EP2097160B1 - Microdevice and method for treating liquid specimens - Google Patents

Description

TECHNICAL FIELD AND PRIOR ART

The invention relates to the field of the treatment of liquid samples, in particular by centrifugation or mixing a drop of liquid.

It applies in particular to the preparation or the purification of biological and chemical samples, to the fields of biomedical diagnosis, molecular biology, the reprocessing of effluents, possibly radioactive (extraction of actinides), and more generally, to all the scientific, technological and industrial fields that involve the selective extraction of macromolecules, organelles, actinides, colloids or solid particles from a liquid sample in the form of a drop or a puddle (liquid inclusions).

The proposed invention also relates to the field of discrete microfluidics, preferably used for continuous microfluidics (in channels) as soon as it is freed from pumps, valves, walls necessary for confining the flow. ... etc.

Indeed, all these elements contribute to parietal physicochemical contaminations as well as intrinsically slow capillary flows despite the high power involved in pumping (significant pressure losses).

Discrete (or digital) micro-fluidics play an increasing role in the development of new micro-systems such as lab on chips, and many analysis steps can be performed in a chain using discrete micro-fluidics.

Molecules of biological or medical interest are for example transported within drops that pass between various analysis steps such as biochemical functionalization, injection of biomolecules by heterogeneous mixing (coalescence of drops), pipetting or localized fragmentation. drops ... etc.

The proposed invention finds many applications in small scale mixing, small scale extraction, small scale centrifugal separation or purification, concentration and then detection of biological targets, pumping in microfluidics, transmission of micro-fluidic movements, the rheological characterization of fluid samples in the form of liquid drops or gels.

The invention also relates to the field of purification of biological samples, and extraction of biological constituents.

The most recognized purification techniques in biology are chromatography, electrophoresis and centrifugation; they are mostly practiced on a macroscopic scale (from a few centimeters to a few meters).

Coupled with powerful detectors, chromatography is the most sensitive analytical technique currently available for assaying a substance in a biological sample.

This analysis technique is certainly one of the most sensitive but its miniaturization is very difficult to implement in particular because of the porous medium that is involved; this is his main disadvantage. The realization of a micro-system integrating the chromatography is random and the preparation upstream of the liquid sample remains in suspense.

Electrophoresis allows selective separation of biological molecules based on their electrical charge.

But the miniaturization of the electrophoresis remains delicate since the medium allowing the migration of the constituents to be analyzed is a very viscous gel. The insertion and then the handling of a gel in a lab- like analysis chain is difficult to implement.

As for the current centrifuges, exploited in biology, biochemistry or in the medical diagnosis to isolate constituents or purify biological samples, they consist of a shaft carrying a special rotor, all driven by a powerful motor. The rotor has locations, symmetrically located on either side of the axis, which can receive small test tubes containing the biological preparations to be analyzed or purified. The whole is enclosed in a tank, sealed during rotation, for security reasons.

• le déséquilibre du rotor à compenser en permanence,
• et la difficulté de miniaturisation puisque l'accélération centrifuge est également proportionnelle au rayon de giration.

The proposed invention is a solution to two problems posed by current centrifuges:

• the imbalance of the rotor to compensate permanently,
• and the difficulty of miniaturization since the centrifugal acceleration is also proportional to the radius of gyration.

The document Y. Fouillet et al. "EWOD digital microfluidics for a lab on a chip", Proceedings of the ASME, 4th Int. Conf. On Nanochannels, Microchannels and Minichannels, June 19-21, 2006, Limerick, Irel and, illustrates a possibility of setting a fluid in motion by implementing electrohydrodynamics (EHD). Electric forces are then used to create tangential electrostatic stresses on drops that are activated on a component of the electrowetting type.

In this type of device, the drop is fixed and the triple line does not move, while internal convection movements are observed.

There is the problem of being able to optimize this phenomenon thanks to a configuration of appropriate electrodes and secondly to implement this phenomenon for different applications.

The document US-A1-2006 / 0132542 describes a device according to the preamble of claim 1.

SUMMARY OF THE INVENTION

The present invention uses the setting in motion of fluid in a drop, which is itself at rest.

The proposed invention applies to liquid inclusions, not in motion as in electrowetting techniques, but at rest (in static position). A liquid inclusion is centered on an EHD chip ("electrohydrodynamic") also object of the invention. This allows to generate an intense and organized movement, or brewing, to the interior of the drop and optionally outside, in the external fluid to the drop, for example if it and the EHD chip are covered with a viscous fluid, the drop being in a static position and not deforming . In particular, there is no block displacement or interfacial deformation of the liquid inclusion. A movement, or displacement, before or after the brewing operation can take place, to bring the drop or the liquid inclusion in the brewing place or to move away after brewing.

The only movement is due to the interface of the drop and the external environment; the particles that constitute this interface move tangentially to it so that it does not deform (there is a scanning movement along the interface).

The geometry of the drop therefore remains fixed and the movement thus generated along the interface is communicated to the internal fluid phases, and possibly external, to the drop by the viscosities specific to each of these fluid phases. Viscosities are a kind of relay of the interfacial tangential impulse.

No electrophoretic gel or porous medium is involved, the centrifugation according to the invention thus allows micro-fluidic miniaturization.

nombre qui mesure la centrifugation rapportée à la pesanteur ou à la gravité, u _φ étant la vitesse de centrifugation) qui doit être atteint : à première vue, plus l'échelle de longueur de l'échantillon liquide est petite (cas des micro-systèmes), plus il semble difficile d'atteindre des intensités de centrifugation significative. La présente invention permet de surmonter cette difficulté et conserve l'essentiel des avantages associés à la centrifugation en tant que technique d'analyse, notamment biologique, tout en permettant sa miniaturisation et les avantages associés :

• la manipulation de petits échantillons biologiques,
• l'implication de faibles volumes de réactifs,
• la portabilité,
• et l'implémentation dans un laboratoire sur puce ou un micro-système basé sur la micro-fluidique digitale.

However, for micro-systems, a problem lies in the number of G $(=\frac{{u_{\phi }}^2}{R}/\mathrm{boy\; Wut},$

number which measures centrifugation relative to gravity or gravity, u _φ being the speed of centrifugation) which must be reached: at first glance, plus the scale of length of the liquid sample is small (case of micro-systems), the more it seems difficult to achieve significant centrifugation intensities. The present invention makes it possible to overcome this difficulty and retains most of the advantages associated with centrifugation as an analysis technique, in particular a biological one, while allowing its miniaturization and the associated advantages:

• handling small biological samples,
• the implication of small volumes of reagents,
• portability,
• and the implementation in a lab-on-a-chip or micro-system based on digital microfluidics.

These advantages are also preserved if it is to apply the invention to the micro-fluidic drop concentration applied to the detection of biological targets.

A device according to the invention is a device for treating a drop of liquid, with the features of claim 1.

According to the invention, the shape of the electrodes makes it possible to promote the existence of fluids, the contours facing the electrodes being neither totally tangent nor totally perpendicular to the triple line.

According to the invention, tangential interfacial motion is induced by the electric field, despite the smallness of the liquid sample, by the application of a tangential electric stress at the interface of a liquid sample, in the zones situated above the electrode interface areas. The only source of energy dissipation, since the liquid inclusion is stabilized in a static position by hooking up its triple line and / or by electrowetting, comes from the volume viscosity (there is no dissipation of energy by triple line displacement). The presence of a solid wall on which the liquid inclusion is deposited or of two solid walls between which the inclusion is sandwiched (capillary bridge), gives rise to a viscous dissipative shear which balances the original interfacial motor term electric.

The angle, strictly between 0 ° and 90 °, between the tangent to the triple line (or its projection) and the edges facing each other of the electrodes, may advantageously be between 40 ° and 50 ° for example equal to substantially 45 °.

The edges of the electrodes facing each other can be for example in the form of zig-zag or logarithmic spiral form.

The electrodes are for example 2, 4, or 8.

Preferably the edges of the electrodes, making, with the projection of the line of contact, an angle strictly between 0 ° and 90 °, alternate with edges of electrodes making an angle of 90 ° with this same projection.

Means may be provided to activate and deactivate, successively, the electrodes. According to a particular embodiment, this activation and deactivation successively in time takes place at high frequency, greater than 100 Hz.

Space separation of the edges of the electrodes facing each other can be alternately (by traversing the electrodes in their plane, in the direction of clockwise or in the opposite direction) a first value and a second value, less than the first.

In addition, means are provided for trapping the triple line, which a drop placed on the device defines with it.

A second set of electrodes may be located opposite, parallel to the first electrodes. For example, this second set of electrodes also forms a device according to the invention.

It is therefore possible to use two EHD chips at the lower and upper ends of a capillary bridge.

A device according to the invention may further comprise a counter-electrode shaped tip.

The invention also makes it possible to produce a pumping device comprising at least one device according to the invention, as described above, and means for bringing a second fluid into contact with a drop of liquid disposed on the device.

