Device For Handling Drops For Biochemical Analysis, Method For Producing Said Device And A System For Microfluidic Analysis

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 subject of this present invention is a drop handling device that is intended for biochemical analysis, a method for the manufacture of such a device, and a microfluidic analysis system using such a device.

Nowadays, the new technologies allow the design of systems of micrometric and nanometric dimensions and with very high levels of complexity. Ideally, these systems come with all sorts of features, and are used in many areas such as biology or biochemistry. In particular, protoemics, an activity associated with the identification and the study of proteins, attempts to use the new technologies to reduce the sampled volumes being manipulated, and to reduce contamination. Generally speaking, the objective is to control the microhandling of the material, before spectrometric analysis for example.

In such microsystems, the problem of controlling the fluid flows arises from a strategic viewpoint, to the extent that the material, such as proteins for example, cannot be manipulated other than in a liquid medium. The invention therefore relates to the area of microfluidics, which more generally concerns flows in systems of micrometric or nanometric dimensions, in which the manipulated sample can subjected to electric fields or to partitioning effects of a complex physical or chemical nature, and in which the high area/volume ratio is very important.

In this area, the reduction in the size of the systems results in a reduction in the volumes and the reaction times or in shorter exchanges, and the ability to integrate several modules with different features such as transportation, treatment, or indeed analysis, all on a single wafer of silicon for example.

In order to transport the liquid, two types of fluidic displacement are generally possible, namely the pumping of a continuous flow, and the displacement of calibrated microvolumes. The displacement of calibrated microvolumes has a certain number of advantages. In fact, it allows very small liquid volumes and allows an appropriate control of the flow of the microvolumes, while continuous-flow pumping is characterised by a constant flow. In addition, this type of displacement allows a variety of synchronisations that allow mixing of the liquids for example. In order to achieve fluidic displacement of the displacement of calibrated microvolumes type, different methods of operation are known, such as by pneumatic action, by acoustic surface waves, by dielectrophoretic effect, by electrowetting, and by electrowetting on dielectric (EWOD). This last a method makes use of a relatively simple technological system and allows control of the flow and the circulation of a calibrated volume of conducting liquid in a network of electrodes.

American patent U.S. Pat. No. 6,565,727, and the publication by Cho et al of “Particle separation and concentration control for digital microfluidic systems”, are known in particular, describing the displacement of drops by electrowetting, as described above. However, the devices described in these publications have a bottom part that includes electrodes and a top part that includes counter-electrodes, with the drop moving between these parts. This top part in particular renders the device more bulky and more complex.

In addition, the manipulated samples are often very precious and in very small quantities. There is therefore a requirement to optimise handling of these samples, by chemical treatment or by interacting with the material during transportation. The known microfluidic displacement systems, whether they necessitate two facing substrates or a single substrate, and whether they use a counter-electrode or not, do not permit this optimisation. In fact, in particular in the publication of Cho et al. entitled “Particle separation and concentration control for digital microfluidic systems”, a device is proposed which allows interaction with the drop physically, by direct interaction between the electrodes and the drop during transportation, and not chemical interaction. This chemical interaction, which is necessary for optimising handling of the samples, is therefore impossible in the device of Cho et al.

In fact, this optimisation is rendered extremely difficult by the fact that the displacement requires one or more tracks in hydrophobic materials in order to limit friction and hysteresis in the displacements. This hydrophobic character of the displacement track in particular prevents chemical treatment of, or interaction with, the material during transportation.

It should be noted here that one is generally more interested in the non-wettability property of the displacement track in relation to any given liquid. When the liquid is aqueous, as is generally the case when one is handling proteins for example, the non-wettability and the wettability in relation to water are the properties of hydrophobicity and hydrophilicity respectively. A hydrophobic material is a non-wetting material in relation to water, and a hydrophilic material is a wetting material in relation to water. Wettability is generally characterised by the angle (θ) of contact between the drop (1) and the surface (2) (see FIGS. 1a to 1d). Use is sometimes made of the wettability coefficient, defined as the cosine of the aforementioned angle. Perfect wettability thus corresponds to a wettability coefficient of 1, and so to θ=0°. Total absence of wettability then corresponds to a wettability coefficient of −1 (minus 1), and so to θ=180°. In what follows, we will therefore speak of wetting material in relation to a liquid for a material whose wettability coefficient in relation to this liquid tends to 1 (without necessarily being equal to 1), as illustrated in FIG. 1a, and we will speak of non-wetting material in relation to a liquid for a material whose wettability coefficient in relation to this liquid tends to −1 (without necessarily being equal to −1) as illustrated in FIG. 1b. FIGS. 1c and 1d illustrate intermediate cases of wettability (θ<90°) or non-wettability (θ>90°) respectively.

The problem posed by the non-wetting materials in relation to a liquid, in particular the hydrophobic materials, also essential to the displacement, is that the surface properties of these materials prevent the creation of surface chemical treatment zones due to the fact that these materials are characterised by a low surface energy. If one tries to functionalise the surface of such materials locally, which would allow chemical treatment of the manipulated liquids, the result is not very reliable, difficult to control and too imperfect. The alternative, consisting of rendering the non-wetting material more rigorous in relation to the liquid, is not an option since it eliminates the ability of the material to favour the transportation of the liquid. It is therefore necessary to use a layer of material which is partially wetting, meaning that it is necessary to maintain the non-wetting character for the displacement, while also creating wetting zones or zones of high wettability for the functionalisation.

