On-Line influenza virus quantification for viral production processes thanks to affinity-based surface plasmon resonance biosensor

Influenza virus seasonal epidemics, associated with the constant threat of new pandemic outbreak, challenge vaccine manufacturers to develop responsive processes that can outreach the limitations of traditional egg-based technology. Recent progress made regarding cell culture bioprocesses allowed for numerous alternative strategies to developed future vaccine candidates, as for example the recombinant HA or Virus—like Particles (VLP) vaccines. However, while cell culture allows for more versatility than ovoculture, regarding process development and monitoring, these alternatives still require optimization to seriously concurrence the traditional process. To drive these developments, WHO and regulatory agencies underlined the need for developing better influenza vaccine potency assays1,2. Actual influenza vaccine formulation and lot release rely on single-radial immunodiffusion (SRID) assay, which requires strain-specific reference sera and antigen reagents. However, the annual preparation of these reagents takes between 2 to 6 months and constitutes a critical bottleneck for the release of vaccine lots3. Additionally, SRID is not implementable for process development as such technique cannot handle in-process low concentrated and non-purified material. We developed an assay for rapid and label-free quantification of influenza hemagglutinin (HA) antigen and influenza virus based on surface plasmon resonance (SPR). The method is based on affinity capture of hemagglutinin antigen by sialic-acid terminated glycans present at the surface of the fetuin-functionalized sensor. Conditions were optimized for the regeneration of the surface, in order to run multiple sequential analyses on a unique sensor. Two types of purified standard were used during the development of the assay. Commercial trivalent inactivated vaccine (“TIV”) has been used for the determination of optimal analytical conditions, while a stock of split inactivated H1N1 virus has been produced and calibrated in our laboratory to study the specific response obtained toward this HA subtype. This assay offers a quantification of influenza hemagglutinin within minutes with a wide dynamic range (30 ng/mL-20 µg/mL). Also, the technique provides a limit of detection (LOD) 100 times lower than SRID, and a better reproducibility than SRID and its potential alternatives recently proposed (1,4,5. Additionally, the applicability of this assay for an on-line vaccine production monitoring has been validated by off-line measurement of influenza H1N1 virus particles derived from cell culture supernatant. Such a test allowed to achieve a LOD of 106 Infectious Viral Particles/mL Thus, our assay provides an innovative tool to evaluate influenza new vaccine bioprocesses, from viral production kinetics in mammalian cell culture to vaccine potency evaluation

Integrated graphene quantum dot decorated functionalized nanosheet biosensor for mycotoxin detection

Decoration of graphene quantum dots (GQDs) on molybdenum disulfide (MoS2) nanosheets serves as an active electrode material which enhances the electrochemical performance of the analyte detection system. Herein, ionic surfactant cetyltrimethylammonium bromide (CTAB)-exfoliated MoS2 nanosheets decorated with GQD material are used to construct an electrochemical biosensor for aflatoxin B1 (AFB1) detection. An antibody of AFB1 (aAFB1) was immobilized on the electrophoretically deposited MoS2@GQDs film on the indium tin oxide (ITO)-coated glass surface using a crosslinker for the fabrication of the biosensor. The immunosensing study investigated by the electrochemical method revealed a signal response in the range of 0.1 to 3.0 ng/mL AFB1 concentration with a detection limit of 0.09 ng/mL. Also, electrochemical parameters such as diffusion coefficient and heterogeneous electron transfer (HET) were calculated and found to be 1.67 x 10(-5) cm(2)/s and 2 x 10(-5)cm/s, respectively. The effective conjugation of MoS2@GQDs that provides abundant exposed edge sites, large surface area, improved electrical conductivity, and electrocatalytic activity has led to an excellent biosensing performance with enhanced electrochemical parameters. Validation of the fabricated immunosensor was performed in a spiked maize sample, and a good percentage of recoveries within an acceptable range were obtained (80.2 to 98.3%)

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio

Développement d un outil d analyse de biomarqueurs pour le diagnostic, le pronostic et le suivi de la réponse au traitement médicamenteux du cancer de la vessie

