Isabelle Le Potier

Stabilization mechanism of oil-in-water emulsions by β-lactoglobulin and gum arabic

Natural biopolymer stabilized oil-in-water emulsions were formulated using β-lactoglobulin (β-lg), gum arabic (GA), and β-lg:GA solutions as an alternative to synthetic surfactants. Emulsions using these biopolymers and their complexes were formulated varying the biopolymer total concentration, the protein-to-polysaccharide ratio, and the emulsification protocol. This work showed that whereas β-lg enabled the formulation of emulsions at concentration as low as 0.5 (w/w)%, GA allowed to obtain emulsions at concentrations equal to or higher than 2.5 (w/w)%. In order to improve emulsion stability, β-lg and GA were complexed through strong attractive electrostatic interactions. GA solution had to be added to previously prepared β-lg emulsions in order to obtain stable emulsions. Interfacial tension and interfacial rheological measurements allowed a better understanding of the possible stabilizing mechanism. β-lg and GA both induced a very effective decrease in interfacial tension and showed interfacial elastic behaviour. In the mixed system, β-lg adsorbed at the interface and GA electrostatically bound to it, leading to the formation of a bi-layer stabilized emulsion. However, emulsion stability was not improved compared to β-lg stabilized emulsion, probably due to depletion or bridging flocculation.

A study of the binding between polymers and peptides, using affinity capillary electrophoresis, applied to polymeric drug delivery systems.

We have investigated the potential of affinity capillary electrophoresis (ACE) to evaluate binding constants between an anionic polydispersed polymer and four peptides. Nonlinear regression and three current linearization methods, the y‐reciprocal, the x‐reciprocal and the double‐reciprocal, were employed for the estimation of the binding constants. The x‐reciprocal and the double‐reciprocal plots indicated the presence of two portions of straight lines for angiopeptin, triptorelin and the thyrotropin releasing hormone (TRH), and therefore the probable existence of a second‐order interaction which causes the deviation from the 1:1 model. Peptide 1 exhibited a unique binding constant of 2.4×106 M–1. In contrast, angiopeptin, triptorelin and TRH exhibited a K1 of 4.0×106, 5.3×106 and 20.2×106 M–1, respectively, and a K2 of 0.4×106, 0.5×106 and 1.4×106 M–1, respectively. The origin of the high scattering of the data points was further investigated. Neither the viscosity, nor the adsorption of the peptides to the capillary wall appeared to be the determining factor of data scattering. Finally, a possible adsorption of the polymer leading to the electroosmotic flow unstability was supposed.

A new CZE method for profiling human serum albumin and its related forms to assess the quality of biopharmaceuticals

We present a new CZE method, which uses a polyethylene oxide‐coated capillary to separate native HSA from more than five of its structural variants. These variants include oxidized, truncated, and cysteinylated forms of HSA which can all be found in biopharmaceutical products. Both CE and MS confirmed the high degree of heterogeneity of HSA preparations. Recovery studies demonstrated that adsorption of HSA on the capillary was significantly reduced under the conditions we developed, which led to a satisfactory repeatability (RSD for migration times and relative peak areas were less than 0.2 and 7.0%, respectively). Assignment of the main peaks was attempted using in vitro degraded/stressed HSA. We used our method to test batch‐to‐batch comparability and detected slight quantitative differences in the proportion of native HSA in batches produced from different fractionation methods.

A quantitative CE method to analyse tau protein isoforms using coated fused silica capillaries

We evaluated the potential of CE to analyse different isoforms of unphosphorylated recombinant tau protein and for separating one phosphorylated tau from the respective unphosphorylated protein. Different capillary coatings such as polyacrylamide, poly-(ethylene oxide) and polybrene (PB) were evaluated to overcome the poor efficiencies obtained with fused-silica capillary. Although peak asymmetry values were quite similar for the three investigated coatings, the peak efficiencies were 35-fold and 5-fold higher with PB coating than with polyacrylamide and poly(ethylene oxide) coatings, respectively. The recovery percentage (over 97%) was satisfactory and confirmed the efficacy of PB coating to limit the adsorption of tau protein to capillary walls. Moreover, PB coating produced higher repeatability for migration times (RSD values <1.2%) in comparison to the neutral coatings. The potential of PB-modified capillary in producing high resolutive separations of one phosphorylated tau isoform from its unphosphorylated counterpart and of a mixture of phosphorylated and unphosphorylated tau peptides was demonstrated with 50 mM phosphate buffer pH 3.0. The separation of unphosphorylated tau isoform 352 (Tau-352) from Tau-352 phosphorylated in vitro by the mitogen-activated protein kinase ERK2, was accomplished in less than 15 min.

