EP1773497B1 - Device for moving and treating volumes of liquid - Google Patents

Abstract

A device for displacing a small volume of liquid under the effect of an electric control, including a first substrate with a hydrophobic surface provided with a first electrical conductor, a second electrical conductor positioned facing the first conductor, and a third conductor, forming with the second conductor, a mechanism for analyzing or heating a volume of liquid.

Description

TECHNICAL FIELD AND PRIOR ART

The invention relates to a device and a method for moving small volumes of liquid, using electrostatic forces to obtain this displacement.

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 movement or manipulation is based on the principle of electrowetting on a dielectric, as described in the article of 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 facing electrodes activated for displacement.

The principle of this type of displacement is synthesized on the Figures 1A - 1C .

A drop 2 rests on a network 4 of electrodes, from which it is isolated by a dielectric layer 6 and a hydrophobic layer 8 ( Figure 1A ).

When the electrode 4-1 located near the drop 2 is activated, the dielectric layer 6 and the hydrophobic layer 8, between this activated electrode and the droplet polarized by an electrode 10, act as a capacitor. The effects of electrostatic charge induce the displacement of the drop on this electrode. The electrode 10 may be a catenary, it then maintains an electrical contact with the drop during its displacement as described in document FR - 2 841 063 ( Figure 2A ).

The drop can thus be moved step by step ( figure 1C ), on the hydrophobic surface 8, by successive activation of the electrodes 4-1, 4-2, etc. and by guiding along the catenary 10.

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.

This type of displacement is increasingly used in devices for biochemical, chemical or biological analyzes, whether in the medical field, or in environmental monitoring, or in the field of quality control.

In some cases, there is the problem of moving and detecting a characteristic of a liquid volume displaced or to be displaced.

There is often the problem of the number of contacts on the chip on which the displacement takes place, as well as the problem of how to bring the drop to be analyzed to a detection zone.

This is particularly the case, but not only, when droplet movement and detection, for example of a product solubilized in this drop, are perfectly dissociated.

There is therefore the problem of finding a new device that makes it easier to move and analyze or treat drops or micro-drops of small volumes of liquid.

SUMMARY OF THE INVENTION

The invention relates to a device for moving a small volume of liquid under the effect of an electrical control, comprising a first hydrophobic surface substrate provided with first electrically conductive means, second electrically conductive means arranged vis-à- screw of the first conductive means, or in correspondence of these first means, or vis-à-vis the portion of the hydrophobic surface which covers the first electrically conductive means, characterized in that it comprises third conductive means forming with the second conducting means of the analysis means or for inducing a reaction or means for heating a volume of liquid.

One of the second and third electrically conductive means may be used in the phase of displacement of the drops of liquids of interest in order to bring the drop onto the desired zone of the first electrically conductive means, the second electrically conductive means being associated with the third means in a couple, for example a pair of electrodes in electrical contact with the drop or the liquid, so as to perform, for example, an electrochemical detection of a redox species present in the drop or drops (two-electrode detection) , or an electrophoretic system, or a heating system or other reactions.

Thus, one of the second and third electrically conductive means has two functions.

First, alone and in combination with the underlying electrodes, a displacement function is provided by energizing the drop for electrowetting.

Then, coupled to the other means among the second and third electrically conductive means, a second function is provided, which is a detection function, for example electrochemical.

The second electrically conductive means will then be either a working electrode or a counter electrode.

These second means will act as both reference electrode and against electrode, the role of the second electrode being a function of that of the first.

According to one embodiment, the second conductive means comprise a catenary or a wire, substantially parallel to the hydrophobic surface.

The catenary or the wire may be buried in the first substrate, at a non-zero distance from the hydrophobic surface, for example between 1 μm and 100 μm or 500 μm.

The third conductive means may also comprise a catenary or a wire, which may be non-buried in the first substrate, at a non-zero distance from the hydrophobic surface, for example between 1 μm and 100 μm or 500 μm.

The two catenaries or wires may be parallel to each other and to the hydrophobic surface.

The two catenaries or wires may not be parallel to each other, but remain parallel to the hydrophobic surface.

One of the catenaries can be buried under the hydrophobic surface.

The catenaries can be directed substantially parallel to each other.

The third conductive means may comprise a plane conductor buried beneath the hydrophobic surface.

The second conductive means may comprise a catenary or a wire buried beneath the hydrophobic surface.

The third conductive means may then also include a catenary or a buried wire, the two buried catenaries being directed substantially parallel to each other.

The third conductive means may comprise a planar electrode buried beneath the hydrophobic surface.

The second conductive means may comprise a buried plane electrode.

The third conductive means may then comprise a buried conductor, of flat or wired form.

The third conductive means may comprise a catenary or a wire directed perpendicularly to the catenary or wire of the second electrically conductive means.

A device as described above may further comprise a second substrate with a hydrophobic surface, this second substrate conferring on the assembly a confined structure.

