JP4763690B2 - Apparatus for handling droplets for biochemical analysis, method for manufacturing said apparatus and microfluidic 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 subject of the present invention is a device for handling droplets intended for biological analysis, a method of manufacturing such a device, and a microfluidic analysis system using such a device.

  Recent new technologies enable very highly complex designs in the micrometer and nanometer dimensions. Ideally, these systems have all kinds of characteristics and are used in many areas, for example biological or biochemical. In particular, proteomics, a work related to identification and protein research, attempts to reduce the volume of processed samples and to use new techniques to reduce impurities. In general, the aim is to control the microhandling of the material, for example before spectroscopic measurements.

  In such a microsystem, unless a material such as a protein can be processed outside of the liquid medium, the problem of controlling the flow of fluid arises from a strategic point of view. Thus, the present invention relates to the field of microfluidics and relates more generally to flows in micrometer or nanometer dimensional systems, in which samples processed in the flow are of electric field or complex physical or chemical properties. It may be subject to partitioning effects and it is very important that the area / volume ratio be high in the flow.

  In this area, the reduction in the size of the system results in different features such as reduced volume and reaction time or shorter exchange and transport, processing or further analysis of, for example, silicon on a single wafer. Leading to the ability to integrate several modules with

  In order to transport liquids, two types of fluid displacement are generally possible: pumping of continuous fluid and movement of calibrated microvolumes. There are some advantages to moving the calibrated microvolume. In fact, while continuous flow pumping is characterized by a constant flow, the movement of a calibrated microvolume allows the liquid volume to be very small and provides adequate control of the microvolume flow. enable. In addition, this type of movement allows various simultaneous progressions that allow, for example, mixing of liquids. To achieve calibrated micro-volume transfer type fluid transfer, by pneumatic action, by acoustic surface waves, by electrophoretic effects, by electrowetting, and by dielectric electrowetting ( The operation of different methods according to EWOD) is known. This last method uses a relatively simple technical system and allows flow control and circulation of a calibrated volume of conductive liquid within the electrode network.

  US Pat. No. 6,565,727 and Cho et al's “Particle separation and concentration control for digital microfluidic systems” are particularly known for describing the movement of droplets by electrowetting as described above. However, the devices described in these publications have a bottom portion containing the electrodes and a top portion containing the counter electrode, and the droplet moves between these portions. This upper part in particular makes the device bulkier and more complex.

  In addition, the samples that are processed are often very expensive and very small. Accordingly, there is a need for optimizing the handling of these samples by chemical processing or by interacting with the material during transport. Already known microfluidic transfer systems, whether they require two opposing substrates or a single substrate and whether they use or do not use a counter electrode In any case, it does not allow optimization. In fact, especially in the publication entitled “Particle separation and concentration control for digital microfluidic systems” by Cho et al, not by chemical interaction but by direct interaction between electrodes and droplets during transport. A device is proposed that allows to physically interact with the droplet. Thus, this chemical interaction required to optimize sample handling is not possible in the Cho et al apparatus.

  In fact, this optimization is made very difficult by the fact that movement requires one or more paths in the hydrophobic material to limit friction and hysteresis in movement. The hydrophobic nature of this transfer path prevents, inter alia, chemical processing of the material during the transport or interaction with the material.

  It should be mentioned that, in general, we are interested in the non-wetting nature of the travel path for a given liquid. For example, as is common when dealing with proteins, when the liquid is aqueous, the non-wetting properties and wetting properties with respect to water are hydrophobic and hydrophilic properties, respectively. Hydrophobic materials are materials that do not wet with water, and hydrophilic materials are materials that wet with water. Wettability is generally characterized by the contact angle (θ) between the droplet (1) and the surface (2) (see FIGS. 1a to 1d). Often a wetting constant defined as the cosine of the aforementioned angle is used. Thus, complete wettability corresponds to a wetting constant of 1 and corresponds to θ = 0 °. And when there is no wettability, it corresponds to a wetting constant −1 (minus 1) and θ = 180 °. In the following, we discuss the wetting material with respect to the liquid for materials that tend to have a wetting constant for this liquid of 1 (not necessarily equal to 1) as illustrated in FIG. 1a and illustrated in FIG. 1b. As will be discussed, wetting materials are discussed with respect to liquids for materials that tend to have a wetting constant for this liquid of -1 (not necessarily equal to -1). FIGS. 1 c and 1 d illustrate intermediate cases of wettability (θ <90 °) or non-wetting (θ> 90 °), respectively.

  The problem presented by non-wetting materials for liquids, especially hydrophobic materials essential for movement, is due to the fact that the surface properties of these materials are characterized by the low surface energy of these materials, It prevents the generation of the surface chemical treatment region. When trying to functionalize the surface of such materials locally to allow chemical treatment of the liquid being processed, the results are very unreliable, uncontrollable and very incomplete . It is not an option to construct the non-wetting material more strictly with respect to the liquid, since it eliminates the performance of materials that prefer liquid transport. It is therefore necessary to use a layer of material that is partially wettable, which also creates a wettable or highly wettable region for functionalization, while maintaining non-wetting for movement It means that.

