Freeze-drying of few microliters of antibody formulations to implement 384-wells homogeneous instant assays.

Enzyme-linked immunosorbent assay protocols have traditionally complex workflows with several intensive wash steps. Analytical tools with both shorter time-to-result and hands-on-time using smaller sample and assays reagents volumes are now investigated. In this context, fluorescence resonance energy transfer (FRET)-based assays are emerging as one of the most promising analytical tools in high-throughput screening (HTS). These immunoassays allow fast quantification of antigens at the nano-gram level in a final assay volume of only a few µL. We used a homogeneous time-resolved FRET (called HTRF) assay to develop a freeze-dried screening and ready-to-use format with only one rehydration step called "instant assay". To assure optimal performance of the developed homogeneous instant assay, we investigated the critical quality attributes by studying the functionality and stability of the critical reagents and fluorophores. The cyclic adenosine 3'-5'-monophosphate (cAMP) was selected as the antigen target. We tested various formulations (with different buffers, sugars, bulking reagents, surfactants and co-solvants) combined with a slow freezing and the use of an aluminium plate holder during the freeze-drying of few microliter of bioreagents. The optimized freeze-drying procedure permits to preserve more than 70% of Ab recognition properties. The developed off-the-shelf homogeneous FRET immunoassay allows direct and fast quantification of cAMP at a nanogram level.

One-step direct immunoassay with horseradish peroxidase as antigen for studying the functionality of antibody surfaces.

Antibody-coated surfaces (Ab surfaces) play a key role in bioanalytical tools developed for biosensors and diagnostics. Therefore, a high and well-defined functional activity of the Ab surface is required. The functional activity of the Ab surface depends on its available binding sites i.e. "the active sites" that are able to capture antigen (Ag). The number of active binding sites strongly depends on the immobilization strategy used to fix the Ab on the solid surface. Determination of layer thickness or surface topology are often used to characterize the Ab surfaces but there is no gold standard method for the study of the functionality of the Ab surfaces. For that purpose, we aim at developing an assay allowing to determine the performances of Ab surfaces. In the present study, anti-horseradish peroxidase antibody (anti-HRP Ab) is used as capture Ab covalently bound to the surface and enzyme HRP as Ag. This direct assay permits, in one-step, to generate a signal utilizing the catalytic properties of HRP. The signal is directly proportional to the amount of HRP bound on the Ab surface, and therefore to the active binding sites of immobilized Ab. The HRP/anti-HRP Ab interactions may be a useful indicator to construct accurate and reproducible active Ab surfaces and also to improve their performances in term of stability and sensitivity. Optimization of the assay parameters and quality of the results are presented. A good repeatability and an acceptable inter-day precision (RSD < 10%) are reported.

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.

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

The 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.

How measurements of rate coefficients at low temperature increase the predictivity of photochemical models of Titan's atmosphere.

The predictivity of photochemical models of Titan's atmosphere depends strongly on the precision and accuracy of reaction rates. For many reactions, large uncertainty results from the extrapolation of rate laws to low temperatures. A few reactions have been measured directly at temperatures relevant to Titan's atmosphere. In the present study, we observed the consequences of the reduced uncertainty attributed to these reactions. The global predictivity of the model was improved, i.e., most species are predicted with lower uncertainty factors. Nevertheless, high uncertainty factors are still observed, and a new list of key reactions has been established.

Investigation of neutron radiation effects on polyclonal antibodies (IgG) and fluorescein dye for astrobiological applications.

Detecting life in the Solar System is one of the great challenges of new upcoming space missions. Biochips have been proposed as a way to detect organic matter on extraterrestrial objects. A biochip is a miniaturized device composed of biologically sensitive systems, such as antibodies, which are immobilized on a slide. In the case of in situ measurements, the main concern is to ensure the survival of the antibodies under space radiation. Our recent computing simulation of cosmic ray interactions with the martian environment shows that neutrons are one of the dominant species at soil level. Therefore, we have chosen, in a first approach, to study antibody resistance to neutrons by performing irradiation experiments at the Applications Interdisciplinaires des Faisceaux d'Ions en Région Aquitaine (AIFIRA) platform, a French ion beam facility at the Centre d'Etudes Nucléaires de Bordeaux-Gradignan in Bordeaux. Antibodies and fluorescent dyes, freeze-dried and in buffer solution, were irradiated with 0.6 MeV and 6 MeV neutrons. Sample analyses demonstrated that, in the conditions tested, antibody recognition capability and fluorescence dye intensity are not affected by the neutrons.

Monte Carlo simulation of the radiation environment encountered by a biochip during a space mission to Mars.

Simulations with a Monte Carlo tool kit have been performed to determine the radiation environment a specific device, called a biochip, would face if it were placed into a rover bound to explore Mars' surface. A biochip is a miniaturized device that can be used to detect organic molecules in situ. Its specific detection part is constituted of proteins whose behavior under cosmic radiation is completely unknown and must be investigated to ensure a good functioning of the device under space conditions. The aim of this study is to define particle species and energy ranges that could be relevant to investigate during experiments on irradiation beam facilities. Several primary particles have been considered for galactic cosmic ray (GCR) and solar energetic particle (SEP) contributions. Ionizing doses accumulated in the biochip and differential fluxes of protons, alphas, neutrons, gammas, and electrons have been established for both the Earth-Mars transit and the journey at Mars' surface. Neutrons and gammas appear as dominant species on martian soil, whereas protons dominate during the interplanetary travel. Depending on solar event occurrence during the mission, an ionizing dose of around a few Grays (1 Gy = 100 rad) is expected.

Comparison of methods for measurement of organic compounds at ultra-trace level: analytical criteria and application to analysis of amino acids in extraterrestrial samples.

