Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the Antiviral Drug Arbidol

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection

Contribution of dynamic and static quenchers for the study of protein conformation in ionic liquids by steady-state fluorescence spectroscopy.

International audienceThe study of protein conformation in ionic liquids (ILs) is crucial to understand enzymatic activity. Steady-state fluorescence is a proven, rapid and easy method to evaluate the protein structure in aqueous solutions, but it is discussed when used in ILs. In this work, the structure of the formate dehydrogenase from Candida boidinii (FDH, EC: 1.2.1.2) in three imidazolium-based ILs (dimethylimidazolium dimethylphosphate [MMIm][Me(2)PO(4)], 1-butyl-3-methylimidazolium acetate [BMIm][CH(3)COO], and dimethylimidazolium methylphosphonate [MMIm][CH(3)HPO(2)(OCH(3))]) is studied by fluorescence spectroscopy. The UV-vis spectroscopic analysis shows that the decrease of the FDH fluorescence is not only due to the high light absorption of these ILs. The Stern-Volmer analysis clearly shows that these ILs are quenchers of the indole fluorescence, while this quenching property is not found when imidazole is used. Fluorescence spectra of the FDH in the presence of the ILs show that a maximal ionic liquid concentration (MILc), which could be used for steady-state fluorescence study, should be defined. Therefore, FDH conformation could not be directly related to the decrease of its fluorescence in ILs. Nevertheless, the structure of the FDH could be evaluated with dynamic and static quenchers like iodide or acrylamide, used below the MILc, demonstrating the relevance of this parameter. The Stern-Volmer constants (K(SV)(Q)), calculated in the presence of the different ILs, demonstrate that these ILs are strong denaturing agents, each one acting with a different mechanism. This report provides a suitable and easy-to-apply method to study any enzyme structures in ILs by steady-state fluorescence

Ionic liquid-inspired cations covalently bound to FDH improve its stability and activity in IL

International audienceIonic liquids (ILs) are important new solvents for electrochemistry and biocatalysis, but dehydrogenases usually do not work in ionic liquids. Adding more than 40 % (v/v) of the water miscible ionic liquid [MMIm][Me2PO4] (MMIm: 1-methyl-3-methyl imidazolium dimethylphosphate) inactivates the formate dehydrogenase (FDH) from Candida boidinii. The grafting of a variety of IL-inspired hydroxylated cations (hydroxyalkyl imidazolium, hydroxylalkyl pyrrolydinium, and cholinium) on the enzyme through lysine coupling was performed to understand the relationship between grafted cation, enzyme activity, and protein structure. As a general trend, the more a cation was kosmotropic (e.g., presenting a high B coefficient), the larger the resulting modifications were. The ability of these enzymes to bind the substrates was studied by fluorescence quenching in the presence of nicotinamide adenine dinucleotide (NAD+) and azide. The dissociation constant for NAD+ was only slightly affected by the grafting of the cations, however, the quenching efficiency was reduced. Azide binding was more affected by the cations. In the presence of 30 % (v/v) [MMIm][Me2PO4], the catalytic efficiency of the wild-type enzyme was reduced by 2.8 fold. In comparison, the catalytic efficiency of the modified FDH was preserved in these conditions and even improved after modification by hydroxypropyl imidazolium. The grafting of the chaotropic cations prevented the unfolding of the FDH due to [MMIm][Me2PO4]

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

SU-8-carbon composite as conductive photoresist for biochip applications.

International audienceA composite photoresist has been developed for the direct photopatterning of electrodes useful as biochip substrates. The material is composed of SU-8 polymer added with graphite carbon filler which enables patterning of conductive thin films (22μm) on both glass substrate and transparency flexible film with a standard UV photolithography protocol. The resolution obtained using the conductive composite compared well with the bare resist, with lateral resolutions of 5 and 10μm for bare and conductive resists, respectively. The obtained electrodes, after an electrochemical pre-treatment, exhibited very good electrochemical behaviors, opening the path to various electrochemical detections and grafting possibilities. In order to demonstrate the potentialities of the developed material in the biosensors and biochips field, DNA probes were electrografted, using diazonium chemistry, directly at the composite photoresist surface. Target oligonucleotide interactions were detected using chemiluminescent labeling and a satisfactory detection limit of 0.25nM target sequence was demonstrated with a detection ranging over three orders of magnitude

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

Polymeric luminol on pre-treated screen-prited electrodes for the design of performant reagentless (bio)sensors.

International audienceA performant reagentless electrochemiluminescent (ECL) system for H2O2 detection based on electropolymerized luminol is first presented. In this work, polyluminol is formed under near-neutral conditions onto pre-treated screen-printed electrodes (SPEs). Pre-treatment conditions of the working electrode surface have been optimized so as to obtain the best ECL responses to H2O2 that were increased by a factor as high as 400. Galvanostatic polymerization has been tested as a new process to form polyluminol films. Good performances were obtained in terms of responses to H2O2, with quite the same linear range as the ones obtained under potentiostatic and potentiodynamic modes.The association of the polyluminol film with an H2O2-producing oxidase has also been investigated, using choline oxidase as a model enzyme. Silica glasses obtained by the sol–gel process have been employed for biomolecule immobilization. Polymeric luminol has been coupled with choline oxidase-immobilizing gel under bilayer or monolayer configurations. In the first case, enzyme has been immobilized in a silica gel formed on a polyluminol film. In the second case, the enzymatic gel formed on SPE was soaked in a solution containing monomeric luminol units, allowing them to diffuse and penetrate throughout the porous gel. Then, electrodeposition was performed to polymerize the luminophore within the silica matrix. In both cases, choline could be detected down to micromolar concentrations

Peroxide detected in imidazolium-based ionic liquids and approaches for reducing its presence in aqueous and non-aqueous environments

International audienceImidazolium-based ionic liquids were discovered to contain micromolar (μM) quantities of a peroxide species. A general approach using catalase (aqueous solution) or a salen–manganese complex (neat IL) for reducing the presence of the peroxide species is described herein