Such a device may comprise a plurality of devices according to the invention.

The invention thus makes it possible to micro-pump secondary flows or to accelerate micro-fluidic flows by placing one (or more) micro-gear (s) consisting of one (or more) liquid inclusion (s) surrounded by a secondary and continuous liquid phase. In "micro pumping" type applications, the present invention is distinguished by the use of a fluid interface which causes a tangential movement of interfacial origin. The flow rate thus obtained is much higher than most current micro-pumps and accidental physicochemical contamination due to the presence of walls is avoided.

The proposed invention also makes it possible to produce apparatus such as a mini-stirrer, or an analytical mini-centrifuge, or a mini-emulsifier, or a micro-centrifuge, or a mini-rheometer. A mini-rheometer measures viscosity and elasticity by measuring or visualizing flow velocity fields.

• il n'est pas nécessaire que le fluide à entraîner soit ionique (contrairement aux micro-pompes électrocinétiques) : dans l'invention proposée, le mécanisme d'entraînement est un cisaillement visqueux d'origine interfaciale et diélectrique,
• dans l'invention proposée, un écoulement peut être pompé qu'il y ait, ou non, des gradients thermiques, chimiques ou ioniques,
• une ou deux parois horizontales suffisent (à comparer aux micro-pompes mécaniques, piézoélectriques ou électrocinétiques) et les sources de contamination physico-chimique sont très réduites.

Among the advantages of producing, in accordance with the invention, a flow using an interposed fluid interface and an array of electrodes, the following may be mentioned:

• it is not necessary that the fluid to be driven is ionic (unlike electrokinetic micro-pumps): in the proposed invention, the drive mechanism is a viscous shear of interfacial and dielectric origin,
• in the proposed invention, a flow can be pumped whether or not there are thermal, chemical or ionic gradients,
• one or two horizontal walls suffice (compared to mechanical micro-pumps, piezoelectric or electrokinetic) and the sources of physico-chemical contamination are very small.

• un caractère non destructif et isotherme : l'inclusion liquide impliquée peut donc contenir des constituants fragiles, dénaturables avec la température ou sous l'effet de forces ioniques,
• la rapidité : avec l'invention, il suffit de quelques secondes ou minutes pour que le brassage ou la centrifugation engendre une sédimentation ou une flottation de constituants,
• une grande simplicité de mise en oeuvre ainsi qu'une possibilité d'asservissement,
• la capacité à engendrer au sein d'une inclusion liquide de taille typiquement millimétrique un mouvement de rotation ou de brassage intense. Le nombre de G atteint dans les expériences effectuées avec des puces selon l'invention, non encore optimales, est de l'ordre de 10 ou 100,
• la puce ainsi que les techniques d'arrachage appliquées à l'apex de l'inclusion liquide proposées dans l'invention permettent la sélection spécifique de constituants après concentration micro-fluidique en vue d'une extraction, d'une analyse ou d'une détection a posteriori.

The proposed invention further has the following advantages:

• a non-destructive and isothermal character: the involved liquid inclusion may therefore contain fragile constituents, which can be denatured with temperature or under the effect of ionic forces,
• the rapidity: with the invention, it only takes a few seconds or minutes for the stirring or centrifugation to cause sedimentation or flotation of constituents,
• a great simplicity of implementation as well as a possibility of servocontrol,
• the ability to generate within a liquid inclusion of typically millimeter size a rotational movement or intense stirring. The number of G reached in the experiments carried out with chips according to the invention, which are not yet optimal, is of the order of 10 or 100,
• the chip as well as the pulling techniques applied to the apex of the liquid inclusion proposed in the invention allow the specific selection of constituents after microfluidic concentration for extraction, analysis or analysis. posterior detection .

The invention also relates to a method of treating a drop of liquid in a surrounding medium according to claim 7.

The applied field is oblique with respect to the liquid droplet - surrounding environment.

The volume of the drop may vary with time.

One or more circulating flows or one or more vortices may be generated in the drop.

The invention also relates to a process for microfluidic concentration by mixing or centrifuging a drop of liquid, in particular for detecting antibodies, or antigens, or proteins or protein complexes, or DNA or RNA , comprising the implementation of a method of forming at least one circulating or vortex flow in said drop of liquid according to a method according to the invention.

A detection step can be performed, after mixing or centrifugation, without displacement of the drop.

A liquid extraction stage of the drop may moreover be provided. Then it is possible to transfer the extracted liquid to a detection zone. The extraction step may be carried out by electrowetting or by emission of droplets from a Taylor cone.

• un rapprochement par déplacement de deux volumes de liquides, destinés à former l'émulsion, l'un par rapport à l'autre, par exemple par électromouillage,
• une étape de mise en oeuvre d'un procédé selon l'invention, tel que décrit ci-dessus.

The invention also relates to the formation of a microemulsion comprising:

• an approximation by displacement of two volumes of liquids, intended to form the emulsion, with respect to each other, for example by electrowetting,
• a step of implementing a method according to the invention, as described above.

A method of pumping a secondary fluid, according to the invention, with a drop of a primary fluid, comprises the implementation of a method of forming at least one circulating or vortex flow in said drop of primary fluid according to a method as described above, and the pumping of the secondary fluid by contact with the primary fluid, the forces present at the primary fluid interface - secondary fluid for driving the secondary fluid.

• la mise en oeuvre d'un procédé de concentration micro-fluidique selon l'invention,
• une désactivation des (au moins) deux premières électrodes, et la formation d'un pont capillaire entre la première surface isolante et une paroi comportant au moins une autre électrode,
• l'activation électrique des premières électrodes et de l'autre électrode, et la coupure du pont capillaire.

An analyte extraction method of a drop of liquid according to the invention comprises:

• the implementation of a microfluidic concentration process according to the invention,
• deactivating (at least) two first electrodes and forming a capillary bridge between the first insulating surface and a wall comprising at least one other electrode,
• the electrical activation of the first electrodes and the other electrode, and the breaking of the capillary bridge.

A particle extraction method according to the invention comprises the implementation of a method according to the invention as described above, the surrounding medium consisting of a second liquid containing particles which have previously sedimented on the surface. interface of the two liquids, then separating, for example by electrowetting, the lateral parts, containing the particles, and a central part of the drop.

BRIEF DESCRIPTION OF THE FIGURES

• The Figures 1A and 1B represent a geometry of the EHD system in the case of electrodes activated by an alternating electric potential difference.
• The figure 2 represents a segmented two-electrode EHD chip.
• The figures 3 and 5 each represent a four-electrode EHD chip with segmented boundaries.
• The figure 4 represents a segmented boundary two-electrode EHD chip.
• The figure 6 represents a drop of water placed on an EHD chip with two electrodes segmented at ± 45 °.
• The Figures 7 to 9 each represent an electrode EHD chip whose internal boundaries are logarithmic spirals.
• The figures 10 and 11 each represent an electrode EHD chip whose internal boundaries are either straight segments or logarithmic spirals.
• The Figures 12A to 12C represent vertical extraction steps using a method according to the invention.
• The Figures 13 and 14 each represent an application of a device according to the invention.
• The Figures 15A to 15D represent extraction steps of another method according to the invention.
• The Figures 16A and 16B each represent a device according to the invention, provided with trapping pads.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

In the remainder of the description, the generic term "constituents" will be used to denote all the potential species that are the subject of the present invention (macromolecules, organelles, actinides, colloids or solid particles).

The invention may in particular implement crosslinked liquid inclusions whose size may for example vary between 10 microns and centimeter.

According to the invention, a liquid inclusion 12 is in a static position, placed symmetrically astride two electrodes 4, 6 (or more, in even or odd numbers), which can be brought to different, continuous or alternating electrical potentials ( Figures 1A, 1B ). These are, for example, electric potentials of the same absolute value but of opposite signs. These electrodes rest on a substrate 3.

To be compatible with electrowetting displacement technology (EWOD technology), the drop may be separated from the electrodes by an insulating layer 10 and possibly by a hydrophobic layer 8. But the device can also function according to the invention without these layers 8, 10 , continuously or alternatively.

The liquid contact line 20 - layer 8 (or layer 10) - ambient medium 22 is called a triple line. This line of contact, in the form of a circle (but not necessarily), does not deform, which is an important contribution, as regards the performance of stirring or centrifugation.

Means 11 make it possible to apply between the two electrodes 4, 6 a difference in potential which gives rise to an oblique electric field with respect to the liquid 12 / liquid 22 or liquid 12 / gas interface 22. This oblique field, that is to say, neither totally tangent nor totally normal to the surface of the liquid inclusion 12, will allow an accumulation of electrical charges at the interface, and the creation of the momentum tangentially at the interface 12/22, amount of movement which, in turn, will cause currents 13, 15 internal to the drop, but no displacement of the drop itself. These currents appear in the plane of the Figure 1A for reasons of clarity, but rather they are oriented in a plane parallel to the plane of the electrodes 4, 6 or layers 8, 10. The oblique nature of the field results from the shape of the electrode edges facing one of the other, as explained later. Between the inter-electrode gap areas, the field is almost zero.