The need for criteria to compare different analytical methods for measuring extraterrestrial organic matter at ultra-trace levels in relatively small and unique samples (e.g., fragments of meteorites, micrometeorites, planetary samples) is discussed. We emphasize the need to standardize the description of future analyses, and take the first step toward a proposed international laboratory network for performance testing.

Antibodies directed against L and D isovaline using a chemical derivatizing reagent for the measurement of their enantiomeric ratio in extraterrestrial samples: first step-production and characterization of antibodies.

Determining the enantiomeric ratio of amino acids in meteorites requires very sensitive and precise measurements. In this study, an immunochemical approach, combined with new chemical derivatizing agents, was investigated for the measurement of the enantiomeric ratio of isovaline. In the initial step, L and D isovaline were derivatized with epsilon-benzyloxycarbonyl-L-lysine-(t-butyl ester)-chloroethylnitrosourea (Z-L-Lys-(OtBu)-CENU). The Z group was hydrolyzed and the resulting isovaline derivatives (L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) were conjugated with protein using glutaraldehyde and reduced with sodium borohydride. Rabbits were immunized with the immunogenic conjugates thus obtained. Antibodies were characterized using many compounds, both derivatized and underivatized, in competitive ELISA tests. These competition experiments performed enabled us to establish the following results: 1) unconjugated L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline were poorly recognized; 2) all related L-Lys(OtBu)-alpha-hydrogenated amino acids (L and D) were not recognized at all, which eliminates the possibility of the measurements being distorted by contamination; 3) only conjugated L-Lys(OtBu)-alpha-amino-isobutyric acid (AIB) was recognized by the antibody, 4) the enantiomeric discrimination of L and D isovaline through their derivatives (diastereoisomeric L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) was in accordance with the measurement of their enantiomeric ratio. Immunopurification was shown to enhance antibody specificity. The strategy employed shows potential for the quantification of meteoritic amino acids.

Detection of martian amino acids by chemical derivatization coupled to gas chromatography: in situ and laboratory analysis.

If there is, or ever was, life in our solar system beyond the Earth, Mars is the most likely place to search for. Future space missions will have then to take into account the detection of prebiotic molecules or molecules of biological significance such as amino acids. Techniques of analysis used for returned samples have to be very sensitive and avoid any chemical or biological contamination whereas in situ techniques have to be automated, fast and low energy consuming. Several possible methods could be used for in situ amino acid analyses on Mars, but gas chromatography would likely be the most suitable. Returned samples could be analyzed by any method in routine laboratory use such as gas chromatography, already successfully performed for analyses of organic matter including amino acids from martian meteorites. The derivatization step, which volatilizes amino acids to perform both in situ and laboratory analysis by gas chromatography, is discussed here.

Chiral determination of amino acids by capillary electrophoresis and laser-induced fluorescence at picomolar concentrations.

In this publication we present results on the determination of enantiomers of amino acids at very low concentrations. A fluoresceine-based chiral dye was synthesized to allow the separation of diastereoisomers of D- and L-amino acids. We used capillary electrophoresis with different non-ionic surfactants (Brij). The separation parameters were optimized and separations of D- and L-isovaline, an unusual terrestrial amino acid, were obtained. The sensitivity limits were also determined using a commercial laser-induced fluorescence detector. The quantitation of these amino acids is very important to understand the process of chiral selection on Earth.

A study of chemical systems using signal flow graph theory: application to Neptune.

Photochemistry of giant planets and their satellites is characterized by numerous reactions involving many chemical species. In the present paper, chemical systems are modeled by signal flow graphs. Such a technique evaluates the transmission of any input into the system (solar flux, electrons...) and gives access to the identification of the most important mechanisms in the chemical system. For a given chemical system, we first evaluate rate coefficients. Then, in order to obtain concentrations of each compound, we integrate the set of continuity equations by Gear's method. Gear's method is chosen rather than another classical method because it is recommended for a system of stiff equations due to the existence of greatly differing time constants. Finally, the technique of signal flow graphs is used. This method is applied to the production of hydrocarbons in the atmospheres of giant planets. In particular, the production of C2H6 in the atmosphere of Neptune from the photodissociation of CH4 is investigated. Different paths of dissociation of CH4 are possible from L alpha radiations. A chemical system containing 14 species and 30 reactions including these different paths of dissociation is integrated. The main mechanism of production of C2H6 is identified and evaluated for each model of dissociation. The importance of various reaction paths as a function of time is discussed.

Numerical simulation of organic compounds formation in planetary atmospheres: comparison with laboratory experiments.

A numerical model of CH4 and CH4-NH3 photochemistry at 147 nm has been developed and results are directly compared with experimental simulations carried out for the same mixtures. Simulations with varying quantities of ammonia and hydrogen show how amines and nitriles can be produce in planetary atmospheres. These comparisons allow one to test schemes of reactions used in photochemical models. In particular, it is shown that the scheme of reactions of CH4 is fairly well consistent with experimental data. On the other hand, the photochemistry of NH3 should be improved.

A study of chemical systems using signal flow graph theory.

Photochemistry of giant planets and their satellites is characterized by numerous reactions involving a lot of chemical species. In the present paper, chemical systems are modeled by signal flow graphs. Such a technique evaluates the transmission of any input into the system (solar flux, electrons ... ) and gives access to the identification of the most important mechanisms in the chemical system. This method is applied to the production of hydrocarbons in the atmospheres of giant planets. In particular, the production of C2H6 in the atmosphere of Neptune from the photodissociation of CH4 is investigated. Different pathways of dissociation of CH4 are possible from L alpha radiation. A chemical system containing 14 species and 30 reactions including these different pathways of dissociation is integrated. The main mechanism of production of C2H6 is identified and evaluated for each model of dissociation. The importance of various reaction pathways as a function of time is presented.