Flowmetering for microfluidics.

Originally designed for chromatography, electrophoresis, and printing technologies, microfluidics has since found applications in a variety of domains such as engineering, chemistry, environmental, and life sciences. The fundamental reason for this expansion has been the development of miniature components, allowing the handling of liquids at the microscale. For the maturation of microfluidic technologies, the need for affordable, reliable, and quantitative techniques to measure flow rates from 1 nL min-1 to 1 mL min-1 appears as a strong challenge. We review herein the different technologies available and those under development, and discuss their sensing principles and industrial maturity. Given the need of traceability of these measurements, we then focus on the developments of primary standards to measure microfluidic flow rates by metrological institutes. We conclude this review with some perspectives and pending challenges for microfluidic flowmeters.

Controlling the distance of highly confined droplets in a capillary by interfacial tension for merging on-demand.

Droplet microfluidics is a powerful technology that finds many applications in chemistry and biomedicine. Among different configurations, droplets confined in a capillary (or plugs) present a number of advantages: they allow positional identification and simplify the integration of complex multi-steps protocols. However, these protocols rely on the control of droplet speed, which is affected by a complex and still debated interplay of various physico-chemical parameters like droplet length, viscosity ratio between droplets and carrier fluid, flow rate and interfacial tension. We present here a systematic investigation of the droplet speed as a function of their length and interfacial tension, and propose a novel, simple and robust methodology to control the relative distance between consecutive droplets flowing in microfluidic channels through the addition of surfactants either into the dispersed and/or into the continuous phases. As a proof of concept application, we present the possibility to accurately trigger in space and time the merging of two confined droplets flowing in a uniform cross-section circular capillary. This approach is further validated by monitoring a conventional enzymatic reaction used to quantify the concentration of H2O2 in a biological sample, showing its potentialities in both continuous and stopped assay methods.

On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation.

Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.

The power of solid supports in multiphase and droplet-based microfluidics: towards clinical applications.

Multiphase and droplet microfluidic systems are growing in relevance in bioanalytical-related fields, especially due to the increased sensitivity, faster reaction times and lower sample/reagent consumption of many of its derived bioassays. Often applied to homogeneous (liquid/liquid) reactions, innovative strategies for the implementation of heterogeneous (typically solid/liquid) processes have recently been proposed. These involve, for example, the extraction and purification of target analytes from complex matrices or the implementation of multi-step protocols requiring efficient washing steps. To achieve this, solid supports such as functionalized particles (micro or nanometric) presenting different physical properties (e.g. magnetic, optical or others) are used for the binding of specific entities. The manipulation of such supports with different microfluidic principles has both led to the miniaturization of existing biomedical protocols and the development of completely new strategies for diagnostics and research. In this review, multiphase and droplet-based microfluidic systems using solid suspensions are presented and discussed with a particular focus on: i) working principles and technological developments of the manipulation strategies and ii) applications, critically discussing the level of maturity of these systems, which can range from initial proofs of concept to real clinical validations.

A simple and low-cost chip bonding solution for high pressure, high temperature and biological applications.

The sealing of microfluidic devices remains a complex and time-consuming process requiring specific equipment and protocols: a universal method is thus highly desirable. We propose here the use of a commercially available sealing tape as a robust, versatile, reversible solution, compatible with cell and molecular biology protocols, and requiring only the application of manually achievable pressures. The performance of the seal was tested with regards to the most commonly used chip materials. For most materials, the bonding resisted 5 bars at room temperature and 1 bar at 95 °C. This method should find numerous uses, ranging from fast prototyping in the laboratory to implementation in low technology environments or industrial production.

[Circulating tumor cells and breast cancer: detection techniques and clinical results]. / Cellules tumorales circulantes et cancer du sein : méthodes de détection et résultats cliniques.

Due to recent technological progresses, Circulating Tumor Cells (CTC) are currently extensively studied in order to define their diagnostic, prognostic and predictive value. We review here the different detection techniques and their clinical results in breast cancer patients.

Epitope extraction technique using a proteolytic magnetic reactor combined with Fourier-transform ion cyclotron resonance mass spectrometry as a tool for the screening of potential vaccine lead peptides.

Epitope extraction technique is based on the specific digestion of a target protein followed by immunoaffinity isolation of a specific recognition peptide. This technique, in combination with mass spectrometry, has been efficiently used for epitope identification. The major goal of this work was to utilize newly developed enzyme and immunoaffinity magnetic reactors for the epitope extraction procedure and confirm the efficiency of this improved system for epitope screening of proteins. Alginic acid-coated magnetite microparticles with immobilized TPCK-trypsin provided high working efficiency with low non-specific adsorption, digestion time in minutes and low frequency of missed cleavages. The sensitivity and specificity of tryptic fragmentation of the beta-amyloid-peptide Abeta (1-40) as a model polypeptide was confirmed by Fourier-transform ion cyclotron resonance mass spectrometry analysis. The Sepharose reactor or immunoaffinity magnetic reactors, both with anti-amyloid-beta monoclonal antibodies, were used for specific isolation and identification of target peptides. In this way, the epitope extraction technique combined with mass spectrometric analysis is shown to be an excellent base for molecular screening of potential vaccine lead proteins.

