EP1781409B1 - Device for handling drops for biochemical analysis, method for producing said device and a system for microfludic analysis - Google Patents

Abstract

A device for handling drops on a displacement by electrowetting plane, including at least one displacement track. The track includes an electrically insulating substrate on the surface of which rest two or more interdigitated conducting electrodes. These electrodes are covered by a dielectric insulating layer, itself covered by a partially-wetting layer. Also, a method for the manufacture of the aforementioned device, in which the creation of the partially-wetting layer includes the creation of a mask in a photosensitive material by the deposition of this material onto a substrate, then a photolithographic stage, followed by development of the photosensitive material, the deposition of a non-wetting material onto the mask, at least one annealing process before dissolution, dissolution of the mask, and at least one annealing process after dissolution. Also, a system for the microfluidic analysis of a liquid sample.

Description

The present invention relates to a drop handling device for biochemical analysis, a method of manufacturing such a device and a microfluidic analysis system using such a device.

Nowadays, new technologies make it possible to design systems of micro and nano size up to high levels of complexity. Ideally, these systems are endowed with all kinds of functionalities, and are used in many fields such as biology or biochemistry. In particular, proteomics, an activity related to the identification and study of proteins, tries to use new technologies to reduce sampled volumes that are handled, and to reduce contamination. The objective is, in a general way, to control the micromanipulation of the material, before spectrometric analysis for example.

In such microsystems, the problematic of the control of fluid flows is strategically important insofar as the material, for example proteins, can not be handled outside a liquid medium. The invention thus relates to the field of microfluidics, which more generally concerns flows in systems of micrometric or nanometric size, in which the sample handled can be subjected to electric fields or wall effects of a physical or chemical nature. complex, and in which the high ratio surface / volume is of great importance.

In this area, the reduction in the size of the systems leads to a decrease in volumes, shorter reaction or exchange times, and the possibility of integrating several modules with different functionalities, for example transport, processing, or again the analysis, all on the same silicon wafer for example.

In order to transport the liquid, two types of fluid displacement are generally possible: the pumping of a continuous flow, and the displacement of calibrated microvolumes. The displacement of calibrated microvolumes has a number of advantages. Indeed, it allows very small volumes of liquids and allows a suitable control of microvolume flow while the continuous flow pumping is characterized by a constant flow. Moreover, this type of movement allows various synchronizations that allow the mixing of liquids for example. To implement a displacement-type fluid displacement of calibrated microvolumes, different modes of action are known: by pneumatic action, by surface acoustic waves, with dielectrophoretic effect, by electrowetting, and by electrowetting on dielectric (EWOD). This last mode of action makes use of a relatively simple technological achievement and allows the control of the flow and the circulation of a calibrated volume of conductive liquid on a network of electrodes.

We know in particular the US patent US 6,565,727 , and the publication of Cho et al. "Particle separation and concentration control for digital microfluidic systems", which describe the movement of drops by electrowetting, as described above. The devices described in these three publications, however, have a lower part incorporating electrodes and an upper part incorporating counter-electrodes, parts between which the drop moves. This upper part makes the device in particular more bulky and more complex.

In addition, the samples handled are often very valuable and in very small quantities. There is therefore a need to optimize the handling of these samples by chemically treating or interacting with the material during its transportation. Known microfluidic displacement systems, whether they require two substrates facing each other or a single substrate, whether they use a counter-electrode or not, do not allow this optimization. Indeed, in particular in the publication of Cho et al. "Particle separation and concentration control for digital microfluidic systems", is proposed a device that makes it possible to interact with the drop physically by direct interaction between the electrodes and the droplet during its transport, and not to interact chemically. This chemical interaction necessary for optimizing the manipulation of samples is therefore impossible in the device of Cho et al.

Indeed, this optimization is made extremely delicate by the fact that the displacement requires one or more tracks in hydrophobic materials to limit friction and hysteresis displacements. This hydrophobic nature of the displacement track notably prevents chemically treating or interacting with the material during its transport.

It should be noted here that we are more generally interested in the property of non-wettability of the displacement track with respect to any liquid. When the liquid is aqueous, as is generally the case when handling proteins, for example, the nonwettability and the wettability with respect to water are respectively the properties of hydrophobicity and hydrophilicity. A hydrophobic material is a non-wetting material with respect to water, and a hydrophilic material is a wetting material with respect to water. The wettability is generally characterized by the angle θ of contact between the drop 1 and the surface 2 (see Figures 1a to 1d ). The wettability coefficient defined as the cosine of the above-mentioned angle is sometimes used. A perfect wettability thus corresponds to a wettability coefficient equal to 1, therefore to θ = 0 °. A total absence of wettability also corresponds to a wettability coefficient equal to -1, therefore to θ = 180 °. Subsequently, we will therefore talk about wetting material with respect to a liquid for a material whose coefficient of wettability by this liquid tends to 1 (without necessarily being equal to 1), as shown in Figure la, and we will talk of non-wetting material with respect to a liquid for a material whose wettability coefficient with respect to this liquid tends to -1 (without necessarily being equal to -1) as illustrated in FIG. figure 1b . The Figures 1c and 1d illustrate intermediate cases of wettability (θ <90 °) or non-wettability (θ> 90 °) respectively.