An EHD chip according to the invention allows a mixture or a centrifugation not via the physical displacement of a drop by electrowetting but by the emergence of movements 13, 15 in the internal fluid to the drop and, optionally, in the external fluid to gout. These movements are generated by a viscous friction tangential to the surface of the considered inclusion.

The only movement is due to the interface; the particles that make up the interface move tangentially to it so that it does not deform (scanning movement along the interface).

The invention thus makes it possible to produce, within liquid inclusions 12, using electrohydrodynamics (EHD), a micro-flow 13, 15 or a drainage, or a mixture (or mixing) of controlled intensity, or centrifugation.

As explained later, it is possible to generate a single vortex, ie a single centrifugation. This will be particularly interesting for targeted applications such as the preparation of biological samples, the purification of samples, or the extraction of constituents (such as macromolecules (DNA, RNA, proteins, etc.), analytes, colloids , solid particles ... etc).

The nature, the thickness, the technological implementation of the layers 8, 10 are for example similar to those of the EWOD technology, such as for example described in the article by Y.Fouillet et al. cited above or in the document WO 2006/005880 or FR 2 841 063 .

The invention operates with various pairs of fluids 12/22 such as water / air, water / oil, water / chloroform .... The ambient medium 22 is preferably rather insulating (air, oil ...).

The drop 12 and the ambient medium 22 (gas or liquid) have different dielectric and resistive properties: different dielectric permittivities and / or different electrical conductivities; by way of example, mention may be made of the water / air or water / oil pairs, whose properties of dielectric permittivity and / or of electrical conductivity have the desired differences. For example with the water / oil pair or the water / air pair, the permittivity and conductivity jump is fully sufficient because the water is very strongly polarized (relative permittivity 80).

When a voltage is applied between the two electrodes 4, 6, a spreading of the drop 12 is initially observed due to the presence of the forces associated with electrowetting.

For a given AC or DC voltage the drop spreads and its shape does not change anymore. This voltage may for example vary from 0.1 V to 100 V or a few hundred V, for example 500 V.

By electrowetting, the drop is kept centered or astride the different electrodes. It is thus possible to use holding studs, as explained below.

At the drop interface 12 - medium 22, there is a vectorial equality between viscous stress jump and tangential electric stress jump. This equality reflects a balance in every point of the interface, a balance that has three components, projected according to the normal unit vector n at the interface and following two unit vectors tangent to this interface, t ₁ and t ₂ .

The normal component at the interface (also called the normal momentum balance) helps to position the inclusion stably.

The stirring or centrifugation results in particular from the tangential components of the preceding equilibrium (tangential momentum balances) and more particularly from the tangential component along the tangent t ₁ to the line 20 of contact of the liquid inclusion 12 concerned.

The nature and the intensity of the mixture resulting from the internal currents 13, 15 can be controlled by controlling the vorticity level, the number and the size of the micro- or mini-vortex (s) generated within from the liquid inclusion.

It is therefore possible to generate recirculating flows (or vortices) in controlled number and intensity in and around a liquid inclusion 12 deposited in a fixed position on an electrohydrodynamic chip. The liquid inclusion is not deformed during the process.

A stirring according to the invention, by electrohydrodynamics, was observed under a microscope ( figure 6 ) with a drop 12 of water under air and selective tracer balls (diameter 30mm) of the interface (density: 0.3). The drop is placed symmetrically astride two electrodes 4, 6 isolated from the drop of water by a thin dielectric film 10 (diagram of FIG. Figure 1A ).

In the experiments carried out in the air, the tangential component at the origin of the fluid movement is simplified because the air 22 around the drop is considered in first approximation as neutral; this component is written explicitly at the interface in the form, $${\mathrm{\epsilon }}^{\mathrm{water}}{\mathrm{E}}_{\mathrm{r}}{\mathrm{E}}_{\mathrm{\phi }}={\mathrm{\eta }}^{\mathrm{water}}\left(r\frac{\mathrm{\partial }}{\mathrm{\partial }\mathrm{r}}\left(\frac{{\mathrm{u}}_{\mathrm{\phi }}}{\mathrm{r}}\right)\right)$$

The geometry of the drop of water 12 is close to a truncated sphere, the normal n is oriented along the radial coordinate r, the tangents t ₁ and t ₂ are oriented along the longitude Φ and co-latitude θ, respectively. The dielectric permittivity ε ^water and the dynamic viscosity η ^water in the drop of water 12 are much greater than their equivalents in the air 22 around the drop. The brewing movement, symbolized by the azimuthal component of the velocity, u _φ , always remains tangential to the surface of the liquid inclusion and thus does not generate its displacement or its interfacial deformation.

According to (1), the tangential electrical stress at the interface is written: $${\mathrm{\tau }}_{\mathrm{r\phi }}\mathrm{=}{\mathrm{\epsilon }}^{\mathrm{water}}{\mathrm{E}}_{\mathrm{r}}{\mathrm{E}}_{\mathrm{\phi }}\mathrm{,}$$

This constraint is the motor of the stirring in the internal and external fluids to the drop or the liquid inclusion; it is proportional to the product of the two main components of the electric field at the interface in the vicinity of the nip: the normal and tangential components, E _r and E _φ respectively. Therefore, for an electric field E = E _r n + E _φ t ₁ available between the electrodes 4, 6, the mixing or centrifugation engine will be maximized if there is equality between the two components involved: E _r = E _φ = E / √2. It is therefore preferable to choose an angle close to 45 ° between the border drawn by the inter-electrode space 14, 16 and the tangent t ₁ to the circular contact line (or the projection on the plane of the electrodes of this line of contact). contact).

According to one embodiment of the electrodes, the latter are separated from each other by an electrically insulating contour 16 in the form of a zig-zag: the segments are alternated at approximately 45 ° for a drop of water, as illustrated. in Figures 1B, 2 or 3 .

The periodicity (spatial) of the alternation, λ, can be optimized: we prefer to take: $$\mathrm{R}\mathrm{/}\mathrm{10}\mathrm{<}\mathrm{\lambda }\mathrm{<}\mathrm{R}\mathrm{,}$$

Or $$\mathrm{R}=\mathrm{ray\; of\; gout}$$

Typically, R may vary between, for example, 0.1 mm and 10 mm.

λ can therefore be between, for example, 0.01 mm and 1 mm.

More generally, as indicated in Figure 1B or α the angle formed between the normal to the triple line 20 (contained in the so-called wetting plane), or its projection on the plane of the electrodes, and the edges 14, 16 of the electrodes. The absolute value of α is strictly between 0 ° and 90 °. An optimum configuration corresponds to an angle close to 45 °.

As described below, this angle constraint is compatible with electrode edges having shapes such as, for example, zig-zag, or spiral.

où le symbole a est un facteur homothétique.An envelope calculation makes it possible to take into account the angular stress α and leads to boundaries 14, 16 of electrodes in the form of a logarithmic spiral (or equiangular spiral). The median line separating the electrodes in their plane, or in the plane of the EHD chip, is described in polar coordinates by: $$\mathrm{\rho }\mathrm{=}\mathrm{at}\mathrm{.}\exp \left(\frac{\mathrm{\theta }}{\mathrm{tan\alpha }}\right)$$

where the symbol a is a homothetic factor.

On the Figure 1B is represented a point M of polar coordinates ρ and θ in a plane parallel to the plane defined by the electrodes 4, 6.

In the case of a drop of water surrounded by air (or vacuum) and placed on an EHD chip optimized in this way, it can be shown that the optimal angle α is close to ± 45 ° ( Figures 2, 3 ).

In the particular case where the number of electrodes is even, the drop is arranged astride the electrodes. Locally, that is to say for two adjacent electrodes it is disposed on both sides of a direction Δ around which the electrode edges (zig-zag or spiral) oscillate, or which represents an average position electrode edges (see the direction Δ on the Figures 1B, 2 , 7 , but also the directions Δ and Δ 'in figure 3 ).

• le nombre de G est accru,
• le déséquilibre statique de l'échantillon liquide sous l'effet de l'électromouillage peut être inhibé dès lors que la période de rotation du champ électrique est beaucoup plus petite que l'échelle de temps associée à la déformation interfaciale engendrée par électromouillage.

A possible instability of the static position of the liquid inclusion 12 can be countered by means of a sufficiently fast rotating electric field (at more than 100 Hz), obtained by the successive activations and deactivations of the electrodes 4, 6 with which the sample interacts: indeed, the liquid sample is then subjected to a motive electrical stress which sweeps its periphery (the successive applications of a stress of electrical origin in the inter-electrode spaces, distributed along the triple line, can be modeled by a mobile constraint which sweeps the interface near the triple line). If, therefore, the activation and deactivation speeds are sufficiently fast, ie if the contactors used to apply a rotating field are capable of operating at high frequency (> 100 Hz), two advantages appear:

• the number of G is increased,
• the static imbalance of the liquid sample under the effect of electrowetting can be inhibited since the period of rotation of the electric field is much smaller than the time scale associated with the interfacial deformation generated by electrowetting.

