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.

On-line capillary electrophoresis-based enzymatic methodology for the study of polymer-drug conjugates.

This work aims at studying the potentialities of an on-line capillary electrophoresis (CE)-based digestion methodology for evaluating polymer-drug conjugates degradability in the presence of free trypsin (in-solution digestion). A sandwich plugs injection scheme with transverse diffusion of laminar profile (TDLFP) mode was used to achieve on-line digestions. Electrophoretic separation conditions were established using poly-l-Lysine (PLL) as reference substrate. Comparison with off-line digestion was carried out to demonstrate the feasibility of the proposed methodology. The applicability of the on-line CE-based digestion methodology was evaluated for two PLL-drug conjugates and for the four first generations of dendrigraft of lysine (DGL). Different electrophoretic profiles presenting the formation of di, tri, and tetralysine were observed for PLL-drug and DGL. These findings are in good agreement with the nature of the linker used to link the drug to PLL structure and the predicted degradability of DGL. The present on-line methodology applicability was also successfully proven for protein conjugates hydrolysis. In summary, the described methodology provides a powerful tool for the rapid study of biodegradable polymers.

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.

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.

A step further in the SATE mononucleotide prodrug approach.

Synthesis, in vitro anti-HIV activity, stability studies as well as potential for oral absorption of some novel phenyl S-acyl-2-thioethyl (SATE) phosphotriester derivatives of AZT (zidovudine; 3'-azido-2',3'- dideoxythymidine) are reported herein. These mononucleotide prodrugs (pronucleotides) are characterized by the presence of polar (amino or hydroxyl) functions on the SATE biolabile phosphate protections. Whereas pronucleotides incorporating an amino residue in the vicinity of the thioester functionality display low chemical stability, the introduction of one or two hydroxyl groups on the SATE moiety confers high resistance of the resulting prodrugs towards esterase hydrolysis. Thus, one of these pronucleotides, derivative 2, was able to cross a Caco-2 cell monolayer mainly in intact form, probing that its further development is warranted as a possible HIV-pronucleotide candidate.

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

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.

Simultaneous HPLC analysis of hydrophobic prodrugs and their hydrophilic metabolites in biological media without sample preparation. Application to the biotransformation studies of pronucleotides derived from 5-fluorouracil.

Improvements of an "on-line cleaning" HPLC method for analysis of biological samples are presented: (i) the use of cleaning precolumns filled with hydrophobic stationary phases instead of the hydrophilic ones previously used to eliminate the biological matrix: (ii) the combination in the mobile phase of anionic and cationic pairing reagents in order to retain on the precolumn all the metabolites, whatever their hydrophilicity and ionicity are. Such modifications allowed to study the biotransformation of prodrugs of 5-Fluorouracil, designed to act as antitumoral pronucleotides.