It may also further comprise a second substrate with a hydrophobic surface, this second substrate conferring on the assembly a confined structure, the third conductor being buried in the second substrate, under its hydrophobic surface.

The third conductor can then be in the form of catenary or buried wire, or in the form of a buried plane conductor.

In such a device, the surface of the second substrate may be locally perforated to form a contact zone between a drop of liquid positioned between the two substrates and the third conductor.

The second substrate may also be disposed at a distance from the first substrate of between 10 μm and 100 μm or 500 μm.

A device as described above may further comprise a second substrate with a hydrophobic surface, this second substrate conferring on the assembly a confined structure, the second and third conductors being buried in the second substrate, under its hydrophobic surface.

The second and third conductors can then each be in the form of catenary or wire.

• la mise en contact d'une goutte de liquide avec les électrodes d'un dispositif tel que décrit ci- dessus,
• l'application d'une différence de potentiel entre les deuxième et troisième moyens conducteurs.

The invention also relates to a method for treating a drop of liquid, for example by reaction or electrochemical detection or by electrophoresis or Joule effect, or treatment of a cell by cell lysis or by electroporation, comprising:

• bringing a drop of liquid into contact with the electrodes of a device as described above,
• applying a potential difference between the second and third conductive means.

The second electrically conductive means, or both electrodes, can thus for example provide electrophoretic separation and / or a heating function.

In a device according to the invention, the tilting of a displacement configuration to a reaction or reading or heating configuration can be fast, allowing several drops to be processed one after the other, in a continuous flow assay protocol, for example, or for high flow rate analyzes.

BRIEF DESCRIPTION OF THE FIGURES

• The Figures 1A - 1C illustrate the principle of moving a drop on a matrix of electrodes by electrowetting,
• the FIGS. 2A to 2C illustrate an embodiment of the invention,
• the Figures 3A - 9B illustrate other variants and other embodiments of the invention,
• the Figures 10A and 10B illustrate two-dimensional variants of the invention,
• the figure 11 illustrates the detection between two catenaries of the Fe ^{II / III} pair.
• the figure 12 illustrates the electrochemical detection of a species generated by an enzyme.
• the Figures 13a and 13b are schematic representations of an exemplary implementation of a device according to the present invention for calibrating a drop of liquid during different calibration steps;

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

A first embodiment of the invention is illustrated on the Figures 2A and 2B .

A device or microfluidic component according to the invention comprises a lower substrate 20 provided with a matrix 24 of independent electrodes.

Each of these electrodes 24 is electrically connected to a conductor 26.

The electrodes 24 are covered with an insulating layer 28 and a hydrophobic layer 29.

The hydrophobic nature of this layer means that a drop 22 has a contact angle on this layer of greater than 90 °.

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

This device comprises a first catenary 30, allowing electrowetting, and a second catenary 32 forming an electrode pair with the first catenary 30.

The first catenary is located opposite the electrodes 24, or the portion of the hydrophobic surface 29 situated above the electrodes 24.

Supply means 34 connect these various electrodes together.

On the Figures 2A - 2B these supply means can be switched in two ways, using switching means 33.

First, for a displacement of a drop 22, one or more of the electrodes 24 is / are under tension, as well as the catenary 30 this configuration is illustrated in FIG. Figure 2A ; as already explained above, the activation of one of the electrodes 24 will induce a displacement of the droplet 22.

Then, for measurements, a voltage is applied to each of the catenaries 30 and 32, generating a non-zero potential difference between these two catenaries, which can induce an electrochemical reaction in the droplet 22, and / or a heating of this droplet , and / or an electroporation detection or reaction and / or a cell lysis-type reaction in this drop if there is the presence of a cell in the drop.

This configuration is illustrated in Figure 2B .

Optionally, with switching means, or with the aid of second voltage generator means, not shown on the Figures 2A - 2B a voltage can be applied to one or more of the electrodes 24, simultaneously with the voltage applied between the catenaries 30 and 32, which makes it possible to cause, at the same time as the above reaction, a displacement of the drop 22.

The use of two electrodes 30, 32 in the form of catenaries, parallel to each other and to the alignment of the electrodes 24, makes it possible to achieve the desired reaction in the drop at any desired location of this alignment. It is possible to bring the drop on top of any one of the electrodes 24 and to produce the desired reaction by activating a non-zero potential difference between the two catenaries 30 and 32.

One of the two catenaries is therefore bifunctional and can be used for a displacement on the hydrophobic surface 29 or for any reaction electrochemical or any other reaction for which there is a need for two electrodes (for example: electrophoresis, electroporation, cell lysis).

According to a variant, represented on the Figure 2C the second conductor may be arranged in a different direction than the first conductor. For example, the catenary 30 is kept parallel to the alignment of the electrodes 24, while the second catenary is directed substantially perpendicular to the first catenary, but parallel to the plane of the layer 29 and the substrate 20, or ( Figure 2C ) is directed substantially perpendicular to the plane of the layer 29 and the substrate 20.