• les inclusions liquides 12, objet du brassage ou de la centrifugation, ont un volume non constant (diamètres évoluant de 100µm à 10mm),
• la goutte 12 se rétracte, ou croît, sous l'effet d'un changement de phase (transfert de masse interfacial : évaporation / liquéfaction),
• après centrifugation, il peut être utile de prélever une fraction volumique de l'échantillon liquide pour purifier celui-ci (extraction d'un culot ou d'un surnageant), pour extraire des constituants chimique ou bien des analytes...etc. Dans ce cas, il y a rétraction de la goutte après extraction.

The invention can be used for a stable volume 12, but also in the following different situations:

• the liquid inclusions 12, object of the stirring or the centrifugation, have a non-constant volume (diameters evolving from 100 μm to 10 mm),
• the drop 12 shrinks, or increases, under the effect of a phase change (interfacial mass transfer: evaporation / liquefaction),
• after centrifugation, it may be useful to take a volume fraction of the liquid sample to purify it (extraction of a pellet or a supernatant), to extract constituents chemical or analytes ... etc. In this case, there is retraction of the drop after extraction.

The invention therefore remains effective if the volume of the liquid sample 12 is random or if it changes over time under the effect of one or more extractions or under the effect of evaporation, for example.

The invention allows easy integration within a lab-on-a-chip or a micro-system based on the displacement of liquid inclusions. Extraction techniques are proposed in the invention, which can for example implement electrowetting droplet displacement means, EWOD type, such as for example described in WO 2006/005880 or in the article of MG Pollack et al. "Electrowetting based actuation of droplets for integrated microfluidics", Lab Chip, 2002, vol.2, p. 96-101 .

It is possible to evaluate the number of G which the invention makes it possible to obtain as a centrifuge. According to the expression of the electrical driving stress (2), an order of magnitude typical of the speed field is written, for a drop of water in air: $${\mathrm{u}}_{\mathrm{\phi }}\mathrm{~}\frac{{\mathrm{\epsilon }}^{\mathrm{water}}{\mathrm{<figure-callout\; id="2"\; label="E"\; filenames="imgf0001.png,imgf0005.png"\; state="\{\{state\}\}">E</figure-callout>}}^{\mathrm{2}}}{\mathrm{2}{\mathrm{\eta }}^{\mathrm{water}}}\mathrm{\delta }\mathrm{.}$$

If δ denotes the thickness of the fluid on which the amount of movement induced by the electrical stress is dissipated, we have: $$\mathrm{\delta }\mathrm{~}\frac{\mathrm{2}{\mathrm{\eta }}^{\mathrm{water}}{\mathrm{u}}_{\mathrm{\phi }}}{{\mathrm{\epsilon }}^{\mathrm{water}}{\mathrm{<figure-callout\; id="2"\; label="E"\; filenames="imgf0001.png,imgf0005.png"\; state="\{\{state\}\}">E</figure-callout>}}^{\mathrm{2}}}\mathrm{.}$$

où p désigne la distance comprise entre l'axe médian de l'espace inter-électrodes et tout point de la surface de la goutte.An inter-electrode space e equal to 20 μm can be considered. In experiments carried out under a microscope, the potential difference between two electrodes 4, 6 is typically set at 70V. If the surface of the liquid inclusion is sufficiently far from the inter-electrode space (thickness of the coating 8, 10 very large in front of e), the electric field lines emitted by two closely spaced electrodes adopt an axisymmetric geometry, and: $$\mathrm{E}\left(\mathrm{\rho }\right)\mathrm{=}\frac{\mathrm{V}}{\mathrm{\pi \rho }}\mathrm{,}$$

where p denotes the distance between the median axis of the inter-electrode space and any point on the surface of the drop.

Let us consider the example of a millimetric drop of water (R = 1 mm) characterized by a dynamic viscosity η ^water equal to 10 ^-3 Pa and a relative dielectric permittivity of 78.5 (vacuum permittivity: 8.85 pF). Between the line of contact (ρ = 0.1mm) and the apex of the drop (ρ = 1mm), the electric field is divided by a factor of 10.

During visualizations conducted using a CCD camera, a spun or residual trace effect of the particles, corresponding to a complete rotation of the balls, corresponds to a closing time of the order of t ≈ 0.01s. Therefore, for the millimeter drop involved in the experiments, the order of magnitude of the velocity field is experimentally evaluated at: $${\mathrm{u}}_{\mathrm{\phi }}\mathrm{~}\frac{\mathrm{2}\mathrm{\pi R}}{\stackrel{\mathrm{~}}{\mathrm{t}}}\mathrm{\approx }\mathrm{0.6}\mathrm{m}\mathrm{/}\mathrm{s}$$

Finally, according to (5) and (6), the typical scale of length on which the induced momentum diffuses under the effect of the viscosity (or thickness of skin set in motion) varies between δ = 0.35 mm in the vicinity of the nip and δ = 3.5 mm at the apex of the drop.

expression déjà définie ci-dessus) engendré avec deux électrodes peut varier entre 1 pour un gel visqueux et 100 pour de l'eau. C'est le cas notamment pour un échantillon liquide qui a une permittivité diélectrique relative équivalente à celle de l'eau (élevée).Number of $\mathrm{BOY\; WUT}\mathrm{(}\mathrm{=}\frac{{{\mathrm{u}}_{\mathrm{\phi }}}^{\mathrm{2}}}{\mathrm{R}}\mathrm{/}\mathrm{boy\; Wut}\mathrm{,}$

expression already defined above) generated with two electrodes can vary between 1 for a viscous gel and 100 for water. This is particularly the case for a liquid sample that has a relative dielectric permittivity equivalent to that of water (high).

Several parameters allow the control of the nature and the intensity of the fluid movement. It can thus achieve several applications, from mixing to centrifugation.

A first control parameter is the number of electrodes.

With two electrodes 4, 6 vis-à-vis (as on the Figure 1B or 2 ), two sources of motive electric stresses are available and oppose in their effects as to the direction of the induced momentum. Two co-rotating recirculations can thus be born, as illustrated on the figure 4 , described later.

With four electrodes, for similar physical reasons, four recirculations are formed ( figure 5 ).

The number of electrodes can be increased in order to produce a cascade of recirculations and thus to control a mixture that is all the more rapid and efficient, especially if it is a question of mixing chemical or biochemical reagents. The increase in the number of electrodes leads to an increase in the number of inter-electrode spaces and therefore in the number of zones in which an oblique field occurs, motor of the mixing in the drop.

In this case, the net balance in terms of contribution of momentum is increasing. This is particularly the case for the 8-electrode chip of the figure 11 .

A second control parameter is the angle between the nip and the boundaries of the electrodes.

Whether the number of electrodes is even or odd, when the objective is centrifugation, the question arises of how to produce a single rotating flow. For this, a first possibility ( figure 11 ) is based on the controlled cancellation of the azimuth component of the electric field , E _φ , so that locally, the driving stress τ _rφ = ε ^water E _r E _φ vanishes (contact line locally orthogonal to the imposed electric field, t ₁ ⊥ E ). If the angle between the boundary of the electrodes and the normal to the nip is alternately equal to 90 ° and 45 ° (this is the case if we go through the circle 70 of the figure 11 in one direction or the other; this would also be the case figure 10 ), then the only non-zero electrical stresses act in the same direction ( figures 10 , 11 ). By modifying the angle α, the driving stress τ _rφ defined by (2) is modified, and thus also the centrifugation intensity.

A second possibility is based on another control parameter, inter-electrode spacing. To obtain a net non-zero balance of all the electrical motor constraints imposed around the drop on its surface one can impose, once on two, a wider inter-electrode spacing, typically a factor of 10, than the previous or the next, as described later, in connection with the figure 9 .

According to the equations above, the driving stress evolves as the square of the imposed electric field which itself is proportional to the imposed potential difference and inversely proportional to the distance e between the electrodes buried under the insulation film, and inversely proportional to the thickness of the dielectric and hydrophobic films 8, 10.

On the Figures 2 to 5 the electrode boundaries are represented, in plan view, in the form of zig-zag, at 45 ° (see in particular the figure 2 and the triple line 20 ") with the tangent to the triple line 20 of the drop.

On the Figures 2 and 3 the dashed circles 20, 20 ', 20 "represent the triple line 20 which delimits the wetting area between the liquid sample and the surface of the EHD chip, and illustrate the possible variability of the liquid sample volumes 12, various instants t, t + dt, t + n.dt (n> 1) The electric potentials (-) and (+), applied to the various electrodes, are distinguished by their opposite signs.The symbol λ represents the periodicity of the segmentation, each segment being inclined at ± 45 ° (drop of water under air).