Applied to the particular case where the material concerned is hydrophobic, one is particularly aware of two conventional photolithographic techniques for the creation of a partially hydrophobic layer by the creation of openings in a hydrophobic material, where these openings become hydrophilic zones distributed in the hydrophobic layer. In a first technique (FIG. 5), also used by Cho et al. in “Particle separation and concentration control for digital microfluidic systems”, after the deposition of a layer of hydrophobic material onto a substrate, a layer of photosensitive resin is deposited which contains a surface-active agent, a chemical substance used to increase the wettability of a surface in relation to a liquid. This technique in particular poses the problem of definitive pollution of the hydrophobic material and therefore loss of the ability of this material to favour the displacement of a liquid. In the second technique (FIG. 6), after deposition of a layer of hydrophobic material onto a substrate, and before deposition of a photosensitive resin, the layer of hydrophobic material is first subjected to surface modification by means of a plasma, in order to alter its hydrophobic properties, meaning to render it less hydrophobic. This technique also poses the problem of definitive alteration of the surface properties of the hydrophobic material.

With such techniques, either the openings created, and therefore the hydrophilic zones, are not sufficiently distinct and precise, possible with hydrophobic deposits, and as a consequence unsuitable for the creation of chemically functionalised zones, or the hydrophobic zones will have their properties modified and their hydrophobic character diminished, with consequent unsuitability for the displacement of liquid. The same comments apply in the case of application of these techniques for the creation of wetting zones in a layer that is non-wetting in relation to the liquid transported.

There is therefore a requirement for a method which can be used to render a non-wetting transportation track partially wetting in relation to the liquid transported, and in particular partially hydrophilic when the liquid is a solution containing water, so that the ability to transport the drop of liquid is maintained, while also allowing chemical treatment or interaction with this drop during transportation.

More generally, there exists the need for a reliable solution which can be used to overcome the aforementioned drawbacks, in particular the optimisation of displacement and the manufacture of an optimised displacement track.

The purpose of the invention is therefore to overcome these drawbacks. To this end, the invention relates, according to a first aspect, to a device for handling of a drop in a displacement by electrowetting plane, which includes at least one displacement by electrowetting track, and which allows chemical treatment of, or interaction with, the drop simultaneously with its transportation.

The displacement track includes at least two interdigitated electrodes which rest on an electrically insulating substrate and which are covered by an insulating dielectric layer. This assembly of insulating substrate, electrodes, and insulating dielectric layer, is covered with a layer that is partially wetting in relation to the manipulated drops.

In an implementation variant concerning handling of drops containing water, the partially-wetting layer is therefore a partially-hydrophilic layer.

In the remainder of the description, and in order to simplify the description, we will speak of layers or materials that are respectively non-wetting, partially-wetting, or wetting, to mean layers or materials that are respectively non-wetting, partially-wetting, or wetting in relation to the manipulated drops.

In another implementation variant, the device of the invention includes at least one counter-electrode which is separate from the first electrodes. This counter-electrode can be an earth line which will then be located on, under or in the partially-wetting layer.

In an implementation variant, possibly in combination with the preceding one, the device includes a second track positioned opposite to and separated from the first track, so that a space, intended to be filled by an electrically insulating fluid that is non-miscible in relation to the drop transported, is formed between the first and second tracks, with the second track including a non-wetting layer directly in contact with the space thus formed. This non-wetting layer of the second track can possibly be partially wetting. This non-wetting layer is also possibly covered by a top layer which is either electrically insulating, semiconducting, or conducting.

In another implementation variant, the second track includes one or more counter-electrodes located between the non-wetting layer and the top layer. It can also possibly include an insulating dielectric layer which will be located between the said non-wetting layer and the said counter-electrode(s).

Possibly in combination with each of these implementation variants of the device, the partially-wetting layer of the first track and/or of the second track includes non-wetting zones and wetting zones, where the wetting zones are reactive functionalised zones.

In another implementation variant, the device of the invention for handling a drop in a plane includes two tracks separated by a space that is intended to be filled by an electrically insulating fluid which is non-miscible in relation to the drop transported. The first track includes a layer or electrically insulating substrate on which rests at least two interdigitated electrodes. On this assembly rests a non-wetting layer. The second track includes a partially-wetting layer. The partially-wetting layer of the first track and/or of the second track includes non-wetting zones and wetting zones, where the wetting zones are reactive functionalised zones.

In this implementation variant, the first track can possibly also include an insulating dielectric layer located between the electrodes and the non-wetting layer. Possibly also, the device in this implementation variant includes an earth line located on, under or inserted into the non-wetting layer.

In an implementation variant, the second track includes a top layer which is electrically insulating, semiconducting, or conducting.

In combination with each of these implementation variants of the device, the electrically insulating substrate of the first track is preferably transparent, like a glass substrate for example.

