Anne Varenne

Determination of nanoparticle diffusion coefficients by Taylor dispersion analysis using a capillary electrophoresis instrument

The collective diffusion coefficient D(C) of diluted suspensions of positively charged iron oxide maghemite particles was experimentally investigated using a capillary electrophoresis instrument on the grounds of Taylor dispersion theory. Conditions for this approach to be applicable to nanoparticles of mean solid diameter below 10nm were set in this work, enabling precisions on D(C) determination of less than 2% relative standard deviation (RSD). Significantly different D(C) values were thus measured for particle populations differing in solid number mean diameter by only 2 nm. The obtained values were compared to the z-average diffusion coefficient derived from dynamic light scattering (DLS) experiments and used for the calculation of the Stokes radius. The measured diffusion coefficients appeared to be dependent on particle volume fraction and electrolyte ionic strength. These observations were eventually discussed in terms of particle interactions.

Interaction of fucoidan with the proteins of the complement classical pathway.

Fucoidan inhibits complement by mechanisms that so far remain to be unraveled, and the objective of this work was to delineate the mode of inhibition by this sulfated polysaccharide. For that purpose, low molecular weight fractions of algal (Ascophyllum nodosum) fucoidan containing the disaccharide unit [-->3)-alpha-L-Fuc(2SO3(-))-(1-->4)-alpha-L-Fuc(2,3diSO3(-))-(1-->](n) have been studied. Gel co-affinity electrophoresis and a new affinity capillary electrophoresis (ACE) method have been implemented to characterize fucoidan-complement protein complexes. Fucoidan binds C1q, likely to its collagen-like region through interactions involving lysine residues, and then prevents the association of the C1r(2)-C1s(2) subunit, required to form the fully active C1. In addition to C1q, fucoidan forms a complex with the protein C4 as observed by ACE. The fucoidan inhibits the first steps of the classical pathway activation that is of relevance in view of the proinflammatory effects of the subsequent products of the cascade. This study shows that a high level of inhibitory activity can be achieved with low molecular weight carbohydrate molecules and that the potential applicability of fucoidan oligosaccharides for therapeutic complement inhibition is worthy of consideration.

A new application of bioorganometallics: the first simultaneous triple assay by the carbonylmetalloimmunoassay (CMIA) method

The first example of simultaneous triple carbonylmetalloimmunoassay (CMIA), a non-isotopic immunological method, is presented herein. This method, originally developed for mono-assays, uses various transition metal carbonyl complexes as labels and sensitive quantitation of these tracers by Fourier transform infrared (FT-IR) spectroscopy, thanks to the characteristic νCO bands appearing in the 1800–2200 cm−1 region. We show here that this method offers an advance in the challenging field of simultaneous multi-immunoassay, with the example of the assay of three antiepileptic drugs (carbamazepine, phenobarbital and diphenylhydantoin).

A new insight into suction and dilution effects in capillary electrophoresis coupled to mass spectrometry via an electrospray ionization interface. Part I‐Suction effect

The hyphenation of CE with MS is nowadays accepted as a powerful analytical approach. Employing ESI, the most common interface, one challenge is to provide quantitative information, which is quite a difficult task, as it is linked, among other factors, to suction and dilution effects. In the coaxial ESI configuration, the suction effect has been presented in literature as stemming from nebulizing gas (NG) flow rate and drying gas temperature. But as this interface consists in three concentric capillaries, allowing for BGE, sheath liquid (SL) and NG mixing, it is demonstrated herein that other parameters are also involved in this suction effect: the CE capillary protrusion from the interface needle, SL flow rate, and overall BGE flow rate and velocity profile. Whereas NG flow rate is the parameter affecting suction to a greater extent, separation capillary protruding length, SL, and overall BGE flow rate have a significant additional impact on this phenomenon. It is shown that SL flow rate can affect suction differently according to the NG velocity, which may be explained by modification of the Taylor cone geometry. Furthermore, it appears that suction effect is noticeably favored by a parabolic velocity profile of the BGE, again probably due to the Taylor cone shape modification. Finally, the temperature gradient created by the contact between the heated NG and the separation capillary enhances this effect.

