FR2887983A1 - Microfluid preparation device comprising a distributor to distribute liquid in droplets to first concentration, a droplet displacer with electro wetting comprising a substrate with hydrophobic surface, and a droplet evaporator - Google Patents

Abstract

A microfluidic preparation device comprising a hydrophobic surface with 2 electrical conductors, is new. One of the electrical conductors is disposed on a substrate. The 2 electrical conductors are connected to a power supply arrangement for the application of electrostatic forces in liquid droplets. The electrical conductors comprise sequentially arranged electrodes, which are placed on a non-electric conductor face of the substrate, which is disposed in a direction of evaporation path. The microfluid preparation device has hydrophobic surface with two electrical conductors. One of the electrical conductors are disposed on a substrate. The two electrical conductors are connected to power supply arrangement for the application of electrostatic forces in liquid droplets. The electrical conductors comprising sequentially arranged electrodes, which are placed on a non-electric conductor face of the substrate, which is disposed in a direction of evaporation path. The microfluid is obtained from a first volume of liquid comprising a substance with first concentration, of a second volume of liquid. The second volume of liquid comprises the substance with a second concentration. The distributor comprises a hood (701) comprising an orifice (705) for liquid supply placed at right of the electrode (701.1). The electro wetting comprises three electrodes (701.1, 701.2,701.3) of which one electrode (701.3) is for dimensioning and another electrode is used for cutting (701.2). The device further comprises a receiver to collect droplets at outlet, where the collected droplets forms the second volume of liquid at the second concentration. The receiver is used to analyze the second volume of liquid. Blocks contains electrodes, where the size of the electrodes decreases from distributor to receiver or distributor to evaporator. The evaporator imposing a small volume of liquid has to and fro movement on the block so that the size of the liquid volume is greater than a value. The electrical conductors are in a shape of rafter oriented in the droplets displacement direction to form centring, where the centring are formed by star-shaped electrodes. The evaporator comprises: a controller to control the displacement speed of small volume of liquid by successive power supply of electrodes for creating an electrostatic field; a heater, which is formed by catenery electrical conductors (6, 22); an element to apply air fluid current, with less rate of humidity and/or creates a low-pressure zone around small volume of liquid; and a detector for detecting electric current circulation between the two electrical conductors and the contact between the small volume of liquid and the electrical conductors. Independent claims are included for: (1) a microfluid preparation unit; (2) an electronic chip; and (3) a method of preparing the liquid.

Description

MICROFLUIDIC DEVICE FOR PREPARING AT LEAST ONE

  SAMPLE AND METHOD OF PREPARATION IMPLEMENTING THE SAME

ONE SUCH DEVICE

  DESCRIPTION TECHNICAL FIELD AND PRIOR ART

  The invention relates mainly to a device for preparing at least one sample using electrostatic forces and to a preparation process using such a device.

  The invention particularly relates to a discrete microfluidic handling device, or microfluidic drop, for chemical or biological applications.

  One of the most used modes of displacement or manipulation is based on the principle of electrowetting on a dielectric, as described in the article by MG Pollack, AD Shendorov, RB Fair, entitled Electro-wetting-based actuation of droplets for integrated microfluidics, Lab Chip 2 (1) (2002) 96-101.

  The forces used for displacement are electrostatic forces.

  The document FR 2 841 063 describes a device implementing a catenary opposite electrodes activated for displacement.

  The principle of this type of displacement is as follows.

  A drop is based on an electrode array, from which it is isolated by a dielectric layer and a hydrophobic layer.

  When an electrode in proximity to the drop is activated, the dielectric layer and the hydrophobic layer, between this activated electrode and the electrode-polarized drop, act as a capacitor. The effects of electrostatic charge induce the displacement of the drop on this electrode. The electrode may be a catenary, it then maintains an electrical contact with the drop during its movement, as described in document FR - 2 841 063.

  The drop can thus be displaced step by step, on the hydrophobic surface, by successive activation of the electrodes of the electrode matrix and by guiding along the catenary.

  It is therefore possible to move liquids, but also to mix them (by bringing drops of different liquids near), and to perform complex protocols.

  The documents cited above give examples of implementations of adjacent electrode series for handling a drop in a plane.

  To enable the development of such devices or microsystems for applications such as diagnostics, agri-food monitoring or environmental monitoring, it is important to be able to make samples adapted to this type of device. The difficulty lies in collecting samples on a macroscopic scale, while the microsystem operates on a microscopic scale. Indeed, the samples collected for treatment are generally of the order of several milliliters, however the microsystem can not handle such a large volume. In addition, the substances to be analyzed are generally very dilute. Therefore, microscopically sized samples, typically less than one milliliter, are required, and on the other hand, the concentration is high enough to allow detection by current analysis means.

  An evaporationbased microfluidic sample concentration method, GM Walker, DJ Beebe, Lab Chip, 2002, 2, 57-61, is known from an evaporative concentration device, comprising a channel provided at one end with a reservoir of liquid. to concentrate and a collection opening through which is evaporation, disposed at a second end This device does not allow to treat a sufficient volume, and secondly the time required to concentrate a sample of 32% to 93% is 28 minutes, which is too long for a practical application.

  It is also known from Electrowetting-based microfluidics for analysis of peptides and proteins by Matrix-assisted laser desorption / ionization mass spectrometry, A.R. Wheeler, et al., Anal. Chem. 2004, 76, 4833-4838, a device electrically moving a small volume of liquid, so as to position it on a given electrode, evaporating it completely in vacuo and analyzing by mass spectrometry the dry residue obtained. During evaporation, there is appearance of coffee round on the substrate due to non-specific adsorption of the targets on the substrate. (Pattern formation in drying drops, R. Deegan, Physical Review E, 61, 1, 475-485). This device does not make it possible to obtain concentrated samples in liquid form.