In one or more of the preceding variants, the wetting zones are preferably biochemically functionalised and reactive.

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

Thus, the device of the invention advantageously allows handling of a drop of liquid, by transporting it in a plane by electrowetting, on a single track or between two facing tracks, with or without the use of a counter-electrode, while also acting chemically on the drop during its passage through chemically functionalised zones. The desired optimisation is therefore achieved, namely reducing the preparatory treatments to a later analysis in a microsystem, during transportation, in order to avoid contamination and the loss of samples that are very costly and in very small volumes, while also allowing for the aforementioned constraints of microfluidics.

According to a second aspect, the invention relates to a method for the manufacture of the aforementioned device, in which creation of the partially-wetting layer of the first or of the second track is derived from the technique known as “lift off”, used in microelectronics to create patterns in metal. Although this “lift off” technique, as it is commonly known, allows deposition of the non-wetting layer at the last stage, thus avoiding a prejudicial surface treatment, it is not suitable however for the creation of patterns in such a non-wetting material, in particular a hydrophobic material such as a tetrafluoroethyene polymer, since it does not allow the creation of wetting zones that are distinct and precise in this non-wetting material. The invention therefore relates, according to this second aspect, to a method for the manufacture of the aforementioned device, in which the creation of the partially-wetting layer of the first or of the second track includes the following stages: creation of a mask in photosensitive material by deposition of the photosensitive material onto a substrate, followed by photolithography, and then development of the photosensitive material, 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.

In an implementation variant, the temperature of the annealing process before dissolution is lower than the temperature of the annealing process after dissolution.

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

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

The dissolution of the mask can possibly be followed by rinsing.

In another implementation variant, the non-wetting material deposited is a tetrafluoroethyene polymer.

Thus, the method of the invention advantageously allows the creation of a partially-wetting layer which contains wetting zones that are distinct and precise, suitable for chemical functionalisation, and which contains non-wetting zones that retain their enhanced properties of non-wettability, which is necessary for the transportation of drops. In fact, the layer of non-wetting material is deposited at the last stage, is subjected to no surface treatment, and is therefore subjected to no alteration of its surface properties.

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

The preparation means preferably includes one or more loading reservoirs or docks.

The analysis means is also preferably a mass spectrometer, a fluorescence detector, or a detector of UV or IR emissions.

The system according to the invention can possibly be integrated into a microsystem which itself includes one or more laboratory operations that are usually performed manually, and which will be known as microlaboratory operations.

Thus the system according to the invention advantageously allows analysis of the liquid samples, after first preparing the samples and then transporting them by the displacement of calibrated microvolumes to an analyser, by automation of the preparation and transportation tasks, built into a microlaboratory. It therefore advantageously allows the risks of contamination and loss of the material of the sample to be reduced, as well as reducing the reaction times.

Other characteristics and advantages of the invention will appear more clearly and more completely on reading the description that follows of the preferred implementation variants of the method and for creation of the device, provided by way of non-limiting examples and with reference to the following appended drawings:

FIGS. 1a to 1d schematically illustrate the non-wettability or wettability property of a surface in relation to a drop,

FIGS. 2a to 2r schematically represent different implementation variants of the device according to the invention (seen in section perpendicular to the direction of displacement of the drop),

FIG. 3 schematically represents the displacement of a drop on a track of the device according to a first implementation variant,

FIG. 4 schematically represents the displacement of a drop on a track of the device according to a second implementation variant,

FIG. 5 schematically represents the method for the creation of openings in a non-wetting material according to the conventional photolithographic technique using a surface-active agent in resin,

FIG. 6 schematically represents the method for the creation of openings in a non-wetting material according to the conventional photolithographic technique with surface alteration by plasma,

FIG. 7 schematically represents an implementation variant of the method for the creation of openings in a non-wetting material according to the invention,

FIG. 8 schematically illustrates the chemical functionalisation of a wetting zone,

FIG. 9 schematically illustrates the chemical treatment of a drop of a sample during its displacement,

FIG. 10 schematically represents an implementation variant of the system according to the invention.

FIGS. 2a to 2r schematically represent different implementation variants of the device of the invention (seen in section perpendicular to the direction of displacement of the drop).

In these FIGS. 2a to 2n, the device includes at least one track with a substrate 1, preferably but not necessarily transparent, in Pyrex® for example. Above this substrate 1 are located the interdigitated electrodes 2. The notion of interdigitated electrodes will be described later in greater detail with reference to FIGS. 3 and 4.

On these electrodes 2 lies an insulating dielectric layer 3, composed of oxides or polymers for example On this electrically isolating layer 3 lies a non-wetting layer 4, which is rendered partially wetting by the method for the creation of wetting openings 5 in the non-wetting material 4. This method will be described in more detail a little later with reference to FIG. 7.

In the implementation variants of FIGS. 2a to 2d, the device includes a single track composed of layers 1, 2, 3 and 4. The device of FIG. 2a allows the execution of a displacement by electrowetting that does not require counter-electrodes. This displacement will be explained later with reference to FIG. 3. The devices of FIG. 2b each have a counter-electrode in the form of an earth line 6 placed on the partially-wetting layer 4 (in FIG. 2b), inserted into and not covered by the partially-wetting layer 4 (in FIG. 2c), or inserted into and covered by the partially-wetting layer 4 (in FIG. 2d) . For their part, the devices of FIGS. 2b to 2d allow execution of the displacement by electrowetting, with an earth line as the counter-electrode. This displacement will be described later with reference to FIG. 4.

