Influence of Lanthanum on Stern Layer Conductance in the Nanochannel.

In this report, high-frequency electric impedance spectroscopy was performed to investigate ionic transport through nanochannels. Special attention was focused on (i) conductance behaviors depending on the role of cation valence in three background electrolytes (XCln): monovalent 1-1 (K+ and Cl-), divalent 2-1 (Mg2+ and 2Cl-), and trivalent 3-1 (La3+ and 3Cl-), (ii) the effects of proton and bicarbonate ions on bulk and surface conductance, and (iii) the connected microchannel dimension (surface/height ratio aspect) within the nanochannel apparent conductance. The results highlight a net quantitative increase in surface silanol density and a strong decrease in surface ionization degree when lanthanum cations are employed. The results also demonstrate that La3+ strongly interacts with the silica surface, leading to negative values of standard free energy for ion-site interactions and chemical potential for ion-ion correlations in the Stern layer of -0.8 and -10.2 kT, respectively. We ascribed the evolution of surface charge density to the balance between the mole ratios of water molecules and adsorbed cations at equilibrium. We found that La3+ behaves as an acidic cation (Lewis conceptualization) that neutralizes the negative silica surface accompanying water molecule expulsion due to steric hindrance. This study constitutes a new contribution to ion-site interactions and to ion-ion correlation phenomena on the planar silica surface to explain charge inversion observation in micro-nanofluidic devices.

Proteomics Methodologies: The Search of Protein Biomarkers Using Microfluidic Systems Coupled to Mass Spectrometry.

Protein biomarkers have been the subject of intensive studies as a target for disease diagnostics and monitoring. Indeed, biomarkers have been extensively used for personalized medicine. In biological samples, these biomarkers are most often present in low concentrations masked by a biologically complex proteome (e.g., blood) making their detection difficult. This complexity is further increased by the needs to detect proteoforms and proteome complexity such as the dynamic range of compound concentrations. The development of techniques that simultaneously pre-concentrate and identify low-abundance biomarkers in these proteomes constitutes an avant-garde approach to the early detection of pathologies. Chromatographic-based methods are widely used for protein separation, but these methods are not adapted for biomarker discovery, as they require complex sample handling due to the low biomarker concentration. Therefore, microfluidics devices have emerged as a technology to overcome these shortcomings. In terms of detection, mass spectrometry (MS) is the standard analytical tool given its high sensitivity and specificity. However, for MS, the biomarker must be introduced as pure as possible in order to avoid chemical noise and improve sensitivity. As a result, microfluidics coupled with MS has become increasingly popular in the field of biomarker discovery. This review will show the different approaches to protein enrichment using miniaturized devices and the importance of their coupling with MS.

Autoxidation Kinetics of Tetrahydrobiopterin-Giving Quinonoid Dihydrobiopterin the Consideration It Deserves.

In humans, tetrahydrobiopterin (H4Bip) is the cofactor of several essential hydroxylation reactions which dysfunction cause very serious diseases at any age. Hence, the determination of pterins in biological media is of outmost importance in the diagnosis and monitoring of H4Bip deficiency. More than half a century after the discovery of the physiological role of H4Bip and the recent advent of gene therapy for dopamine and serotonin disorders linked to H4Bip deficiency, the quantification of quinonoid dihydrobiopterin (qH2Bip), the transient intermediate of H4Bip, has not been considered yet. This is mainly due to its short half-life, which goes from 0.9 to 5 min according to previous studies. Based on our recent disclosure of the specific MS/MS transition of qH2Bip, here, we developed an efficient HPLC-MS/MS method to achieve the separation of qH2Bip from H4Bip and other oxidation products in less than 3.5 min. The application of this method to the investigation of H4Bip autoxidation kinetics clearly shows that qH2Bip's half-life is much longer than previously reported, and mostly longer than that of H4Bip, irrespective of the considered experimental conditions. These findings definitely confirm that an accurate method of H4Bip analysis should include the quantification of qH2Bip.

Quantification of monoamine biomarkers in cerebrospinal fluid: Comparison of a UHPLC-MS/MS method with a UHPLC coupled to fluorescence detection method.