  In addition, it has been found that the partial evaporation of drops of immobile liquid causes deposits on the substrate. In the case where the drop is on a substrate having a hydrophobic surface and a path of movement by electrowetting, the surface becomes non-hydrophobic, thus preventing the displacement of partially evaporated liquid drops and their recovery for further processing.

  It is also important to be able to control the size of the samples to be processed.

  It is known from the document Capacitive sensing of droplets for microfluidic devices based on thermocapillary actuation, J.Z. Chen, et al., LabChip, 2004, 4, 473-480, a device with a capacitive detector capable of determining the position of a small volume of liquid and its size, however this device is not able to modify the size of the small volume of liquid.

  It is therefore an object of the present invention to provide a preparation device capable of being associated with a microsystem of analysis and making it possible to prepare at least one sample of given concentration and of a given volume adapted to the scale of the microsystem. from a sample of large volume and low concentration corresponds to a collection on a macroscopic scale.

  It is also an object of the present invention to provide a preparation device that avoids the risks of depositing substances on the substrate, in particular the non-specific adsorption of targets on the substrate, causing target losses.

  It is also an object of the present invention to provide a rapid sample preparation device capable of being processed by a microsystem.

  It is also an object of the present invention to provide a device for acting on the size of small volumes of liquid, in order to calibrate them.

  DISCLOSURE OF THE INVENTION The previously stated goals are achieved by a device capable of preparing at least one sample for microsystems, said device achieving a controlled evaporation by electrowetting displacement of small volumes of liquid formed from a large volume sample. , making it possible to obtain a liquid sample of determined substance concentration and of determined size, this size being adapted to microsystems.

  The displacement of the small volumes of liquid has the advantage of increasing the rate of evaporation by renewal of the air layer surrounding the drop, and limits the substance deposits on the hydrophobic surface, to recover the drop and not to loss of material, which would make the sample obtained after evaporation not representative of the macroscopic sample.

  The device according to the present invention also has the advantage of having electrical connections that are better controllable and more easily replaceable, in place of the fluidic connections.

  The main subject of the present invention is a device for preparing, from a first volume of liquid comprising at least one substance at a first concentration of a second volume of liquid comprising said substance at a second concentration, comprising means for dispensing the first volume of liquid in drops of liquid at the first concentration, electrowetting means for moving said drops comprising a first hydrophobic surface substrate provided with at least first electrically conductive means defining a path, at least second electrically conductive means arranged opposite the substrate, the first and second electrically conductive means being connectable to electrical supply means to allow the application of electrostatic forces to the drops of liquid and means for controlling, at least to promote evaporation of said drops.

  The first electrically conductive means may comprise electrodes on a nonelectrically conductive surface of the substrate, arranged in a direction of the evaporation path.

  Preferably, the means for distributing the first volume of liquid form drops of a determined size.

  In an exemplary embodiment, the means for distributing the first volume of liquid comprise a cover disposed at a given height of a hydrophobic substrate and electrowetting displacement means comprising at least three electrodes, including a sizing electrode and a cutting, the cover having a liquid supply port placed in line with the first electrode.

  The device according to the present invention advantageously comprises a means for collecting drops, at the exit of the path, collected drops forming the second volume of liquid at the second concentration.

  The collection means may comprise means for analyzing the second volume of liquid. Advantageously, the first electrically conductive means are formed by a succession of electrodes placed end to end, the size of which decreases from the distribution means, forming the means to promote evaporation. This particular configuration makes it possible to control the drop size by controlling their displacement using electrode sizes. Indeed, an electrode can only move a drop having a maximum size determined. So if the drop is too big, it can not be set in motion by the electrode. Therefore, it is only when the drop has sufficiently evaporated and it has reached a sufficiently small volume that it can be moved by the electrode.

  In an alternative embodiment, the succession of electrodes comprises identical electrode blocks, the successive blocks being composed of decreasing electrodes of the means of distribution to the collecting means.

  The means for promoting evaporation can then force a small volume of liquid at least one movement back and forth on a block as long as the size of the small volume of liquid is greater than a value allowing its displacement by a next block.

  The decrease in the size of the electrodes advantageously follows the law R.sub.ref L.sub.ref - R being the radius of the wetting perimeter, - L being the length of the triple line, - Ref being a value of the radius of the wetting perimeter in a configuration of reference, and - Lref being the length of the triple line in a reference configuration.

  Advantageously, the device according to the present invention comprises means for centering the small volumes of liquid relative to the electrically conductive means.

  In a first embodiment, the electrodes of the first electrically conductive means may have substantially a chevron shape oriented in the direction of movement of the drop so as to form the centering means In a second variant, the centering means are formed by star-shaped electrodes.

  The means for promoting evaporation may also comprise means for controlling the speed of displacement of the small volumes of liquid, by a successive electrical supply of the electrodes, so as to create an electrostatic field moving along the path.

  The means for promoting evaporation advantageously comprise heating means.

  These heating means are, for example formed by the second electrically conductive means and third electrically conductive means. The second and third conductive means are in the form of catenary.

  The means for promoting evaporation may also comprise means capable of applying a current of a pneumatic fluid, for example air, with low humidity and / or means for creating a zone of low pressure around the small volumes of liquid.

  In another embodiment, the means for promoting evaporation comprise means for detecting the flow of an electric current between two electrically conductive means and thus detecting the contact between a small volume of liquid and the electrically conductive means.

  The electrically conductive means are formed, for example by the second and third conductive means.

  The present invention also relates to a preparation assembly comprising at least two preparation devices according to the present invention arranged in series, the devices being connected by the drop collecting means in communication with the dispensing means.