FIG. 2e and those that follow show implementation variants in which a second track is added, formed from a non-wetting layer 7 which itself is covered by a top layer 8 that can be either electrically insulating or electrically semiconducting or indeed electrically conducting. This second track is positioned in relation to the first, with the use of spacers 9 employed to maintain a displacement space 10 that is intended to be filled with an electrically insulating fluid that is non-miscible in relation to the drop transported.

It will be noted that, in order to achieve displacement by electrowetting, the fluid filling the space 10 must actually be electrically insulating. In addition, in order not to interact with the drop transported, the fluid must actually be non-miscible in relation to the liquid. It can be air or oil for example, in the case of a drop of aqueous solution.

In particular, FIGS. 2f to 2h show implementation variants which are respectively based on the devices of FIGS. 2b to 2d, to which a second track is added as described previously.

In the implementation variant of the device of FIG. 2i, the second track also includes one or more counter-electrodes 11 inserted between the non-wetting layer 7 and the top layer 8. There is therefore no earth line, in contrast to the devices of FIGS. 2f to 2h, since the counter-electrode is present in the second track. The displacement method is nevertheless identical to that of FIGS. 2f to 2h.

The implementation variants of FIGS. 2j to 2l (seen in section perpendicular to the direction of displacement of the drop) are derived respectively and directly from the implementation variants of FIGS. 2f to 2h, the only difference being that the non-wetting layer 7 of the second track is rendered partially wetting by the method for the creation of wetting openings 5 in the non-wetting material 7, and which will be described later with reference to FIG. 7.

FIG. 2m describes an implementation variant which is based on that previously described in FIG. 2e, the only difference being that the non-wetting layer 7 of the second track is rendered partially wetting by the method for the creation of wetting openings 5 in the non-wetting material 7, and which will be described later with reference to FIG. 7.

For its part, the implementation variant of FIG. 2n is derived from the implementation variant of FIG. 2i, the only two differences being that the non-wetting layer 7 of the second track is rendered partially wetting by the creation of wetting openings 5 in the non-wetting layer 7 according to the method which will be described later with reference to FIG. 7, and in order to allow the biochemical functionalisation of these wetting openings 5 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-electrode(s) 11.

The implementation variant described in FIG. 2o concerns a device with two tracks. The first track differs from the first track of the previous implementation variants in that the non-wetting layer 4 of which it is composed is not partially wetting, so that no wetting opening is created in this non-wetting layer 4. In addition, this implementation variant requires no insulating dielectric layer between the interdigitated electrodes 2 and the non-wetting layer 4 in the case where this non-wetting layer 4 is itself electrically insulating. This is the case in particular for a hydrophobic layer, in a material such as a tetrafluoroethylene polymer. In practice however, such a material is actually electrically insulating only if the thickness of the layer is substantial (a thickness of the order of a micrometre). In addition, in the case of FIG. 2o where the thickness of the non-wetting layer 4 is not sufficient, it is possible to insert, between the layer of interdigitated electrodes 2 and the non-wetting layer 4, an insulating dielectric layer of the same type as the layer 3 in the other figures.

On the non-wetting layer 4, there is located an earth line 6 acting as a counter-electrode. In this implementation variant, the second track is identical to that of the implementation variants of FIGS. 2j to 2m.

In the respective implementation variants of FIGS. 2p and 2q, the non-wetting layer 4 is not partially wetting since it has no openings (5). These implementation variants are therefore derived respectively from the variants of FIGS. 2k and 2l, with the aforementioned difference (layer 4 totally non-wetting, while in the variants of FIGS. 2k and 2l, it is partially wetting).

Finally, the implementation variant of FIG. 2r again uses the displacement method of FIGS. 2a, 2e and 2m, meaning without the use of a counter-electrode, and, as in the variants of FIGS. 2o to 2p, has a non-wetting layer 7 in the second track which is partially wetting with the presence of the wetting openings 5, and a non-wetting layer 4 in the first track which is totally non-wetting since it has no wetting opening. In addition, as in the variant of FIG. 2o, this implementation variant requires no insulating dielectric layer between the interdigitated electrodes 2 and the non-wetting layer 4 in the case where this non-wetting layer 4 is itself electrically insulating, this being the case in particular for a hydrophobic layer, in a material such as a tetrafluoroethylene polymer. Here again however, in practice, such a material is actually electrically insulating only if the thickness of the layer is substantial (a thickness of the order of a micrometre) . In addition, in the case of FIG. 2r where the thickness of the non-wetting layer 4 is not sufficient, it is possible to insert, between the layer of interdigitated electrodes 2 and the non-wetting layer 4, an insulating dielectric layer of the same type as layer 3 in the other figures.