Inborn errors of monoamine neurotransmitter metabolism are rare genetic diseases classified as catecholamine and serotonin metabolism disorders or neurotransmitter transportopathies. To diagnose these orphan diseases, monoamine metabolites have been identified and validated as cerebrospinal fluid (CSF) biomarkers: 5-hydroxy-tryptophane, 5-hydroxy-indol-acetic acid, 3-ortho-methyl-DOPA, homovanillic acid, and 3-methoxy-4-hydroxyphenylglycol. The present work presents a UHPLC-MS/MS method developed for the quantification of these metabolites in CSF and compares it with a previously described UHPLC with fluorescence detection (UHPLC-FD) method. MS/MS detection was performed in positive electrospray ionization and multiple reaction monitoring mode. The UHPLC-MS/MS and UHPLC-FD methods were validated in terms of accuracy, linearity, precision and matrix effect. The lower limits of quantification (LLOQ) ranged between 0.5 and 10 nm and between 1 and 5 nm for the UHPLC-MS/MS method and the UHPLC-FD one, respectively. We verified the applicability of both methods by analyzing 30 CSF samples. The measured concentrations were comparable with the reference values described in the literature. The two methods allowed pathological samples to be distinguished from healthy ones for clinical diagnosis. UHPLC-MS/MS and UHPLC-FD methods exhibited very close LLOQs. As the UHPLC-MS/MS method is more selective, it allows faster analysis with a run time of 6 min per run vs. 10 min for the UHPLC-FD method.

A rapid and sensitive method for the quantification of dopamine and serotonin metabolites in cerebrospinal fluid based on UHPLC with fluorescence detection.

Inborn errors of dopamine and serotonin metabolism are diseases caused by deficiencies in enzymes belonging to metabolic pathways. The specific diagnosis of these inborn illnesses is based on the identification and quantification of biomarkers in cerebrospinal fluid (CSF), especially: 5-hydroxy-tryptophane (5-HTP), 5-hydroxy-indol-acetic acid (5-HIAA), 3-ortho-methyl-DOPA (3-OMD), homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). In the present work, we propose a novel ultrahigh performance liquid chromatography (UHPLC) method coupled to fluorescence detection (FD) to quantify simultaneously the five dopamine and serotonin metabolites. This method efficiently separates the five molecules in less than 10 min. A complete validation of the proposed method was performed in terms of accuracy, linearity, precision, and lower limit of quantification (LLOQ). Depending on the compound, the obtained LLOQs are between 1 nM and 5 nM, thus allowing to measure concentrations as low as in CSF samples. We also verified the method applicability by analyzing 10 CSF samples in triplicates. The obtained results showed satisfactory repeatability and an ability of this method to clearly distinguish healthy samples from pathologic samples, hence, demonstrating, the method suitability for diagnosing inborn errors of dopamine and serotonin metabolism. Therefore, the proposed UHPLC-FD method appears as a reliable alternative to the current gold standard for the quantification of these biomarkers, which is based on UHPLC coupled to electrochemical detection (ECD).

Predicting ion concentration polarization and analyte stacking/focusing at nanofluidic interfaces.

We report on the investigation of electropreconcentration phenomena in micro-/nanofluidic devices integrating 100 µm long nanochannels using 2D COMSOL simulations based on the coupled Poisson-Nernst-Planck and Navier-Stokes system of equations. Our numerical model is used to demonstrate the influence of key governing parameters such as electrolyte concentration, surface charge density, and applied axial electric field on ion concentration polarization (ICP) dynamics in our system. Under sufficiently extreme surface-charge-governed transport conditions, ICP propagation is shown to enable various transient and stationary stacking and counter-flow gradient focusing mechanisms of anionic analytes. We resolve these spatiotemporal dynamics of analytes and electrolyte ICP over disparate time and length scales, and confirm previous findings that the greatest enhancement is observed when a system is tuned for analyte focusing at the charge, excluding microchannel, nanochannel electrical double layer (EDL) interface. Moreover, we demonstrate that such tuning can readily be achieved by including additional nanochannels oriented parallel to the electric field between two microchannels, effectively increasing the overall perm-selectivity and leading to enhanced focusing at the EDL interfaces. This approach shows promise in providing added control over the extent of ICP in electrokinetic systems, particularly under circumstances in which relatively weak ICP effects are observed using only a single channel.

Electropreconcentration diagrams to optimize molecular enrichment with low counter pressure in a nanofluidic device.

Concentration polarization (CP)-based focusing electrokinetics nanofluidic devices have been developed in order to simultaneously detect and enrich highly diluted analytes on-a-chip. However, stabilization of focal points over long time under the application of the electric field remains as a technical bottleneck. If pressure-assisted preconcentration methods have been proposed to stabilize propagating modes at low inverse Dukhin number (1/Du≪1) , these recent protocols remain laborious for optimizing experimental parameters. In this paper, "electric field E/counter-pressure P" diagrams have been established during pressure-assisted electro-preconcentration of fluorescein as a model molecule. Such E/P diagram allows direct observation of the region for which the optimal counter-pressure P leads to a stable focusing regime. This region of stable focusing is shown to vary depending of the nanoslit length (100 µm < Lnanoslit < 500 µm) and the nature of the background electrolyte (KCl and NaCl). Longer nanoslits (500 µm) produce stabilization at low counter-pressure P, whereas NaCl offers a narrower region of stable focusing in the E/P diagram compared to KCl. Finally, the ability of such pressure-assisted protocol to concentrate negatively charged proteins has been tested with a more applicative protein, i.e., ovalbumin. The corresponding E/P diagram confirms the existence of the stable focusing regime at both low electric field E (≤20 V) and counter-pressure P (≤0.4 bar). With an enrichment factor as high as 70 after 2 min for ovalbumin at a concentration of 10 µM, such pressure-assisted nanofluidic electro-preconcentration protocol appears very promising to concentrate and detect biomolecules.