The displacement of the drop 22 of liquid takes place in the same manner as above, while a reaction or heating is induced by establishing a non-zero potential difference between the electrodes 30 and 32.

A variant of the device described above is represented in Figures 3A and 3B , on which numerical references identical to those of Figures 2A - 2C designate identical or similar elements.

One of the catenaries is still located above the substrate (here the catenary 30, but it could be the catenary 32). Another electrode 40, here a catenary, is buried in the substrate 20, for example under the hydrophobic layer 29. This buried electrode can be flat, instead of being a catenary.

For a displacement of a droplet 22, one or more of the electrodes 24 is / are under tension, as well as, for example, the catenary 30. It could also be the electrode 40 that is energized in place of the catenary 30; this configuration is illustrated in figure 3A ; as already explained above, the activation of one of the electrodes 24 will induce a displacement of the droplet 22.

Then, for measurements, a voltage is applied between the catenaries 30 and 40, generating a potential difference between these two catenaries, which can induce an electrochemical reaction / detection in the drop 22, and / or a heating of this drop, and / or an electroporation reaction and / or a cell lysis type reaction of cells present in the drop.

This configuration is illustrated in figure 3B .

Again, displacement and reaction or heating can be simultaneous, using adequate switching means or second voltage generating means.

Yet another variant of this device is represented in Figures 4A and 4B , on which numerical references identical to those of Figures 2A - 2C designate identical or similar elements.

None of the catenaries is no longer located above the substrate. On the other hand, two catenaries 50 and 52 are buried in the substrate 20, for example under the hydrophobic layer 29.

The Figure 4A represents a longitudinal view of the device, on which only one of the two buried catenaries is visible, hiding the second, while the Figure 4B represents a sectional view AA 'of the device, on which the two buried catenaries 50, 52 are visible, above an electrode 24-1 which hides the other electrodes of the network 24. On this Figure 4B the voltage generating means 34 and the switching means 33 are also shown.

For a displacement of a drop 22, one or more of the electrodes 24 is / are under tension, as well as, for example, the catenary 52; this configuration is illustrated in Figures 4A and 4B ; as already explained above, the activation of one of the electrodes 24 will induce a displacement of the droplet 22.

Then, for measurements, a voltage is applied to each of the catenaries 50 and 52 using the means 34 and 33 (situation not shown in the figures), generating a non-zero potential difference between these two catenaries, which can inducing a heating of this drop, and / or an electroporation reaction and / or a cell lysis type reaction of this drop.

The invention also relates to other embodiments, particularly of the confined type, with an upper substrate.

Thus, according to another embodiment, it is possible to produce a so-called closed system device, with an upper substrate that confines the drop.

Such an embodiment is illustrated in figure 5 , on which numerical references identical to those of Figures 2A - 2B designate identical or similar elements.

An upper substrate 120 comprises a hydrophobic layer 129, for example Teflon. Like the layer 29, it is in contact with the droplet 22.

The two conductors 30, 32, are located in this example between the two substrates 20, 120 and are both in direct contact, mechanical and electrical, with the drop 22.

The operation of this type of device is the same as that explained above in connection with the Figures 2A and 2B , the only difference residing in the confinement of gout.

On the figure 5 , the device is shown in the displacement position of the drop, a reaction or heating being induced by switching means 33 switching. Here again, displacement and reaction or heating can be induced simultaneously, by appropriate switching means or by means of a second voltage source.

According to a variant of this embodiment, one of the two conductors making it possible to induce a reaction in the drop can be buried in the lower substrate 20.

For example, on the figure 6 , on which numerical references identical to those of Figures 2A - 2C y designate identical or similar elements, one of the catenaries is still located above the substrate (here the catenary 30, but this could be catenary 32). Another electrode 60, for example a catenary, is buried in the substrate 20, for example under the hydrophobic layer 29, leaving only the conductor 30 in mechanical and electrical contact with the drop.

This embodiment allows a displacement of the drop using the conductors 24 and the conductor 30, and the induction of a reaction with the application of a difference in voltages between the conductors 60 and 30 (which is represented on the figure 6 ).

The buried electrode 60 may have the shape of either a linear conductor or a catenary, or the shape of a plane conductor.

When it has the shape of a linear conductor, it can be oriented in a direction not necessarily parallel to the direction of the catenary 30, as illustrated in FIG. figure 6 on which the two catenaries are substantially perpendicular; and the advantage of this structure is that only one drop at a time is in electrical contact with the two electrodes. Or the two electrodes 30, 60 may be parallel to each other (for example as illustrated in FIGS. Figures 3A and 3B ), which makes it possible to achieve the desired reaction at any place above the electrodes 24. The same advantage is offered when the buried electrode 60 has the shape of a plane conductor.

For a displacement of a droplet 22, one or more of the electrodes 24 is / are under tension, as well as the catenary 30; as already explained above, the activation of one of the electrodes 24 will induce a displacement of the droplet 22.