FIG. 3 schematically represents the displacement of a drop on a track of the device according to an implementation variant. This figure breaks down into two parts. In the top part (diagrams A, B and C), in the interests of simplification and so as to facilitate the explanation, the representation of the device is a representation from above and partial in that it shows neither the non-wetting or partially wetting layer nor the dielectric insulating layer, located between the drop (15) and electrodes 1, 2, 3 and 4. In the bottom part (diagrams A′, B′ and C′), the representation of the device is a representation in section from the side, in the direction of displacement of the drop.

More precisely, the device is of the same type as that of FIG. 2a, meaning with a single track. However, the following explanations concerning the displacement of the drop are applicable more generally to the cases of FIGS. 2a, 2e, 2m and 2r, meaning displacement on a track with interdigitated electrodes, without counter-electrodes, and possibly with a second upper plane.

The device therefore requires several interdigitated electrodes (1, 2, 3, 4) which rest on an electrically insulating substrate 10 that is possibly transparent. On the layer of interdigitated electrodes is located a dielectric insulating layer 11 and a non-wetting layer 12. This non-wetting layer 12 can be partially wetting according to the configuration in which one finds oneself (see FIG. 2 concerned), which does not alter the following explanations concerning the displacement. The drop 15 is initially on electrode 2 (stage A). By creating a potential difference between electrode 3 and electrodes 1, 2 and 4, the drop moves onto electrode 3 (stage B). In order to move it onto electrode 4, a potential difference is created between electrode 4 and electrodes 1, 2 and 3. And so on.

FIG. 4 schematically represents the displacement of a drop on a track of the device according to another implementation variant. Here again, the figure breaks down into two parts. In the top part (diagrams A, B and C), again in the interests of simplification and in order to facilitate explanation as for FIG. 3, the representation of the device is a representation from above, and partial in that it shows neither the non-wetting or partially wetting layer nor the dielectric insulating layer, located between the drop 15 and electrodes 1, 2, 3 and 4. In the bottom part (diagrams A′, B′ and C′), the representation of the device is a representation in section from the side, in the direction of displacement of the drop.

More precisely, the device presented corresponds to a device with a single track and an earth line as the counter-electrode, as previously described at FIG. 2b. However, the following explanations concerning the displacement of a drop on this device are also applicable to the cases of FIGS. 2c, 2d, 2f, 2g, 2h, 2j, 2k, 2l, 2o, 2p, and 2q.

The device includes a layer of interdigitated electrodes (1, 2, 3, 4) which rest on an electrically and possibly transparent insulating substrate 10. Above this layer of electrodes lies a dielectric insulating layer 11. Above this dielectric insulating layer 11 lies a non-wetting layer 12. This layer is possibly partially wetting, depending on the configuration in which one finds oneself (see FIG. 2). Above this non-wetting layer 12 (which is possibly partially wetting) is located an earth electrode earth line.

The drop 15 is initially on electrode 2 (stage A). By creating a potential difference between electrode 3 and electrodes 1, 2, and 4 and the earth electrode, the drop moves onto electrode 3 (stage B). In order to move the drop onto the electrode 4, a potential difference is created between electrode 4 and electrodes 1, 2, and 3 and the earth electrode, and so on.

If the earth electrode or earth line is replaced by a counter-electrode located in an upper plane (the case of FIGS. 4i and 4n), the previous explanations concerning FIG. 4 still apply.

The method used to render 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. 7, with a reminder of previous designs with reference to FIGS. 5 and 6.

FIG. 5 schematically represents the stages of the method for the creation of an opening in a non-wetting material, thus rendering it partially-wetting, using the conventional photolithographic technique with a surface-active agent. At stage (a), a layer of non-wetting material 2 is deposited on a substrate 1. At stage (b), a layer of resin 3 containing a surface-active agent is deposited on the non-wetting layer 2. The surface-active agent is used to increase the wettability of the non-wetting layer in relation to the resin, and therefore the adherence of the resin to this layer. At stage (c), the photolithographic stage proper, layer 3 is subjected to UV radiation. If layer 3 is in resin said to be positive, then the ultraviolet radiation leads to rupturing of the macromolecules of the exposed zones, conferring on these zones increased solubility to the development solvent that will be used at stage (d), while in contrast, the non-insulated parts will be polymerised. This is therefore what happens, with the result of the development stage (d). The development of the resin is accompanied by an attack on the exposed non-wetting material and therefore the appearance of zones or openings 4 in the non-wetting layer 2 (stage (e)). This technique can be accompanied by definitive alteration of the surface properties of the non-wetting material due to the use of the surface-active agent in resin.

FIG. 6 schematically represents the stages of the method for the creation of openings in a non-wetting material using the conventional photolithographic plasma technique. This technique differs from the preceding one in that it includes a complementary stage that consists of subjecting the non-wetting layer 2 to plasma-argon radiation (stage (b)) before deposition of the resin layer. It is this radiation that will alter the surface properties of the non-wetting layer 2, while in the preceding technique (FIG. 5), it is the presence of the surface-active agent in resin that plays this role. The following stages (c), (d), (e), and (f)) are respectively the same as stages (b), (c), (d) and (e) of FIG. 5. The conclusion is the same as that for the conventional photolithographic technique with a surface-active agent, namely that there can be definitive alteration of the surface properties of the non-wetting layer 2.