On-a-chip tryptic digestion of transthyretin: a step toward an integrated microfluidic system for the follow-up of familial transthyretin amyloidosis.

A microfluidic microreactor for trypsin mediated transthyretin (TTR) digestion has been developed as a step towards the elaboration of a fully integrated microdevice for the detection of a rare and disabling disease, the familial transthyretin amyloidosis (ATTR) which is related to specific TTR mutations. Therefore, an enzymatic microreactor coupled to an analytical step able to monitor the mutation of TTR on specific peptide fragments would allow an accurate monitoring of the treatment efficiency of ATTR. In this study, two types of immobilized trypsin microreactors have been investigated: a new miniaturized, microfluidic fluidized bed packed with trypsin functionalized magnetic particles (MPs), and a thiol-ene (TE) monolith-based chip. Their performances were first demonstrated with N-benzoyl-dl-arginine-4-nitroanilide hydrochloride BApNA, a low molecular weight substrate. High reaction yields (75.2%) have been reached within 0.6 min for the TE-based trypsin microreactor, while a lower yield (12.4%) was obtained for the micro-fluidized bed within a similar residence time. Transposition of the optimized conditions, developed with BApNA, to TTR digestion in the TE-based trypsin microreactor was successfully performed. We demonstrated that the TE-chip can achieve an efficient and reproducible digestion of TTR. This has been assessed by MS detection. In addition, TTR hydrolysis led to the production of a fragment of interest allowing the therapeutic follow-up of more than twenty possible ATTR mutations. High sequence coverage (90%), similar to those obtained with free trypsin, was achieved in a short time (2.4 min). Repeated experiments showed good reproducibility (RSD = 6.8%). These promising results open up the route for an innovative treatment follow-up dedicated to ATTR.

Logic digital fluidic in miniaturized functional devices: Perspective to the next generation of microfluidic lab-on-chips.

Microfluidics has emerged following the quest for scale reduction inherent to micro- and nanotechnologies. By definition, microfluidics manipulates fluids in small channels with dimensions of tens to hundreds of micrometers. Recently, microfluidics has been greatly developed and its influence extends not only the domains of chemical synthesis, bioanalysis, and medical researches but also optics and information technology. In this review article, we will shortly discuss an enlightening analogy between electrons transport in electronics and fluids transport in microfluidic channels. This analogy helps to master transport and sorting. We will present some complex microfluidic devices showing that the analogy is going a long way off toward more complex components with impressive similarities between electronics and microfluidics. We will in particular explore the vast manifold of fluidic operations with passive and active fluidic components, respectively, as well as the associated mechanisms and corresponding applications. Finally, some relevant applications and an outlook will be cited and presented.

Localized Mechanical Stress Promotes Microtubule Rescue.

Microtubule dynamics rely on the properties of tubulin and are regulated by microtubule-associated proteins. GTP-tubulin assembles into hollow polymers, which can depolymerize upon GTP hydrolysis. Depolymerizing microtubules may stop shrinking and resume growth. Such rescues are regulated by microtubule-associated proteins like CLIP-170 and the CLASPs [1, 2]. Microtubule domains prone to rescues contain discrete regions (previously termed "GTP islands") that retain a GTP-tubulin-like conformation in the main body of the microtubule [3]. However, the exact nature of these domains and the mechanisms controlling their occurrence and distribution are largely unknown. Here we show that collisions between growing microtubules and mechanical obstacles (including other microtubules) in vitro result in the higher abundance of GTP-like islands in stressed microtubule regions. Furthermore, these islands were found to be efficiently generated by both lateral contacts and mechanical constraints applied to the main body of the microtubules. They were also particularly prominent where shifts in the number of protofilaments occur in the microtubule lattice. GTP-like islands and rescues frequently co-occurred at microtubule intersections in vitro and in living cells, both in crossing and in crossed microtubules. We also observed that CLIP-170 recognizes GTP-like islands in vivo and is retained at microtubule crossings. Therefore, we propose that rescues occur via a two-stage mechanism: (1) lattice defects determine potential rescue-promoting islands in the microtubule structure, and (2) CLIP-170 detects these islands to stimulate microtubule rescue. Our results reveal the interplay between rescue-promoting factors and microtubule architecture and organization to control microtubule dynamics.