Then, for measurements, a voltage is applied to each of the catenaries 30 and 60, generating a potential difference between these two catenaries, which can induce an electrochemical reaction in the drop 22, and / or a heating of this drop, and or an electroporation reaction and / or a cell lysis type reaction of this drop. This configuration is illustrated in figure 6

According to yet another variant of this embodiment, one of the two conductors for inducing a reaction in the drop can be buried in the upper substrate 120.

For example, on the figure 7 , on which numerical references identical to those of Figures 2A - 2C y designate identical or similar elements, one of the catenaries is still located above the substrate (here the catenary 30, but it could be the catenary 32).

Another electrode 70, for example a catenary, is buried in the substrate 120, for example under the hydrophobic layer 129, leaving only the conductor 30 in mechanical and electrical contact with the drop.

This embodiment allows a displacement of the drop using the conductors 24 and the conductor 30, and the induction of a reaction with the application of a difference in voltages between the conductors 70 and 30.

The buried electrode 70 may have the shape of either a linear conductor or a catenary, or the shape of a plane conductor.

When in the form of a linear conductor, it may be oriented in a direction not necessarily parallel to the direction of the catenary 30 (as shown in FIG. figure 7 , on which the two catenaries are substantially perpendicular), or the two conductors may be parallel to each other (for example, as illustrated in FIGS. Figures 3A and 3B ), which makes it possible to achieve the desired reaction at any place above the electrodes 24. The same advantage is offered when the buried electrode 70 has the shape of a plane conductor.

For a displacement of a droplet 22, one or more of the electrodes 24 is / are under tension, as well as the catenary 30; this configuration is illustrated in figure 7 ; as already explained above, the activation of one of the electrodes 24 will induce a displacement of the droplet 22.

Then, for measurements, a voltage is applied to each of the electrodes 30 and 70, generating a non-zero potential difference between them, which can induce an electrochemical reaction in the drop 22, and / or a heating of this drop, and or an electroporation reaction and / or a cell lysis-type reaction in this drop.

According to another variant, each of the two conductors for inducing a reaction in the drop is buried in one of the substrates.

So, on the figure 8A , on which numerical references identical to those of Figures 2A - 2C y denote identical or similar elements, one of the catenaries is buried in the substrate 20, for example under the hydrophobic layer 29.

The other electrode 130, for example a catenary, is buried in the substrate 120, for example over the hydrophobic layer 129.

None of the drivers are in mechanical contact with the drop.

This embodiment allows a displacement of the drop using the conductors 24 and the conductor 50 and the induction of a reaction with the application of a difference in voltages between the conductors 130 and 50.

Each of the buried electrodes 50, 130 may have the shape of either a linear conductor or a catenary, or the shape of a plane conductor.

When both have the shape of a linear conductor, they can be oriented in directions that are not necessarily parallel to each other (as illustrated in FIG. figure 7 , on which the two catenaries are substantially perpendicular), or the two conductors may be parallel to each other (for example as illustrated in FIG. figure 8A ) which makes it possible to carry out the desired reaction or detection at any place above the electrodes 24. The same advantage is offered when one of the two buried electrodes has the shape of a plane conductor (in particular that of the substrate 120) while the other has the shape of a linear conductor aligned above the electrodes 24, or when the two electrodes each have the shape of a plane conductor.

For a displacement of a droplet 22, one or more of the electrodes 24 is / are under tension, as well as the electrode 50; this configuration is illustrated in figure 8A ; as already explained above, the activation of one of the electrodes 24 will induce a displacement of the droplet 22.

Then, for measurements, a voltage is applied to each of the electrodes 130 and 50, generating a non-zero potential difference between them, which can induce heating in the droplet 22, and / or an electroporation and / or a cell lysis-type reaction in this drop if there are cells in the drop.

According to a variant of this embodiment, illustrated on the Figure 8B , on which numerical references identical to those of Figures 2A - 2C y denote identical or similar elements, one of the buried conductors, for example the conductor 130 of the upper substrate 120, is locally in physical contact with the drop 22 due to an opening 127 made in the hydrophobic layer 129, for example by lithography and etching of this layer 129.

• une réaction électrochimique dans la goutte 22 lorsqu'elle est en contact direct avec l'électrode 130 par l'ouverture 127,
• et/ou, quelle que soit la position de la goutte par rapport à l'ouverture 127, un chauffage de cette goutte et/ou une réaction d'électroporation et/ou une réaction de type lyse cellulaire dans cette goutte s'il y a des cellules dans cette goutte.

In this case, for measurements, a voltage is applied to each of the electrodes 130 and 50, generating a potential difference between these two electrodes, which can induce:

• an electrochemical reaction in the drop 22 when in direct contact with the electrode 130 through the opening 127,
• and / or, regardless of the position of the drop relative to the opening 127, a heating of this drop and / or an electroporation reaction and / or a cell lysis-type reaction in this drop if there is cells in this drop.

One can have a variant in which the opening is made in the layer 29 of the lower substrate, for a contact between the drop 22 and the conductor 50.