  The present invention also relates to a preparation assembly, comprising at least two devices according to the present invention, able to operate simultaneously, said devices being fed by the same tank. These devices can be arranged in parallel.

  This set may include a unique collection means for the various paths.

  This collection means can be placed in communication with the distribution means a following preparation device.

  The present invention also relates to an electronic chip, comprising a substrate provided with at least one device according to the present invention and / or an assembly according to the present invention.

  According to the present invention, the preparation of a sample of given concentration and given volume from a macroscopic sample comprises the steps of forming small volumes of liquid from the macroscopic sample and moving these small volumes. by electrowetting.

  This preparation process according to the present invention has the advantage of increasing the evaporation rate of the small volumes of liquid and consequently of increasing the speed of preparation of the samples of given concentration and volume adapted to a microsystem. In addition, it avoids or at least advantageously reduces the risk of deposit formation on the surface of the device, and therefore loss of substance, as found in Pattern formation in drying drops, RD Deegan Physical Review E, 61, 1, 475-485.

  The subject of the present invention is also a process for preparing, from a first volume of liquid comprising at least one substance at a first determined concentration, at least one sample in liquid form at a second volume comprising said substance at a first concentration. second determined concentration, said method using a device according to the present invention or an assembly the present invention, comprising the steps of: - distribution of the first volume in the form of drops on moving means by electrowetting, - moving the drops of liquid by applying a potential difference between the first and second conductive means to reach the second volume of liquid having the second concentration.

  The type of displacement of the small volume of liquid can be varied depending on the size of the small volume of liquid during its movement.

  In addition, simultaneously with the movement of the small volume, it can be provided a heating of the drop and / or a low pressure drop and / or a pneumatic fluid flow around the drop.

  The method according to the present invention may also comprise a step of discharging heavier particles contained in a drop.

  By way of example, the first volume may be between 1 ml and several milliliters and the second volume may be between 100 nl and 5 μl.

DESCRIPTION OF THE DRAWINGS

  The present invention will be better understood from the following description and the appended drawings, in which: FIG. 1 is a diagrammatic representation in plan view of a first embodiment of a device according to FIG. present invention; - Figure 1A is a schematic representation in partial cross section of the device of Figure 1; - Figure 1B is a schematic representation of an alternative embodiment of a device of Figure 1; - Figure 1C is a schematic representation of the drop distribution means of determined sizes; - Figure 2 is a schematic representation of a device according to an alternative embodiment; FIGS. 3A to 3D are diagrammatic representations of a first example of a second embodiment of a device according to the present invention for calibrating a drop of liquid during the various stages of evaporation; FIGS. 4A to 4C are diagrammatic representations of a second example of the second embodiment of a device according to the present invention for calibrating a drop of liquid during the various evaporation stages; FIGS. 4d and 4e are exemplary embodiments of a chip comprising devices according to the present invention; FIGS. 5A and 5B are diagrammatic representations of a third example of the second embodiment of a device according to the present invention for calibrating a drop of liquid during the various calibration steps; FIG. 6 is a schematic representation of another embodiment of a device according to the present invention; - Figure 7 is a top view of a detail of the device according to the present invention.

  DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS In FIGS. 1 and 1A, a device for microfluidic preparation of at least one sample according to the present invention can be seen.

  A device, or microfluidic component, according to the invention comprises a lower substrate 2, provided with an independent matrix of electrodes 4.

  Each of these electrodes 4 is electrically connected to a conductor 3, and can be electrically powered independently of each other, by a not shown addressing means.

  The electrodes 4 are covered with an insulating layer 5 and a hydrophobic layer 7, forming a displacement surface 26.

  The hydrophobic character of this layer means that a drop has a contact angle on this layer greater than 90.

  A single layer can combine these two functions, for example a parylene layer.

  This device also comprises a first catenary 6, forming a counterelectrode of the electrowetting electrodes 4, connected to an electric source 11 (FIG. 1B), allowing electrowetting, that is to say the modification of the wetting angle of the drop deposited on the substrates by applying an electric field between the catenary 6 and the electrode matrix 4. The catenary, the electrode matrix and the insulating layer 54 form a capacitor.

  In the example shown, the counter electrode is formed by a suspended catenary, but a buried counter electrode, that is to say formed in the substrate may also be suitable.

  A small volume of liquid B is represented on the surface of the hydrophobic layer 7.

  In FIG. 1, a path 8 formed by the set of electrodes 4 and catenary 6 can be seen. The device allows, by successive supply of electrodes 4, a displacement of a small volume of liquid B on path 8, the speed of movement of the small volume B being adjustable according to the supply sequence of the electrodes 4.

  The path 8 comprises in the example shown an inlet 10 and an outlet 12. The inlet 10 is connected to a dispensing means 14 small volumes of liquid comprising a reservoir 16 and forming means 18 of small volumes, arranged in outlet of the tank 16.

  The forming means 18 comprise means for moving a calibrated volume of liquid, formed for example by an electrode path as described above. The liquid dispensed at the outlet of the tank 16 is carried by the electrode path 8 in the form of a drop of determined volume.

  These means 18 (FIG. 1C) for forming calibrated drops comprise, in the example shown, a cover 701 disposed at a determined height above a substrate 2, delimiting a confined space, the substrate being provided with a hydrophobic surface and a path of electrowetting electrodes. This path comprises, for example three aligned electrodes 703.1 to 703.3. It could also include more than three. The counterelectrode is not shown.

  The cover has a feed port 705 disposed above an end of the electrode path, allowing a supply of liquid from for example a tank.

