Optical Nanoimpacts of Dielectric and Metallic Nanoparticles on Gold Surface by Reflectance Microscopy: Adsorption or Bouncing?

International audienceOptical modeling coupled to experiments show that a microscope operating in reflection mode allows imaging, through solutions or even a microfluidic cover, various kinds of nanoparticles, NPs, over a (reflecting) sensing surface, here a gold (Au) surface. Optical modeling suggests that this configuration enables the interferometric imaging of single NPs which can be characterized individually from local change in the surface reflectivity. The interferometric detection improves the optical limit of detection compared to classical configurations exploiting only the light scattered by the NPs. The method is then tested experimentally, to monitor in situ and in real time, the collision of single Brownian NPs, or optical nanoimpacts, with an Au-sensing surface. First, mimicking a microfluidic biosensor platform, the capture of 300 nm FeOx maghemite NPs from a convective flow by a surface-functionalized Au surface is dynamically monitored. Then, the adsorption or bouncing of individual dielectric (100 nm polystyrene) or metallic (40 and 60 nm silver) NPs is observed directly through the solution. The influence of the electrolyte on the ability of NPs to repetitively bounce or irreversibly adsorb onto the Au surface is evidenced. Exploiting such visualization mode of single-NP optical nanoimpacts is insightful for comprehending single-NP electrochemical studies relying on NP collision on an electrode (electrochemical nanoimpacts)

In-capillary immuno-preconcentration with circulating bio-functionalized magnetic beads for capillary electrophoresis

International audienceThis study reports on the conception of magneto-Capillary Electrophoresis (magneto-CE), an approach integrating immuno-capture on circulating bio-functionalized magnetic beads into a unique capillary for preconcentration and electrokinetic separation. This hybrid mode is an evolution of in-capillary magnetic bead-based operation from static cluster format to dynamic configuration where beads are allowed to controllably circulate inside a CE capillary for interaction improvement. To implement the magneto-CE operation, a purpose-made instrument was constructed, allowing visual observation of the movement of the magnetic beads. We applied a new methodological strategy for determination of the amyloid β peptide (Aβ 1–42), which is as an established biomarker for molecular diagnosis of Alzheimer's disease (AD). The methodology is based on magneto-immuno-capture of fluorescently labeled Aβ 1–42 followed by a chemical elution with a basic solution prior to CE separation with laser induced fluorescent (LIF) detection. The superiority of this dynamic configuration of magneto-CE was demonstrated for this target analyte, with sample pretreatment and separation being performed in-capillary without any delay in between and without any waste of pretreated sample, which otherwise would not be the case with offline/batch-wise operation

In-capillary immuno-preconcentration with circulating bio-functionalized magnetic beads for capillary electrophoresis

This study reports on the conception of magneto-Capillary Electrophoresis (magneto-CE), an approach integrating immuno-capture on circulating bio-functionalized magnetic beads into a unique capillary for preconcentration and electrokinetic separation. This hybrid mode is an evolution of in-capillary magnetic bead-based operation from static cluster format to dynamic configuration where beads are allowed to controllably circulate inside a CE capillary for interaction improvement. To implement the magneto-CE operation, a purpose-made instrument was constructed, allowing visual observation of the movement of the magnetic beads. We applied a new methodological strategy for determination of the amyloid beta peptide (A beta 1-42), which is as an established biomarker for molecular diagnosis of Alzheimer's disease (AD). The methodology is based on magneto-immuno-capture of fluorescently labeled A beta 1-42 followed by a chemical elution with a basic solution prior to CE separation with laser induced fluorescent (LIF) detection. The superiority of this dynamic configuration of magneto-CE was demonstrated for this target analyte, with sample pretreatment and separation being performed in-capillary without any delay in between and without any waste of pretreated sample, which otherwise would not be the case with offline/batch-wise operation. (C) 2019 Elsevier B.V. All rights reserved

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Support immunologique pour biocapteur (caractérisations physico-chimiques et biologiques)