According to yet another variant of this device, the two electrodes are both located either in the lower substrate or in the upper substrate. None of the electrodes are located in mechanical contact with the drop.

The case of two electrodes buried in the lower substrate is similar to the case described above in connection with the Figures 4A - 4B , to which an upper substrate 120 such as that of the figure 6 would be added to confine the drop 22.

The case of two electrodes buried in the upper substrate is illustrated on the Figures 9A - 9B , on which numerical references identical to those of Figures 2A - 2C designate identical or similar elements.

Two catenaries 130 and 132 are buried in the substrate 120, for example under the hydrophobic layer 129.

The Figure 9A represents a longitudinal view of the device, on which only one of the two buried catenaries is visible, hiding the second.

The Figure 9B represents a sectional view BB 'of the device, on which the two buried catenaries 130, 132 are visible, above an electrode 24-1 which hides the other electrodes of the network 24.

For a displacement of a drop 22, one or more of the electrodes 24 is / are under tension, as well as, for example, the catenary 130; as already explained above, the activation of one of the electrodes 24 will induce a displacement of the droplet 22.

Then, for measurements, a voltage is applied to each of the catenaries 130 and 132, generating a potential difference between these two catenaries, which can induce heating of this drop, and / or an electroporation reaction and / or a cell lysis reaction in this drop (this configuration is illustrated in FIG. Figures 9A and 9B ).

The invention can be implemented with a row of electrodes 24, thus a linear arrangement of these electrodes.

These electrodes may however, in the context of the invention, be arranged according to any scheme, and in particular in 2 dimensions.

Another aspect of the invention is therefore represented by the Figures 10A and 10B on which numerical references identical to those of Figures 2A - 2C designate identical or similar elements.

On the figure 10A , the substrate 20 supports an array of electrodes 24, distributed in rows and columns, covered with an insulating layer 28 and a hydrophobic layer 29.

Several pairs of micro-catenaries 30, 32 are paralleled along the lines of electrodes.

These micro-catenaries can be positioned at a given distance from the surface of the substrate by means of spacers 70.

In this way, it is possible to work in parallel on several electrode lines, and to move several drops by previously described methods.

The spacer technique can also be used in conjunction with the other embodiments to maintain a catenary at a predetermined distance from the hydrophobic layer 29.

Another aspect of the invention is shown in the figure 10B .

The substrate 20 supports an array of electrodes 24, distributed in rows and columns, covered with a thin insulating layer 28 and a hydrophobic layer 29.

A first series of micro-catenaries 30, 32 is paralleled along the lines of electrodes.

These micro-catenaries are positioned at a given distance from the surface of the substrate by means of spacers 70.

A second series of micro-catenaries 130, 132 is paralleled but placed perpendicularly to the series of micro-catenaries 30, 32, that is to say in the direction of the columns of electrodes 24.

These micro-catenaries are positioned at a given distance from the surface of the substrate by means of spacers 72.

The spacers 70 and 72 may be of different heights. Thus, it is possible to move drops in two perpendicular directions.

With regard to the reaction or heating to be induced in a drop of liquid, these 2D embodiments function in the same manner as described above in connection with the Figures 2A-9B _: Activation of two neighboring electrodes 30,32 or 130,132 induces a potential difference between these two electrodes and a heating reaction or in the liquid of the drop.

The electrodes of these 2D embodiments are connected to switching means, not shown on the Figures 10A and 10B but in a manner analogous to that described above in connection with the preceding figures.

• une ou deux électrodes enterrées pour une ou plusieurs lignes et/ou colonnes d'électrodes 24,
• un deuxième substrat de confinement, muni d'une surface hydrophobe, avec éventuellement, là encore, une ou deux électrodes enterrées pour une ou plusieurs lignes et/ou colonnes d'électrodes 24. La surface hydrophobe de ce deuxième substrat peut être munie d'ouvertures de contact telle que l'ouverture 127 de la figure 8B.

These 2D embodiments can also implement the following features, taken alone or in combination:

• one or two buried electrodes for one or more rows and / or columns of electrodes 24,
• a second confinement substrate, provided with a hydrophobic surface, with, where appropriate, one or two buried electrodes for one or more rows and / or columns of electrodes 24. The hydrophobic surface of this second substrate may be provided with contact openings such as the opening 127 of the Figure 8B .

In general, in the embodiments using buried conductor (s), the economy is made of a wired wiring step; in addition (the wetted surface is only located on the hydrophobic surfaces 29 and 129) are then best used the wetting properties of the corresponding layer 29, 129.

Typically, the distance between the conductors 30, 32 ( Figures 2A - 3B , 5 - 7 ) on the one hand and the hydrophobic surface 29 is for example between 1 micron and 100 microns or 500 microns.

The catenaries 30, 32 are for example in the form of son diameter between 10 microns and a few hundred microns, for example 200 microns. These wires may be gold or aluminum wires or tungsten or other conductive materials.