  The cover is positioned at a precise distance from the substrate, for example by means of a spacer 707 formed for example of photoresist deposited by spinning, by deposition of photosensitive film or a polymer.

  These means 18 operate in the following manner.

  The electrodes 701.1 to 703.3 are activated, the liquid contained in the reservoir is then drawn to form a liquid finger on the three electrodes 701.1 to 701.3. Then the central electrode 701.2, said cutoff electrode is deactivated by cutting off the power supply. Due to the hydrophobic nature of the surface of the substrate, the finger is cut in two, a drop of calibrated size is formed on the third electrode 701.3, called the sizing electrode. since it determines the size of the drop.

  The size of the drops formed depends on the distance between the cap and the substrate and the size of the sizing electrode 701.3. For example, for a distance between the substrate and the cover of 100 μm, and 800 μm square electrodes of side the drop formed has a volume of 64 nl.

  These means make it possible to manufacture drops of definite size reproducible with a CV <3, the CV being the coefficient of variation which corresponds to the standard deviation of the distribution expressed as a percentage of the distribution average.

  Other means of the state of the art may be suitable for producing the formation of calibrated drops, for example a pump or a capillary.

  The path 8 extends, in the example shown, zigzag to optimize the size of the device. But a path in a straight line or spiral, for example, is not beyond the scope of the present invention.

  In the example shown, the electrodes 4 are of a single size, for moving small volumes of liquid of different volume, for example between 0.5 pl and 2 pl.

  The device also comprises at the output 12 of the path 8, a means 20 for collecting small displaced volumes. The means 20 are, for example, a single reservoir 21 as shown, to gather all the small volumes in a large volume, a capillary or a means of analysis of each drop collected.

  When moving the small volume on the path 8, there is an evaporation phenomenon which increases with an increase in the speed of movement v of the small volume. Indeed, the displacement of the drop allows a renewal of the air layer near the drop, and a withdrawal of the water vapor contained in the air. The speeds of movement are of the order of a few mm / s to a few cm / s.

  On the other hand, evaporation is more efficient when working on a large volume divided into smaller volumes, than by working directly on the entire large volume, since the total evaporation surface is increased.

  In addition, the displacement of the drop of liquid during its movement avoids or at least reduces the deposits of substance on the substrate that can damage the device. Indeed, these deposits may make the substrate partially hydrophilic.

  In addition, these deposits cause losses of substances and therefore loss of information between the starting macroscopic volume and the arrival volume for treatment by a microsystem.

  According to the present invention, it is intended to control the size of the dispensed drops, it is thus possible to obtain a sample having a volume V2 less than a volume V1 of concentration Cl of the starting sample and having a determined concentration C2 in substances, C2 being higher Cl concentration in volume V1.

  The present invention advantageously makes it possible to quickly switch from a large so-called macroscopic volume to a small, so-called microscopic volume.

  For example, a macroscopic volume may be between 1 ml and several milliliters and a microscopic volume may be between 100 nl to 5 pl. These values are not limiting and larger or smaller volumes can be processed by the device according to the present invention.

  The volume V2 at the concentration C2 is adapted to be processed by a microsystem.

  The device according to the present invention makes it possible to significantly accelerate the evaporation of the liquid. It has, for example, been found, under similar conditions elsewhere, an evaporation of a drop of liquid up to 30% faster, when it moves, the evaporation of a drop still.

  The device shown in FIG. 1 makes it possible to make a single evaporation cycle. If a new evaporation cycle is desired, the reservoir 16 is emptied, then the liquid is transferred from the collection means 20 to the reservoir 16 for a new evaporation cycle.

  A device could be provided allowing successive evaporation cycles continuously until the desired concentration is obtained.

  For this, the device would include a path of electrodes forming a closed loop, on which we would inject drops to concentrate, these drops would move on the loop until reaching a given concentration and a given volume. The number of turns could be calibrated according to the initial concentration of the drop.

  It is also possible to envisage using a common tank 16-21 in which the liquid is distributed in the form of a drop, then partially evaporated, then collected in the tank 18-21, and then again divided in the form of a drop, until the desired concentration.

  One can also consider having separate tanks 16 and 21, in communication through a valve. The collected liquid from the collection tank 21 is transferred to the distribution tank 16, for example by means of a pump or by electrowetting. These alternative embodiments have the advantage of allowing space savings since we are still working on the same evaporation path.

  It is also possible to accelerate the evaporation further by associating with the device previously described, for example a heating means, for example of the double-catenary type.

  In FIG. 1B, one can see such a heating means comprising the first catenary 6 and a second catenary 22 forming a pair of electrodes with the first catenary 6.

  The first catenary 6 is located opposite the electrodes 4, or the portion of the hydrophobic surface 7 situated above the electrodes 4.

  When a voltage is applied to each of the catenaries 6 and 22, generating a non-zero potential difference between these two catenaries, a heating of this drop is induced. Thus simultaneously with the movements of the drop, it is heated, accelerating its evaporation.

  In the example shown, the catenary 22 is permanently powered. So we have a permanent heating. But we can provide a controlled switch to feed the second catenary only for specific periods, allowing a displacement of the drop without heating.

  It is also possible to heat the drop without moving it.

  We then obtain a very great control of evaporation.

  One of the two catenaries 6,22 is bifunctional and can be used for a displacement on the hydrophobic surface 7 and / or for a temperature rise of the drop.

  It is also possible to bury one or both catenaries in the substrate, for example under the hydrophobic layer. The buried electrode or electrodes can be flat, instead of being catenaries.

  It is also possible to perform the evaporation in a low pressure environment or to provide air renewal means to reduce the moisture of the pneumatic gas, for example air surrounding the drop.

