Antoine Pallandre

Pressure-assisted selective preconcentration in a straight nanochannel.

We investigate the preconcentration profiles of a fluorescein and bovine serum albumin derivatized with this fluorescent tag in a microfluidic chip bearing a nanoslit. A new preconcentration method in which a hydrodynamic pressure is added to both electroosmotic and electrophoretic contributions is proposed to monitor the location of the preconcentration frontline. A simple predictive model of this pressure-assisted electropreconcentration is proposed for the evolution of the flow profile along this micro/nano/microfluidic structure. We show with a small analyte such as fluorescein that the additional hydrostatic pressure mode enables to stabilize the concentration polarization (CP) effect, resulting in better control of the cathodic focusing (CF) peak. For BSA (bovine serum albumin), we exhibit that the variation of the hydrodynamic pressure can have an even more drastic effect on the preconcentration. We show that, depending on this hydrodynamic pressure, the preconcentration can be chosen, either in the cathodic side or in the anodic one. For the first time, we prove here that both anodic focusing (AF) and cathodic focusing (CF) regimes can be reached in the same structures. These results also open new routes for the detection and the quantification of low abundance biomarkers.

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.

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.