The buried electrode is obtained by depositing and then etching a thin layer of a metal selected from Au, Al, Ito, Pt, Cu, Cr, etc. using conventional microtechnology technologies. The thickness is from a few tens of nm to a few microns. The width of the pattern is from a few μm to a few nm (flat electrodes).

When two substrates 20, 120 are used ( Figures 5 - 9B ), they are separated by a distance of, for example, 10 μm and 100 μm or 500 μm.

Whatever the embodiment considered, a drop of liquid 22 will have a volume of between, for example, 1 nanolitre and a few microliters, for example between 1 nl and 5 μl or 10 μl.

In addition, each of the electrodes 24 will for example have a surface of the order of a few tens of μm ² (for example 10 μm ² ) up to 1 mm ² , depending on the size of the drops to be transported, the spacing between adjacent electrodes being for example between 1 .mu.m and 10 .mu.m.

The structuring of the electrodes 24 can be obtained by conventional microtechnology methods, for example by photolithography. The electrodes 24 are made by depositing a metal layer (Au, Al, ITO, Pt, Cr, Cu, ...) by photolithography.

The substrate is then covered with a dielectric layer of Si ₃ N ₄ , SiO ₂ , ... Finally, a deposit of a hydrophobic layer is performed, such as a teflon deposit made by spinning.

Methods for producing chips incorporating a device according to the invention may be directly derived from the methods described in the document FR - 2 841 063 : Instead of making a catenary row of electrodes, it is two, or is made a buried plane conductor and a catenary.

Conductors, and in particular buried catenaries, may be made by depositing a conductive layer and etching this layer in the appropriate pattern of conductors, before deposition of the hydrophobic layer.

An example of electrochemical detection of a redox species will be given. This detection is carried out using a device according to the invention, for example the device of the Figures 2A - 2B .

A drop of 1 μl of a solution of ferri / potassium ferrocyanide (10 ^-2 M) is deposited on the hydrophobic surface 29.

This drop is in contact with the two catenaries 30, 32.

During the measurement, the catenary 30 used for displacement plays the role of working electrode while the second electrode 32 acts as counter-electrode and reference electrode.

An electrochemical measurement is then carried out in potential cyclic voltammetry between -400mV and + 300mV relative to the reference electrode;

As shown in figure 11 a conventional redox system Fe ^II / Fe ^III is obtained.

More generally, electrochemistry makes it possible to describe the chemical phenomena coupled with reciprocal exchanges of electrical energy.

The electrochemical reaction that occurs at the surface of an electrode is the result of the transfer of electric charge across the interface between it and an electroactive species (in one direction or the other).

In general, two electrodes (working electrode and counter-electrode) are immersed in an electrolytic solution containing an electroactive species.

A third electrode (reference electrode) is used to reference the potential of the working electrode.

• dans les électrodes, le courant circule par déplacement des électrons (porteurs de charges),
• aux interfaces électrode/liquide, le courant circule par le biais de réactions rédox qui s'y déroulent (transfert d'électrons entre électrode et solution ou espèce rédox),
• dans la solution, le courant circule par déplacement des ions (porteurs de charges).

Thus, when two electrodes are connected by a non-infinite resistance circuit (the electrolyte is conductive), the non-zero current flows in the electrochemical cell. This circulation involves three different mechanisms:

• in the electrodes, the current circulates by displacement of the electrons (charge carriers),
• at the electrode / liquid interfaces, the current flows through redox reactions that take place there (electron transfer between electrode and solution or redox species),
• in the solution, the current flows by displacement of the ions (charge carriers).

• l'une des électrodes du dispositif joue le rôle d'électrode de travail,
• l'autre, la seconde électrode, joue à la fois le rôle de contre-électrode et d'électrode de référence.

It is also possible to carry out this electrochemical measurement between two electrodes, for example the electrodes of one of the devices as described above in relation to the Figures 2A - 2B , 3A - 3B , 5 - 7 , 8B , 10A - 10B :

• one of the electrodes of the device acts as a working electrode,
• the other, the second electrode, acts as both a counter-electrode and a reference electrode.

Electrophoresis is a known method for separating charged species. Indeed charged molecules in an electric field will begin to migrate to the opposite charge electrode. The migration rate will depend on the charge / mass ratio of the molecule, which effectively separates molecular species of different charges / mass.

The electrodes of a device according to the invention, especially as described above in connection with the Figures 2A - 10B can be used to induce such an electrophoresis reaction in a drop of liquid.

• soit par contact, les électrodes chauffant et transférant la chaleur au liquide de la goutte 22,
• soit en faisant passer un courant entre les deux électrodes, en utilisant le liquide de la goutte comme une résistance qui s'échauffe par effet Joule. Dans ce cas, il n'est pas nécessaire qu'un contact direct, mécanique, soit établi entre le liquide de la goutte et au moins une des électrodes Ce type de chauffage peut être induit par exemple dans la configuration des figures 9A et 9B.