The method of the invention, now described with reference to FIG. 7, is therefore a method for the manufacture of one or more tracks of the device described previously, in which the creation of the partially-wetting layer first includes a stage for the creation of a mask in photosensitive material by the deposition of a layer of this material 2 onto a substrate 1 (stage (a)), then photolithography (stage (b)), and development of the photosensitive material (stage (c)). In the implementation variant described in FIG. 7, a negative resin is used as the photosensitive material, meaning one in which the UV radiation leads to a polymerisation of the insulated zones, leading to increased solubility of the zones not exposed in the developer. It is therefore the zones not exposed at stage (b) that disappear at stage (c), while the zones insulated at stage (b) remain present at stage (c) and are identified by the number 2. The choice of a negative resin does not limit the invention in any way. The functioning of the method of the invention is exactly the same with the use of a positive resin.

The stage (c) is followed by stage (d) for the deposition of a layer of non-wetting material 3.

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

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

Stage (d) for deposition of the non-wetting material 3 is followed by a first annealing stage. Depending the material chosen (tetrafluoroethylene polymer for example), the annealing process can be at 50° C., and can last for 5 minutes. Preferably, but not necessarily, this annealing process is followed by another complementary annealing process. This second annealing process can then be performed at a temperature of 110° C., also for 5 minutes.

In the particular case of a hydrophobic material such as a tetrafluoroethylene polymer, very little solvent remains in the material at this stage. However a second annealing stage will be needed after dissolution of the resin mask 2 (stage (e). In fact, at the annealing temperatures of the hydrophobic material, the resin polymerises, thus rendering it difficult to remove. The consequence of this can be to leave traces of resin on the substrate. These traces may well be difficult or even impossible to remove during the following dissolution stage, and this can alter the surface properties of the partially-wetting layer (partially hydrophilic in the case of wettability in relation to water). The openings may well not be perfectly non-wetting (or hydrophobic for non-wettability in relation to water) and the zones that are not open may well not be perfectly non-wetting (hydrophobic). This is why, before proceeding to this second annealing stage, the resin will first be dissolved, in the acetone for example, for 30 to 40 seconds for example. Preferably, but not necessarily, this dissolving stage is followed by a rinsing stage, in alcohol for example.

Finally, the second annealing stage is performed, at 170° C. for example (according to the material chosen) for 5 minutes, the result of which is to cause any the solvent that may be present in the hydrophobic material to disappear totally. In order to obtain a uniform surface and maximum adherence of the non-wetting material on the substrate, another complementary annealing process can be effected, at 330° C. for 15 minutes for example.

Thus, the method of the invention advantageously allows the creation of a partially-wetting layer in a non-wetting material. This result is achieved by the creation of openings in the non-wetting material, which then become wetting zones, suitable for chemical or biochemical functionalisation. The zones that are not open remain perfectly non-wetting, and therefore retain their enhanced properties of non-wettability which are necessary for the transportation of drops. In particular, the fact that the layer of non-wetting material is deposited at the last stage of the method, in contrast to previous designs, means that this material will not be subjected to such surface treatment (a technique using a surface-active agent, or using a plasma-argon).

The device of the invention therefore includes at least one layer which is rendered partially wetting by the creation of wetting openings in a non-wetting layer, as explained previously. It will be possible to activate and functionalise these wetting zones chemically (FIG. 8) so as to then react with the manipulated drop (FIG. 9). Use will therefore be made of the principle for displacement of the drop as explained previously in order to activate the zones that are still not functionalised, using a drop containing an agent that allows functionalisation.

It can be seen, in particular in FIG. 8 (method of representation identical to that of the top part of FIGS. 3 and 4, from above and partial, meaning without the insulating dielectric and non-wetting layers respectively, between the interdigitated electrodes and the drop) that a drop containing an agent that allows the functionalisation 15, starting from electrode 1, moves to electrode 2 over a functionalisable zone 5, and then arrives at electrode 3 after having activated and functionalised the zone 5 chemically.

In FIG. 9 (a method of representation identical to that of the top part of FIGS. 3 and 4, from above and partial, meaning without the insulating dielectric and non-wetting layers respectively, between the interdigitated electrodes and the drop), it can be seen how a drop 15 moving on the track rests firstly on electrode 1 and then passes on to electrode 2, above which lies the functionalised zone 5, and arrives, changed, at electrode 3, after reaction with the functionalised zone.

FIG. 10 schematically represents an implementation variant of the system according to the invention. The system includes one or more means 1 for preparation of the liquid sample to be analysed, one or more devices 2 for handling of drops according to the invention and as explained previously, and one or more means 3 for analysis on exiting. The preparation means 1 can include one or more loading reservoirs or docks for example. The analysis means 3 can be a mass spectrometer, a fluorescence detector or a UV light detector for example. The device 2 according to the invention, at the heart of this system, is coupled upstream with the preparation means (s) 1, and downstream with the analysis means (s) 3.

The system according to the invention can be thus possibly be integrated into a microsystem that itself includes one or more laboratory operations usually effected manually. Such a system is known as a microlaboratory.