The problem posed by the non-wetting materials with respect to a liquid, in particular the hydrophobic materials, which are otherwise essential for the displacement, is that the surface properties of these materials prevent the creation of chemical surface treatment zones by the These materials are characterized by low surface energy. If we try to functionalize locally the surface of such materials, which would chemically treat the liquids handled, the result is unreliable, difficult to control and too imperfect. The alternative of making the non-wetting material more rough with respect to the liquid is not conceivable because it makes the ability of the material to favor the transport of the liquid lose. It is therefore necessary to use a layer of material that is partially wetting, that is to say that it must maintain the non-wetting character for movement, while creating wetting or high wettability for functionalization.

Applied to the particular case where the material in question is hydrophobic, two conventional photolithographic techniques are known for creating a partially hydrophobic layer by creating openings in a hydrophobic material, the openings becoming hydrophilic zones distributed in the hydrophobic layer. In a first technique ( figure 5 ), also used by Cho et al. in "Particle separation and concentration control for digital microfluidic systems", after deposition of a layer of hydrophobic material on a substrate, a layer of photoresist is deposited which contains a surfactant, a chemical product for increasing the wettability of a surface against a liquid. This technique poses the particular problem of the final pollution of the hydrophobic material and therefore the loss of the ability of this material to promote the movement of a liquid. In the second technique ( figure 6 ), after depositing a layer of hydrophobic material on a substrate, and before depositing a photoresist, the layer of hydrophobic material is first subjected to a surface modification using a plasma, to modify its hydrophobic properties, that is to say to make it less hydrophobic. This technique also poses the problem of permanently modifying the surface properties of the hydrophobic material.

With such techniques, the openings created, and therefore the hydrophilic zones, are not sufficiently sharp and precise, with possible hydrophobic deposits, and consequently an unsuitability for the creation of chemically functionalized zones, the hydrophobic zones having their properties modified. and their decreased hydrophobicity, with the consequent maladaptation to the displacement of liquid. The same observations can be made in the case of an application of these techniques for producing wetting zones in a non-wetting layer with respect to the transported liquid.

There is therefore a need for a method which makes it possible to render a non-wetting transport track partially wetting with respect to the transported liquid, in particular partially hydrophilic when the liquid is a solution containing water, so that the capacity transporting the drop of liquid is maintained, while allowing the chemical treatment or the interaction with this drop during its transport.

More generally, there is a need for a reliable solution that overcomes the aforementioned drawbacks, including the optimization of displacement and the manufacture of an optimized displacement track.

It is therefore the object of the invention to overcome these disadvantages. To this end, the invention relates, in a first aspect, to a device for handling drops on an electrowetting displacement plane which comprises at least one electrowetting displacement track and which makes it possible to chemically treat or interact with the droplet. simultaneously with its transport.

The displacement track comprises at least two interdigitated electrodes which rest on an electrically insulating substrate and which are covered by an insulating dielectric layer. This set insulating substrate, electrodes, insulating dielectric layer, is covered with a partially wetting layer vis-à-vis the manipulated drops.

In an alternative embodiment concerning the handling of drops containing water, the partially wetting layer is therefore a partially hydrophilic layer.

In the remainder of the description, and for the sake of simplicity, the term "non-wetting, partially wetting or wetting" layer or material will be used for a respectively non-wetting, partially wetting or wetting layer or material. manipulated drops.

The device of the invention comprises, in another variant embodiment, at least one counter electrode distinct from the first electrodes. This counter-electrode may be a ground line which will then be located on, under or in the partially wetting layer.

In an alternative embodiment, possibly in combination with the previous one, the device comprises a second track positioned opposite to and separated from the first track so that a space, intended to be filled by an immiscible electrically insulating fluid opposite the transported drop, is formed between the first and second tracks, the second track comprising a non-wetting layer directly in contact with the space thus formed. This non-wetting layer of the second track is possibly partially wetting. This non-wetting layer is also possibly covered with an upper layer which is either electrically insulating, semiconductive, or conductive.

In another embodiment, the second track comprises one or more counter-electrodes located between the non-wetting layer and the upper layer. It optionally comprises an insulating dielectric layer which will be located between said non-wetting layer and said one or more counter electrodes.

Optionally, in combination with each of these alternative embodiments of the device, the partially wetting layer of the first track and / or the second track comprises non-wetting areas and wetting areas, the wetting areas being reactive functionalized areas.

In another variant embodiment, the device for handling drops in a plane of the invention comprises two tracks separated by a space intended to be filled by an immiscible electrically insulating fluid with respect to the drop carried. The first track comprises an electrically insulating layer or substrate on which at least two interdigitated electrodes are based. On this set rests a non-wetting layer. The second track includes a partially wetting layer. The partially wetting layer of the first track and / or the second track includes non-wetting areas and wetting areas, the wetting areas being reactive functionalized areas.

Optionally, in this variant embodiment, the first track also comprises an insulating dielectric layer between the electrodes and the non-wetting layer. Optionally also, the device in this embodiment variant comprises a ground line located on, under or inserted into the non-wetting layer.

In an alternative embodiment, the second track comprises an electrically insulating, semiconductive, or conductive upper layer.

In combination with each of these embodiments of the device, the electrically insulating substrate of the first track is preferably transparent, such as a glass substrate.

Preferably, in one or more of the preceding embodiments, the wetting zones are biochemically functionalized and reactive.

These wetting zones are preferably openings in non-wetting areas. Preferably, the non-wetting material constituting the non-wetting layer and / or the non-wetting areas of the partially wetting layer is a tetrafluoroetylene polymer.