L objectif de mon doctorat, réalisé dans le cadre d une collaboration entre le laboratoire des protéines et nanotechnologies en sciences séparatives (institut Galien Paris-Sud) et le groupe Micro et Nano Système (institut d électronique fondamentale) était d étudier l influence des monocouches autoassemblées, sur l activité biologique du bio-récepteur dans une perspective de développement de biocapteur. Dans ce projet, nous avons choisi les organo-silanes qui peuvent se lier de manière covalente sur le silicium. Deux silanes (7-octenyl trichlorosilanes et le (3-aminopropyl)triethoxysilane) ont été étudiés, leur impact en terme de nature et de stabilité sur la fonctionnalité du bio-récepteur, des immunoglobulines G de souris, ont été évalué. Dans un premier temps, nous nous sommes intéressés à la fonctionnalité des anticorps greffés sur une mono couche auto-assemblée composée de 7-octenyltrichlorosilane (OTS) présentant à sa surface un groupement carboxylique. Une caractérisation spectroscopique par XPS et infra-rouge à transformé de Fourrier (FTIR) a tout d abord été effectuée afin de vérifier la présence de ces groupements carboxyliques. L homogénéité de la surface a été évaluée par AFM. Nous avons ensuite immobilisé ces anticorps, sur ces supports, de manière covalente et une étude topographique par AFM a été menée pour mesurer la répartition de ces anticorps. L orientation des anticorps greffés a été évaluée à l aide d immuno-essais. Ensuite, nous avons comparé l APTES, permettant l obtention de plaques de silicium fonctionnalisé avec des groupements aminés à leur surface, avec l OTS. Nous avons notamment comparé la capacité de capture des anticorps immobilisés sur ces deux types de silanes. Dans la dernière partie, l impact du vieillissement d un support immunologique préparé chimiquement en utilisant l APTES a été évaluée sur le plan physico-chimique et biologique.The aim of my PhD thesis, conducted as part of a collaboration between the laboratory of protein separation sciences and nanotechnology (Paris-Sud Galen Institute) and the Micro and Nano System (basic electronics institute) group was to study the influence of self-assembled monolayers on the biological activity of bioreceptor toward biosensor development. In this project, we choose the organosilanes that can bind covalently to the silicon. Two silanes (7-octenyl trichlorosilane(OTS) and 3- aminopropyltriethoxysilane (APTES)) were studied., Their impact on the stability and the functionality of bio- receptor , model mouse immunoglobulin G (IgG), were evaluated. Spectroscopic characterization by XPS and infra- red Fourier transformed (FTIR) was first carried out to assess that the silanized surface exhibit carboxylic groups. The homogeneity of the surfaces was measured by AFM. Then, IgG were immobilized on these supports, covalently and a topographic AFM study was conducted to measure the distribution of these antibodies. The orientation of the grafted antibody was investigated by immune-enzymatic assays. We have also evaluated the binding capacity of the IgG immobilized on both surfaces. Then, the impact of aging on APTES surface was evaluated by spectroscopics and biological methods.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Apport de nouvelles phases stationnaires dans le contrôle qualité des peptides à visée thérapeutique

La production de peptides-médicaments et de protéines recombinantes est en plein essor dans l industrie pharmaceutique. La synthèse des peptides en phase solide conduit à la formation de substances apparentées de structure proche du peptide d intérêt, d où la nécessité d un contrôle qualité rigoureux. En ce qui concerne les protéines recombinantes, leur contrôle qualité est généralement effectué par l établissement d une carte peptidique, qui correspond à l analyse des fragments peptidiques issus de la digestion enzymatique de la protéine à l aide d une méthode séparative. Pour la détection des substances apparentées ou issus de la dégradation des peptides, des méthodes analytiques capables de distinguer des formes structurellement très proches doivent être proposées pour le contrôle qualité, à toutes les étapes du développement du produit. La chromatographie liquide haute performance (CLHP) en phase inverse est la méthode la plus utilisée pour la détermination de la pureté des peptides synthétiques ou l établissement de la carte peptidique d une protéine. Le but de ce travail de thèse était d évaluer l apport de nouvelles phases stationnaires dans l analyse des peptides. Différents types de phases stationnaires particulaires ou monolithiques ont été étudiés : phases mixtes, d interactions hydrophiles et d échange d ions. Des peptides de caractère physicochimique différents ou de structure proche (les enképhalines) ont été étudiés. L application à la séparation d un peptide d intérêt (dalargine) de ses impuretés de synthèse a été réalisée.The production of peptide-drug and recombinant proteins is growing in the pharmaceutical industry. The solid phase synthesis of peptides leads to the formation of related substances of similar structure of the target peptide, hence the need for rigorous quality control. Regarding the recombinant proteins, their quality control is generally performed by peptide mapping, which corresponds to the analysis of peptide fragments resulted from the enzymatic digestion of the protein. For the detection of related substances or degradation products of peptides, analytical methods able to distinguish similar structurally products should be proposed for quality control at all stages of product development. Reversed phase high-performance liquid chromatography (HPLC-RP) is the most common method used either for determining the purity of synthetic peptides or for the peptide mapping of a protein. The aim of this thesis was to evaluate the contribution of new stationary phases in peptide analysis. Different types of particulate or monolithic stationary phases were studied: mixed, hydrophilic interaction and ion-exchange phases. Peptides of different physico-chemical properties or of closed structure (enkephalins) have been studied. Application to the separation of a target peptide (dalargin) from its synthetic impurities has been performed.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Integration of microcoils for on-chip immunosensors based on magnetic nanoparticles capture

Immunoassays using magnetic nanoparticles (MNP) are generally performed under the control of permanent magnet close to the micro-tube of reaction. Using a magnet gives a powerful method for driving MNP but remains unreliable or insufficient for a fully integrated immunoassay on lab-on-chip. The aim of this study is to develop a novel lab-on-chip concept for high efficient immunoassays to detect ovalbumin (Biodefense model molecule) with microcoils employed for trapping MNP during the biofunctionalization steps. The objectives are essentially to optimize their efficiency for biological recognition by assuring a better bioactivity (antibodies-ovalbumin), and detect small concentrations of the targeted protein (~10 pg/mL). In this work, we studied the response of immunoassays complex function of ovalbumin concentration. The impact of MNP diameter in the biografting protocol was studied and permitted to choose a convenient MNP size for efficient biorecognition. We realized different immunoassays by controlling MNP in test tube and in microfluidic device using a permanent magnet. The comparison between these two experiments allows us to highlight an improvement of the limit of detection in microfluidic conditions by controlling MNP trapping with a magnet. Keywords: Bacteria, Lab-on-chip, ELISA, Magnetic nanoparticles, Ovalbumin, Microcoils, Fluorescent microscop