Two examples of functionalisation will now be described, on the basis of an example of implementation of the device of the invention that includes a substrate in Pyrex®, conducting interdigitated electrodes in nickel with a thickness of about one hundred nanometres, a layer of about one micrometre of SU8 resin deposited by centrifuging, and a dielectric insulating layer. Finally, the device includes a hydrophobic layer in tetrafluoroethylene polymer, also deposited by centrifuging, on the resin layer previously mentioned.

Example of an Affinity Reactor:

The zones not covered by the hydrophobic layer will undergo a surface treatment that is intended to convert them into a reactive surface, such as a Streptavidine grafted NH2 support.

Thus, with such a device, including such functionalised zones, a drop of liquid containing proteins for example, and moving in the path of electrodes over a functionalised zone, will find that its molecules of interest (certain proteins such as biotine for example) with an affinity for the surfaces previously grafted during the functionalisation, fix onto these surfaces. When the chemical reaction has ended, the drop continues on its path in the device. In what follows, the passage of a special mixture (a denaturing buffer mixture for example) in these zones, allows the molecules of interest to be liberated (by destruction of the non-covalent interactions for example) and draws them along with it. Such a device is therefore used to isolate and separate molecules of interest.

Example of a Digestion Reactor:

In the device, the zones not covered by the hydrophobic layer will undergo a surface treatment with the aim of converting them into reactive surfaces, such as a trypsine grafted NH2 support for example.

Thus, in such a device with such functionalised zones, a drop of liquid moving in the path of electrodes is immobilised in a functionalised 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 (peptides obtained by tryptidic digestion for example). In what follows, the drop continues on its path in the device. Such a device therefore allows the analysis of long chains of molecules for example, by prior cutting using specific enzymes, with a view to analysis by mass spectrometry.

The device, the method, and the system of the invention, therefore allow implementation of the basic elements of a microsystem that is intended to move microdroplets from one functionalised zone to another, in an architecture which lends itself readily to integration, upstream or downstream, with other complementary functions. It is therefore possible to design specialised Microsystems that differ from each other only by the chaining and the nature of the biochemical operations effected.

All of the above description is given by way of an example, and does not limit the invention in any way. In particular, the choice of a material in tetrafluoroethylene polymers for the non-wetting or partially wetting layer does not limit the invention. A tetrafluoroethylene polymer is a suitable choice in the sense that it is actually non-wetting, in particular, but not only, in relation to water, and therefore hydrophobic. More generally, one is always looking for a non-wetting material which is biocompatible (does not adsorb any of the material transported, does not mix with the material transported, does not provoke chemical reactions, and does not leech material) . It must therefore be neutral in the light of the preceding explanations, and also display a homogeneity of its surface properties.

Likewise, the choice of silicon or Pyrex® for the substrate does not in any way limit the invention. This is also the case for the choice of a positive or negative resin in the context of the method for the manufacture of the device of the invention. It will also be noted, still in the context of the method for the manufacture of the device of the invention, that the temperatures and times of the annealing stages of the method do not limit the invention, and are essentially a function of the non-wetting material chosen. In addition, the use of acetone for dissolving and of alcohol for rinsing, does not limit the invention. Any other product suitable for dissolving and rinsing can be used.

Furthermore, the examples of displacement in a given direction, mentioned in this description, do not limit the invention. It is naturally possible to envisage a displacement matrix that allows the drop to be moved anywhere on the track. The displacement options depend essentially on the geometric layout of the electrodes. A matrix of electrodes can in fact be used to achieve a displacement of the matricial type. Also, the shape of the electrodes in the examples of this description does not in any way limit the invention. Any other shape allowing interdigitation of the electrodes will be suitable.

In addition, the list of the examples of means for the preparation of the displacement device upstream, in an integrated system such as the system of the invention, is naturally not exhaustive, and therefore does not limit the invention. This also applies to the list of means for analysis of the displacement device downstream.

Finally, the examples of functionalisation of the wetting zones of the partially-wetting layer, and the examples of treatment of the drop by these functionalised zones, given in this description, do not limit the invention. Generally, in fact one is interested in the separation, the sorting or the cutting of molecules, whatever they may be. Other handlings by chemical and/or biochemical reactions can also be envisaged.

Claims

1. A device for handling drops on a displacement by electrowetting plane, including at least one track, wherein said track comprises:

an electrically insulating substrate with a top surface,

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

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

a partially-wetting layer with a bottom surface and a top surface, resting by its bottom surface on the top surface of the said dielectric insulating layer.

2. A device according to claim 1, further comprising at least one counter-electrode separate from said first electrodes.

3. A device according to claim 2, wherein said separate counter-electrode is an earth line located on or under the top surface of, or inserted into, the said partially-wetting layer.

4. A device according to claim 1, further comprising a second track positioned opposite to and separated from the first track, so that a space is formed between said first and second tracks, with said second track including a non-wetting layer with a bottom surface on one side of said space and a top surface on the other side.

5. A device according to claim 4, wherein said non-wetting layer of said second track is partially wetting.

6. A device according to claim 4, wherein said second track includes a top layer that is electrically insulating, semiconducting or conducting, located on one side of the top surface of the said non-wetting layer.