Thus, the device of the invention advantageously makes it possible to manipulate a drop of liquid, by transporting it on a plane by electrowetting, on a single track or between two tracks facing each other, with or without the use of a counter-electrode, while acting chemically on the drop as it passes over chemically functionalized zones. The desired optimization is thus obtained: concentrate the preparatory treatments for a subsequent analysis in a microsystem, during transport, to avoid contamination and losses on very expensive samples and in small volumes, while taking into account the aforementioned constraints microfluidics.

According to a second aspect, the invention relates to a method of manufacturing the aforementioned device, in which the creation of the partially wetting layer of the first or second track is derived from the so-called "lift off" technique used in microelectronics for create metal patterns. This technique of the "lift off" as it is known, if it allows the deposition of the non-wetting layer in the last step, thus avoiding a detrimental surface treatment, is however not suitable for creating patterns in such a way. non-wetting material, in particular a hydrophobic material, such as a tetrafluoroetylene polymer, because it does not allow to obtain sharp and precise wetting zones in this non-wetting material. The invention thus relates, according to this second aspect, to a manufacturing method of the aforementioned device, in which the creation of the partially wetting layer of the first or second track comprises the following steps: creation of a mask of photosensitive material by depositing the photosensitive material on a substrate, photolithography, then revealing the photosensitive material; depositing a non-wetting material on the mask; at least one annealing before dissolution; dissolution of the mask; at least one annealing after dissolution.

In an alternative embodiment, the annealing temperature before dissolution is lower than the annealing temperature after dissolution.

In another variant of implementation, the first annealing before dissolution is followed by at least one other annealing at a temperature greater than that of the first annealing.

In another variant, possibly in combination with the previous one, the first annealing after dissolution is followed by at least one other annealing at a temperature higher than that of the first annealing.

Optionally, the dissolution of the mask is followed by rinsing.

In another variant embodiment, the nonwetting material deposited is a tetrafluoroetylene polymer.

Thus, the method of the invention advantageously allows the creation of a partially wetting layer which contains sharp and precise wetting zones, adapted to a chemical functionalization, and which contains non-wetting areas which retain their high nonwetting properties necessary for transport of drops. Indeed, the layer of non-wetting material is deposited in the last step and does not undergo surface treatment, so does not undergo any change in its surface properties.

The invention finally relates, according to a third aspect, to a microfluidic analysis system of a liquid sample which comprises at least one means for preparing the sample, coupled to at least one device for handling of gout according to the invention and as mentioned above, itself coupled to at least one means of analysis.

Preferably, the preparation means comprises one or more tanks or loading docks.

Also preferably, the analysis means is a mass spectrometer, a fluorescence detector, a UV emission detector, or IR.

The system according to the invention is optionally integrated into a microsystem that integrates itself one or more laboratory operations usually performed manually, and that will be called micro-laboratory.

Thus, the system according to the invention advantageously makes it possible to analyze liquid samples after preparation of the samples and then transport by displacement of calibrated microvolumes to an analyzer, by automating the preparation and transport tasks integrated into a microlaboratory. It thus advantageously makes it possible to reduce the risks of contamination and loss of material of the sample, and to reduce the reaction times.

• figures la à 1d : illustre schématiquement la propriété de non mouillabilité ou de mouillabilité d'une surface vis-à-vis d'une goutte,
• figures 2a à 2r : représentent schématiquement différentes variantes de réalisation du dispositif selon l'invention (vues en coupe perpendiculaire au sens de déplacement de la goutte),
• figure 3 : représente schématiquement le déplacement d'une goutte sur une piste du dispositif selon une première variante de réalisation,
• figure 4 : représente schématiquement le déplacement d'une goutte sur une piste du dispositif selon une deuxième variante de réalisation,
• figure 5 : représente schématiquement le procédé de création d'ouvertures dans un matériau non mouillant selon la technique photolithographique classique utilisant un surfactant dans la résine,
• figure 6 : représente schématiquement le procédé de création d'ouvertures dans un matériau non mouillant selon la technique photolithographique classique avec modification de surface par plasma,
• figure 7 : représente schématiquement une variante de mise en oeuvre du procédé de création d'ouvertures dans un matériau non mouillant selon l'invention,
• figure 8 : illustre schématiquement la fonctionnalisation chimique d'une zone mouillante,
• figure 9 : illustre schématiquement le traitement chimique d'une goutte d'un échantillon au cours de son déplacement,
• figure 10 : représente schématiquement une variante de réalisation du système selon l'invention.

Other characteristics and advantages of the invention will appear more clearly and in a complete manner on reading the following description of the preferred variants of implementation of the method and of the embodiment of the device, which are given as non-exemplary examples. and with reference to the following appended drawings:

• FIGS. 1a to 1d: schematically illustrates the property of non-wettability or wettability of a surface with respect to a drop,
• Figures 2a to 2r : schematically represent different embodiments of the device according to the invention (sectional views perpendicular to the direction of movement of the drop),
• figure 3 : schematically represents the displacement of a drop on a track of the device according to a first variant embodiment,
• figure 4 represents schematically the displacement of a drop on a track of the device according to a second variant embodiment,
• figure 5 : schematically represents the process of creating openings in a non-wetting material according to the photolithographic technique classic using a surfactant in the resin,
• figure 6 : schematically represents the process of creating openings in a non-wetting material according to the conventional photolithographic technique with plasma surface modification,
• figure 7 : schematically represents an alternative embodiment of the method for creating openings in a non-wetting material according to the invention,
• figure 8 : schematically illustrates the chemical functionalization of a wetting zone,
• figure 9 : illustrates schematically the chemical treatment of a drop of a sample during its displacement,
• figure 10 : schematically represents an alternative embodiment of the system according to the invention.