The electrodes of a device according to the invention, especially as described above in connection with the Figures 2A - 10B , can also be used as a heating resistor:

• either by contact, the electrodes heating and transferring the heat to the liquid of the droplet 22,
• either by passing a current between the two electrodes, using the liquid of the drop as a resistance that heats by the Joule effect. In this case, it is not necessary for a direct mechanical contact to be established between the liquid of the drop and at least one of the electrodes. This type of heating can be induced, for example, in the configuration of the Figures 9A and 9B .

The invention makes it possible to implement detections or electrochemical reactions, when at least one of the two electrodes is in physical contact with the drop.

It also makes it possible to carry out electrophoresis reactions, or to heat the liquid of the droplet 22.

The invention can also be applied to electroporation methods, which make it possible to open or modify the membrane of a cell (which is then the droplet 22) and thus to bring into the cell other chemicals , transported by means of the electrodes as described above, or brought manually, for example by means of a pipette.

It can also be applied to cell lysis methods, which make it possible to burst the membrane of a cell, for example with a difference in voltages, applied to the two electrodes 30, 32, of approximately a few volts, for example approximately 100 V / mm.

A first example of electrochemical detection of a redox species has been given in connection with the figure 11 .

A second example concerns the electrochemical detection of a species generated by an enzyme.

A first reaction mixture is prepared as follows: 50 mM phosphate-citrate buffer, pH 6.5 (10 ml), o-phenylene diamine (OPD, 20 mg) and hydrogen peroxide (4 μl).

A second mixture is prepared as follows: MilliQ water (9 μl) and "horse radish" peroxidase (1 μl at 20 μM). A drop of 0.5 .mu.l of the first mixture is converged on the chip to a drop of 0.5 .mu.l of the second mixture by applying a voltage of 50V. During this movement only the catenary 30 intervenes. After 5 minutes of reaction at room temperature and protected from light, the product of the enzymatic reaction is detected by differential pulsed voltammetry using as the pair of electrodes the catenaries 30 and 32, the catenary 30 serving as electrode of and the catenary 32 serving both against electrode and reference electrode. Thus, a redox peak is obtained at -480mV corresponding to the reduction of the enzymatic product generated (see figure 12 ).

A second example concerns the displacement of a drop followed by a localized variation of electro-controlled pH.

For some applications, a drop of a reaction medium is moved and then the pH is varied to stop or start a reaction. Here, this pH is electrochemically varied using the invention.

A drop of buffered solution (PBS pH 7.4) containing a colored indicator, cresol red 1 mM, is deposited on the chip and then moved on it by applying a voltage of 50V. A potential of -1.4V for 10 s is then applied between the two catenaries, 30 and 32, thus causing the hydrolysis of water and the generation of OH ^- ions. These OH ions ^- make the solution basic, hence the appearance of a red indicator color with a pH greater than 8.8. When the voltage is cut off, the buffer then compensates for the pH and the red color disappears.

On the Figures 13a and 13b , one can see a device according to the present invention, using the two catenaries 30, 32, and allowing a control of the size of the drops. These two catenaries are arranged at different heights relative to the substrate.

The second catenary 32 allows a heating of a drop of liquid or small volume of liquid 22 by Joule contact or effect. Heating by heat transfer is preferred because the current flow in the drop may be 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, the current flow between the catenaries 30, 32 can advantageously make it possible to determine an order of magnitude of the drop size, making it possible to further control the evaporation. When a drop is present and is in contact with the two catenaries 30, 32, a small current flows between the two catenaries. The detection of this current informs the presence of a drop 22 of sufficient size to come into contact, in the example shown, with the second catenary 32. 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 30 and 32, 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 the catenaries 30 and 32, it is known that h is less than d.

On the figure 13a at first the drop 22 has a height h greater than d and puts the two catenaries 30, 32 in electrical contact.

After partial evaporation of the drop 22, h is less than d, there is no more electrical contact between these 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, one can move the drop on an evaporation path in one direction and the other until no current is no longer detected between the two catenaries. We will then know that the size of the drop is less than a given value. Displacement favors evaporation, thus speeding up the process. It is also possible to leave the drop in place, and let the liquid evaporate until there is no more contact between the drop 22 and the catenary 32.

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

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 envisage catenary secondaries arranged transversely to the first catenary (as on the figure 10B for example) discretely 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.