  Two conventional photolithographic techniques for creating a partially hydrophobic layer by forming openings in the hydrophobic material are particularly known, applied in special cases where the relevant material is hydrophobic. These openings become hydrophilic regions dispersed in the hydrophobic layer. In the first technique (FIG. 5), it is also used in Cho et al's “Particle separation and concentration control for digital microfluidic systems”, after depositing a layer of hydrophobic material on top of the substrate and then wetting the surface with respect to the liquid A layer of a photosensitive resin containing a chemical, a surfactant, is used that is used to increase the viscosity. This technique presents, inter alia, the problem of final contamination of the hydrophobic material and, consequently, the reduction of the performance of this material, which is advantageous for liquid movement. In the second technique (FIG. 6), a layer of hydrophobic material is first formed by a plasma process to change its hydrophobic properties after deposition of the hydrophobic material layer on top of the substrate and before deposition of the photosensitive resin. Which means that the hydrophobicity of the layer is reduced. This technique also presents the problem of ultimately changing the surface properties of the hydrophobic material.

With such a technique, both the openings that are formed and the resulting hydrophilic regions are not sufficiently clear and precise, and consequently chemically modified, that is possible with hydrophobic deposition. Inappropriate for the generation of regions, or hydrophobic regions are altered in their properties, and hydrophobicity is reduced, resulting in instability with respect to liquid movement. Similar explanations apply for the application of these techniques to the creation of wetted areas in layers that are non-wetting with respect to the transported liquid.
US Pat. No. 6,565,727

  Accordingly, there is a need for a method that can be used to partially wett a non-wetting transport path with respect to the liquid being transported, especially if the liquid is water so that the ability to transport droplets is retained. Partially hydrophilic when in solution, while also allowing chemical treatment during transport or interaction with droplets.

  More generally, there is a need for a reliable solution that can be used to overcome the aforementioned drawbacks, particularly the optimization of movement and the production of optimized movement paths.

  The object of the present invention is therefore to overcome these drawbacks. To achieve this object, the present invention, in a first aspect, relates to an apparatus for the handling of liquid in movement by an electrowetting surface, which comprises at least one movement by an electrowetting path, Allows chemical treatment or interaction with droplets simultaneously with their transport.

  The travel path includes at least two interdigitated electrodes that are disposed on an electrically isolated substrate and are covered by an insulating dielectric layer. The insulating substrate, electrode, and insulating dielectric substrate assembly is covered with a layer that is partially wettable with respect to the droplet being manipulated.

  In embodiments relating to the handling of droplets containing water, the partially wettable layer results in a partially hydrophilic layer.

  For the purpose of description reminders and for the sake of brevity, we mean layers or materials that are each non-wetting, partially wettable, or wettable with respect to the manipulated droplets. Layers or materials that are each non-wetting, partially wettable, or wettable.

  In other embodiments, the device of the present invention includes at least one counter electrode independent of the first electrode. The counter electrode may be a ground wire that is placed on top, below or inside the partially wettable layer.

  Certain embodiments may be combined with the previous embodiments, where the device is immiscible with the liquid to be transported and the spaces intended to be filled by an electrically insulating fluid are first and first. As a result, a second path including a non-wetting layer is formed, including a second path disposed opposite to the first path and independent of the first path, so as to be formed between the two paths. Direct contact with the space. This non-wetting layer of the second path may be partially wettable. This non-wetting layer may be covered by a top layer that is either electrically insulating, semi-conductive, or conductive.

  In other embodiments, the second path includes one or more counter electrodes disposed between the non-wetting layer and the top layer. It may also include an insulating dielectric layer that will be disposed between the non-wetting layer and the counter electrode.

  Although may be combined with these device embodiments, the partially wettable layer of the first path and / or the second path includes a non-wetting region and a wetting region, the wetting region being a reactive functionalized region. is there.

  In other embodiments, the apparatus of the present invention for handling in-plane droplets is separated by a space intended to be filled with an electrically insulating fluid that is immiscible with the transported droplets. Including two routes. The first path includes a layer having at least two interdigitated electrodes disposed thereon, or an electrically insulating substrate. A non-wetting layer is located on this assembly. The second path includes a partially wettable layer. The partially wettable layer of the first path and / or the second path includes a non-wetting region and a wetting region, the wetting region being a reactive functionalized region.

  In this embodiment, the first path may include an insulating dielectric layer disposed between the electrode and the non-wetting layer. The device of this embodiment may include a ground wire that is inserted and placed above, below or within the non-wetting layer.

  In certain embodiments, the second path includes an electrically insulative, semiconductor, or conductive top layer.

  When combined with each of these embodiments of the device, the electrically insulating substrate of the first path is preferably transparent, such as a glass substrate.

  In one or more embodiments described above, the wettability region is preferably biochemically functionalized and reactive.