Derivatization strategies for CE-LIF analysis of biomarkers: Toward a clinical diagnostic of familial transthyretin amyloidosis

We report three derivatization strategies for CE analysis with LIF detection (CE‐LIF) of two synthetic peptides mimicking the wild and mutated fragments of interest for the diagnosis of familial transthyretin amyloidosis. The precapillary derivatization of the peptides with three optical tags, 5‐carboxytetramethylrhodamin succinimidyl ester (TAMRA‐SE), naphtalene‐2,3‐dicarboxyaldehyde (NDA), and 3‐(2‐furoyl)quinoline‐2‐carboxyaldehyde (FQ) has been investigated by CE‐LIF detection and MS. Results provide evidence that high reaction yields have been reached whereas the multitagging phenomenon has occurred for both NDA and TAMRA‐SE labeling procedures. The derivatization and electrokinetic separation of a mixture of the two peptides of interest for the pathology diagnosis (22‐aa peptides that differ only from one amino acid) were achieved using both approaches. The highest resolution with a value of 2.5 was obtained with TAMRA‐SE labeled derivatives whereas NDA gave the best detection sensitivity (LOD of 2.5 μM). The validation of the developed methods showed a good linearity (R ≥ 0.997) between the peak area of the labeled derivatives and the peptide concentration for both NDA and FQ labeling procedures. The intraday RSDs of A and the migration times were less than 3.8 and 2.2%, respectively.

Investigating of labelling and detection of transthyretin synthetic peptide derivatized with naphthalene-2,3-dicarboxaldehyde

Labelling and detection of a synthetic peptide (PN) mimicking a tryptic fragment of interest for the diagnosis of familial amyloidal polyneuropathy have been investigated optically and electrochemically. We decided to covalently label naphtalene-2,3-dicarboxyaldehyde (NDA), a fluorogenic and electroactive molecule on PN. First, the optimization of the labelling chemical reaction was performed by capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). The analytical parameters such as separation efficiency and peak area were considered to propose this optimized derivatization reaction. The results obtained allowed us to establish the pH and ionic strength of the derivatization buffer, the molar ratio between NDA and PN and the reaction time of the labelling. Optimal conditions are obtained when [NDA]/[PN]=40, buffer pH of 9, buffer ionic strength of 70 mM and reaction time of 15 min. Second, differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were also used to characterize NDA-labelled PN and different electroinactive amino acids (histidine, lysine, serine, threonine) which are in the PN sequence. The electrochemical detection experiments demonstrated that the labelled biomolecules could be also easily detected at low concentration. Moreover, the derivatization reaction could be followed to describe more precisely the labelling process of these biomolecules. Optimal conditions for labelling are obtained when [NDA]total/[CN(-)] ratio =1 and [NDA]total/[amino acid or peptide]=100 with a buffer having a pH=9 on a glassy carbon electrode. In all cases, an obvious oxidation peak for the N-2-substituted-1-cyanobenz-[f]-isoindole derivative (CBI) has been observed at 0.5-0.7 V/SCE. The multi-labelling of PN and lysine were shown with DPV. We presumed this result to occur because of the shouldered shape of the DPV peak shape. These experiments confirm that NDA can be used as a derivative agent for PN, allowing for electrochemical and fluorescence detections with a limit of detection of labelled PN estimated at 0.2 µM and 5 µM, respectively.

Electrophoretic mobility measurement by laser Doppler velocimetry and capillary electrophoresis of micrometric fluorescent polystyrene beads

Many studies have been made and techniques developed to measure the mobility of particles and molecules by laser Doppler velocimetry and capillary electrophoresis. We propose here to evaluate and compare these two measurement techniques for their ability to characterize various fluorescent polystyrene beads as a function of the buffer pH. The repeatability of electrophoretic mobility determination by the two techniques in buffer at different pHs (neutral to alkaline) was first examined and compared. The accuracy of the determination was then evaluated. A wide range of beads which varied in their size (diameters ranging from 270 to 1000 nm), surface functional groups (NH2, COOH, and neutral), and the presence or absence of surfactants or incorporated dye molecules were investigated in order to perform a comprehensive study. The results indicated that apart from large amino beads (with a diameter over 800 nm), capillary electrophoresis generally gave better or similar relative standard deviations for most polystyrene beads, which could be attributed to a stronger adsorption of these beads onto the silica capillary surface in CE. Beads with neutral pH were more difficult to measure accurately with both methods. We also concluded that capillary electrophoresis measurements are not accurate for amino beads in the pH range of this study. However, both methods were capable of distinguishing polystyrene beads with different sizes or surface groups. We found that dye molecules introduced in beads did not alter their electrophoretic mobility values. Taken together, the data and discussion provide a guide to choose the right technique to characterize any given set of functional particles precisely and with the highest accuracy.