  The present invention also makes it possible to obtain samples having a very precise concentration, when it is intended to collect partially evaporated drops in a single large volume. Indeed, the displacement of the drops on the same path one after the other, may allow a next drop to collect on the displacement surface a particle possibly lost by a previous drop, as reported in Integrated chemical / biochemical sample collection, preconcentration and analysis on a digital microfluidic labon-chip platform, RB Fair, et al., SPIE Optics East, Philadelphia, Oct. 25-28,2004.

  It is also possible to obtain heating of small volumes of liquid by resistive or inductive heating means or a Peltier effect module, these devices being known to those skilled in the art.

  We will now explain the operation of the sample preparation device according to the present invention.

  A volume of liquid V1 to be analyzed, containing in diluted form at least one substance at a concentration C1, for which a sample of volume V2 having a given concentration C2 is to be analyzed, V2 being less than V1, is placed in the reservoir 16. C2 being greater than cl.

  The means 18 for dispensing drops, produce and bring by electrowetting at the entrance of the path 8, drops of liquid of determined size.

  The drops, by activation of the electrodes 4 according to determined sequences, are displaced on the path at a speed set according to the concentration of the desired substance or the volume of the expected drop at the outlet. The drops are then partially evaporated, this is shown schematically by the arrow 24.

  The drops may also be heated simultaneously as previously described.

  At the outlet, the partially evaporated drops are collected and form the liquid volume V2 at the concentration C2 for further processing, for example an analysis.

  In Fig. 2, a preferred exemplary embodiment of a sample preparation assembly having preparation devices according to the present invention can be seen. This set makes it possible to simultaneously evaporate several drops of liquid, which makes it possible to further increase the rate of evaporation.

  Indeed, as previously described, the fact of working on small volumes makes it possible to increase the speed of evaporation, since the total evaporation surface is greater than for a large drop, this rate of evaporation of the volume sample V1 is all the more increased as the evaporation takes place in parallel.

  The assembly according to the present invention advantageously comprises a plurality of evaporation paths 108 and means 114 allowing a multiple distribution of drops, making it possible to supply simultaneously all the paths 108. But it would be possible to provide distribution means dedicated to each path. .

  The dispensing means 114 are for example in the form of a liquid stream 129 having small volume distribution orifices 130, each placed at an inlet 110 of a path 108. These means can also be formed in a manner similar to the means 18 shown in Figure 1C, or by a capillary or a pump.

  The device also comprises a means 120 for collecting small volumes partially evaporated in a single volume.

  This collection means 120, in turn, provides a means for dispensing a small volume of liquid on a path 108 '. Another collection means (not shown) is provided at the exit of the path 108 'to allow the subsequent treatment of the drop.

  This preparation set makes it possible to prepare a sample quickly by working simultaneously on several evaporation paths.

  In the example shown, the set comprises three paths 108, but it may include as many as necessary, depending on the volume of liquid to be treated and / or treatment time to be respected. It is also possible to provide more than one step of simultaneous evaporation, if the concentration to be obtained must be very high compared to the initial concentration or if the reduction of the desired volume is important.

  In the example shown, the paths 108, 108 'are rectilinear and aligned but can be provided in any other form, and arranged differently.

  The additional means for accelerating evaporation, such as heating, evaporation under vacuum and the air flow are also applicable to this embodiment.

  Preferably, the electrodes are of decreasing size in the main direction of movement, that is to say means for dispensing drops to the collection means. This particular geometry of the electrode path makes it possible, as will be explained in the remainder of the description, to control the displacement of the liquid drop according to its size, in fact an electrode of given size can not move a drop of size. given maximum.

  We will now explain the operation of such a set.

  The volume of liquid C1 at concentration C1 moves in the vein 129 in the direction of the arrow 140, and form the drops B1, B2, B3 at an inlet of each evaporation path 108.

  The drops B1 to B3 move simultaneously on the paths 108, by applying an activation control of the electrodes composing the paths in the direction of the arrow 142.

  The drops B1 to B3 of reduced size arrive at the exit of the paths 108, they are then collected by the means 120, to form a volume V1 'of concentration Cl'.

  The means 120 then forms drops B 'from V1' the entrance of the path 108.

  The drops B 'then move according to the arrow 146.

  At the end of the path 146, the partially evaporated drops B 'are collected and form a volume V2 of concentration C2.

  For example, to evaporate a drop of 2 p3, 1300s are necessary against to evaporate a drop of 20 pl, 4800 seconds, it appears that the invention allows to evaporate ten drops of liquid 2 pl simultaneously instead of one drop of 20 pl is very advantageous.

  In order to be able to further control the evaporation of the liquid drops, it is possible to perform a volume control or the dimensions of a drop. Thus it will be possible to modify the evaporation procedure, if for example the measured volume is greater than that expected. For this purpose, capacitive or resistive measurement may be carried out, or optical detection or any other technique known to those skilled in the art may be carried out.

  In an electrowetting device, the surface of a conductive drop in contact with the substrate and the buried electrode forms a capacitance; the contact is made between the drop and the catenary. The equivalent capacity between the buried electrode and the drop is a function of the volume of the drop, which depends on the surface covered by the drop on the substrate. This capacity therefore varies with a change in volume of the drop placed above the electrode. Thus it is possible from this measure of ability to determine the size of the drop.

  One can also consider determining this size by optical detection, observing laterally the drop with a mirror, the image thus formed to determine the size of the drop.

  The device according to the present invention also advantageously comprises means for controlling the displacement of the drops according to their size.

  In FIGS. 3A to 3D, we can see a first example of a second embodiment of the device according to the present invention making it possible to further increase the control of evaporation.

  The device, represented in FIGS. 3A to 3D, comprises an electrode path 208 (partially shown) formed of a succession of electrodes 204 of decreasing size between an input and an output (not shown).