The figure 2 is an example of an EHD chip according to the invention, with two electrodes 4, 6 with segmented boundaries, and the figure 3 is an example of an EHD chip according to the invention, with four electrodes 4, 6, 24, 26 with segmented boundaries.

On the Figures 4 and 5 the circle (thick line) delimits the contact line 20 of the liquid sample 12. The symbols E, E _t and q _ε respectively designate the electric field in the inter-electrode space, the component of this field tangential to the triple line, and the electrical charge accumulated on the surface of the fluid sample under the effect of the normal jump of the electric field and the electrical characteristics (conductivity, dielectric permittivity).

The figure 4 is an example of an EHD chip according to the invention, with two electrodes 4, 6 with segmented boundaries. Two co-rotating vortices 13, 16 (dashed) are potentially generated.

On the figure 5 an EHD chip according to the invention has four electrodes 4, 6, 24, 26 with segmented boundaries. Four co-rotating vortices (dashed) are potentially generated.

The figure 6 represents a drop of water 12 placed on an EHD chip 2 according to the invention, with two electrodes segmented at ± 45 ° (structure of the figure 2 ). Hollow microbeads of effective density, ρ = 0 .3, are used as tracers in the interface. In the center of the two vortices, we actually find the presence of two packets 23, 25 of micro-beads agglomerated by centripetal effect ( figure 4 ).

As illustrated by this experiment, it is more generally possible to isolate beads, functionalized or not, at the heart of the vortex on the surface of a drop of water subjected to a stirring according to the invention. The proposed invention can thus be applied to the preparation of biological or medical samples, to the isolation of analytes for analysis or to microfluidic concentration purification. in the heart or on the periphery of one or more vortices if it is a more evolved brewing.

Isolated constituents can also be extracted within a vortex in view of their subsequent elimination, biochemical characterization or detection.

In the context of the extraction of constituents (extractants) from a liquid phase donor to a liquid or gaseous receiving phase, the proposed invention can accelerate the interfacial transfer of extractants by producing a mixture in the phase donor fluid if it takes the form of a drop.

On the Figures 7 and 8 are represented chips according to the invention, respectively two or four electrodes 4, 6, 24, 26 optimized to take into account the volume variability of the liquid samples: the internal boundaries 30, 30 ', 32, 32' of the electrodes are logarithmic spirals. The line 20 of contact (dashed) is circular. The electric potentials (-) (+) are distinguished by their opposite signs: to two adjacent electrodes are applied opposite signs (except for an odd number of electrodes, for centrifugation, but this except for the rotating field).

• prendre en compte la variabilité volumique des échantillons liquides : les frontières internes 30, 30', 32, ,32', 34, 34', 36, 36' des électrodes sont des spirales logarithmiques,
• et forcer la présence d'un seul vortex dans l'objectif d'une centrifugation.

The EHD chip of the figure 9 has eight electrodes optimized for:

• take into account the volume variability of the liquid samples: the internal boundaries 30, 30 ', 32, 32', 34, 34 ', 36, 36' of the electrodes are logarithmic spirals,
• and force the presence of a single vortex for the purpose of centrifugation.

The thicker spirals 30 ', 32', 34 ', 36' signal an electrode boundary separation gap wider than the spirals 30, 32, 34, 36. The nip 20 (dashed) is circular. The electric potentials (-) and (+) are distinguished by the opposite signs of two neighboring electrodes. The electrodes delimited by the electrode boundaries are alternately a positive potential and a negative potential.

In general, the alternation of wider inter-electrode zones and less wide inter-electrode zones makes it possible to significantly reduce, in the wider areas, the level of electrical stresses that otherwise would oppose electrical stresses. motor generated by the smaller inter-electrode areas.

• prendre en compte la variabilité volumique des échantillons liquides : les frontières internes des électrodes sont alternativement des segments droits et des spirales logarithmiques,
• et forcer la présence d'un seul vortex dans l'objectif d'une centrifugation.

In figures 10 and 11 , the chip EHD has respectively four electrodes 4, 6, 24, 26 and 8 electrodes 4, 6, 24, 26, 44, 46, 64, 66 optimized for:

• take into account the volume variability of the liquid samples: the internal boundaries of the electrodes are alternately straight segments and logarithmic spirals,
• and force the presence of a single vortex for the purpose of centrifugation.

The electric potentials (-) and (+) are distinguished by their opposite signs. The circle more. thick suggests cutting the electrodes to stabilize the contact line in a fixed position.

Indeed, each electrode, brought to a certain potential, may itself be cut locally in a circular contour (segmented electrode). This cutting makes it possible to create an artificial roughness facilitating the attachment of the line of contact of the drop.

On the other hand, the portion of the electrode outside the contact line 20 can be deactivated, which can also stabilize the triple line by non-wetting.

On the figure 11 , the spirals are, unlike the figure 10 , extended towards the center, which manifests itself in a reverse direction of centrifugation for the smallest liquid inclusions. The inversion boundary is symbolized by the dotted circle 70.

The structures described above with the figures 10 and 11 allow a trapping of the triple line, because of the circular cutting of the electrodes. Alternatively, one can also provide circular roughness or micrometric studs implanted vertically around the triple line. This technique of the pads is moreover applicable to structures other than those of figures 10 and 11 , in particular to all other device structures according to the invention explained in the present application.

Another interesting variant consists in stabilizing the position of the liquid sample by means of a difference in localized wettability at the level of the triple line. For this purpose, it is necessary to allow the zone outside the triple line to be hydrophobic (either by nature or by coating a hydrophobic film) while the inner zone is hydrophilic, either by nature or by EWOD activation, or by deposition of a hydrophilic film.

The Figures 16A and 16B represent pads 80, for example of resin. They are preferably positioned furthest from the inter-electrode spaces, or in the inter-electrode spaces for which the component Et is to be suppressed; these are the larger inter - electrode spaces than their neighbors or the inter - electrode spaces that are locally orthogonal to the triple line.

The pads 80 are made for example by photolithography of a thick resin layer (for example of thickness between 10 microns and 100 microns).

In the case of figure 16A , the pads 80 make it possible to automatically center the drop in the center of the spiral.

In the case of figure 16B , they allow to automatically center the drop in the center of the spiral, and each is placed astride two electrodes where locally the electrohydodynamic stress is removed.

A trapping of the triple line makes it possible to ensure the balance of the contact line 20 and to avoid any effect that may disturb the cohesion of the liquid sample 12 to be analyzed or treated. It also makes it possible to reinforce the stability of the static position of the drop 12.

A chip according to the invention can be made with known technologies, for example as described in the document Fouillet et al., 2006, already cited in the introduction to this request or in the document WO 2006/005880 or FR 2 841 063 .

In embodiments using more than two electrodes, the drop is centered on the intersection of the internal edges of the electrodes ("O" point on the electrodes). figures 3 , 5 , 8-11 . In the case of two electrodes with logarithmic spiral edges ( figure 7 ), the drop is centered on the intersection O of the two spirals.

Rather than considering a liquid inclusion placed on a single chip, it is possible to consider a liquid inclusion sandwiched between two chips linked to two superposed horizontal walls. As in the case where we increase the number of electrodes, we will double the actuation capabilities. However, the interfacial electrical stresses only induce the momentum on a fluid thickness of a few millimeters. The viscous friction increases proportionally to the inverse of the distance separating the two horizontal walls.

The invention can be applied to extract analytes concentrated at the apex of a liquid inclusion 12 under the effect of centrifugal or centripetal forces.

The Figures 12a-12c represent a three-stage extraction with two superposed horizontal walls: the lower horizontal wall is equipped with an EHD chip 2 according to the invention (according to one of the embodiments described in the present application) and the upper horizontal wall is equipped an electrode 200, which is optionally an EHD chip according to the invention.

1. i) étape de centrifugation sur la paroi horizontale inférieure équipée de la puce EHD 2 (figure 12a), par activation de cette puce, et désactivation de l'électrode de la paroi supérieure. Il en résulte une centrifugation dans l'inclusion liquide 12 déposée sur la puce, avec création des vortex 13, 15 ; Cette première étape permet de favoriser la concentration de constituants à l'apex (surnageant) ou au fond, sur le pourtour de l'échantillon liquide (culot), selon qu'ils sont sensibles aux forces centripètes ou centrifuges, respectivement.
2. ii) il y a ensuite désactivation électrique sur la paroi inférieure 2, pendant un laps de temps conduisant à la formation d'un pont capillaire 110 avec la paroi supérieure équipée d'une électrode 200 qui est désactivée (figure 12b) ; il y a alors démouillage relatif au niveau de la paroi inférieure 2 ;
3. iii) l'étape précédente est suivie d'une réactivation électrique de la puce EHD 2 et de l'électrode supérieure 200 (figure 12c) pour la mise en oeuvre de l'électromouillage et l'extraction spécifique d'un surnageant 123 (dans la goutte supérieure 122) et d'un culot (goutte inférieure 120). On coupe le pont capillaire 110 (technique décrite dans A. Klingner et al., Self Excited Oscillatory dynamics of capillary bridges in Electric Fields, Applied physics Letters, Vol.82, 2003, p. 4187-4189 ) en deux inclusions indépendantes, chacune étant rattachées aux parois inférieure et supérieure. Deux situations peuvent alors se présenter: si les constituants 123 sont moins denses que le liquide de l'échantillon, l'inclusion supérieure contient le surnageant à analyser (cas de la figure 12c) ; et si les constituants 123 sont plus denses que la liquide de l'échantillon, c'est l'inclusion inférieure qui contient le culot à analyser.