Injection and flow control system for microchannels.

In spite of considerable efforts, flow control in micro-channels remains a challenge owing to the very small ratio of channel/supply-system volumes, as well as the induction of spurious flows by extremely small pressure or geometry changes. We present here an inexpensive and robust system for flow control in a microchannel system, based on a dynamic control of reservoir pressures at the end of each channel. This system allows flow equilibration with a time constant smaller than one second, and is also able to maintain stable flux from stopped flow to many microl min(-1) range over several hours. It is robust to changes in ambient pressure and temperature. This system further includes a feature for sub-microliter sample injection during the experiment. We quantify flow control in elastomer and thermoplastic channels, and demonstrate the impact on one application of the system, namely the reproducible, automated separation of large DNA by electrophoresis in a self-organized magnetic bead matrix in a microchannel.

New block-copolymer thermoassociating matrices for DNA sequencing: effect of molecular structure on rheology and resolution.

A new family of matrices for DNA sequencing by capillary electrophoresis is presented. These matrices combine easy injection with high sieving performances, due to thermal switching between a low and a high viscosity state through a modest increase in temperature (approximately 20 degrees C). They are constructed from a hydrophilic polymer backbone with grafted lower critical solution temperature (LCST) side chains. The comb-like LCST copolymers are characterized in terms of size of the polymer backbone, the size of LCST side chains and the grafting densities. The dependance of rheological behavior and electrophoretic performance of these copolymers is correlated with their microstructure. Without complete optimization, a resolution of order 0.5, corresponding to a very reasonable limit for read length with current base calling softwares, could be achieved for segments around 800 bases differing by 1 base in less than one hour in a commercial ABI 310 apparatus.

Direct imaging of single-molecules: from dynamics of a single DNA chain to the study of complex DNA-protein interactions.

Recent years have seen significant advances in the characterization and manipulation of individual molecules. The combination of single-molecule fluorescence and micromanipulation enables one to study physical and biological systems at new length scales, to unravel qualitative mechanisms, and to measure kinetic parameters that cannot be addressed by traditional biochemistry. DNA is one of the most studied biomolecules. Imaging single DNA molecules eliminates important limitations of classical techniques and provides a new method for testing polymer dynamics and DNA-protein interactions. Here we review some applications of this new approach to physical and biological problems, focusing on videomicroscopy observations of individual DNA chains extended in a shear flow. We will first describe data obtained on the stretching, relaxation and dynamics of a single tethered polymer in a shear flow, to demonstrate that the deformation of sheared tethered chains is partially governed by the thermally driven fluctuations of the chain transverse to the flow direction. Next, we will show how single-molecule videomicroscopy can be used to study in real time DNA folding into chromatin, a complex association of DNA and proteins responsible for the packaging of DNA in the nucleus of an eukaryotic cell.

Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy.

Fluorescence videomicroscopy and scanning force microscopy were used to follow, in real time, chromatin assembly on individual DNA molecules immersed in cell-free systems competent for physiological chromatin assembly. Within a few seconds, molecules are already compacted into a form exhibiting strong similarities to native chromatin fibers. In these extracts, the compaction rate is more than 100 times faster than expected from standard biochemical assays. Our data provide definite information on the forces involved (a few piconewtons) and on the reaction path. DNA compaction as a function of time revealed unique features of the assembly reaction in these extracts. They imply a sequential process with at least three steps, involving DNA wrapping as the final event. An absolute and quantitative measure of the kinetic parameters of the early steps in chromatin assembly under physiological conditions could thus be obtained.

Dynamics of a tethered polymer in shear flow.

The dynamics of a single polymer tethered to a solid surface in a shear flow was observed using fluorescently labeled DNA chains. Dramatic shear enhanced temporal fluctuations in the chain extension were observed. The rate of these fluctuations initially decreased for increasing shear rate gamma; and increased above a critical gamma;. Simulations revealed that these anomalous dynamics arise from a continual recirculating motion of the chain or cyclic dynamics. These dynamics arise from a coupling of the chain velocity in the flow direction to thermally driven fluctuations of the chain in the shear gradient direction.

The effect of blob size and network dynamics on the size-based separation of polystyrenesulfonates by capillary electrophoresis in the presence of entangled polymer solutions.