  An electrode 204 of determined size may cause the displacement of a liquid droplet of size between a lower limit and an upper limit determined by the size of the electrode and the nature of the liquid component of the drop, any drop larger than the upper limit, can not be moved by said electrode. For example, a drop of 2p1 can move on 800pm2 electrodes. This drop can not be moved by smaller electrodes, it can not be moved until it reaches a volume of 0, 5 p1.

  The size of the drop is understood to mean the volume of this drop.

  The term "electrode size" refers to the surface of the electrode on which the drop is moving. Thus, considering the path 208, a drop B of volume V1 moves on the electrodes of the path 208 in the direction of the arrow 236. by successive activations of the electrodes 204. The electrode 204.1 is activated (state I), the drop B comes on the electrode 204.1, the next electrode 204.2 is deactivated (state 0) (FIG. 3A).

  The electrode 204.2 is then activated (state I), or it is of insufficient size to allow a displacement of the volume V1. Drop B does not move (Figure 3B).

  As long as the volume of the drop B is greater than a volume V2, which represents the upper limit of a drop volume displaceable by the electrode 204.2, the drop B does not move.

  The drop B evaporates to reach the volume V2 (FIG. 3C) The drop B can then be displaced by the electrode 204.2 which is in the activated state (state I), the electrode 204.1 being in the state disabled (Figure 3C).

  The drop comes on the electrode 204.2 (FIG. 3D). The displacement of the drop on the following electrodes continues according to the diagram previously described up to the exit of the path 208. There is then an autocalibration of the size of the drop.

  The choice of successive sizes of the electrodes 204 is advantageously in accordance with a homothetic rule between the contact radius of the drop and the length of the activated triple line.

  The contact radius corresponds to the radius of the wetting surface (in the example shown in disc form) of the drop in contact with the solid substrate.

  The triple line is the line of contact common to the three parts namely the drop, the solid substrate and the surrounding air. The activated triple line is the part of the triple line located above an activated electrode, that is to say the part of the triple line on which the displacement force is exerted.

  The electrodes advantageously follow the following rule: ## EQU1 ## where L is the length of the triple line, - R ref is a value of the radius of the wetting perimeter in a reference configuration, - L ref being a value of the length of the triple line in a reference configuration, which corresponds to a configuration in which the displacement takes place? that is to say for a given liquid and a given potential, an electrode size and a drop volume.

  The shape of the electrodes is not limited to a rectangular shape but to any shape, for example square, disc or polygonal.

  In FIGS. 4A to 4C, a second example of the second embodiment having additional means of evaporation can be seen.

  As for the device of FIGS. 3A to 3D, the device of FIGS. 4A to 4C comprises an electrode path 308 partially shown, (1) whose size decreases in the direction of movement (arrow 336). However, unlike the electrodes 204, the electrodes 304 are not of strictly decreasing size.

  Indeed, the path comprises successive blocks D1, D2, D3 of substantially identical electrodes 304.1, 304, 244.3 respectively.

  In the example shown, each block has four identical electrodes, but a greater or smaller number of electrodes are not outside the scope of the present invention. In addition, a different number of electrodes can be provided for each block.

  The size of the electrodes may also advantageously follow rule (1) above.

  We will now explain the operation of this device.

  As for the previous device, the volume drop B VD1 moves in the direction of the arrow 336 on the electrode block D1, until it arrives in front of an electrode 304.2.1, the first block electrode D2 in direction 336, of size too small to be able to move the drop B. The displacement in the direction of the arrow 336 beyond the block D1 is stopped. However this time, it is advantageously provided to keep in motion the drop B by imposing a movement back and forth along the arrows 336 and 337 on the block D2.

  For this, the electrodes 304.1 are activated one after the other in a direction 337 and then in another direction 336. This continuous movement causes accelerated evaporation of the drop B. When the drop is on the electrode 304.1.4, last electrode of the block D1 in the direction 336, adjacent to the electrode 304.2.1 of the block D2, the electrode 304.2.1 is activated. If the volume of the drop is less than or equal to VD2, which is less than VD1, the drop passes on the block D2. Otherwise the drop continues back and forth until its volume is less than or equal to VD2.

  The displacement of the drop continues from block to block until the collection means.

  For example, a drop of 2p1 can quickly move back around large 800pm2 electrodes. This drop is too big to move on smaller electrodes. After a while, because of its displacement, the drop evaporated to have, for example, a volume of 0.5 p1. The drop is now small enough to move on the next pad of electrodes.

  At the same time, the forward and backward movement can be provided with heating and / or low pressure and / or air flow.

  In Fig. 7, an advantageous form of the electrode path can be seen. It is preferable that the small volumes of liquid are centered on the electrodes 4 so that the drop remains in a configuration close to the reference configuration. For this, it is possible to provide electrodes comprising at least a central portion 28 in the form of a chevron so as to center the drop on the electrode path. Thus the drop moves substantially always at the same place of each electrode.

  It is also possible to provide electrodes 4 in the form of a star, in order to refocus the drop on the electrode. Indeed, electrowetting has the effect of spreading the liquid at the activated electrodes, which is reflected here by a liquid position to maximize the surface facing the electrode. This results in a collecting effect of the liquid in the center of the electrode.

  FIG. 4d shows a top view of a chip comprising two devices according to the present invention 500, 510. It is also possible to see the electrical connections 520 of each of the electrodes of the different blocks allowing individual control thereof. .

  FIG. 4e shows another example of a chip according to the present invention, comprising devices according to the present invention 600, 610. The device 600 is similar to that shown in Figures 4A to 4C, the device 610 is similar to that shown in Figures 3A to 3B. The chip also comprises an evaporation device according to the present invention without autocalibration, formed of a path of electrodes of identical size. These devices have the advantage of not requiring a catenary for the counter electrodes.