Computational study of velocity profile obtained in microfluidic channel bearing a fluidic transistor: Toward highly resolved electrophoretic separation

The present work is a computational study of velocity profiles in microfluidic channels bearing field flow effect transistors (FFET). In particular, this work investigates perturbations and distortions of the sample band during electrophoretic transport in a rectangular separation channel. The EOF heterogeneity and its induced pressure render the predictions of the analytical performances rather complex. In this context, we propose a systematic numerical inquiry that focuses on the distribution of the velocities for several geometries and EOF modulations. We compare the calculated parabolic velocity profiles to the bare glass microchips. Here, the reported parabolic velocity profiles are coherent with recent experimental results that have been published elsewhere. From the presented equations, in such active hybrid microfluidic chip that integrates a FFET gate layer, separation can be optimized by playing on the gate coverage ratio. The flow fields obtained from analytical models allow further investigations about the efficiency and resolution during electrophoresis. The resulting induced pressure gradient and the associated band broadening underline the need to optimize the resolution in the detriment of the efficiency in such active microfluidic chips.

Study of Surface Charge Instabilities by EOF Measurements on a Chip: A Real-Time Hysteresis and Peptide Adsorption Based Methodology

This paper describes the measurement of the electroosmotic mobility (EOF) in a Wheatstone fluidic bridge (μFWB) as a direct probe of the surface instability. The variation of EOF known as one major contribution of the electrokinetic migration has been determined with a real-time measurement platform after different conditionings on chips. We also scan the pH of the background electrolytes with three different ionic strengths to evaluate the dependencies of the EOF as a function of the pH. A hysteresis methodology has been developed for probing the surface charge instabilities. EOF mobility has been recorded during on-a-chip electrophoresis to estimate the effect of such instability on the analytical performance. As expected, our experimental curves show that a decrease in the ionic strength increases the surface charge stability of the hybrid microchip. This result demonstrates that ionic exchanges between the surface and the fluid are clearly involved in the stability of the surface charge. With this original method based on real-time EOF measurement, the surface state can be characterized after hydrodynamic and electrophoresis sequences to mimic any liquid conditioning and separation steps. Finally, as a demonstrative application, isotherms of the adsorption of insulin have been recorded showing the change in surface charge by unspecific adsorption of this biomolecule onto the microfluidic channels wall. These methodologies and findings could be particularly relevant to investigating various analytical pathways and to understanding the molecular mechanisms at solid/liquid interfaces.

Determination of the isomeric forms proportion of fluorogenic naphthalene-2,3-dicarboxaldehyde in a binary mixture of water:methanol using electrochemical methods

The electrochemical response of the fluorogenic label naphthalene-2,3-dicarboxyaldehyde (NDA) in a binary mixture of water/methanol was characterized with cyclic voltammetry (CV) and differential pulse voltammetry (DPV) electrochemical techniques. Naphthalene-2,3-dicarboxyaldehyde does exist in three isomeric forms in aqueous solution: the unhydrated dialdehyde (DA), the acyclic monohydrated (MA) and the cyclic hemiacetal (HAC). The study underlines that the proportion of each of them varies according to the working pH. At low and high pH, the dialdehyde form is in larger proportion than the acyclic monohydrated form. Conversely at intermediate pH, the concentration of the acyclic form is in greater proportion than the dialdehyde form. These results allowed us to determine the optimal pH of 9 for which the labeling of biomolecules could be more efficient due to the base catalyzed regeneration of the unhydrated form. At this pH, the data processing from the analysis of measured currents and estimation of diffusion coefficients of each form according to the semi-empirical models of Wilke-Chang, Scheibel, Reddy-Doraiswamy and Lusis-Ratcliff allowed us to obtain the concentration of dialdehyde (0.28 mM), acyclic monohydrated (0.57 mM) and cyclic hemiacetal monohydrated (0.15 mM) forms starting from 1mM naphthalene-2,3-dicarboxyaldehyde.