7. A device according to claim 4, wherein said second track includes one or more counter-electrodes located between said non-wetting layer and the said top layer.

8. A device according to claim 7, wherein said second track includes a dielectric insulating layer located between said non-wetting layer and the said counter-electrode(s).

9. A device according to said partially-wetting layer of said first track and/or of the said second track includes non-wetting zones and wetting zones, with said wetting zones being reactive functionalised zones.

10. A device for handling drops between two displacement by electrowetting planes, including two tracks separated by a space, the device comprising: where said partially-wetting layer of the said first track and/or of said second track includes non-wetting zones and wetting zones, and where the said wetting zones are reactive functionalised zones.

the first track includes: an electrically insulating substrate with a top surface, at least two first electrodes with a top surface and a bottom surface, resting by their bottom surface on said top surface of said electrically insulating substrate, with each of said first electrodes being interdigitated with at least one other of these said first electrodes, a non-wetting layer with a bottom surface and a top surface, located on one side of the top surface of the said first electrodes,

the second track includes: a partially-wetting layer with a top surface and a bottom surface,

11. A device according to claim 10, wherein said first track includes a dielectric insulating layer located between the top surface of the said first electrodes and the bottom surface of the said non-wetting layer.

12. A device according to claim 10, further comprising an earth line located on or under the top surface of, or inserted into, the said non-wetting layer.

13. A device according to claim 10, wherein said second track includes a layer that is electrically insulating, conducting or semiconducting, located on one side of the top surface of the said non-wetting layer.

14. A device according to claim 1, wherein said electrically insulating substrate of said first track is transparent.

15. A device according to claim 14, wherein said electrically insulating substrate of said first track is a glass substrate.

16. A device according to claim 9, wherein said wetting zones are openings in non-wetting zones.

17. A device according to claim 9, wherein said wetting zones are biochemically functionalised and reactive.

18. A device according to claim 1, wherein said non-wetting layer and/or said non-wetting zones of said partially-wetting layer, are non-wetting in relation to water and therefore hydrophobic, and said wetting zones are wetting in relation to water and therefore hydrophilic.

19. A device according to claim 1, wherein said non-wetting layer and/or said non-wetting zones of the said partially-wetting layer are in tetrafluoroethylene polymer.

20. A method for the manufacture of the device according to claim 1, in which the creation of said partially-wetting layer of said first track or the said second track includes:

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

at least one annealing stage before dissolution,

a stage for the dissolution of the said mask,

at least one annealing stage after dissolution.

21. A method according to claim 20, wherein the annealing temperature of said annealing stage before dissolution is lower than the annealing temperature of said annealing stage after dissolution.

22. A method according to claim 20, wherein said stage for the deposition of a non-wetting material onto said mask is a stage for the deposition of a tetrafluoroethyene polymer.

23. A system for the microfluidic analysis of a liquid sample, characterised in that it includes:

at least one means for preparing the liquid sample with at least one outlet, at least one drop handling device according to claim 1, coupled by one of its inlets to one of the outlets of the said preparation means, and with at least one outlet,

at least one analysis means coupled by one of its inlets to one of the outlets of the said drop handling device.

24. A system according to claim 23, wherein said preparation means includes one or more loading reservoirs or docks.

25. A system according to claim 23, wherein said analysis means is a mass spectrometer, a fluorescence detector, or a UV light detector.

26. A system according to claim 23, wherein the system is integrated into a microlaboratory.

27. A device according to claim 15, wherein said wetting zones are openings in non-wetting zones.

28. A device according to claim 27, wherein said wetting zones are biochemically functionalised and reactive.

29. A device according to claim 28, wherein said non-wetting layer and/or said non-wetting zones of said partially-wetting layer, are non-wetting in relation to water and therefore hydrophobic, and said wetting zones are wetting in relation to water and therefore hydrophilic.

30. A device according to claim 29, wherein said non-wetting layer and/or said non-wetting zones of the said partially-wetting layer are in tetrafluoroethylene polymer.

31. A method for the manufacture of the device according to claim 10, in which the creation of said partially-wetting layer of said first track or the said second track includes:

a stage 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 stage for the deposition of a non-setting material onto the said mask,

at least one annealing stage before dissolution,

a stage for the dissolution of the said mask,

at least one annealing stage after dissolution.

32. A method according to claim 31, wherein the annealing temperature of said annealing stage before dissolution is lower than the annealing temperature of said annealing stage after dissolution.

33. A method according to claim 31, wherein said stage for the deposition of a non-wetting material onto said mask is a stage for the deposition of a tetrafluoroethyene polymer.

34. A system for the microfluidic analysis of a liquid sample, characterised in that it includes:

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

at least one drop handling device according to claim 10, coupled by one of its inlets to one of the outlets of the said preparation means, and with at least one outlet,

at least one analysis means coupled by one of its inlets to one of the outlets of the said drop handling device.

35. A system according to claim 34, wherein said preparation means includes one or more loading reservoirs or docks.

36. A system according to claim 34, wherein said analysis means is a mass spectrometer, a fluorescence detector, or a UV light detector.

37. A system according to claim 34, wherein the system is integrated into a microlaboratory.