Charge-based characterization of nanometric cationic bifunctional maghemite/silica core/shell particles by capillary zone electrophoresis.

In view of employing functionalized nanoparticles (NPs) in the context of an immunodiagnostic, aminated maghemite/silica core/shell particles were synthesized so as to be further coated with an antibody or an antigen via the amino groups at their surface. Different functionalization rates were obtained by coating these maghemite/silica core/shell particles with 3‐(aminopropyl)triethoxysilane and 2‐[methoxy(polyethyleneoxy)propyl]‐trimethoxysilane at different molar ratios. Adequate analytical performances with CE coupled with UV‐visible detection were obtained through semi‐permanent capillary coating with didodecyldimethyl‐ammonium bromide, thus preventing particle adsorption. First, the influence of experimental conditions such as electric field strength, injected particle amount as well as electrolyte ionic strength and pH, was evaluated. A charge‐dependent electrophoretic mobility was evidenced and the separation selectivity was tuned according to electrolyte ionic strength and pH. The best resolutions were obtained at pH 8.0, high ionic strength (ca. 100 mM), and low total particle volume fraction (ca. 0.055%), thus eliminating interference effects between different particle populations in mixtures. A protocol derived from Kaisers original description was performed for quantitation of the primary amino groups attached onto the NP surface. Thereafter a correlation between particle electrophoretic mobility and the density of amino groups at their surface was established. Eventually, CE proved to be an easy, fast, and reliable method for the determination of NP effective surface charge density.

Capillary electrophoresis determination of the binding affinity of bioactive sulfated polysaccharides to proteins: study of the binding properties of fucoidan to antithrombin

The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. An affinity capillary electrophoresis method has been developed to evidence the complex formation and to determine the binding properties between an anticoagulant polysaccharide of marine origin, fucoidan, and a potential target protein, antithrombin. This method is a variant of zonal electrophoresis in the mobility shift format. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant for the polysaccharide/antithrombin complex. The results indicate that fucoidan binds to antithrombin in a 1:1 stoichiometry and with an affinity depending on the molecular weight of the polysaccharide. For heparin, the binding constant obtained similarly is in accordance with the literature. This is the first report showing the implementation of a capillary electrophoresis method contributing to the mechanistic understanding of the biological activities of fucoidan and providing evidence for the complex formation between fucoidan and the protein inhibitor of the coagulation antithrombin.

Identification and determination of inorganic anions in real extracts from pre- and post-blast residues by capillary electrophoresis

Fast, selective, and sensitive analysis of inorganic anions is compulsory for the identification of explosives in post-blast or environmental samples. For the last twenty years, capillary electrophoresis (CE) has become a valuable alternative to ion chromatography (IC) for the analysis of inorganic-based explosives because of its low running costs and its simplicity of use. This article focuses on the development and validation of a CE method for the simultaneous analysis of 10 anions (chloride, nitrite, nitrate, thiosulphate, perchlorate, chlorate, thiocyanate, carbonate, sulphate, and phosphate) which can be found in post-blast residues, plus for the first time azide anion, possibly present in the composition of detonators, and the internal standard (formate) in 20 min total runtime. Intermediate precisions were 2.11% for normalized areas and 0.72% for normalized migration times. Limits of detection close to 0.5 ppm for all anions were obtained with the use of preconcentration techniques, thanks to a fast and simple sample preparation allowing the analysis of a large variety of matrices with the developed generic CE method. The matrix effects were statistically studied for the first time in the explosive field for different matrices, containing interfering anions and cations, sometimes at high levels. In fact, no significant matrix effect occurred (tests with blank matrix extracts of soil, cloth, glass, plastic, paper, cotton, and metal). Finally, analyses of real post-blast residues and real detonator extracts were performed. The CE results were compared with those obtained with the IC method used routinely and showed excellent correlation.