  These wettability regions are preferably openings of non-wettable regions. The non-wetting material that constitutes the non-wetting region of the non-wetting layer and / or the partial wetting layer is preferably a tetrafluoroethylene polymer.

  Thus, the device of the present invention chemically acts on a droplet in its path through a chemically functionalized region while using electrowetting on a single path or two opposing paths. Between, the handling of the droplets is advantageously made possible by transporting the droplets on a plane, with or without the use of a counter electrode. While taking into account the aforementioned limitations in microfluidics, the desired optimization is achieved, i.e. avoiding the contamination of impurities and the loss of expensive and very small volume samples during transport. Reduce pre-processing for later analysis in the microsystem.

  According to a second aspect, the present invention relates to a method of manufacturing the aforementioned device, wherein the generation of a partially wettable layer of the first or second path is used in microelectronics to pattern a metal. Derived from a technique known as “lift off”. Although this commonly known exfoliation technique allows the deposition of a non-wetting layer in the final stage and consequently avoids detrimental surface treatments, this is not the case with hydrophobic materials such as such non-wetting materials, especially tetrafluoroethylene polymers. It is not a technique suitable for pattern formation in a conductive material. This is because this technique does not allow the formation of clear and precise wettability regions within the non-wettable material. Accordingly, the present invention relates to a method for manufacturing the aforementioned device according to this second aspect, wherein the formation of the partially wettable layer of the first or second path comprises the following steps: light on the substrate Formation of a mask of photosensitive material by depositing a sensitive material, subsequent photolithography and subsequent development of the photosensitive material, deposition of non-wetting material on top of the mask, at least one annealing treatment prior to dissolution, mask And at least one annealing treatment after dissolution.

  In some embodiments, the temperature of the annealing process before melting is lower than the temperature of the annealing process after melting.

  In another embodiment, after the first annealing process before melting, another annealing process is subsequently performed at a temperature higher than the temperature in the first annealing process.

  Other embodiments may be combined with the previous embodiments, and after the first annealing treatment after dissolution, at least one other annealing treatment is performed at a temperature higher than the temperature of the first annealing treatment.

  A rinsing step may be performed after dissolution of the mask.

  In other embodiments, the deposited non-wetting material is a tetrafluoroethylene polymer.

  As a result, the method of the present invention includes a clear and rigorous, suitable for chemical functionalization, wettability region, and a non-wettable region that retains the strong non-wetability necessary for droplet transport. Advantageously enables the production of partially wettable layers, including In fact, the layer of non-wetting material is deposited at the final stage and is not surface treated, and as a result is not subject to changes in its surface properties.

  The present invention, in a third aspect, comprises at least one sample preparation means finally combined with at least one droplet handling device according to the invention, and, as mentioned, at least one analysis means, It relates to a coupled system for microfluidic analysis of sample liquids.

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

  The analytical technique is preferably a mass spectrometer, a fluorescence detector, or a UV or IR emission detector as well.

  The system according to the invention is usually implemented manually and may be integrated inside a microsystem, which itself contains one or more experimental operations, known as microlaboratory operations.

  As a result, the system according to the invention automates the preparation and transport tasks incorporated in the microlab after the first preparation of samples and their transport to the analyzer by subsequent movement of the calibrated microvolume. This advantageously allows analysis of liquid samples. As a result, the risk of contamination and the loss of sample material are advantageously made possible as well as reducing the reaction time.

  Other features and advantages of the present invention will be more apparent upon reading the description according to the preferred embodiment of the method and of the formation of the apparatus, provided by way of non-limiting illustration, and provided with reference to the attached drawings below. It will be clearer and more complete.

  In FIGS. 2a to 2n, the device comprises at least one path in the substrate 1 and is preferably (although not necessary) transparent, for example Pyrex. On the substrate 1, the interdigitated electrodes 2 are arranged. The concept of interdigitated electrodes is described in more detail later with reference to FIGS.

  An insulating dielectric layer 3 made of, for example, an oxide or a polymer is disposed on these electrodes 2. A non-wetting layer 4 is disposed on the electrically insulating layer 3, and the non-wetting layer 4 is partially wettable using a method of creating a wettable opening 5 in the non-wetting material 4. To be. This method is described in more detail later with reference to FIG.

  In the embodiment of FIGS. 2a to 2d, the device comprises a single path consisting of layers 1, 2, 3 and 4. The apparatus of FIG. 2a allows performing electrowetting movements that do not require a counter electrode. This movement will be described later with reference to FIG. The device of FIG. 2b is in the form of a ground wire 6 placed on a partially wettable layer 4 (FIG. 2b), in a form of being inserted into the partially wettable layer 4 and not covered by the layer ( 2c), or partially inserted into the wettable layer 4 and covered by the layer (FIG. 2d), each having a counter electrode. For those parts, the device of FIGS. 2b to 2d makes it possible to carry out electrowetting movements with the ground wire as the counter electrode. This movement will be described later with reference to FIG.