The implementation steps are then as follows:

1. i) centrifugation step on the lower horizontal wall equipped with the EHD chip 2 ( figure 12a ), by activation of this chip, and deactivation of the electrode of the upper wall. This results in centrifugation in the liquid inclusion 12 deposited on the chip, with the creation of vortices 13, 15; This first step makes it possible to promote the concentration of constituents at the apex (supernatant) or at the bottom, around the periphery of the liquid sample (pellet), depending on whether they are sensitive to centripetal or centrifugal forces, respectively.
2. ii) there is then electrical deactivation on the bottom wall 2, for a period of time leading to the formation of a capillary bridge 110 with the upper wall equipped with an electrode 200 which is deactivated ( figure 12b ); there is then relative dewetting at the level of the bottom wall 2;
3. iii) the preceding step is followed by electrical reactivation of the EHD chip 2 and the upper electrode 200 ( figure 12c ) for the implementation of the electrowetting and the specific extraction of a supernatant 123 (in the upper drop 122) and a pellet (lower drop 120). The capillary bridge 110 is cut (technique described in A. Klingner et al., Self Excited Oscillatory Dynamics of Capillary Bridges in Electric Fields, Applied Physics Letters, Vol.82, 2003, p. 4187-4189 ) in two independent inclusions, each being attached to the lower and upper walls. Two situations can then arise: if the constituents 123 are less dense than the sample liquid, the upper inclusion contains the supernatant to be analyzed (case of figure 12c ); and if the constituents 123 are denser than the sample liquid, it is the lower inclusion which contains the pellet to be analyzed.

The formation of a cone at the apex of a liquid inclusion under the effect of the convergence of electric field lines is known from the following documents: Taylor, GI, 1964, Disintegration of water drops in an electric field, Proc. R. Soc. A, 280, pp. 383-397 ; Ramos, A. & Castellanos, A., 1994, Conical points in liquid-liquid interfaces to electric fields, Phys. Letters A, 184, pp. 268-272 ; Ganan-Calvo, A., 1997, Cone-jet analytical extension of Taylor's electrostatic solution and the asymptotic universal scaling laws in electrospraying, Phys. Rev. Letters, 79, 2, pp. 217-220 . The emergence of a Taylor cone may also be useful for extracting isolated analytes at the apex of a liquid sample after brewing or centrifugation according to the invention. In this case, the liquid sample is placed on an EHD chip as proposed in the invention. At a sufficiently close distance, close to the associated capillary length, a counter-shaped electrode is located in the opposing wall, as explained in the articles cited above in this paragraph.

The operation can take place in three stages.

The first step is to centrifuge the liquid sample to cause the microfluidic concentration of target constituents.

The second step is to modify this action for a short time while carrying all the electrodes of the lower chip at the same potential while the upper electrode in the form of tip is brought to a very different potential.

• soit un pont capillaire se forme avec la paroi supérieure et dans ce cas, la déstabilisation du pont capillaire peut être facilitée en activant une zone plus large d'électrodes au niveau de la paroi supérieure ; on est donc ramené à la technique précédente,
• soit il y a éjection d'une ou de plusieurs gouttes (électro-spray, comme expliqué dans les articles de Taylor, Ramos et Ganan-Calvo cités ci-dessus). Auquel cas, ou bien les constituants sédimentent et se retrouvent concentrés sous la forme d'un culot dans la goutte inférieure résiduelle, ou bien ils surnagent et sont alors contenus dans la ou les gouttes éjectées par le cône de Taylor. Si ces gouttes ne coalescent pas tout de suite (elles possèdent une charge électrique semblable), leur fusion peut être facilitée ultérieurement par électromouillage le long de la paroi supérieure.

Due to the lengthening of the liquid sample and the subsequent formation of a Taylor cone under the influence of electric field lines, two scenarios can arise:

• either a capillary bridge is formed with the upper wall and in this case the destabilization of the capillary bridge can be facilitated by activating a wider area of electrodes at the upper wall; we are thus brought back to the previous technique,
• either ejection of one or more drops (electro-spray, as explained in the articles of Taylor, Ramos and Ganan-Calvo cited above). In which case, or the constituents sediment and are concentrated in the form of a pellet in the residual bottom drop, or they supernatant and are then contained in the droplet or drops ejected by the Taylor cone. If these drops do not coalesce immediately (they have a similar electrical charge), their fusion can be facilitated later by electrowetting along the top wall.

The figure 13 represents a micro-pump using for example a four-electrode EHD chip (as for example on the figure 10 ; but another number of electrodes is possible).

A fluid inlet 72 makes it possible to introduce a secondary fluid 12 'into a cavity or a reactor 74 containing an EHD device according to the invention, here with 4 electrodes. Liquid inclusion primary 12 undergoes a treatment as already described above, without overall displacement. The surface forces in motion cause the secondary fluid 12 'by viscosity as described above, in accordance with the invention.

A micro-pump according to the invention can be applied to a cooling process in microelectronics (for processors), or the dispensing of small quantities of drugs (pharmacology, galenic), or the micro-propulsion of objects (in exploration Space).

Thanks to the physical mechanism used in the invention (the electro-hydrodynamics), the speed range allowing mixing is considerably wider compared to conventional micropumps. The invention makes it possible in particular to reach a speed of at least 0.1 m / s or 1 m / s.

If we denote by (p) and (s) the primary 12 and secondary 12 'fluids, the relation (1) must be completed and written explicitly: $${\left[{\mathrm{E}}_{\mathrm{\phi }}\right]}_{\mathrm{i}}\left({\mathrm{\epsilon }}^{\mathrm{p}}{\left[{\mathrm{E}}_{\mathrm{r}}\right]}_{\mathrm{p}\mathrm{,}\mathrm{i}}\mathrm{-}{\mathrm{\epsilon }}^{\mathrm{s}}{\left[{\mathrm{E}}_{\mathrm{r}}\right]}_{\mathrm{s}\mathrm{,}\mathrm{i}}\right)\mathrm{=}{\mathrm{\eta }}^{\mathrm{p}}{\left[\mathrm{r}\frac{\mathrm{\partial }}{\mathrm{\partial }\mathrm{r}}\left(\frac{{\mathrm{u}}_{\mathrm{\phi }}}{\mathrm{r}}\right)\right]}_{\mathrm{p}\mathrm{.}\mathrm{i}}\mathrm{-}{\mathrm{\eta }}^{\mathrm{s}}{\left[\mathrm{r}\frac{\mathrm{\partial }}{\mathrm{\partial }\mathrm{r}}\left(\frac{{\mathrm{u}}_{\mathrm{\phi }}}{\mathrm{r}}\right)\right]}_{\mathrm{s}\mathrm{.}\mathrm{i}}\mathrm{,}$$

The index i indicates that the quantity is evaluated at the interface, on the side of the primary fluid (p) or the secondary fluid (s). The drive of the secondary fluid is therefore more effective than its viscosity is low and yet higher than that of the primary fluid (η ^p <η ^s ).

It is also possible, from a first drop 12, to cause stirring or centrifugation in another drop by viscous drive even if the latter has a dielectric permittivity or electrical conductivity similar to those of the continuous liquid phase constitutive of the external medium. In particular, it is possible to create a micro-gear with a continuous liquid phase and at least two drops. In such a micro-gear, the ratio of reduction or amplification is programmable by adjusting the viscosity or diameter ratios between continuous liquid phase and drops.

On the figure 14 there is shown a micro-fluidic gear involving for example two EHD chips 200, 202, preferably optimized (for example of the four-electrode type: figure 10 ), with their respective liquid inclusions 12, 112, one of characteristics: diameter d1 and viscosity μl and the other of characteristics: diameter d3 and viscosity μ3. More EHD chips and liquid embedding can be implemented. A secondary liquid phase 212, viscosity μ2, circulates between the primary liquid inclusions 12, 112 through the movements of the latter, one in the direction of clockwise, the other in the opposite direction.

This technique, implementing the joint use of a continuous liquid phase 212 based on several liquid samples 12, 112 each activated by a chip 2, 202 similar to those proposed in the invention, leads to an increase in intensity. brewing or centrifugation within the liquid samples. The flow is more intense outside and inside the drops.

Similarly, it is possible to induce a movement of a primary fluid phase (p) to a tertiary fluid phase (t) via a viscous secondary phase (s). In which case, the tertiary fluid phase can be mixed or centrifuged, even if its dielectric permittivity does not allow the emergence of electrical motor stresses at the interface which surrounds it ( figure 14 ).