The Figures 2a to 2r schematically represent different embodiments of the device of the invention (sectional views perpendicular to the direction of movement of the drop).

In these Figures 2a to 2n , the device comprises at least one track with a substrate 1, preferably but not necessarily transparent, for example Pyrex ^© . Above this substrate 1 are the interdigital electrodes 2. The notion of interdigital electrodes will be specified later with reference to the figures 3 and 4 .

On these electrodes 2, there is an insulating dielectric layer 3, consisting for example of oxides or polymers. On this insulating electrical layer 3 is a non-wetting layer 4, which is rendered partially wetting by the method of creating wetting apertures 5 in the non-wetting material 4, a process which will be described in more detail a little later with reference to the figure 7 .

In the variant embodiments of Figures 2a to 2d , the device comprises a single track consisting of layers 1, 2, 3 and 4. The device of the figure 2a makes it possible to implement a displacement by electrowetting that does not require counter-electrodes, a displacement which will be explained later with reference to the figure 3 . The devices of figures 2b each have a counter-electrode in the form of a line of mass 6 placed on the partially wetting layer 4 ( figure 2b ), inserted in and not covered by the partially wetting layer 4 ( Figure 2c ), or inserted into and covered by the partially wetting layer 4 ( figure 2d ). The devices of Figures 2b to 2d enable them to implement the electrowetting displacement with a ground line for counter-electrode, a displacement which will be described later with reference to the figure 4 .

The figures 2e and following show alternative embodiments in which is added a second track formed of a non-wetting layer 7 itself covered with an upper layer 8 which can be either electrically insulating or electrically semiconductive or electrically conductive. This second track is placed opposite the first, with use of shims 9 to maintain a displacement space 10 to be filled with an immiscible electrically insulating fluid with respect to the drop carried.

It should be noted that, to implement an electrowetting displacement, the fluid filling the space 10 must effectively be electrically insulating. Moreover, in order not to interact with the drop carried, the fluid must actually be immiscible with respect to the liquid. It may be for example air or oil, in the case of a drop of aqueous solution.

In particular, Figures 2f to 2h show alternative embodiments which are respectively based on the devices of the Figures 2b to 2d to which we add a second track as described above.

In the variant embodiment of the device of the figure 2i , the second track further comprises one or more counter-electrodes 11 inserted between the non-wetting layer 7 and the upper layer 8. Therefore, no ground line is used, unlike the devices of FIGS. Figures 2f to 2h since the counter-electrode is present in the second track. The mode of travel is, however, identical to that of Figures 2f to 2h .

The variant embodiments of the Figures 2j to 2l (sectional views perpendicular to the direction of displacement of the drop) respectively derive directly from the variants of embodiment of the Figures 2f to 2h , with the following difference: the non-wetting layer 7 of the second track is rendered partially wetting by the method of creating wetting openings 5 in the non-wetting material 7 which will be described later with reference to the figure 7 .

The figure 2m describes an alternative embodiment based on that previously described in the figure 2e with the following difference: the non-wetting layer 7 of the second track is rendered partially wetting by the method of creating wetting openings 5 in the non-wetting material 7 which will be described later with reference to the figure 7 .

The variant embodiment of the figure 2n is derived from the variant embodiment of the figure 2i with the following two differences: the non-wetting layer 7 of the second track is rendered partially wetting by creating wetting openings 5 in the non-wetting layer 7 according to the method which will be described later with reference to the figure 7 ; and, to allow the biochemical functionalization of these wetting apertures without interactions with the counter-electrode (s) 11, an insulating dielectric layer 12 similar to that present in the first track is inserted between the partially wetting layer 7 and the counter-seal (s). electrodes 11.

The variant embodiment described in the figure 2o relates to a device with two tracks. The first track differs from the first track of the preceding embodiments in that the non-wetting layer 4 which constitutes it is not partially wetting: no wetting opening is created in this non-wetting layer 4. Moreover, this variant embodiment does not require an insulating dielectric layer between the interdigital electrodes 2 and the non-wetting layer 4 in the case where this non-wetting layer 4 is itself electrically insulating. This is particularly the case for a hydrophobic layer made of a material such as a tetrafluoroethylene polymer. However, in practice, such a material is effectively electrically insulating only if the thickness of the layer is important (thickness of about one micrometer). Also, in the cases of figure 2o where the thickness of the non-wetting layer 4 is not sufficient, it is possible to insert between the interdigital electrode layer 2 and the non-wetting layer 4 an insulating dielectric layer of the type of the layer 3 of the other figures.

On the non-wetting layer 4 is a ground line 6 acting as a counter-electrode. In this variant embodiment, the second track is identical to that of the variants of embodiment of Figures 2j to 2m .

In the respective embodiments of the Figures 2p and 2q the non-wetting layer 4 is not partially wetting since it does not comprise the openings 5. These variant embodiments are therefore derived respectively from the variants of the Figures 2k and 2l , with the aforementioned difference (layer 4 totally non-wetting, whereas in the variants of Figures 2k and 2l it is partially wetting).