Claims (36)

1. A device for displacing a small volume of liquid under the effect of an electric control, including a first substrate with a hydrophobic surface (29) provided with electrically conducting means (24), second electrically conducting means (30, 50, 130) positioned facing the first conducting means, characterized in that it includes third conducting means (32, 40, 60, 70, 130, 132), forming with the second conducting means, analysis means or means for inducing a reaction or means for heating a volume of liquid, the first electrically conducting means and the second electrically conducting means activating displacement of the volumes of liquid by electrowetting.
2. The device according to claim 1, the second conducting means including a catenary or a wire (30, 50, 130), substantially parallel to the hydrophobic surface.
3. The device according to claim 2, the catenary or the wire being non-buried in the first substrate, at a non-zero distance from the hydrophobic surface.
4. The device according to claim 3, the distance being between 1 µm and 100 µm or 500 µm.
5. The device according to claim 2, 3 or 4, the third conducting means (32, 40, 60, 70, 130, 132) also including a catenary or a conducting wire.
6. The device according to claim 5, the catenary or the wire being non-buried in the first substrate, at a non-zero distance from the hydrophobic surface.
7. The device according to claim 6, the distance being between 1 µm and 100 µm or 500 µm.
8. The device according to claim 5, 6 or 7, both catenaries or wires being parallel to each other and to the hydrophobic surface (29).
9. The device according to any of claims 5 to 7, both catenaries or wires not being parallel to each other, but being parallel to the hydrophobic surface (29).
10. The device according to any of claims 2 to 5, one of the catenaries (40, 50) being buried under the hydrophobic surface (29).
11. The device according to claim 10, the catenaries being directed substantially parallel to each other.
12. The device according to claim 2, 3 or 4, the third conducting means including a planar conductor buried under the hydrophobic surface (29).
13. The device according to claim 1 or 2, the second conducting means including a catenary or a buried wire (50) under the hydrophobic surface (29).
14. The device according to claim 13, the third conducting means (52) also including a catenary or a buried wire, both buried catenaries being directed substantially parallel to each other.
15. The device according to any of claims 1 to 4, the third conducting means including a planar electrode buried under the hydrophobia surface (29).
16. The device according to claim 1, the second conducting means including a buried planar electrode.
17. The device according to claim 16, the third conducting means including a buried conductor with a planar or wire shape.
18. The device according to any of claims 1 to 4, the third conducting means including a catenary or a wire directed perpendicularly to the catenary or wire of the second electrically conducing means.
19. The device according to any of claims 1 to 18, further including a second substrate (120) with a hydrophobic surface (129), this second substrate giving a confined structure to the whole.
20. The device according to any of claims 1 to 4 or 13 or 16, further including a second substrate with a hydrophobic surface (129), this second substrate giving a confined structure to the whole, the third conductor (70, 130) being buried in the second substrate, under its hydrophobic surface (129).
21. The device according to claim 20, the third conductor either being in the form of a catenary or a buried wire, or in the form of a buried planar conductor.
22. The device according to claim 20 or 21, the surface of the second substrate being vocally apertured in order to form a contact area (127) between a drop of liquid positioned between both substrates and the third conductor.
23. The device according to any of claims 19 to 22, the second substrate being positioned at a distance from the first substrate, between 10 µm and 100 µm or 500 µm.
24. The device according to claim 1, further including a second substrate with a hydrophobic surface (129), this second substrate giving a confined structure to the whole, the second and third conductors (130, 132.) being buried in the second substrate, under its hydrophobic surface (129).
25. The device according to claim 24, the second and third conductors each being in the form of a catenary or wire.
26. The device according to any of claims 1 to 25, the hydrophobic surface of the first substrate and/or that of the second substrate being in Teflon.
27. A method for treating a drop (22) of liquid by an electrochemical redaction including:

    - putting a drop (22) of liquid into contact with the electrode of a device according to any of claims 1 to 13, or 16 to 21,

    - applying a potential difference between the second and third conducting means.
28. A method for treating a drop (22) of liquid by electrophoresis including:

    - putting a drop (22) of liquid into contact with the electrodes of a device according to any of claims 1 to 26,

    - applying a potential difference between the second and third conducting means.
29. A method for treating a cell by cell lysis including:

    - putting a cell into contact with the electrode of the device according to any of claims 1 to 26,

    - applying a potential difference between the second and third conducting means.
30. A method for heating a drop (22) of conducting liquid by the Joule effect including:

    - putting a drop of liquid into contact with the electrically conducting means of a device according to any of claims 1 to 26,

    - applying a potential difference between the second and third conducting means,
31. A method for controlling or calibrating the size of a drop (22), including:

    - putting a drop of liquid into contact with the second and third electrically conducting means (30, 32) of a device according to any of claims 1 to 26,

    - having a current flow between the second and third electrically conducting means,

    - evaporating the drop until said current no longer flows between the second and the third electrically conducting means.
32. The method according to claim 31, further including displacement of the drop by electrowetting during evaporation.
33. A method for treating a cell by electroporation including:

    - putting a cell into contact with the electrode of a device according to any of claims 1 to 26,

    - applying a potential difference between the second and third conducting means.
34. A device for calibrating a drop of liquid including a device according to any of claims 1 to 26, and means for controlling a current flowing between the second and the third electrically conducting means.
35. The device according to claim 34, the second and the third electrically conducting means each including a catenary, both catenaries being positioned at different heights relatively to the hydrophobic surface.
36. The device according to claim 35, further including at least an additional catenary, positioned at a distance from the hydrophobic surface, different from the distance between this surface and the two previous catenaries.

Images