  Figures after FIG. 2e are covered by an upper layer 8, which can be either electrically insulative, electrically semiconductor, or electrically conductive. An embodiment formed from a non-wetting layer 7 with a second path added is shown. This second path uses a spacer 9 that is used to hold a moving space 10 that is intended to be filled with an electrically insulating fluid that is immiscible with the transported droplets. , Arranged in relation to the first path.

  In order to achieve movement by electrowetting, the fluid filling the space 10 must actually be electrically insulating. In addition, the fluid must actually be immiscible with the liquid in order not to interact with the droplets being transported. For example, it may be air or oil if the droplet is an aqueous solution.

  In particular, FIGS. 2f to 2h each show an embodiment based on the apparatus of FIGS. 2b to 2d, with a second path added as described above.

  In the device embodiment of FIG. 2 i, the second path also includes one or more counter electrodes 11 inserted between the non-wetting layer 7 and the top layer 8. Thus, in contrast to the device of FIGS. 2f to 2h, there is no ground wire. This is because the counter electrode exists in the second path. Nevertheless, the movement method is the same as the method of FIGS. 2f to 2h.

  The embodiment of FIGS. 2j to 2l (seen in the part perpendicular to the direction of droplet movement) is obtained directly from the embodiment of FIGS. 2f to 2h, respectively, and only the non-wetting layer 7 of the second path. However, the only difference is that it is made partially wettable using the method of creating the wettable opening 5 in the non-wetting material 7 and will be described later with reference to FIG.

  FIG. 2 m describes an embodiment based on the embodiment previously described in FIG. 2 e, just by a method for the non-wetting layer 7 of the second path to create a wetting opening 5 in the non-wetting material 7. The only difference is that it is partially wettable and will be described later with reference to FIG.

  On the other hand, the embodiment of FIG. 2n is derived from the embodiment of FIG. 2i, except that the non-wetting layer 7 of the second path wets the non-wetting layer 7 according to the method described later with reference to FIG. In order to be made partially wettable by creating permeable openings 5 and to allow biochemical functionalization of these wettable openings 5 without interaction with the counter electrode 11 In addition, the only difference is that an insulating dielectric layer 12 similar to the one existing in the first path is inserted between the partially wettable layer 7 and the counter electrode 11.

  The embodiment described in FIG. 2o relates to two paths. The non-wetting layer 4 constituting the first path is not partially wettable, there is no wettability opening generated in the non-wetting layer 4, and the first path of the above-described embodiment is different from the first path. Different. In addition, in this embodiment, the non-wetting layer 4 does not require an insulating dielectric layer between the interdigitated electrode 2 and the non-wetting layer 4 if it is electrically insulating itself. This is particularly the case for the hydrophobic layer, and the material is tetrafluoroethylene polymer or the like. In practice, however, such materials are electrically insulating only if the layer thickness is sufficient (thickness on the order of micrometers). In addition, in the case of FIG. 2o, when the thickness of the non-wetting layer 4 is insufficient, the same type as that of the layer 3 in other figures is provided between the layer of the interdigitated electrode 2 and the non-wetting layer 4. It is possible to insert an insulating dielectric layer.

  On the non-wetting layer 4, a ground wire 6 serving as a counter electrode is disposed. In this embodiment, the second path is the same as in the embodiment of FIGS. 2j to 2m.

  In each of the embodiments of FIGS. 2p and 2q, the non-wetting layer 4 is not partially wettable because it does not have an opening 5. As a result, these embodiments are derived from the embodiments of FIGS. 2k to 2l respectively, and the differences described above (layer 4 is partially wettable in the embodiments of FIGS. Are all non-wetting.

  Finally, the embodiment of FIG. 2r utilizes the movement method of FIGS. 2a, 2e and 2m, which means that no counter electrode is used, and as in the embodiments of FIGS. 2o to 2p, the wettability There is a non-wetting layer 7 in the second path that is partially wettable in the presence of the opening 5, and the non-wetting layer 4 in the first path does not have a wettable opening and is completely non-wetting. It is wettability. In addition, as in the embodiment of FIG. 2o, this embodiment is used when the non-wetting layer 4 is itself electrically insulating, especially when it is a hydrophobic layer, such as a tetrafluoroethylene polymer. No insulating dielectric layer is required between the interdigitated electrodes 2 and the non-wetting layer 4. In practice, however, such materials are electrically insulating only if the thickness of the layer is sufficient (thickness order is micrometers). In addition, in the case of FIG. 2r where the thickness of the non-wetting layer 4 is insufficient, an insulating dielectric of the same type as the layer 3 in the other figures between the interdigitated electrode layer 2 and the non-wetting layer 4 It is possible to insert a body layer.

  FIG. 3 schematically illustrates the movement of a droplet on the path of a device according to an embodiment. This figure is divided into two parts. In the upper part (FIGS. A, B and C), the device is represented from above and partly to facilitate simplification and explanation, the droplet (15) and the electrodes 1, 2, 3 and None of the non-wetting or partially wettable layers or dielectric insulating layers disposed between 4 are shown. In the lower part (Figures A ', B' and C '), the device is represented by the part from the side of the direction of droplet movement.