Finally, the variant embodiment of the figure 2r resumes the mode of movement of Figures 2a, 2e and 2m , that is to say without the use of a counter-electrode, and, as in the variants of the Figures 2o to 2p , has a non-wetting layer 7 in the second track which is partially wetting with the presence of wetting openings 5, and a non-wetting layer 4 in the first track which is completely non-wetting since it has no wetting opening. Moreover, as in the variant of figure 2o this variant embodiment does not require an insulating dielectric layer between the interdigital electrodes 2 and the non-wetting layer 4 in the case where this non-wetting layer 4 is itself electrically insulating, which is particularly the case for a hydrophobic layer, material such as a tetrafluoroethylene polymer. However, here again, in practice, such a material is effectively electrically insulating only if the thickness of the layer is important (thickness of about one micrometer). Also, in the cases of figure 2r where the thickness of the non-wetting layer 4 is not sufficient, it is possible to insert between the interdigital electrode layer 2 and the non-wetting layer 4 an insulating dielectric layer of the type of the layer 3 of the other figures.

The figure 3 schematically represents the displacement of a drop on a track of the device according to an alternative embodiment. This figure is broken down into two parts. In the upper part (diagrams A, B and C), for the sake of simplification and to facilitate explanation, the representation of the device is a representation in top view and partial in that it does not show the non-wetting layer or partially wetting or the insulating dielectric layer, located between the drop 15 and the electrodes 2a, 2b, 2c and 2d. In the lower part (diagrams A ', B' and C '), the representation of the device is a representation in section from the side, in the direction of movement of the drop.

More precisely, the device is of the type of that of figure 2a , that is to say with only one track. However, the following explanations concerning the displacement of gout are applicable more generally to the cases of Figures 2a, 2e , 2m and 2r , that is to say a displacement on a track with interdigital electrodes, without counter-electrodes, possibly with a second upper plane.

The device therefore requires several interdigitated electrodes (2a, 2b, 2c, 2d) which rest on an electrically insulating substrate 1, possibly transparent. On the layer of interdigitated electrodes, there is an insulating dielectric layer 3 and a non-wetting layer 4. This non-wetting layer 4 can be partially wetting according to the configuration in which one finds oneself (see figure 2 concerned), which does not change the following explanations concerning displacement. The drop 15 is initially on the electrode 2a (step A). By creating a potential difference between the electrode 2c and the electrodes 2a, 2b and 2d, the drop moves on the electrode 2c (step B). To move it on the electrode 2d, a potential difference is created between the electrode 2d and the electrodes 2a, 2b and 2c. And so on.

The figure 4 schematically represents the displacement of a drop on a track of the device according to another embodiment variant. Here again, the figure is divided into two parts. In the upper part (diagrams A, B and C), with the same concern for simplification and facilitation of the explanation as for the figure 3 , the representation of the device is a representation in top view and partial in that it does not show either the non-wetting layer or partially wetting or insulating dielectric layer, located between the drop 15 and the electrodes 2a, 2b, 2c and 2d. In the lower part (diagrams A ', B' and C '), the representation of the device is a representation in section from the side, in the direction of movement of the drop.

More specifically, the device presented corresponds to a device with a single track and a ground line 6 as a counter-electrode 6, as previously described in FIG. figure 2b . However, the following explanations concerning the displacement of a droplet on this device are also applicable to the cases of FIGS. 2c, 2d, 2f, 2g, 2h, 2d, 2k, 2d, 2d, 2d, 2d .

The device comprises a layer of interdigital electrodes (2a, 2b, 2c, 2d) which rest on an electrically insulating and optionally transparent substrate 1. Above this layer of electrodes is an insulating dielectric layer 3. Above this insulating dielectric layer 11 is a non-wetting layer 4. This layer is optionally partially wetting, depending on the configuration in which it is located ( see figures 2 ). Above this non-wetting layer 4 (possibly partially wetting), there is an electrode of mass 6 or ground line 6.

The drop 15 is initially on the electrode 2a (step A). By creating a potential difference between the electrode 2c and the electrodes 2a, 2b, 2d and the ground electrode 6, the drop moves on the electrode 2c (step B). To move the drop on the electrode 2d, a potential difference is created between the electrode 2d and the electrodes 2a, 2b, 2c and the ground electrode 6, and so on.

If one replaces the ground electrode 6, or ground line 6, with a counter-electrode located in a higher plane (in the case of FIGS. 4i and 4n), the preceding explanations concerning the figure 4 still apply.

The method making it possible to partially wetting the non-wetting layer of one of the tracks of the device of the invention will now be described with reference to FIG. figure 7 , with a reminder of the state of the art with reference to figures 5 and 6 .

The figure 5 schematically represents the steps of the creation process opening in a non-wetting material, which renders it partially wetting, according to the conventional photolithographic technique with surfactant. In step (a), a layer of non-wetting material 4 is deposited on a substrate 1. In step (b), a layer of resin 20 containing a surfactant is deposited on the non-wetting layer 4. The surfactant allows to increase the wettability of the non-wetting layer with respect to the resin, so the attachment of the resin on this layer. In step (c), photolithographic step itself, the layer 20 is subjected to UV radiation. If the layer 20 is in so-called positive resin, the ultraviolet radiation causes a rupture of the macromolecules of the exposed zones, which gives these zones an increased solubility to the developing solvent which will be used in step (d), while the non insolated on the opposite will have polymerized. This is what happens with the result of the revelation step (d). The revelation of the resin is accompanied by an attack of the exposed non-wetting material and thus the appearance of the zones or openings 5 in the non-wetting layer 4 (step (e)). This technique is accompanied by the risk of permanently modifying the surface properties of the non-wetting material due to the use of the surfactant in the resin.