  It should be noted that one can associate on the same chip, the devices of Figure 1 and one or more sets of Figure 2 to the devices of Figures 3 and 4.

  In Figure 6, we can see a sample preparation device according to the present invention to have samples of size even better controlled. The device of FIG. 6 comprises a means of collection and distribution 420 of drops of liquid, interposed in the evaporation path. This means 420 is disposed at one end of a plurality of first evaporation paths 408 and collects the partially evaporated drops moving in the direction 436. Then the means 420 will distribute on a plurality of second paths 408 ', drops of size calibrated.

  In the example shown, the drops are harvested at the output of the second paths for further processing. Thus, the means 420 form a buffer tank allowing an intermediate calibration of the drops, avoiding the appearance of variation on relatively long evaporation paths, since the formation by electrowetting of drops from, for example, distribution means 18 shown in FIG. 1C from a reservoir, is reproducible with a CV <2%.

  In FIGS. 5A and 5B, a device according to the present invention can be seen, using the second catenary 22 as described with reference to FIG. 1B, allowing a control of the size of the drops.

  The second catenary 22 allows a heating of a drop of liquid or small volume of liquid by Joule contact or effect. Heating by heat transfer is preferred because the flow of current in the drop is too dependent on its content, for example its salt concentration. Heating by transfer means heating by contact, the electrodes heat because of their internal resistance, transferring heat to the liquid of the drop.

  In addition, the flow of current can also denature the substances in solution, which could distort any subsequent analysis.

  However, current flow between the catenaries can advantageously make it possible to determine an order of magnitude of the size of the drop, making it possible to further control the evaporation. When a drop is present and is in contact with both catenaries, a weak current flows between the two catenaries. The detection of this current informs the presence of a drop of sufficient size to come into contact, in the example shown, with the second catenary 22. This detection makes it possible to determine an approximate size of the drop.

  In the example shown, the second catenary is disposed substantially parallel to the substrate at a distance d. The drop has a height h. When h is at least equal to d, a current flows between the catenaries 6 and 22, which makes it possible to deduce that the height h is at least greater than d. On the contrary, in the case where no current flows between catenaries 6 and 22, it is known that h is less than d.

  In FIG. 5A, at first the drop B has a height h greater than d and puts the two catenaries in electrical contact.

  After partial evaporation of the drop B, h is less than d, there is no more electrical contact between the catenaries.

  This two-catenary system has the advantage of allowing both to heat to accelerate evaporation and to allow a calibration of the drops. Indeed, it is possible to connect the detection of the current to the displacement electrodes 4. Thus, the drop is moved on the evaporation path in one direction and the other until no current is detected between the two catenaries. We will then know that the size of the drop is less than a given value.

  It is also possible to provide third, fourth catenaries arranged at increasingly smaller distances from the substrate. This plurality of catenaries may allow the use of the microfluidic device for drops of different sizes, a control of the size of the drop over the entire evaporation path by detecting a continuous decrease in the volume of the drop, or a very fine determination of the size of the drops.

  These catenaries can also be arranged in parallel, at the same height as the travel catenary but on the side and at different distances.

  It is also possible to consider second catenary arranged transversely to the first catenary in a discrete manner and at increasingly smaller distances from the substrate. The size control is then carried out in an ad hoc manner, when the drop meets a second catenary. The detection of a current can then generate a command to prolong the evaporation of the drop to reduce the volume of the drop.

  The devices according to the present invention are for example made by conventional microtechnology technologies, for example by photolithography.

  Methods for producing chips incorporating a device according to the invention can be directly derived from the processes described in document FR-2 841 063.

  The electrodes forming the first electrically conductive means may be made by deposition of a metal layer (for example a metal selected from Au, Al, ITO, Pt, Cr, Cu) by photolithography. The substrate is then covered with a dielectric layer, for example Si3N4 or SiO2. Finally a deposit of a hydrophobic layer is performed, such as a teflon deposit made by spinning.

  In the case of the embodiment with catenaries, a two-dimensional (2D) set of these catenaries can be realized above the 2D set of electrodes.

  Such a device for moving drops can implement a two-dimensional array of electrodes that will allow, step by step, to move liquids in or on a plane, to mix them, to achieve complex protocols.

  We will now describe a method for preparing a sample at a given concentration of at least one substance, using the device according to the present invention.

  An example of a preparation method according to the present invention comprises the following steps: - distribution of the volume V1 in the form of drops on electro-displacement displacement means 8, 108, 208, 308, 408, displacement of the drops of liquid by application of a potential difference between the first and second conductive means until reaching a volume of liquid V2 having a concentration C2.

  The method allows the preparation of a small volume sample by collecting a large number of small, partially evaporated volumes.

  During the second step, simultaneously with the movement of the small volume, it can provide a heating of the drop and / or low pressure of the drop and / or a flow of air flow around the drop.

  When moving the drop, it may also be provided a volume control of said drop by a current measurement according to the device of Figures 5A and 5B. If the drop has a large volume, its displacement can be modified, for example by imposing back and forth movements to increase its evaporation The device according to the present invention and the preparation process can limit the adsorption not specific targets on the substrate. In addition, it is possible to take advantage of the ratio of the densities between the drop and the suspended targets, it is thus possible to discharge in a given location of the substrate of the heavier unwanted particles.

  The device according to the present invention is particularly applicable to biochips requiring low volumes for analysis, this device also applies to the concentration for sample preparation in general, for example chemical concentrations in volatile solvents.