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.

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 channel's wall. These methodologies and findings could be particularly relevant to investigating various analytical pathways and to understanding the molecular mechanisms at solid/liquid interfaces.

Neutral polymers as coatings for high resolution electrophoretic separation of Aß peptides on glass microchips.

This study reports a comparison of the performances of two neutral polymers, poly ethylene-oxide (PEO) and poly(dimethylacrylamide-co-allyl glycidyl ether) (EpDMA), in glass microchips to achieve zone electrophoresis separation of several truncated forms of beta amyloid (Aß) peptides, sharing very similar structures. The peptides were derivatized by FluoProbes 488 NHS to allow their fluorescence detection. Two protocols based either on PEO or EpDMA led to good pH stabilities in addition to a significant reduction of the electroosmotic flow. These two polymer coatings allowed repeatable analyses and high resolution for the simultaneous analysis of three Aß peptides, Aß 1-38, Aß 1-40 and Aß 1-42, considered as potential biomarkers of Alzheimer's disease. A recovery study showed that EpDMA was superior in reducing the adsorption of the Aß peptides on the coated inner wall. Finally, the separation method relying on the EpDMA coated microchips was validated as linear using a calibration curve and the LOD was estimated to be close to 200 nM. Despite very short migration distances, different N-terminal or C-terminal truncated Aß peptides, corresponding to promising biomarker combinations for the future diagnostic, were fully resolved. The method was successfully applied to detect these peptides in spiked cerebrospinal fluid and has provided a first achievement towards the development of a microsystem that would integrate preconcentration and separation steps.

Improved electrochemical detection of a transthyretin synthetic peptide in the nanomolar range with a two-electrode system integrated in a glass/PDMS microchip.

An alternative to a three-electrode set-up for electrochemical detection and analysis in microfluidic chips is described here. The design of the electrochemical sensor consists of the surface of the glass substrate covered with a PDMS block which bears the microfluidic channels. A band microelectrode which acts as a working electrode surrounded by a large counter electrode is obtained at the micrometric level to propose a simple and efficient sensing area for on-a-chip analysis. The counter-electrode with a surface area about 22-fold greater than the working-microelectrode can also be considered as a pseudo reference since its current density is low and thus limits the potential variations around the rest potential. To this purpose, the [Fe(III)(CN)6]³â»/[Fe(II)(CN)6]4⁻ redox couple was used in order to set a reference potential at 0 V since both electrodes were platinum. The electrochemical microchip performance was characterized using differential pulse voltammetric (DPV) detection and quantification of the optically multi-labelled transthyretin synthetic peptide mimicking a tryptic fragment of interest for the diagnosis of familial transthyretin amyloidosis (ATTR). The limit of detection of the peptide by the working microelectrode was 25 nM, a value 100-fold lower than the one reported with conventional capillary electrophoresis coupled with laser-induced fluorescence under the same analytical conditions.

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.

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.

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.

A "dry and wet hybrid" lithography technique for multilevel replication templates: Applications to microfluidic neuron culture and two-phase global mixing.

A broad range of microfluidic applications, ranging from cell culture to protein crystallization, requires multilevel devices with different heights and feature sizes (from micrometers to millimeters). While state-of-the-art direct-writing techniques have been developed for creating complex three-dimensional shapes, replication molding from a multilevel template is still the preferred method for fast prototyping of microfluidic devices in the laboratory. Here, we report on a "dry and wet hybrid" technique to fabricate multilevel replication molds by combining SU-8 lithography with a dry film resist (Ordyl). We show that the two lithography protocols are chemically compatible with each other. Finally, we demonstrate the hybrid technique in two different microfluidic applications: (1) a neuron culture device with compartmentalization of different elements of a neuron and (2) a two-phase (gas-liquid) global micromixer for fast mixing of a small amount of a viscous liquid into a larger volume of a less viscous liquid.

Ionic and mass transport in micro-nanofluidic devices: a matter of volumic surface charge.

The shape and the surface charge of microchannels are critical parameters for ionic and mass transport in microfluidic systems. A great number of studies and developments have been carried out in order to optimize these features separately. We propose to consider them together within a new fundamental parameter for microfluidics, that we named the Volumic Surface Charge (VSC), which is the ratio of the surface charge to the section height in planar microchannels. The non-linear effects induced by rapid VSC variations can result in selective preconcentration processes, which can be used for a simultaneous preconcentration and separation of biomolecules within simple straight channels. In this review, we first present 3 different techniques that we developed to tune the VSC either by surface chemical patterning, integration of polarisable interfaces or geometrical constrictions. The proof of concept of the selective preconcentration using VSC variations will be presented on the basis of experimental results obtained with fluorescent probes and numerical simulations.