The figure 6 schematically represents the steps of the method of creating openings in a non-wetting material according to the conventional photolithographic technique with plasma. This technique differs from the previous one in that it comprises an additional step of subjecting the non-wetting layer 4 to plasma-argon radiation (step (b)) before the deposition of the resin layer 20. which will modify the surface properties of the non-wetting layer 4, whereas in the previous technique ( figure 5 ), it is the presence of surfactant in the resin that plays this role. The following steps ((c), (d), (e), (f)) are respectively the same as steps (b), (c), (d) and (e) of the figure 5 . The conclusion is the same as for the conventional photolithographic technique with surfactant, namely that there is a risk of definitive modification of the surface properties of the non-wetting layer 4.

The process of the invention, now described with reference to the figure 7 , is therefore a method of manufacturing one or more tracks of the device described above, wherein the creation of the partially wetting layer first comprises a step of creating a mask of photosensitive material by depositing a layer of this material On a substrate 1 (step (a)), photolithography (step (b)), and revealing the photosensitive material (step (c)). In the implementation variant described in this figure 7 a negative resin is used as the photosensitive material, that is to say for which the UV radiation causes a polymerization of the insolated zones resulting in increased solubility of the unexposed areas in the developer. It is therefore the areas not exposed to step (b) that disappear in step (c), while the areas exposed to step (b) remain present in step (c) and are marked by the number 20. The choice of a negative resin is of course not limited to the invention. The considerations of the process of the invention are exactly the same in the case of the use of a positive resin.

Step (c) is followed by a step (d) of depositing a layer of non-wetting material 4.

• résine AZ 4562,
• révélation dans AZ 351 B.

By way of example, it is possible to use for the photolithographic step a resin with the following parameters:

• AZ resin 4562,
• revelation in AZ 351 B.

The step (d) of deposition of the non-wetting material 4 is followed by a first annealing step. Depending on the material chosen (tetrafluoroethylene polymer for example), it is possible to anneal at 50 ° C. for 5 minutes. Preferably, but not necessarily, this annealing is followed by another complementary annealing. This other annealing can then be carried out at a temperature of 110 ° C. for 5 minutes also.

In the particular case of a hydrophobic material such as a tetrafluoroethylene polymer, at this stage, very little solvent remains in the material. But it will take a second annealing step after dissolution of the resin mask 20 (step (e) .In fact, at the annealing temperatures of the hydrophobic material, the resin polymerizes which makes it difficult to remove. leave traces of resin on the substrate. These traces may be difficult or impossible to remove during the next dissolution step, which may change the surface properties of the partially wetting layer (partially hydrophilic in the case of wettability with respect to water ): the openings 5 may not be perfectly non-wetting (or hydrophobic for non-wettability with respect to water) and unopened areas may not be perfectly non-wetting (hydrophobic). Therefore, before proceeding to this second annealing step, we will first dissolve the resin for example in acetone, for example for 30 to 40 seconds. Preferably, but not necessarily, this dissolution step is followed by a rinsing step for example with alcohol.

Finally, the second annealing step is carried out, for example (depending on the material chosen) at 170 ° C. for 5 minutes, which has the effect of completely removing the solvent that may be present in the hydrophobic material. Optionally, to obtain a uniform surface and a maximum adhesion of the non-wetting material on the substrate, another complementary annealing is carried out, for example at 330 ° C. for 15 min.

Thus, the method of the invention advantageously makes it possible to create a partially wetting layer in a non-wetting material. This result is achieved by creating openings 5 in the non-wetting material, which become wetting areas, adapted to chemical or biochemical functionalization. The unopened areas remain perfectly non-wetting and thus retain their high nonwetting properties necessary for the transport of drops. In particular, the fact that the layer of non-wetting material is deposited in the last stage of the process, unlike the state of the art, makes it possible not to subject this surface treatment material (technique using a surfactant, or using a plasma-argon).

The device of the invention therefore comprises at least one layer made partially wetting by creating wetting openings in a non-wetting layer, as explained above. These wetting zones will be able to be activated and chemically functionalized ( figure 8 ) then react with the manipulated gout ( figure 9 ). We will therefore use the principle of displacement of the drop as explained above to activate the areas not yet functionalized with a drop 15 containing an agent for functionalization.

We see in particular on the figure 8 (representation mode identical to that of the upper part of figures 3 and 4 in top view and in partial view, that is to say without the respectively insulating and non-wetting dielectric layers between the interdigitated electrodes 2a, 2b, 2c and taste 15) that a drop containing an agent allowing the functionalisation 15 to leave of the electrode 2a moves on the electrode 2b, above a functionalizable zone 5 and then arrives on the electrode 2c after having activated and chemically functionalized the zone 5.

In the figure 9 (representation mode identical to that of the upper part of figures 3 and 4 , in top view and partially, that is to say without the dielectric layers respectively insulating and non-wetting between the interdigital electrodes 2a, 2b, 2c and the drop 15), we see how a drop 15 moving on the track is first on the electrode 2a and then passes on the electrode 2b above which is the functionalized zone 5, and arrives, modified, on the electrode 2c after reaction with the functionalized zone.