This work focuses on the separation of standard polystyrenesulfonates (PSS), with molecular masses (Mr) between 16 and 990 x 10(3) in capillaries filled with semidilute (entangled) linear hydrophilic polymers. Contrary to cross-linked chemical gels, which produce permanent networks, solutions of linear polymers lead to dynamic networks. The analytical performances and migration mechanisms are discussed on the basis of experiments performed in solutions of linear polyethyleneoxides and derivatized celluloses of various molecular masses. The influence of the mesh size and of the lifetime of the obstacles of the separating network has been investigated in detail. The mesh size is assimilated to the blob size of the separating polymer and is a decreasing function of its concentration. The lifetime of the obstacles of the network, identified with the reptation time of the polymer chain, characterizes its dynamics. This characteristic time increases with both the molecular weight of the separating polymer and its concentration. Its impact was first examined at fixed blob size. Then, the influence of the blob size was studied while keeping the reptation time of the network constant. By doing so, the existence of interactions between the solute and the separating polymer or between the solute and capillary wall can be more safely assessed. It appears that the reptation time of the mesh has a large influence on the electrophoretic mobility of the PSSs under a threshold value, which is of the order of magnitude of the time taken by the PSS to migrate on the blob size. Also shown are separations using networks made up with mixtures of polyethyleneoxides of the same nature and same mass concentration, but of very different molecular masses. This latter approach allows one to adapt the viscosity of the solution and the dynamics of the network, keeping the blob size constant.

Acting on actin: the electric motility assay.

We have developed a novel technique which allows one to direct the two dimensional motion of actin filaments on a myosin coated sheet using a weak electric field parallel to the plane of motion. The filament velocity can be increased or decreased, and even reversed, as a function of orientation and strength of the field. PMMA (poly(methylmethacrylate)) gratings, which act as rails for actin, allow one for the first time to explore three quadrants of the force velocity diagram. We discuss effective friction, duty ratio and stall force at different myosin densities. A discontinuity in the velocity force relationship suggests the existence of dynamical phase transition.

High resolution capillary electrophoretic separation of oligonucleotides in low-viscosity, hydrophobically end-capped polyethylene oxide with cubic order.

A triblock self-associating polymer with the structure n-dodecane-poly(ethylene oxide)-n-dodecane and a very low polydispersity has been used as a matrix to separate a sample of single-stranded oligonucleotides containing Pd(A)25-30 and Pd(A)40-60. Above a concentration of 4%, this associative polymer forms a micellar network with cubic order and a well-defined micellar spacing, in which the dodecane micellar cores are bridged by polyoxyethylene segments. This medium combines a low viscosity with excellent resolution of oligonucleotides. This work confirms that associative polymers are potentially powerful media for separation in capillary electrophoresis, and argues in favor of the use of monodisperse products presenting a high-order in the physical gel state.

Micropreparative capillary electrophoresis of DNA by direct transfer onto a membrane.

We have developed a new technique for the collection of DNA fragments separated by capillary electrophoresis, by direct transfer from the capillary outlet to a positively charged membrane. Transfer and post-run detection of two different nonradioactively labeled DNA standards, ranging in size from 150 bp to 2 kbp and 120 bp to 23 kbp are presented, and discussed. Capillary electrophoresis with direct blotting presents several advantages over the blotting from gels: the separation is faster and requires less manual steps, the resolution is higher, and each DNA fragment is collected into a very concentrated spot on the membrane due to the small surface of the capillary outlet and to a design of the collection device inducing a refocusing of field lines across the hybridization membrane. Therefore, very small amounts of DNA (in the pg range) can be detected. This fraction collection makes further analysis of the sample possible, e.g. by hybridization, thus suppressing one of the major present limitations of the capillary electrophoresis technique for DNA analysis.

Reptation theories of electrophoresis.

In this review, we present the main aspects of the reptation theory, which has provided an essential insight into the processes at work during DNA electrophoretic separation in gels. We avoid mathematical developments, and rely as much as possible on an intuitive description. We first present the original biased reptation model, which assumes that the DNA threads its way as a "worm" of fixed length among the fibers of the gel. We then introduce a more recent version, the model of Biased Reptation with Fluctuations (BRF), which allows for longitudinal flexibility along the DNA. We then propose a quantitative comparison with experiments performed in constant field, and discuss the application of reptation theories to pulsed field techniques either with crossed fields or with field inversion. We also discuss at some length the different experiments that led to a criticism of reptation ideas, such as orientation measurements and videomicroscopy. Finally, we use these experiments together with various computer simulations developed recently for gel electrophoresis, to propose a more realistic qualitative description of DNA motion in gels, and we discuss what elements in this motion are relevant to reptation and what processes are not included in present analytical models.