Les avancées technologiques, notamment en protéomique, ont permis d améliorer la compréhension des aspects physiopathologiques de certaines maladies mais également de découvrir de nouveaux biomarqueurs. Ces derniers ont un fort potentiel au regard de la médecine personnalisée, que ce soit à l étape de diagnostic, de traitement ou de suivi. Dans ce contexte, le projet DIPROMON a pour objectif d améliorer les stratégies de médecine personnalisée dans le cancer de la vessie. Pour cela, un concept de stratification des patients sera élaboré grâce à l analyse de profils de biomarqueurs. La stratification sera notamment utile pour la surveillance des récidives du cancer de la vessie et pour le choix de la thérapie selon la prédiction de réponse au traitement. Même si la majorité des patients est atteinte de tumeurs superficielles, le taux de récidive est élevé et concerne entre 50 et 70% des patients. La surveillance régulière des patients en est une des conséquences principales. Les travaux présentés dans ce mémoire concernent la mise au point d un test multiparamétrique haut-débit pour la détection et la quantification des biomarqueurs, qui fera partie de la plate-forme technologique élaborée dans le projet DIPROMON. Le développement de la puce à protéines a débuté avec trois interleukines (IL6, IL8 et IL10) détectées et quantifiées par une méthode d immunoessai en sandwich. Le test a subi plusieurs étapes d optimisation mais la validation analytique n est pas encore complète. L outil d analyse final se complexifiera lors de l ajout d autres marqueurs sélectionnés par les partenaires du projet. Il nécessitera ensuite une validation clinique, notamment par une étude pilote.The advent of technologies, particularly in the field of proteomics, has improved the understanding of disease pathophysiology as well as the discovery of new biomarkers. Biomarkers show a high potential in personalized medicine in various stages such as diagnosis, treatment and follow-up. In this context, the DIPROMON project aims to improve personalization strategies in bladder cancer. The concept of patient stratification will be developed through the analysis of biomarker profiles. Stratification will be especially useful for monitoring recurrence of bladder cancer and for tailoring therapeutic interventions. Although the majority of patients develops superficial tumors, recurrence rate is high and affects 50 to 70% of patients. One of the main consequences of recurrence is regular monitoring for most patients. The work presented in this report relates to the development of a high-throughput and multiplex test for detection and quantification of biomarkers, which will be part of the technological platform developed in the DIPROMON project. The development of the protein chip started with three interleukins (IL6, IL8 and IL10) which are detected and quantified by a sandwich immunoassay. The test has undergone several optimization steps but the analytical validation is not completed yet. The final analysis tool will be more complex as other markers selected by the project partners will be added. It will then require a clinical validation, including a pilot study.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

A label-free ultrasensitive microfluidic surface Plasmon resonance biosensor for Aflatoxin B-1 detection using nanoparticles integrated gold chip

The Surface Plasmon resonance (SPR) based label-free detection of small targeted molecules is a great challenge and require substantial signal amplification for the accurate and precise quantification. The incorporation of noble metal nanoparticles (NPs) like gold (Au) NPs for the fabrication of SPR biosensor has shown remarkable impact both for anchoring the signal amplification and generate plasmonic resonant coupling between NPs and chip surface. In this work, we present comparative studies related to the fabrication of self-assembled monolayer (SAM) and the influence of AuNPs on Au chip for Aflatoxin B-1 (AFB(1)) detection using SPRi apparatus. The SAM Au chip was sequentially modified by EDC-NHS crosslinkers, grafting of protein-A and finally interaction with anti-AFB(1) antibodies. Similar multilayer chip surface was prepared using functionalized lipoic acid AuNPs deposited on SAM Au chips followed by in situ activation of functional groups using EDC-NHS crosslinkers, grafting of protein-A and immobilization of anti-AFB(1) antibodies. This multilayer functionalized AuNPs modified Au chip was successfully utilized for AFB(1) detection ranging from 0.01 to 50 nM with a limit of detection of 0.003 nM. When compared to bare self-assembled Au chip which was shown to exhibit a limit of detection of 0.19 nM and a linear detection ranging from 1 to 50 nM, the AuNPs modified Au chip was proven to clearly be a better analytical tool. Finally, validation of the proposed biosensor was evaluated by spiked wheat samples and average recoveries (93 and 90.1%) were found to be acceptable

Immobilisation de biomolécules par la technique du transfert de macromolécules dans le PDMS pour le développement de biopuces