The figure 10 schematically represents an alternative embodiment of the system according to the invention. The system comprises one or more means 100 for preparing the liquid sample to be analyzed, one or more drop-handling devices 200 according to the invention and as explained above, and one or more means 300 for analyzing the output. . The means 100 of preparation may comprise for example one or more tanks or loading docks. The analysis means 300 may for example be a mass spectrometer, a fluorescence detector or a UV emission detector. The device 200 according to the invention, at the heart of this system, is coupled upstream with the means or means 100 of preparation, and downstream with the means 300 of analysis.

The system according to the invention can thus be optionally integrated in a microsystem that itself integrates one or more laboratory operations usually performed manually. Such a system is called microlaboratory.

Two examples of functionalization will now be described on the basis of an exemplary embodiment of the device of the invention comprising a substrate of Pyrex ^©, nickel conductive electrodes interdigitated with a thickness of one hundred nanometers, a layer about one micrometer of resin SU8 deposited by centrifugation, insulating dielectric layer. Finally, the device comprises a hydrophobic layer of tetrafluoroethylene polymer, also deposited by centrifugation, on the previously mentioned resin layer.

Example of an affinity reactor:

The areas not covered by the hydrophobic layer will undergo a surface treatment to transform them into a reactive surface, for example a grafted-NH 2 support Streptavidin.

Thus, with such a device comprising such functionalized zones, a drop of liquid containing proteins for example, and moving in the path of electrodes on a functionalized zone, will see its molecules of interest (certain proteins such as biotin for example). ) having an affinity for previously grafted surfaces during functionalization, attach to these surfaces. When the chemical reaction is complete, the drop continues its way in the device. Thereafter, the passage of a specific mixture (for example a denaturing buffer mixture) on these zones makes it possible to release the molecules of interest (by destroying the non-covalent interactions for example) and carries them with him. Such a device thus makes it possible to isolate and separate molecules of interest.

Example of a digestive reactor

In the device, the areas not covered by the hydrophobic layer will undergo a surface treatment in order to transform them into reactive surfaces, for example trypsin-grafted support-NH2.

Thus, in such a device with such functionalized zones, a drop of liquid moving in the electrode path is immobilized on a functionalized zone, and certain molecules of interest (proteins for example). will react with the grafted surfaces. The result of such a reaction will be to cut the molecules (for example peptides obtained by tryptic digestion). Subsequently, the drop continues its path in the device. Such a device therefore makes it possible, for example, to analyze long chains of molecules by preliminary cutting by means of specific enzymes, for mass spectrometry analysis.

The device; the method, and the system of the invention, therefore make it possible to produce the basic elements of a microsystem intended to move microdrops from one functionalized zone to another, in an architecture which lends itself perfectly to integration. upstream or downstream with other complementary functions. We can thus design specialized microsystems distinguished only from each other by the sequence and the nature of the biochemical operations performed.

The whole of the description above is given by way of example, and is not limiting of the invention. In particular, the choice of a tetrafluoroethylene polymer material for the non-wetting or partially wetting layer is not limiting of the invention. A tetrafluoroethylene polymer is a suitable choice in that it is effectively non-wetting, especially, but not only, with respect to water, therefore hydrophobic. More generally, we will always focus on a non-wetting material, which is biocompatible (does not adsorb material transported, does not mix with the transported material, does not cause chemical reactions, does not release material). It must therefore be neutral with regard to the preceding explanations, and also present a homogeneity of its properties on the surface.

Similarly, the choice of silicon or Pyrex ^© for the substrate is of course not limit the invention. This is also the case of the choice of a positive or negative resin in the context of the manufacturing process of the device of the invention. It will also be noted, again in the context of the manufacturing process of the device of the invention, that the temperatures and times of the annealing steps of the process are not limiting of the invention, and are essentially a function of the nonwetting material chosen. Also, the use of acetone for dissolution and an alcohol for rinsing, is not limiting of the invention. Any other product suitable for dissolution and rinsing may be used.

In addition, the examples of displacement in a given direction, mentioned in this description, are not limiting of the invention. One can of course consider a displacement matrix to move the drop anywhere on the track. The displacement possibilities depend essentially on the geometrical arrangement of the electrodes. An array of electrodes makes it possible to obtain a displacement of matrix type. Also, the shape of the electrodes in the examples of this description is of course not limiting of the invention. Any other form allowing the interdigitation of the electrodes is suitable.

Moreover, the list of examples of preparation means upstream of the displacement device, in an integrated system such as the system of the invention, is of course not exhaustive, and therefore not limiting of the invention. The same is true for the list of analysis means downstream of the displacement device.

Finally, the examples of functionalization of the wetting zones of the partially wetting layer, and the examples of treatment of the drop by these functionalized zones, given in this description, are not limiting of the invention. Generally, we will focus on the separation, sorting or cutting of any kind of molecules. Other manipulations by chemical and / or biochemical reactions are possible.

