Toxicity Assays in Nanodrops Combining Bioassay and Morphometric Endpoints

BACKGROUND: Improved chemical hazard management such as REACH policy objective as well as drug ADMETOX prediction, while limiting the extent of animal testing, requires the development of increasingly high throughput as well as highly pertinent in vitro toxicity assays. METHODOLOGY: This report describes a new in vitro method for toxicity testing, combining cell-based assays in nanodrop Cell-on-Chip format with the use of a genetically engineered stress sensitive hepatic cell line. We tested the behavior of a stress inducible fluorescent HepG2 model in which Heat Shock Protein promoters controlled Enhanced-Green Fluorescent Protein expression upon exposure to Cadmium Chloride (CdCl(2)), Sodium Arsenate (NaAsO(2)) and Paraquat. In agreement with previous studies based on a micro-well format, we could observe a chemical-specific response, identified through differences in dynamics and amplitude. We especially determined IC50 values for CdCl(2) and NaAsO(2), in agreement with published data. Individual cell identification via image-based screening allowed us to perform multiparametric analyses. CONCLUSIONS: Using pre/sub lethal cell stress instead of cell mortality, we highlighted the high significance and the superior sensitivity of both stress promoter activation reporting and cell morphology parameters in measuring the cell response to a toxicant. These results demonstrate the first generation of high-throughput and high-content assays, capable of assessing chemical hazards in vitro within the REACH policy framework

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

3D-4D printed objects : new bioactive material opportunities

International audienceOne of the main objectives of 3D printing in health science is to mimic biological functions. To reach this goal, a 4D printing might be added to 3D-printed objects which will be characterized by their abilities to evolve over time and under external stimulus by modifying their shape, properties or composition. Such abilities are the promise of great opportunities for biosensing and biomimetic systems to progress towards more physiological mimicking systems. Herein are presented two 4D printing examples for biosensing and biomimetic applications using 3D-printed enzymes. The first one is based on the printing of the enzymatic couple glucose oxidase/peroxidase for the chemiluminescent detection of glucose, and the second uses printed alkaline phosphatase to generate in situ programmed and localized calcification of the printed object

Biomolecules immobilization using the aryl diazonium electrografting

International audienceThe electrografting of aryl-diazonium was proven to be an efficient way to immobilize antibodies, oligonucleotides and enzymes onto conductive supports. Biomolecules chemically functionalized with aniline derivates or diazotated derivates could be used to build complex architectures acting as sensing layers for biosensors, biochips or others bioelectronic devices. Additionally, the use of SPR offers new opportunities to characterize the grafted surfaces as well as to develop label-free assays. Here, we give an overview of our group achievements in the field during the last 7 years. We highlight the applications of these functionalized surfaces in multiparametric sandwich assays, in the label free detection and imaging of macromolecular interactions by SPRi and in the immobilization of dehydrogenase via its cofactor

Development of an elastography bench for MR exam of small samples

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Oligonucleotide solid-phase synthesis on fluorescent nanoparticles grafted on controlled pore glass

International audienceOligonucleotide solid-phase synthesis is now possible on nano-sized particles, thanks to the use of controlled pore glass-nanoparticle assemblies. We succeeded in anchoring silica nanoparticles (NPs) inside the pores of micrometric glass via a reversible covalent binding. The pore diameter must be at least six times the diameter of the nanoparticle in order to maintain efficient synthesis of oligonucleotides in the synthesizer. We demonstrated that the pores protect NP anchoring during DNA synthesis without decreasing the coupling rate of the phosphoramidite synthons. This bottom-up strategy for NP functionalization with DNA results in unprecedented DNA loading efficiency. We also confirmed that the DNA synthesized on the nanoparticle surface was accessible for hybridization with its complementary DNA strand

Oligonucleotide solid-phaOligonucleotide solid-phase synthesis on fluorescent nanoparticles grafted on controlled pore glass

International audienceOligonucleotide solid-phase synthesis is now possible on nano-sized particles, thanks to the use of controlled pore glass-nanoparticle assemblies. We succeeded in anchoring silica nanoparticles (NPs) inside the pores of micrometric glass via a reversible covalent binding. The pore diameter must be at least six times the diameter of the nanoparticle in order to maintain efficient synthesis of oligonucleotides in the synthesizer. We demonstrated that the pores protect NP anchoring during DNA synthesis without decreasing the coupling rate of the phosphoramidite synthons. This bottom-up strategy for NP functionalization with DNA results in unprecedented DNA loading efficiency. We also confirmed that the DNA synthesized on the nanoparticle surface was accessible for hybridization with its complementary DNA stran

Toxicity measured by novel high content endpoints.

<p>The Dose response curves for a selection of High Content Analysis parameters upon Arsenate, Cadmium and Paraquat exposure are presented. 3a, Cell Area (in µ²m). 3b. shape index ( = measured cell perimeter ²/4 п² R², R is the minimum calculated radius). 3c. Roundness ( = R² п/area); 3d. EGFP-Gray Level (GL) intensity. At doses greater than 100 µM, too few cells remained to be considered for statistic analysis (grey shading). As on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000163#pone-0000163-g002" target="_blank">figure 2</a> the concentration of the chemicals on x-axis is plotted in log scale and the 0 M control has been replaced by 10⁻⁷ M value. For each parameter the values for IC50parameter were calculated by linear regression on the linear phases of the curves of two independent experiments and are displayed below the graphs when applicable (significant response to toxic insult, NA not applicable otherwise).</p

Multiplexed toxicity assay in drops.

<p>The ‘Cell-on-Chip’ device was used to obtain four hundred independent HepG2 stress inducible fluorescent cell based assay measurements using two <i>hsp</i> promoter containing clones and four toxic at ten doses. The experiments were performed in quintuplicate measurements. 1a. Cell dispensing with sciFlexarrayer robot arrayer. 1b Zoom on the assembled mosaic of images corresponding to A10 clone after 6 h exposure to ten doses of Arsenate in quintuplicate (columns); the Hsp induction is monitored by the green EGFP signal, cell nucleus is stained in blue by Hoechst and cell cytoplasm is stained in red by Phalloïdin. 1c: Heterogeneity in cell response is illustrated by an example of Hsp response to 5 10⁻⁵ M Arsenate exposure. Scale bar represents 500 µm. Fully automated image capture with a 10× objective and dedicated image analysis were performed using the same detection protocols by IMSTAR Pathfinder™ Cellscan system. All cells were individually segmented (contour highlighted in white) to extract information (signal intensity, morphology) on every single cell within each drop.</p