An innovative technology based on the direct immobilization of proteins and DNA spots during the PDMS polymerization was developed during this work. The introduced technology was named macromolecules to PDMS transfer and allowed the fabrication of functionalized PDMS surfaces containing active immobilized DNA sequences and proteins for molecular recognition. Then, taking advantage the available PDMS properties, two microfluidics systems were designed and used for the chemiluminescent detection of proteins interactions. Finally, an extension of the possibilities offered by this approach was demonstrated through the replacement of PDMS with a solution of poly(methylmethacrylate) (PMMA) polymerizable to produce PMMA biochips.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Adding biomolecular recognition capability to 3D printed objects : 4D printing

International audienceThree-dimensional (3D) printing technologies will impact the biosensor community in the near future, at both the sensor prototyping level and the sensing layer organization level. The present study aimed at demonstrating the capacity of one 3D printing technique, digital light processing (DLP), to produce hydrogel sensing layers with 3D shapes that are unattainable using conventional molding procedures. The first model of the sensing layer was composed of a sequential enzymatic reaction (glucose oxidase and peroxidase), which generated a chemiluminescent signal in the presence of glucose and luminol. Highly complex objects with assembly properties (fanciful ball, puzzle pieces, 3D pixels, propellers, fluidic and multicompartments) with mono-, di-, and tricomponents configurations were achieved, and the activity of the entrapped enzymes was demonstrated. The second model was a sandwich immunoassay protocol for the detection of brain natriuretic peptide. Here, highly complex propeller shape sensing layers were produced, and the recognition capability of the antibodies was elucidated. The present study opens then the path to a totally new field of development of multiplex sensing layers, printed separately and assembled on demand to create complex sensing systems

Multiplex microarray ELISA versus classical ELISA, a comparison study of pollutant sensing for environmental analysis.

International audienceThe present study describes the development, optimization and performance comparison of three ELISAs and one multiplex immunoassay in a microarray format. The developed systems were dedicated to the detection of three different classes of pollutants (pesticide, explosive and toxin) in water. The characteristics and performances of these two types of assays were evaluated and compared, in order to verify that multiplex immunoassays can replace ELISA for multiple analyte sensing. 2,4-Dichlorophenoxyacetic acid, 2,4,6-trinitrotoluene and okadaic acid were chosen as model targets and were immobilized in classical microtiter plate wells or arrayed at the surface of a microarray integrated within a classical 96-well plate. Once optimized, the classical ELISAs and microarray-based ELISA performances were evaluated and compared in terms of limit of detection, IC50, linearity range and reproducibility. Classical ELISAs provided quite good sensitivity (limit of detection down to 10 μg L(-1)), but the multiplex immunoassay was proven to be more sensitive (limit of detection down to 0.01 μg L(-1)), more reproducible and an advantageous tool in terms of cost and time expenses. This multiplex tool was then used for the successful detection of the three target molecules in spiked water samples and achieved very promising recovery rates

High-Throughput Multiplexed Competitive Immunoassay for Pollutants Sensing in Water

International audienceThe present study described the development and evaluation of a new fully automated multiplex competitive enabling the of five water pollutants (okadaic acid (OA), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), 2.4-dichlorophenoxyacetic acid (2,4-D), 2,4,6-trinitrotoluene (TNT), and 1,3,5trinitroperhydro-1,3,5-triazine (RDX)). The technology is taking advantage of an optical-clear pressure-sensitive adhesive on which biomolecules can be immobilized and that can be integrated within a classical 96-well format. The optimization of the microarray composition and cross-reaction was performed using an original approach where probe molecules (haptens) were conjugated to different carriers such as protein (bovine serum albumin or ovalbumin), amino-functionalized latex beads, or dextran polymer and arrayed at the surface of the adhesive. A total of 17 different probes were then arrayed together with controls on the adhesive surface and screened toward their specific reactivity and cross-reactivity. Once optimized, the complete setup was used for the detection of the five target molecules (less than 3 h for 96 samples). Limits of detection of 0.02, 0.01, 0.01, 100, and 0.02 pg were found for OA, atrazine, 2,4-D, TNT, and RDX, respectively. The proof of concept of the multiplex competitive detection (semiquantitative or qualitative) of the five pollutants was also demonstrated on 16 spiked samples