New insight into suction and dilution effects in CE coupled to MS via an ESI interface. II ― Dilution effect

The hyphenation of CE with MS is nowadays accepted as a powerful analytical approach. As far as ESI, the most common interface, is concerned, one challenge is to provide the most sensitive as well as quantitative information, which is quite a difficult task, as it is linked, among other factors, to suction and dilution effects. In the coaxial ESI configuration, it has been previously demonstrated that suction effect depends on many parameters inherent to the ESI interface geometry, the prevailing ones being the CE capillary protrusion from the interface needle, the sheath liquid (SL) and the overall BGE flow rates and velocity profile. In this paper, dilution effect is studied, as the CE electrolyte is mixed with SL at the interface. Considering peak intensity and efficiency, this effect was studied as a function of the various parameters of the interface (capillary protrusion from the SL tube, nebulizing gas, SL and CE electrolyte flow rates) or of the source (skimmer and ESI voltages, drying gas flow rate and temperature). It appears that the dilution effect seems slightly lower than what can be anticipated from the proportions of the liquid flow rates. This study also indicates that suction effect has to be considered first to better understand the dilution phenomenon, as suction effect leads to an increase in peak intensity, before a dilution effect appears.

Separation and quantitation of milk whey proteins of close isoelectric points by on-line capillary isoelectric focusing—Electrospray ionization mass spectrometry in glycerol–water media

On-line coupling between CIEF and ESI/MS based on the use of bare fused-silica capillaries and glycerol-water media, recently developed in our laboratory, has been investigated for the separation of milk whey proteins that present close pI values. First, a new rinsing procedure, compatible with MS detection, has been developed to desorb these rather hydrophobic proteins (α-casein (α-CN), bovine serum albumin (BSA), lactoferrin (LF)) from the inner capillary wall and to avoid capillary blockages. Common hydrochloric acid washing solution was replaced by a multi-step sequence based on the use of TFA, ammonia and ethanol. To achieve the separation of major whey proteins (β-lactoglobulin A (β-LG A), β-lactoglobulin B (β-LG B), α-lactalbumin (α-LA) and BSA, which possess close pI values (4.5-5.35), CIEF parameters i.e. carrier ampholyte nature, capillary partial filling length with ampholyte/protein mixture and focusing time, have been optimized with respect to total analysis time, sensitivity and precision on pI determination. After optimization of sheath liquid composition (80:20 (v/v) methanol-water+1% HCOOH), quantitation of β-LG A, β-LG B, α-LA and BSA was performed. The limits of detection obtained from extracted ion current (EIC) and single ion monitoring (SIM) modes were in the 57-136 nM and 11-68 nM range, respectively. Finally, first results obtained from biological samples demonstrated the suitability of CIEF-MS as a potential alternative methodology to 2D-PAGE to diagnose milk protein allergies.

Size-based characterization of nanometric cationic maghemite particles using capillary zone electrophoresis

Size‐sorted maghemite (γ‐Fe2O3) particle populations of number mean solid diameters ranging from 6 to 10 nm were suspended and directly characterized in their stabilizing acidic, citrated or basic aqueous media using CZE coupled with UV detection. Analytical conditions were optimized in order to ensure reliable mobility measurements of these ferrofluids in their anionic and cationic forms. Particular interest has been paid to the investigation of the positively charged ferrofluids since cationic colloids have received little attention so far. A strategy for capillary wall modification was chosen in order to prevent particle adsorption while preserving high analytical performances. The influence of experimental conditions such as particle volume fraction, injection volume, electric field strength and electrolyte nature on electrophoretic profiles and measured electrophoretic mobilities was evaluated. A size‐dependent electrophoretic mobility was demonstrated and discussed in terms of the ratio of the particle radius to Debye length with reference to existing models (Henry, etc.). Although these nanometric particle distributions lie in a very narrow size range, partial separation was obtained with selectivity varying as a function of electrolyte ionic strength.