  More particularly, the device is of the same type as in FIG. 2a, which means it has a single path. However, the subsequent description of droplet movement is more generally applicable to the embodiments of FIGS. 2a, 2e, 2m and 2r, meaning movement on a path with interdigitated electrodes and facing It may have no electrode and may have a second top plane.

  Thus, the device requires several electrodes (1, 2, 3, 4) disposed on the substrate 10, which may be electrically insulating and transparent. A dielectric insulating layer 11 and a non-wetting layer 12 are disposed on the interdigitated electrode layer. This non-wetting layer 12 may be partially wettable according to various arrangements (see related FIG. 2), which does not change the following description of movement. The droplet 15 is initially on the electrode 2 (stage A). By creating a potential difference between the electrode 3 and the electrodes 1, 2 and 4, the droplet moves to the top of the electrode 3 (stage B). In order to move it to the top of the electrode 4, a potential difference is created between the electrode 4 and the electrodes 1, 2 and 3. And so on.

  FIG. 4 schematically illustrates the movement of a droplet on the path of an apparatus according to another embodiment. Again, the figure is divided into two parts. In the upper part (FIGS. A, B and C), for simplicity, the representation of the device is from above and in part for ease of explanation with respect to FIG. Neither the non-wettable or partially wettable layer disposed between the electrodes 1, 2, 3, and 4 nor the dielectric insulating layer is shown. In the lower part (FIGS. A ', B' and C '), the device is represented by the part from the side with respect to the direction of droplet movement.

  More particularly, the device shown corresponds to a device having a single path and a ground wire as a counter electrode, as previously described in FIG. 2b. However, the subsequent description of droplet movement on this device is equally 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) disposed on a substrate 10, which may be electrically insulating and transparent. A dielectric insulating layer 11 is disposed on the electrode layer. A non-wetting layer 12 is disposed on the dielectric insulating layer 11. This layer may be partially wettable depending on various arrangements (see FIG. 2). An earth electrode ground wire is disposed on the non-wetting layer 12 (which may be partially wettable).

  The droplet 15 is initially on the electrode 2 (stage A). By creating a potential difference between the electrode 3, the electrodes 1, 2 and 4, and the ground electrode, the droplet moves to the top of the electrode 3 (stage B). In order to move the droplet to the upper part of the electrode 4, a potential difference is generated between the electrode 4, the electrodes 1, 2 and 3, and the ground electrode.

  The ground electrode or ground wire is replaced by a counter electrode arranged in the top plane (in the case of FIGS. 4i and 4n), and the above description with respect to FIG. 4 still applies.

  The method used to partially wett one non-wetting layer of the path of the device of the present invention will be described with reference to the previous design with respect to FIGS. 5 and 6 and with reference to FIG. .

  FIG. 5 schematically represents the steps of a method of creating an opening in a non-wetting material and consequently making it partially wettable using conventional photolithographic techniques using a surfactant. In step (a), a layer of non-wetting material 2 is deposited on the substrate 1. In step (b), a layer of resin 3 containing a surfactant is deposited on the non-wetting layer 2. Surfactants are used to increase the wettability of the non-wetting layer in relation to the resin and consequently to increase the adhesion of the resin to this layer. In step (c), layer 3, which is suitable for the photolithographic step, is exposed to UV radiation. If the resin layer 3 is positive, the UV radiation will lead to the destruction of the polymer in the exposed areas, increasing the solubility of these areas in the developer used in step (d). On the other hand, the uncovered parts are polymerized. This is what happens in the development stage (d). The development of the resin is accompanied by a strong action on the exposed non-wetting material and consequently the appearance of the regions or openings 4 in the non-wetting layer 2 (step (e)). This technique is achieved by a distinct change in the surface properties of the non-wetting material due to the use of surfactants in the resin.

  FIG. 6 schematically represents method steps for the creation of openings in non-wetting materials using conventional photolithographic plasma technology. This technique differs from that described above in that it includes a complementary step consisting of subjecting the non-wetting layer 2 to plasma-argon radiation (step (b)) prior to the deposition of the resin layer. In the technique described above (FIG. 5), it is this radiation that alters the surface properties of the non-wetting layer 2 while this role is the presence of the surfactant in the resin. The subsequent steps (c), (d), (e) and (f) are the same as steps (b), (c), (d) and (e) in FIG. The conclusion is the same as for conventional photolithographic techniques with surfactants, i.e. a clear change in the surface properties of the non-wetting layer 2.

  The method of the present invention described in connection with FIG. 7 results in a method relating to the production of one or more paths of the aforementioned device, which method produces a partially wettable layer, First, generation of a mask of photosensitive material by deposition of this layer of material 2 on top of the substrate 1 (step (a)), subsequent photolithography implementation (step (b)), and development of the photosensitive material Including the step of performing (step (c)). In the embodiment described in FIG. 7, the negative resin, which means that UV radiation results in polymerization in areas not covered, is used as a light sensitive resin and the solubility of areas not exposed in the developer. Leads to an increase in As a result, the area that was covered in step (b) remains in step (c) and is represented by the number 2, whereas it is not exposed in step (b) and the step ( It is an area excluded in c). The selection of the negative resin does not limit the present invention in any way. The functionalization of the method of the present invention is exactly the same as in the use of positive resins.