Claims (30)

  A preparation device, from a first volume of liquid comprising at least one substance at a first concentration of a second volume of liquid comprising said substance at a second concentration, comprising means for distributing the first volume of liquid drops of liquid at the first concentration, displacement means by electrowetting said drops comprising a first hydrophobic surface substrate provided with at least first electrically conductive means (4,204,304) defining a path, at least second electrically conductive means (6) arranged opposite the substrate, the first and second electrically conductive means (4,204,304) being connectable to electrical supply means to allow the application of electrostatic forces to the drops of liquid and means for promoting evaporation said drops.   2. Device according to claim 1, wherein the first electrically conductive means (4,204,304) comprise electrodes on a non-electrically conductive surface of the substrate, arranged in a direction of the evaporation path.   3. Device according to claim 1 or 2, wherein the means for dispensing the first volume of liquid form drops of a determined size.   4. Device according to one of claims 1 to 3, wherein the means for dispensing the first volume of liquid comprises a cap (701) disposed at a predetermined height of a hydrophobic substrate and electrowetting moving means comprising at least three electrodes (701.1,701.2,701.3), including a design electrode (701.3) and a cut-off electrode (701.2), the cover having a liquid supply port (705) located in line with the first electrode (701.1) .   5. Device according to one of claims 1 to 4, also comprising a collection means (20) drops, at the outlet (12) of the path, collected drops forming the second volume of liquid at the second concentration.   6. Device according to the preceding claim, wherein the collection means (20) comprises means for analyzing the second volume of liquid. 7. Device according to any one of the preceding claims, wherein the first (4,204,304) electrically conductive means (6) are formed by a succession of electrodes placed end to end, whose size decreases from the distribution means, forming the means to promote evaporation.   8. Device according to the preceding claim, wherein the succession of electrodes comprises blocks (D1, D2, D3) of identical electrodes, the successive blocks being composed of decreasing electrodes, distribution means by means of collection. .   9. Device according to the preceding claim, wherein the means for promoting evaporation are able to impose a small volume of liquid at least one back and forth movement on a block (D1, D2, D3) as long as the size the small volume of liquid is greater than a value allowing its displacement by a next block (D2, D3).   10. Device according to claim 8 or 9, wherein the decrease in the size of the electrodes follows the law R Réf L Lref R being the radius of the wetting perimeter, L being the length of the triple line, Ref being a value of the radius the wetting perimeter in a reference configuration, and - Lref being the length of the triple line in a reference configuration.   11. Device according to any one of the preceding claims, comprising means for centering small volumes of liquid relative to the electrically conductive means (4,6,22).   12. Device according to the preceding claim in combination with claim 2, wherein the electrodes of the first electrically conductive means have substantially a chevron shape oriented in the direction of movement of the drop so as to form the centering means 13. Device according to claim 11, wherein the centering means are formed by star-shaped electrodes.   14. Device according to any one of the preceding claims, wherein the means for promoting evaporation also comprise means for controlling the speed of displacement of small volumes of liquid, by a successive electrical supply of the electrodes, so as to create an electrostatic field moving along the path.   15. Device according to one of the preceding claims, wherein the means for promoting evaporation also comprise heating means.   16. Device according to the preceding claim, wherein the heating means are formed by the second electrically conductive means (6) and third electrically conductive means (22).   17. Device according to the preceding claim, wherein the second (6) and third (22) conductive means are in the form of catenary.   18. Device according to any one of the preceding claims, wherein the means for promoting evaporation also comprise means capable of applying a current of a pneumatic fluid, for example air, with low humidity and / or means for creating a zone of low pressure around small volumes of liquid.   19. Device according to any one of the preceding claims, wherein the means for promoting evaporation comprise means for detecting the flow of an electric current between two electrically conductive means and thus detecting the contact between a small volume of liquid and the electrically conductive means.   20. Device according to the preceding claim in combination with claims 18 or 19, wherein the electrically conductive means are formed by the second and third conductive means.   Preparation assembly comprising at least two preparation devices according to any one of the preceding claims placed in series, the devices being connected by means of drop collecting means in communication with the dispensing means.   22. Preparation assembly, comprising at least two devices according to any one of claims 1 to 20, operable simultaneously, said devices being fed by the same reservoir (114).   23. Assembly according to the preceding claim, comprising a collection means (120) unique for the various paths.   24. Assembly according to the preceding claim, wherein the collection means (120) is adapted to be placed in communication with the distribution means a following preparation device.   25. An electronic chip, comprising a substrate provided with at least one device according to any one of claims 1 to 20 and / or an assembly according to any one of claims 21 to 24.   26. A method for preparing, from a first volume (V1) of liquid comprising at least one substance at a first determined concentration, at least one sample in liquid form at a second volume (V2) comprising said substance at a first second determined concentration, said method using a device according to one of claims 1 to 20 or an assembly according to one of claims 21 to 24, comprising the steps of: - distribution of the first volume (V1) in the form of drops on electrowetting means (8, 108, 208, 308, 408) for moving the drops of liquid by applying a potential difference between the first and second conducting means to reach the second volume of liquid (V 2) having the second concentration (C2).   27. Method according to the preceding claim, wherein the type of displacement of the small volume of liquid is changed depending on the size of the small volume of liquid during its movement.   28. The method according to the preceding claim, wherein simultaneously with the movement of the small volume, a heating of the drop and / or a low pressure of the drop and / or a stream of pneumatic fluid around the drop are provided.   29. A method according to any one of claims 26 to 28, comprising a step of discharging heavier particles contained in a drop. 20   30. Method according to one of claims 26 to 29, wherein the first volume (V1) is between 1 ml and several milliliters and the second volume (V2) is between 100 nl and 5 pl.