The primary phase is for example a liquid sample placed on a chip according to the present invention. Surrounded by a secondary liquid, a movement of electrical origin is generated at the p / s interface which propagates within the secondary liquid via the viscosity.

• Soit il est impossible d'y engendrer des contraintes électriques motrices et auquel cas le brassage interne créé au sein de l'inclusion tertiaire est d'origine purement visqueuse ; (7) se simplifie sous la forme, $${\mathrm{\eta }}^{\mathrm{p}}{\left[\mathrm{r}\frac{\mathrm{\partial }}{\mathrm{\partial }\mathrm{r}}\left(\frac{{\mathrm{u}}_{\mathrm{\varphi }}}{\mathrm{r}}\right)\right]}_{\mathrm{p}\mathrm{,}\mathrm{i}}\mathrm{=}{\mathrm{\eta }}^{\mathrm{s}}{\left[\mathrm{r}\frac{\mathrm{\partial }}{\mathrm{\partial }\mathrm{r}}\left(\frac{{\mathrm{u}}_{\mathrm{\varphi }}}{\mathrm{r}}\right)\right]}_{\mathrm{s}\mathrm{,}\mathrm{i}}\mathrm{.}$$
  
• Soit il est possible d'engendrer à l'aide de contraintes électriques motrices un brassage interne à l'intérieur de l'inclusion liquide tertiaire ; auquel cas celle-ci est posée sur une puce EHD, et le brassage interne est engendré non seulement par l'intermédiaire des contraintes électriques motrices mais aussi par entraînement visqueux à l'interface, du fait que l'écoulement du fluide secondaire est également dû au rôle moteur de l'inclusion liquide primaire.

Therefore, at the s / t interface, there are two cases:

• Either it is impossible to generate motor electrical stresses and in this case the internal stirring created within the tertiary inclusion is of purely viscous origin; (7) simplifies itself in the form, $${\mathrm{\eta }}^{\mathrm{p}}{\left[\mathrm{r}\frac{\mathrm{\partial }}{\mathrm{\partial }\mathrm{r}}\left(\frac{{\mathrm{u}}_{\mathrm{\phi }}}{\mathrm{r}}\right)\right]}_{\mathrm{p}\mathrm{,}\mathrm{i}}\mathrm{=}{\mathrm{\eta }}^{\mathrm{s}}{\left[\mathrm{r}\frac{\mathrm{\partial }}{\mathrm{\partial }\mathrm{r}}\left(\frac{{\mathrm{u}}_{\mathrm{\phi }}}{\mathrm{r}}\right)\right]}_{\mathrm{s}\mathrm{,}\mathrm{i}}\mathrm{.}$$
  
• Either it is possible to generate by means of electric motor constraints an internal stirring inside the tertiary liquid inclusion; in which case it is placed on an EHD chip, and the internal stirring is generated not only by means of the electrical drive constraints but also by viscous drive at the interface, because the flow of the fluid secondary is also due to the driving role of the primary liquid inclusion.

A device of the micro-gear type according to the invention may comprise a series of inclusions, each resting on an EHD chip and connected to one another via the secondary liquid: in this case, such a micro-fluidic micro-gear amplifying the internal flows. and external to the inclusions is close to an amplification system. The secondary fluid and the fluid of each of the drops or inclusions have different dielectric permittivities and / or different electrical conductivities.

Applied sequentially, this embodiment achieves a large number of G within one of the liquid inclusions participating in the chain ( figure 14 ). The viscosity ratios of the fluids, the diameter ratios of the various inclusions involved, the number and the level of the electrical motor stresses applied to the various interfaces are all parameters that contribute to the overall amplification of the flows and that can be adjusted to optimize the flow. system.

• soit d'une ou plusieurs gouttes posée sur une ou plusieurs puces (figures 13 ou 14),
• soit d'un pont capillaire piégé entre deux puces (figure 12a-12c),
• La présente invention peut donc être utilisée pour mettre en mouvement un fluide secondaire dans le contexte de la micro-fluidique continue. Une micro-pompe selon l'invention peut comporter une seule inclusion de liquide noyée dans un fluide secondaire (figure 13), ou bien plusieurs inclusions liquides noyées dans un fluide secondaire (figure 14). Ce dernier peut être mis en mouvement par un mécanisme d'engrenage qui peut être qualifié d'engrenage micro-fluidique à frottement visqueux interfacial.

The present invention therefore makes it possible to generate a volume movement within a sufficiently viscous liquid sample via an electrical stress (or constraints) exerted on its surface. If the liquid sample 12 is surrounded by another liquid 22, also viscous, the amount of movement induced by the surface electrical stress diffuse not only in the liquid internal to the liquid sample 12 but also in the external fluid 22. It is therefore possible to drive a secondary fluid in motion by means of a primary fluid adopting the shape:

• either one or more drops placed on one or more chips ( Figures 13 or 14 )
• a capillary bridge trapped between two chips ( figure 12a-12c )
• The present invention can therefore be used to set in motion a secondary fluid in the context of continuous microfluidics. A micro-pump according to the invention may comprise a single inclusion of liquid embedded in a secondary fluid ( figure 13 ), or several liquid inclusions embedded in a secondary fluid ( figure 14 ). The latter may be set in motion by a gear mechanism which may be referred to as an interfacial viscous friction micro-fluidic gear.

• centrifugation ou concentration micro-fluidique,
• fragmentation ou arrachage local d'une partie de l'inclusion liquide pour sélectionner puis manipuler ou éliminer des constituants concentrées localement à l'issue de l'étape précédente (par exemple, un surnageant concentré par effet centripète à l'apex d'une inclusion liquide).

Another embodiment of a method according to the invention comprises the steps of:

• centrifugation or microfluidic concentration,
• local fragmentation or tearing of part of the liquid inclusion to select and then manipulate or remove constituents concentrated locally at the end of the previous step (for example, a centripetal-centrally concentrated supernatant at the apex of an inclusion liquid).

A particular embodiment of this method is illustrated in the Figures 15A-15D . In these figures, the surrounding medium 22 consists of a second liquid, for example a second drop, immiscible with the first, containing particles 23. These particles 23 will gradually settle on the interface 12-22 ( figure 15C ). The setting in motion of this interface, according to the invention, thus with the aid of electrodes having the characteristics already described above, without displacement of the drop 12, causes a displacement of the particles 23 along the interface 12-22 and their grouping on the edges of the drop 12.

Finally ( figure 15D ) the lateral parts, containing the particles 23, are separated from the central part of the drop 22, for example by electrowetting cutoff, one or more of the electrodes located between the side portion (s) and the central electrodes being deactivated.

On the Figures 15A-15D , the two drops are represented between on the one hand a substrate 3 on which is formed a device according to the invention and on the other hand a 3 'confinement substrate.

Microscale rheological instrumentation is an area of application of the invention. Micro-rheometers based on electrokinetics are currently in the development phase ( Juang, Yi-I, 2006, Electrokinetics-based Micro Four-Roll Mill, http://www.chbmeng.chio-state.edu/facultypages/leeresearch/154RollMill.htm ). The proposed invention, which is based on electrodynamics, makes it possible, for example, to generate four or two vortices within a liquid or gelled sample in order to obtain a purely elongational or purely sheared flow. Measurements of viscoelastic parameters can therefore be made with the invention using speed measurements made for example by video acquisition.

A device according to the invention can be included in new micro-systems or on-chip laboratories, for the purpose of preparing biological samples before further analysis steps.

Applications of the invention to this biological field will be described.

Most known techniques for detecting biological targets have an important defect: all of them previously require purification and more generally preliminary preparation of the biological samples to be analyzed.

With regard to the detection of pathogenic viruses by extraction of DNA segments, the technique that refers to is PCR; this consists of a process of amplification of the DNA strands present in a liquid sample. PCR is commonly developed in micro-systems ( Kopp-MU; de Mello-AJ; Manz-A, 1998, Chemical amplification: Continuous-flow PCR on a chip, Science, 280, 5366, pp.1046-1048 ; Zhan-Z; Dafu-C; Zhongyao-Y; Li-W. Biochip for PCR amplification in silicon, 2000, 1st Annual International IEEE-EMBS Special Topic Conference on Microtechnology in Medicine and Biology. Proceedings (Cat No.00EX451). IEEE, Piscataway, NJ, USA, pp. 25-28 ). After a relatively large number of these thermal cycles, the DNA concentration is sufficient to allow detection. Among the disadvantages of PCR are i) the duration associated with the amplification process, ii) the background noise related to the fact that the polymerase can amplify nonspecific DNA segments present in the liquid sample represents the second major disadvantage of PCR, and especially, iii) as for most detection techniques, PCR requires the preparation or purification of biological samples.