  After step (c), step (d) relating to the deposition of a layer of non-wetting material 3 is carried out.

According to an example relating to the photolithographic stage, it is possible to use a resin with the following parameters:
-Resin AZ4562
-AZ351B developer

  After step (d) relating to the deposition of non-wetting material 3, a first annealing step is performed. Depending on the material selected (eg, tetrafluoroethylene polymer), the annealing process may be 50 ° C. and may last for 5 minutes. Preferably, but not necessary, this annealing process is followed by another complementary annealing process. This second annealing process may then be performed at a temperature of 110 ° C. for 5 minutes. In the special case of hydrophobic polymers such as tetrafluoroethylene polymer, there is little solvent left in the material at this stage. However, the second annealing step is required after the resin mask 2 is dissolved (step (e)). In fact, at the annealing temperature of the hydrophobic material, the resin polymerizes, making it difficult to remove. As a result, there is a possibility that a resin mark on the substrate remains. These traces can be difficult or even impossible to remove during the following dissolution stages, which is the surface of the partially wettable layer (partially hydrophilic in the case of wettability with water) There is a possibility of changing the characteristics. The opening may not be completely non-wetting (or hydrophobic, which is non-wetting with respect to water), and the unopened region may not be completely non-wetting (hydrophobic). This is because the resin is first dissolved in, for example, 30 to 40 seconds, for example in acetone, before proceeding with the second annealing step. Preferably, although not necessary, a rinsing step after this dissolution step is performed, for example with alcohol.

  Finally, a second annealing step is carried out, for example at 170 ° C. (depending on the material selected), for 5 minutes, so that any solvent that may be present in the hydrophobic material , Completely excluded. Other complementary annealing processes, for example 15 minutes at 330 ° C., can be effective to obtain a uniform surface and a state of maximum non-wetting material deposition on the substrate.

  As a result, the method of the present invention advantageously enables the creation of a partially wettable layer in a non-wetting material. This result is achieved by the creation of openings in non-wetting materials that are suitable for chemical or biochemical functionalization, where they become wettable regions. The unopened area remains completely non-wetting, and as a result retains the strong non-wetting required for droplet transport. In particular, in contrast to the aforementioned design, the fact that a layer of non-wetting material is deposited at the last stage of the method, this material uses such a surface treatment (surface active agent, or plasma-argon) Technology).

  Accordingly, the device of the present invention includes at least one layer that has been partially wetted by the creation of a wettable opening in the non-wetting layer, as described above. It is possible to chemically activate and functionalize (FIG. 8) these wettability regions to react with subsequently manipulated droplets (FIG. 9). As a result, as described above, the principle of droplet movement is used to activate non-functionalized regions using droplets containing agents that allow functionalization.

  8 (partially shown from above in the same manner of expression as in the upper part of FIGS. 3 and 4, wherein the insulating dielectric layer between the interdigitated electrode and the droplet and In the absence of each non-wetting layer), the droplets containing the agent enabling the functionalization 15 start from the electrode 1 and move over the functionalized region 5 to the electrode 2, It can be seen that the electrode 3 is reached after being chemically activated and functionalized.

  FIG. 9 (shown in a similar manner to the upper part of FIGS. 3 and 4, partially from above, with an insulating dielectric layer and non-wetting between the interdigitated electrode and the droplet In this way, it is assumed that the droplet 15 moving on the path is first located on the electrode 1 and then has a functionalized region 5 on top of it. It can be seen that after proceeding to 2 and reacting with the functionalized region, it reaches the electrode 3 and changes.

  FIG. 10 schematically represents an embodiment of the system according to the invention. The system comprises one or more means 1 for the preparation of the liquid sample to be analyzed, one or more devices 2 as described above for handling droplets according to the invention, and one or more means for analysis at the outlet. 3 is included. The preparation means 1 may comprise for example one or more loading reservoirs or docks. The analysis means 3 may include, for example, a mass spectrometer, a fluorescence detector, or a UV light detector. The device 2 according to the invention, the heart of the invention, is connected to the preparation means 1 upstream and the analysis means 3 downstream.

  The system according to the present invention may thus be integrated within one or more microsystems, including laboratory operations that are usually performed manually.

  An example of functionalization is based on an embodiment of the device of the present invention comprising a Pyrex substrate, an interdigitated electrode of nickel and about 100 nanometers thick, about 1 micrometer of SU8 resin deposited by centrifugation. A layer, and a dielectric insulating layer, described herein. Finally, the device includes a hydrophobic layer of tetrafluoroethylene polymer deposited by centrifugation on the aforementioned resin layer.

