcasing an accessible approach to measure the binding constants and kinetics.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Nanoparticles/chemistry , Peptide Fragments/chemistry , Peptide Fragments/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Dynamic Light Scattering , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Substrate Specificity
6.
Molecules ; 26(9)2021 May 04.
Article in English | MEDLINE | ID: covidwho-1238922

ABSTRACT

Quercetin is a poorly water-soluble flavonoid with many benefits to human health. Besides the natural food resources that may provide Quercetin, the interest in delivery systems that could enhance its bioavailability in the human body has seen growth in recent years. Promising delivery system candidates are represented by Solid Lipid Nanoparticles (SLNs) which are composed of well-tolerated compounds and provide a relatively high encapsulation efficiency and suitable controlled release. In this study, Quercetin-loaded and negatively charged Solid Lipid Nanoparticles were synthesized based on a coacervation method, using stearic acid as a core lipid and Arabic Gum as a stabilizer. Samples were qualitatively characterized by Dynamic light scattering (DLS), Zeta Potential, Surface infrared spectroscopy (FTIR-ATR), and Time of flight secondary ion mass spectrometry (ToF-SIMS). Encapsulation efficiency, drug release, and antioxidant effect against ABTS•+ were evaluated in vitro by UV-VIS spectrophotometry.


Subject(s)
Drug Carriers/chemistry , Lipids/chemistry , Nanoparticles/chemistry , Quercetin/pharmacology , Antioxidants/pharmacology , Delayed-Action Preparations , Dynamic Light Scattering , Particle Size , Spectrometry, Mass, Secondary Ion , Spectroscopy, Fourier Transform Infrared , Static Electricity , Time Factors
7.
Int J Nanomedicine ; 16: 2689-2702, 2021.
Article in English | MEDLINE | ID: covidwho-1186650

ABSTRACT

BACKGROUND: The COVID-19 pandemic is requesting highly effective protective personnel equipment, mainly for healthcare professionals. However, the current demand has exceeded the supply chain and, consequently, shortage of essential medical materials, such as surgical masks. Due to these alarming limitations, it is crucial to develop effective means of disinfection, reusing, and thereby applying antimicrobial shielding protection to the clinical supplies. PURPOSE: Therefore, in this work, we developed a novel, economical, and straightforward approach to promote antimicrobial activity to surgical masks by impregnating silver nanoparticles (AgNPs). METHODS: Our strategy consisted of fabricating a new alcohol disinfectant formulation combining special surfactants and AgNPs, which is demonstrated to be extensively effective against a broad number of microbial surrogates of SARS-CoV-2. RESULTS: The present nano-formula reported a superior microbial reduction of 99.999% against a wide number of microorganisms. Furthermore, the enveloped H5N1 virus was wholly inactivated after 15 min of disinfection. Far more attractive, the current method for reusing surgical masks did not show outcomes of detrimental amendments, suggesting that the protocol does not alter the filtration effectiveness. CONCLUSION: The nano-disinfectant provides a valuable strategy for effective decontamination, reuse, and even antimicrobial promotion to surgical masks for frontline clinical personnel.


Subject(s)
Anti-Infective Agents/pharmacology , Disinfectants/pharmacology , Masks , Metal Nanoparticles/chemistry , Silver/pharmacology , Animals , Anti-Infective Agents/administration & dosage , Anti-Infective Agents/chemistry , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/prevention & control , COVID-19/transmission , Chick Embryo , Disinfectants/administration & dosage , Disinfectants/chemistry , Disinfection/methods , Dynamic Light Scattering , Equipment Reuse , Humans , Influenza A Virus, H5N1 Subtype/drug effects , Masks/virology , Metal Nanoparticles/administration & dosage , Microbial Sensitivity Tests , Silver/chemistry , Spectroscopy, Fourier Transform Infrared , Textiles , X-Ray Diffraction
8.
Biosens Bioelectron ; 179: 113099, 2021 May 01.
Article in English | MEDLINE | ID: covidwho-1086796

ABSTRACT

The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30° and 60°, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30° could statistically differentiate 10, 103, and 105 copies/µL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60° could statistically differentiate 105 copies/µL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.


Subject(s)
COVID-19 Nucleic Acid Testing/instrumentation , COVID-19/diagnosis , Dynamic Light Scattering/instrumentation , Emulsions/chemistry , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/instrumentation , SARS-CoV-2/isolation & purification , Biosensing Techniques/economics , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/methods , Dynamic Light Scattering/economics , Dynamic Light Scattering/methods , Equipment Design , Humans , Limit of Detection , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Smartphone , Time Factors
9.
Proc Natl Acad Sci U S A ; 117(22): 11875-11877, 2020 06 02.
Article in English | MEDLINE | ID: covidwho-260636

ABSTRACT

Speech droplets generated by asymptomatic carriers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are increasingly considered to be a likely mode of disease transmission. Highly sensitive laser light scattering observations have revealed that loud speech can emit thousands of oral fluid droplets per second. In a closed, stagnant air environment, they disappear from the window of view with time constants in the range of 8 to 14 min, which corresponds to droplet nuclei of ca. 4 µm diameter, or 12- to 21-µm droplets prior to dehydration. These observations confirm that there is a substantial probability that normal speaking causes airborne virus transmission in confined environments.


Subject(s)
Air Microbiology , Betacoronavirus/physiology , Coronavirus Infections/transmission , Pneumonia, Viral/transmission , Saliva/virology , COVID-19 , Dynamic Light Scattering , Fomites/virology , Humans , Pandemics , Particle Size , SARS Virus , SARS-CoV-2 , Saliva/chemistry , Speech , Viral Load
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