Claims (25)

1. A device for handling drops on a displacement by electrowetting plane, including at least one track, characterised in that the said track includes:

   an electrically insulating substrate (1) with a top surface,

   at least two first conducting electrodes (2, 2a to 2d) with a top surface and a bottom surface, resting by their bottom surface on the said top surface of the said electrically insulating substrate, with each of the said first electrodes (2, 2a to 2d) being interdigitated with at least one other of these said first electrodes (2, 2a to 2d),

   a dielectric insulating layer (3) with a bottom surface and a top surface, resting by its bottom surface on the said top surface of the said first electrodes,

   a partially-wetting layer (4) with a bottom surface and a top surface, resting by its bottom surface on the top surface of the said dielectric insulating layer, said partially-wetting layer (4) consisting in a non-wetting material layer comprising wetting openings (5).
2. A device according to claim 1, characterised in that it includes at least one counter-electrode (6) separate from the said first electrodes (2, 2a to 2d).
3. A device according to claim 2, characterised in that the said separate counter-electrode (6) is an earth line (6) located on or under the top surface of, or inserted into, the said partially-wetting layer (4).
4. A device according to anyone of claims 1 to 3, characterised in that it includes a second track positioned opposite to and separated from the first track, so that a space (10) is formed between the said first and second tracks, with the said second track including a non-wetting layer (7) with a bottom surface on one side of the said space and a top surface on the other side.
5. A device according to claim 4, characterised in that the said non-wetting layer of the said second track is a partially-wetting layer consisting in a non-wetting material layer comprising wetting openings (5).
6. A device according to either of claims 4 and 5, characterised in that the said second track includes a top layer (8) that is electrically insulating, semiconducting or conducting, located on one side of the top surface of the said non-wetting layer (7).
7. A device according to any of claims 4 to 6, characterised in that the said second track includes one or more counter-electrodes (11) located between the said non-wetting layer and the said top layer (8).
8. A device according to claim 7, characterised in that the said second track includes a dielectric insulating layer (12) located between the said non-wetting layer (7) and the said counter-electrode(s) (11).
9. A device for handling drops between two displacement by electrowetting planes, including two tracks separated by a space (10), characterised in that:

   the first track includes:

   an electrically insulating substrate with a top surface,

   at least two first electrodes (2, 2a to 2d) with a top surface and a bottom surface, resting by their bottom surface on the said top surface of the said electrically insulating substrate (1), with each of the said first electrodes (2, 2a to 2d) being interdigitated with at least one other of these said first electrodes (2, 2a to 2d),

   a non-wetting layer (4) with a bottom surface and a top surface, located on one side of the top surface of the said first electrodes (2, 2a to 2d),

   the second track includes :

   a partially-wetting layer (7) with a top surface and

   a bottom surface,

   where the said partially-wetting layer (7) of said second track consisting in a non-wetting material layer comprising wetting openings (5).
10. A device according to claim 9, characterised in that the said first track includes a dielectric insulating layer (3) located between the top surface of the said first electrodes (2, 2a to 2d) and the bottom surface of the said non-wetting layer (4).
11. A device according to either of claims 9 and 10, characterised in that it includes an earth line (6) located on or under the top surface of, or inserted into, the said non-wetting layer.
12. A device according to any of claims 9 to 11, characterised in that the said second track includes a layer (12) that is electrically insulating, conducting or semiconducting, located on one side of the top surface of the said non-wetting layer (7).
13. A device according to any of claims 1 to 12, characterised in that the said electrically insulating substrate of the said first track is transparent.
14. A device according to claim 13, characterised in that the said electrically insulating substrate of the said first track is a glass substrate.
15. A device according to any of claims 1 to 14, characterised in that the said wetting zones formed by the wetting openings (5) in the non-wetting material of the partially-wetting layer (4, 7) of first and/or second track are reactive functionalised zones.
16. A device according to claim 15, characterised in that the said wetting zones are biochemically functionalised and reactive
17. A device according to any of claims 1 to 16, characterised in that the said non-wetting layer (4, 7) and/or the said non-wetting zones of the said partially-wetting layer (4, 7), are non-wetting in relation to water and therefore hydrophobic, and in that the said wetting zones are wetting in relation to water and therefore hydrophilic.
18. A device according to any of claims 1 to 17, characterised in that the said non-wetting layer (4, 7) and/or the said non-wetting zones of the said partially-wetting layer (4, 7) are in tetrafluoroethylene polymer.
19. A method for the manufacture of the device according to any of claims 1 to 18, in which the creation of the said partially-wetting layer (4, 7) of the said first track or the said second track, consisting in a non-wetting material layer comprising wetting openings (5) ; includes:

    a step for the creation of a mask in a photosensitive material, by deposition of the said photosensitive material onto a substrate, then photolithography, and then development of the said photosensitive material,

    a step for the deposition of a non-wetting material onto the said mask,

    at least one annealing stage before dissolution,

    a step for the dissolution of the said mask,

    at least one annealing step after dissolution.
20. A method according to claim 19, characterised in that the annealing temperature of the said annealing step before dissolution is lower than the annealing temperature of the said annealing stage after dissolution.
21. A method according to any of claims 19 and 20, characterised in that the said stage for the deposition of a non-wetting material onto the said mask is a step for the deposition of a tetrafluoroethyene polymer.
22. A system for the microfluidic analysis of a liquid sample, characterised in that it includes:

    at least one means (100) for preparing the liquid sample with at least one outlet,

    at least one drop handling device (200) according to any of claims 1 to 18, coupled by one of its inlets to one of the outlets of the said preparation means (100), and with at least one outlet,

    at least one analysis means (300) coupled by one of its inlets to one of the outlets of the said drop handling device (200).
23. A system according to claim 22, characterised in that the said preparation means includes one or more loading reservoirs or docks.
24. A system according to any of claims 22 and 23, characterised in that the said analysis means is a mass spectrometer, a fluorescence detector, or a UV light detector.
25. A system according to any of claims 22 to 24, characterised in that it is integrated into a microlaboratory.

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