The ELISA is another technique, widespread detection, immunoanalysis type or viral load determination by nucleic acid assay, intended to detect and / or assay an antigen present in a fluid biological sample. The ELISA test, practiced in homogeneous or heterogeneous phase, has the advantage of being fast and inexpensive. But again, the biological samples must first be subject to a minimum purification step.

Among the techniques aimed at developing an alternative to PCR, detection without amplification is a sensitive technique while allowing the reduction of the detection time. The principle of non-amplification detection relies on the capture of target DNA segments, however few they may be.

A first technique consists in hybridizing the target DNA segments with functionalized paramagnetic nanobeads responsible for vectorizing these segments to a functionalized solid interface for detection purposes. This concentration process can be based on a magnetic process, the target DNAs are eluted (by increasing the temperature above 50 ° C.) and hybridize on the functionalized solid surface before the detection phase ( Marrazza, G., Chianella, I. and Mascini, M., 1999, Disposable DNA Electrochemical Sensor for Hybridization Detection, Biosensors & Bioelectronics, 14, 1, pp. 43-51 ; Lenigk, R, Carles, M, Ip, NY & Sucher, NJ., 2001, Surface Characterization of a silicon-chip-based DNA microarray, Langmuir, 17, 8, pp. 2497-2501 ). The concentration of the beads can also be accelerated by thermal Marangoni effect on the surface of a drop (Ginot, F., Achard, J.L., Drazek, L. & Pham, P., September 12, 2001, Method and device for isolation / or determination of an analyte; patent application FR 01 11883 ). These methods, however, face the problem of nonspecific adsorption of certain magnetic beads to solid walls. The sensitivity reached is no longer the expected one.

The present invention makes it possible to accelerate the hybridization kinetics while being compatible with a miniaturization constraint. It also makes it possible to concentrate by centrifugation the functionalized beads for a more sensitive detection. It is then applied as explained in the document FR 01 11883 .

Another possibility is to hybridize strands of target DNA at a liquid / gas or liquid / liquid interface functionalized by probes ( Picard, C. & Davoust, L., 2005, Optical investigation of a wavy age interface, Colloids & Surfaces A: Physichem. Eng. Aspects, 270-271, pp. 176-181 ; Picard, C. & Davoust, L., 2006, Dilational rheology of an air-water interface functionalized by biomolecules: the role of surface diffusion, Rheologica Acta, 45, pp. 1435-1528 ) then to use, if necessary, a microfluidic concentration process to increase the local densification of the hybridized target / probe complexes, and thus allow a more sensitive local detection (Berthier, J. & Davoust, L., 2003, Method of concentrating macromolecules or agglomerates of molecules or particles; patent application WO 2003/080209 ). Micro-mechanical type detection based on the modification of the rheological properties of the fluid interface during the hybridization process is also possible (Picard & Davoust, 2005, cited above). This technique, like the previous ones, comes up against a difficulty of micro integration within a lab on chip as well as the prior need to prepare the biological sample.

The present invention can be applied in two stages: it can be used to purify / prepare a liquid biological sample and then be used one last time by allowing a microfluidic type concentration.

By allowing centrifugation within a liquid sample 12 ( Figure 1A ), the invention allows selective localization of {receptor-bound analyte} complexes in order to further increase the detection performance.

An application of the invention therefore is especially the microfluidic concentration by mixing or centrifugation for facilitated detection of antibodies, antigens, protein or protein complexes, DNA or RNA. In this case the fluids used are based on aqueous solutions. The ambient environment may be air or a pure oil. The detection can be carried out directly in situ at the level of the concentration zone or be the subject of a subsequent step after extraction by selective tearing of said concentration zone.

The invention also makes it possible to improve the performance of PCR or PMCA for the detection of DNA or proteins. After the microfluidic concentration step, using a device according to the invention and according to the centrifugation method according to the present invention, applied either to target DNA segments adsorbed directly to the functionalized interface From the liquid inclusion (a drop of aqueous solution) to functionalized microbeads, it is possible to specifically collect the concentration zone by electrowetting or by emission of droplets from a Taylor cone, as already explained here. -above.

It is also possible to overcome the PCR and achieve an ultrasensitive detection by repeatedly applying the EHD centrifugation according to the present invention to liquid inclusions successively extracted. Indeed, an EHD chip according to the invention can be optimized in order to take into account a variability of sample volumes (for example by a logarithmic spiral-shaped electrode chip, as illustrated in FIGS. Figures 7-11 ).

A microemulsion can also be achieved by promoting the coalescence of two electrowetting displacement inclusions and then producing a mixture using the present invention. PCR can then be performed directly on the emulsion thus obtained. The emulsion may also make it possible to eliminate certain unnecessary components by adsorption at the interfaces for biological purification.

Another example of application is the following. Two immiscible liquid inclusions can fuse with each other by the electrowetting technique, as described in the Y.Fouillet document already mentioned above. The invention then makes it possible to generate a two-phase mixture such as a foam or an emulsion (micro-foam, microemulsion), in order to facilitate sequencing, or the purification of biomolecules or even the extraction of colloids by capture at liquid / gas (foam) or liquid / liquid (emulsion) interfaces.

Claims (14)

1. A processing device of a drop (12) of liquid, deposited on the device and fixed relatively to the latter, comprising:

   - at least two first electrodes (4, 6, 24, 26) forming a plane and having edges (14, 16) facing each other, such that the contact line (20) of the drop (12), has a tangent forming, when projected onto the plane of the electrodes, an angle strictly comprised between 0° and 90° with the edges facing each other of the electrodes, characterised by:

   - means (11) allowing application between the two first electrodes (4, 6, 24, 26) of a potential difference which gives rise to an oblique electric field, relatively to the liquid/surrounding interface, in way to create vortex (13, 15) in the drop (12) of liquid;

   - means (30', 32', 34', 36', 80) to trap the contact line (20) defined by the drop deposited on the device.
2. The device according to claim 1, the angle being comprised between 40° and 50°.
3. The device according to claim 1 or 2, the edges of the electrodes facing each other having a zigzag shape or a logarithmic spiral shape.
4. The device according to any of claims 1 to 3, the edges of the electrodes forming with the projection of the contact line, an angle strictly comprised between 0° and 90°, alternating with electrode edges forming an angle of 90° with the projection of this same circular contact line (20) and/or the separation spaces of the edges (14, 16) of the electrodes facing each other having alternately a first value and a second value that is less than the first.
5. A pumping device including at least one device according to any of claims 1 to 4, and means for bringing a second fluid (12') into contact with a drop (12) of liquid positioned on the device.
6. The device according to any of claims 1 to 5, further including an insulating layer (10).
7. A method for processing a drop (12) of liquid in a surrounding medium (22), having relatively to each other different dielectric properties and/or different resistivities, including:

   - positioning the drop on a device including at least two first electrodes (4, 6, 24, 26) having edges (14, 16) facing each other, so that the projection of the contact line (20) of the drop (12) on the plane containing the electrodes has a tangent forming with these electrode edges an angle strictly comprised between 0° and 90°,

   - applying an oblique electric field relatively to the liquid/surrounding medium interface, in way to create a circulating flow (13, 15) inside the drop (12) of liquid, a triple line (20), being a contact line between the drop of liquid, the surface device on which the drop of liquid is positioned and the surrounding medium, being fixed.
8. The method according to claim 7, the electric field applied between the two first electrodes (4, 6, 24, 26) generated a single circulating flow or a single vortex in the drop (12).
9. A microfluidic concentration method by mixing or centrifuging a drop of liquid, notably for detecting antibodies, or antigens, or proteins or protein complexes, or DNAs or RNAs, including the application of a method for forming at least one circulating flow or vortex (13, 15) in said drop (12) of liquid, in accordance with a method according to any of claims 7 to 8.
10. The method according to claim 9, a detection step being carried out, after mixing or centrifuging, without displacement of the drop, including a step for extracting liquid from the drop, for example by carried out by electrowetting or by emitting droplets from a Taylor cone, and including eventually a step for transferring extracted liquid towards a detection area.
11. A method for forming a microemulsion including:

    - bringing two volumes of liquids closer by displacing them relative to each other, for example by electorwetting,

    - a step for applying a method according to any of claims 7 to 10.
12. A method for pumping a secondary fluid (12') by a drop of primary fluid (12), including the application of a method for forming at least one circulating flow or vortex (13, 15) in said drop (12) of primary fluid, in accordance with a method according to any of claims 7 to 11.
13. A method for extracting an analyte from a drop of liquid including:

    - the application of microfluidic concentration method according to claim 9,

    - a deactivation of the at least two first electrodes, and the formation of a capillary bridge (110) between the first insulating surface (10) and a wall including at least a second electrode (200),

    - the electric activation of the first electrodes and of the second electrode (200), and the cutting of the capillary bridge.
14. A method for extracting particles including the application of a method according to any of claims 7 to 13, the surrounding medium consisting of a second liquid containing particles (23) which have settled beforehand on the interface of the two liquids, and then the separation of the side portions, containing the particles (23), and of a central portion of the drop (22), for example by cutting by means of electrowetting.

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