Gaëlle Coussot

Aminated dendritic surfaces characterization: a rapid and versatile colorimetric assay for estimating the amine density and coating stability

AbstractThe functionalization of surfaces with amino groups is used in many application areas such as in industrial biocatalytic processes for the development of medical biomaterials and in the environment for removing pollutants from water. Amino group density and grafting stability are often related to functionalized material performances; thus, their characterizations are of prime importance. The determination of amino density and grafting stability on polymeric material (e.g. polypropylene, polystyrene and cylco olefin copolymer) is often time consuming and sometimes presents technical constraints, more particularly with non-flat materials. In this paper, we report a novel colorimetric assay using the Coomassie Brilliant Blue dye for both amino density determination and grafting stability measurement. The assay named ADECA for “Amino Density Estimation by Colorimetric Assay” is sensitive, rapid, robust and versatile. We demonstrate that ADECA makes the evaluation of aminated materials performances possible for numerous material compositions, formats and chemistries used for grafting. Our study focuses on dendrigraft of poly-l-lysine and poly(amidoamine) dendrimers dendritic materials. FigureAnimated surfaces characterization

Investigation of Low-Energy Proton Effects on Aptamer Performance for Astrobiological Applications

Biochips are promising instruments for the search for organic molecules in planetary environments. Nucleic acid aptamers are powerful affinity receptors known for their high affinity and specificity, and therefore are of great interest for space biochip development. A wide variety of aptamers have already been selected toward targets of astrobiological interest (from amino acids to microorganisms). We present a first study to test the resistance of these receptors to the constraints of the space environment. The emphasis is on the effect of cosmic rays on the molecular recognition properties of DNA aptamers. Experiments on beam-line facilities have been conducted with 2 MeV protons and fluences much higher than expected for a typical mission to Mars. Our results show that this irradiation process did not affect the performances of DNA aptamers as molecular recognition tools.

Irradiation effects on antibody performance in the frame of biochip-based instruments development for space exploration

Several instruments based on immunoassay techniques have been proposed for life-detection experiments in the framework of planetary exploration but few experiments have been conducted so far to test the resistance of antibodies against cosmic ray particles. We present several irradiation experiments carried out on both grafted and free antibodies for different types of incident particles (protons, neutrons, electrons and 12C) at different energies (between 9 MeV and 50 MeV) and different fluences. No loss of antibodies activity was detected for the whole set of experiments except when considering protons with energy between 20 and 30 MeV (on free and grafted antibodies) and fluences much greater than expected for a typical planetary mission to Mars for instance. Our results on grafted antibodies suggest that biochip-based instruments must be carefully designed according to the expected radiation environment for a given mission. In particular, a surface density of antibodies much larger than the expected proton fluence would prevent significant loss of antibodies activity and thus assuring a successful detection.

SATE (aryl) phosphotriester series. II. Stability studies and physicochemical parameters

Abstract The stability of phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and various aryl residues derived from L-tyrosine was evaluated in biological media. The results demonstrate that such compounds give rise to intracellular delivery of the parent mononucleotide through esterase and phosphodiesterase hydrolytic steps, successively.

A gold standard method for the evaluation of antibody-based materials functionality: Approach to forced degradation studies.

&NA; The scope of this paper is to present a gold standard method to evaluate functional activity of antibody (Ab)‐based materials during the different phases of their development, after their exposure to forced degradations or even during routine quality control. Ab‐based materials play a central role in the development of diagnostic devices, for example, for screening or therapeutic target characterization, in formulation development, and in novel micro(nano)technology approaches to develop immunosensors useful for the analysis of trace substances in pharmaceutical and food industries, clinical and environmental fields. A very important aspect in diagnostic device development is the construction of its biofunctional surfaces. These Ab surfaces require biocompatibility, homogeneity, stability, specificity and functionality. Thus, this work describes the validation and applications of a unique ligand binding assay to directly perform the quantitative measurement of functional Ab binding sites immobilized on the solid surfaces. The method called Antibody Anti‐HorseRadish Peroxidase (A2HRP) method, uses a covalently coated anti‐HRP antibody (anti‐HRP Ab) and does not need for a secondary Ab during the detection step. The A2HRP method was validated and gave reliable results over a wide range of absorbance values. Analyzed validation criteria were fulfilled as requested by the food and drug administration (FDA) and European Medicines Agency (EMA) guidance for the validation of bioanalytical methods with 1) an accuracy mean value within +15% of the nominal value; 2) the within‐assay precision less than 7.1%, and 3) the inter‐day variability under 12.1%. With the A2HRP method, it is then possible to quantify from 0.04 × 1012 to 2.98 × 1012 functional Ab binding sites immobilized on the solid surfaces. A2HRP method was validated according to FDA and EMA guidance, allowing the creation of a gold standard method to evaluate Ab surfaces for their resistance under laboratory constraints. Stability testing was described through forced degradation studies after exposure of Ab‐surfaces to storage, pH and aqueous‐organic solvent mixture stresses.

A methodological approach for the thermal stability and stress exposure studies of a model antibody

The anti-horseradish peroxidase (HRP) antibody is conventionally used in immunohistochemistry. More recently, it has been used as the key element in a gold standard method to evaluate the functionality of antibody-based materials. However, few information are available about its melting temperature and its stability after exposition to laboratory stress conditions including freeze-drying and freeze-thawing cycles. The aim of this study was to evaluate the effects of these environmental constraints on the anti-HRP antibody in order to further use it as a reference in quality control and in the development of new antibody-based materials. In the developed method, the anti-HRP antibody is covalently immobilized onto a solid surface. After the direct recognition of its antigen HRP, the signal is proportional to the number of antibody active binding sites. The method was successfully utilized to accurately evaluate the anti-HRP antibody melting temperature (Tm was 73.5 ± 0.2 °C). The method is a rapid and reliable tool with minimal cost for studying the anti-HRP antibody stability to solvent stress, freeze-thawing cycles, and freeze-drying process. The obtained information may be useful for routine analysis or in the development of antibody-based materials. This can be also proposed as an easy way to control antibody freeze-drying process.