[Example of affinity reactor]
Regions not covered by the hydrophobic layer undergo a surface treatment intended to convert the surface to a reactive surface, such as streptavidin-grafted NH ₂ support.

  Thus, in such a device comprising such a functionalized area, for example a droplet containing a protein and moving over the functionalized area of the electrode pathway was grafted during the previous functionalization. We find that the molecules of interest (for example proteins such as biotin) that have an affinity for the surface are immobilized on top of these surfaces. After the chemical reaction is complete, the droplet travels a path through the device. The pathway of a special mixture (eg, denaturing buffer mixture) of these regions then frees the molecule of interest (eg, by breaking non-covalent interactions) and attracts the molecule along it. Such a device is consequently used to separate and identify molecules of interest.

[Example of digestion reactor]
In the device, the areas not covered by the hydrophobic layer are surface treated for the purpose of converting them into a reactive surface, for example NH ₂ alone grafted with trypsin.

  As a result, in such a device having such a functionalized region, droplets traveling in the electrode path are immobilized in the functionalized region and molecules of interest (eg, proteins) are grafted. Reacts with the surface. The result of such a reaction is to cleave the molecule (eg, a peptide obtained by trypsin digestion). The droplet then travels its path through the device. Thus, such a device allows analysis of long-chain molecules, for example, by cutting with a specific enzyme in advance for analysis by a mass spectrometer.

  Thus, the apparatus, method and system of the present invention is a structure that facilitates integration and allows microdroplets to be moved from one functionalized area to another, either upstream or downstream, having other complementary functions. Allows implementation of the basic elements of a microsystem intended to move to Thus, it is possible to design special microsystems that differ from one another only by the nature of the chain and the biochemical manipulation performed.

  All descriptions so far are given by way of example and do not limit the invention in any way. In particular, the selection of the tetrafluoroethylene polymer material for the non-wetting layer or the partial wetting layer does not limit the invention. Tetrafluoroethylene polymer is a suitable choice in the sense that it is actually non-wetting and, in particular, but not exclusively, it is hydrophobic with respect to water. More generally, seeking a non-wetting material that is usually bi-compatible (does not adsorb to any transported material, does not mix with the transported material, and causes a chemical reaction) Without clinging to the material). Therefore, it should be neutral in view of the foregoing description, indicating the uniformity of its surface properties.

  Similarly, the choice of whether the substrate is silicon or Pyrex does not limit the present invention. The same applies to the selection of positive or negative resins in the context of the method for manufacturing the device of the present invention. Further, in the context of the method of manufacturing the device of the present invention, the temperature and time of the annealing stage of the method does not limit the present invention and they are essentially a function of the non-wetting material selected. In addition, using acetone for dissolution and alcohol for rinsing does not limit the invention. Other materials suitable for dissolution and rinsing may be used.

  Further, the illustration of movement in a given direction mentioned in this description does not limit the invention. It is of course possible to recognize a movement matrix that allows droplets to be moved anywhere on the path. The choice of movement depends essentially on the electrode geometry. The matrix of electrodes may actually be used to achieve a matrix type movement. Similarly, the exemplary electrode shapes in this description do not limit the present invention in any way. Any other shape that allows interdigitation of the electrodes is suitable.

  In addition, in an integrated system such as the system of the present invention, it is self-evident that exemplary listings for mobile device upstream preparation are not complete and therefore do not limit the present invention. This also applies to the list of means relating to analysis downstream of the mobile device.

  Finally, the examples of functionalization of the wettability regions of the partially wettable layer and the treatment of the droplets with these functionalized regions given in this description do not limit the invention. In general, in fact, whatever they might be, they are interested in separating, sorting, or cleaving molecules. Other treatments by chemical and / or biochemical reactions are recognized as well.

1a to 1d schematically describe the non-wetting or wetting properties associated with the droplet. 2a to 2r schematically show different embodiments of the device according to the invention (indicated by the part perpendicular to the direction of droplet movement). 1 schematically shows the movement of a droplet on the path of a device according to a first embodiment. 4 schematically shows the movement of a droplet on the path of a device according to a second embodiment. 1 schematically illustrates a method for creating an opening in a non-wetting material by conventional photolithographic techniques using a surface activator in a resin. 1 schematically illustrates a method for the creation of openings in non-wetting materials by conventional photolithographic techniques using plasma surface transformation. 2 schematically illustrates an embodiment of a method relating to the creation of openings in a non-wetting material according to the present invention. The chemical functionalization of the wettability region will be schematically described. The chemical treatment of the sample droplet during movement is schematically described. 1 schematically shows an embodiment of a system according to the invention.

Explanation of symbols

1 Substrate 2 Interdigitated Electrode 3 Insulating Dielectric Layer 4 Non-Wettable Layer 5 Opening

Claims (26)

Including at least one route,
The route is
An electrically insulating substrate having an upper surface;
At least two first conductive electrodes having a top surface and a bottom surface, wherein the bottom surface is disposed on a top surface of the electrically insulating substrate, each of the first electrodes being A first conductive electrode alternately mated with at least one of the other first electrodes;
A dielectric insulating layer having a bottom surface and a top surface, the bottom surface being disposed on the top surface of the first electrode;
Has a bottom surface and an upper surface, seen containing a partially wettable layer its bottom surface is disposed on the upper surface of the dielectric insulating layer, and
The partially wettable layer, a non-wetting region and wettability region characterized by containing Mukoto, handling droplets in movement by electrowetting plane device.   The apparatus of claim 1, comprising at least one counter electrode spaced from the first electrode.   3. The grounded wire, wherein the spaced counter electrode is a ground wire that is inserted above, below, or inside the top surface of the partially wettable layer. The device described.   A second path facing and spaced apart from the first path so that a space is formed between the first path and the second path, the second path including a non-wetting layer; 4. A device according to any one of the preceding claims, characterized in that the bottom surface of the wettable layer is on one side of the space and the top surface is on the other side.   The apparatus of claim 4, wherein the non-wetting layer of the second path is partially wettable.   The second path is electrically insulative, semiconducting, or conductive and includes an upper layer disposed on one of the upper surfaces of the non-wetting layer. Item 6. The device according to Item 4 or 5.   The device according to any one of claims 4 to 6, wherein the second path includes one or more counter electrodes disposed between the non-wetting layer and the upper layer. .   8. The device of claim 7, wherein the second path includes a dielectric insulating layer disposed between the non-wetting layer and the counter electrode. Wherein the pre-Symbol wettability region of the first path and / or said second path is a function of the area of the reactive Apparatus according to any one of claims 1 to 8. Including two paths separated by space,
The first route is
An electrically insulating substrate having an upper surface;
A top surface and a bottom surface, the bottom surface being at least two first electrodes disposed on the top surface of the electrically insulating substrate, each of the first electrodes being the other A first electrode that is alternately fitted with at least one of the first electrodes;
A non-wetting layer having a bottom surface and a top surface and disposed on one surface of the top surface of the first electrode;
The second route is
Including a partially wettable layer having a top surface and a bottom surface;
The partially wettable layer of the first path and / or the second path includes a non-wettable region and a wettable region, and the wettable region is a reactive functionalized region;
A device that handles droplets between two locations by an electrowetting plane.   11. The device of claim 10, wherein the first path includes a dielectric insulating layer disposed between a top surface of the first electrode and a bottom surface of the non-wetting layer. .   12. A device according to claim 10 or 11, characterized in that it comprises a ground wire which is arranged above, below or above the upper surface of the non-wetting layer.   The said second path is electrically insulative, conductive, or semiconductive, and includes a layer disposed on one surface of the upper surface of the non-wetting layer. The apparatus according to any one of 10 to 12.   14. Apparatus according to any one of the preceding claims, characterized in that the electrically insulating substrate of the first path is transparent.   15. The apparatus of claim 14, wherein the electrically insulating substrate in the first path is a glass substrate.   The device according to any one of claims 9 to 15, characterized in that the wettability region is an opening in the non-wettable region.   The device according to any one of claims 9 to 16, characterized in that the wettability region is biochemically functionalized and reactive.   The non-wetting layer and / or the non-wetting region of the partially wettable layer has a hydrophobic property that is non-wetting with respect to water and the wettability layer has a hydrophilic property that is wettable with respect to water. Device according to any one of the preceding claims, characterized in that it comprises a device. The non-wetting region of the non-wetting layer and / or the partially wettable layers, characterized in that it is a Tetorafuruo Roe Chirenporima Apparatus according to any one of claims 1 18. Generation of the partially wettable layer of the first path or the second path;
Forming a mask of the photosensitive material by depositing the photosensitive material on top of the substrate, then performing photolithography, and then developing the photosensitive material;
Depositing a non-wetting material on top of the mask;
At least one stage of annealing treatment prior to dissolution;
Dissolving the mask;
And a step of at least one annealing process after the dissolution, device manufacturing method according to any one of claims 1 to 19.   21. The method of claim 20, wherein the annealing temperature in the annealing stage before melting is lower than the annealing temperature in the annealing stage after melting.   The method according to claim 20 or 21, characterized in that the step of depositing the non-wetting material on top of the mask is the step of depositing a tetrafluoroethylene polymer. At least one means for preparing a liquid sample having at least one outlet;
20. At least one droplet handling device according to any one of claims 1 to 19, wherein one of the inlets is connected to one of the outlets of the preparation means and has at least one outlet;
A system for microfluidic analysis of a liquid sample, characterized in that it comprises at least one analysis means connected to one of the outlets of the droplet handling device by one of its inlets.   24. The system of claim 23, wherein the method of preparation includes one or more loading reservoirs or docks.   25. System according to claim 23 or 24, characterized in that the analysis means is a mass spectrometer, a fluorescence detector or a UV light detector.   26. A system according to any one of claims 23 to 25, characterized in that it is incorporated within a micro laboratory.

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