KR20070053165A - Apparatus for drip manipulation for biochemical analysis, methods for manufacturing the apparatus and systems for micro flow 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

DEVICE FOR HANDLING DROPS FOR BIOCHEMICAL ANALYSIS, METHOD FOR PRODUCING SAID DEVICE AND A SYSTEM FOR MICROFLUIDIC ANALYSIS}

The present invention is directed to a device for drop operation for biochemical analysis, a method of making the device and a system for micro flow analysis using such a device.

Today, new technologies are making it possible to plan micro or nanometer size systems up to critical levels of complexity. Ideally these systems have all sorts of functionality and are used in many fields such as biology and biochemistry. In particular, one protein genetic information, which is active in the identification and study of proteins, attempts to use new techniques to reduce the sample volume to be manipulated and to reduce contamination. In general, the purpose is to control the fine manipulation of materials, for example prior to spectrometer analysis.

In a strategic way in such microsystems, the problem of controlling the flow of fluid is presented as long as a substance such as a protein cannot be manipulated without a liquid support. Thus, the present invention allows the sample to be manipulated to achieve wall-adhesive effects of electric fields or physical or chemical complexes, and flow in more generally micrometer or nanometer size systems, where high surface / volume ratios are of great importance. It refers to the field of one microflow that is related to.

In this field, a reduction in system size results in a reduction in volume, for example on a same silicon wafer, to change or shorten the reaction time, and to move several modules, e.g. This creates the possibility of incorporation into the same other functionality.

In order to move the liquid, two types of transposition are generally possible: pumping of continuous flow and displaced with microvolume. Pre-weighed translocations have several advantages. In practice, this allows very small volumes of liquid and enables control to be applied to the microvolume flow, while pumping continuous streams is characterized by continuous flow. In addition, this type of dislocation allows for a variety of synchronizations to enable, for example, liquid mixtures. Various modes of action are known for realizing a pre-measured fluid volume of a microvolume: by aerodynamic action, by acoustic waves on the surface for dual electrophoresis, electrowetting and electric By wetting (EWOD). The last mode of action allows for a relatively simple technical realization and enables control of the flow and circulation of the metered volume leading to the liquid on the network of electrodes.

In particular known publications, US Pat. No. 6.565.727 and Cho et al., "Particle Separation and Concentration Digital Control of Systems for Microfluidics," describe the translocation of droplets by electrophoresis as described above. However, the device described in these publications shows a small portion of coalescing to the electrode and a large portion of coaling to the counter-electrode remaining between the droplets moving. More than this makes the device more complicated and more complicated, in particular.

In addition, the sample to be manipulated is sometimes very precious and very small. Thus, there is a need to optimize the manipulation of these samples during chemical processing or during interaction with the material between their movements. Known pre-electron microfluidic systems that require two opposing substrates or only one substrate, with or without counter-electrodes, do not enable these optimizations. Indeed, especially in Cho et al., "Particle Separation and Concentration Digital Control of Microfluidic Systems," it is possible to interact physically with droplets by direct interaction between electrodes and droplets between their movements without chemical effects. We propose device to do. The chemical interactions necessary for the optimization of the manipulation of the sample are therefore not possible in a device such as Cho.

Indeed, this optimization is quite subtly due to the fact that the displace requires one or more hydrophobic material tracks to limit wear and hysteresis to the displace. In particular, the hydrophobic nature of the anterior track prevents chemical treatment or prevents material interaction between movements.

It should be appreciated here that it is generally more interesting for the nonabsorbent properties of the pretreatment track for the unpurified liquid. For example, when the liquid is aqueous, as is generally the case with proteins being engineered, the non-absorbency and absorbency to water are properties of hydrophobicity and hydrophilicity, respectively. Hydrophobic materials are materials that are not wet with water, and absorbent materials are materials that are wet with water. In general, the absorbance is characterized by the contact angle θ (see FIGS. 1A-1D) between the droplet 1 and its surface 2. Sometimes the above mentioned angle cosines are used to define the absorbance coefficients. Thus, the complete absorbance corresponds to the absorbance coefficient 1, thus θ = 0 °. In addition, the complete absence of absorbance corresponds to the absorbance coefficient -1, thus θ = 180 °. Thus, in the following, reference will be made to the wetted material for the liquid for the substance, as described with reference to FIG. 1A, for which the coefficient of absorption is directed towards 1 (not necessarily required to be 1), and FIG. 1B. As described with reference to this liquid, we will refer to materials that do not get wet for liquids for which the coefficient of absorption tends to -1 (which need not necessarily be -1). 1C and 1D illustrate the intermediate case of absorption (θ <90 °) or nonabsorption (θ> 90 °), respectively.

The problem that arises from materials that are not immersed for liquids, especially hydrophobic materials that are additionally necessary for transposition, is that the properties of the surface of these materials create a chemical treatment area of the surface from the fact that these materials are characterized by the low energy of the surface. It is to be prevented from. If one attempts to localize the surface of such material, which makes it possible to chemically treat the liquid being manipulated, the result is very unreliable, not easily controlled and too incomplete. An alternative configuration for constructing a rough material that does not get wet with the liquid is impossible because it causes the material's ability to support the movement of the liquid to be lost. Thus it is necessary to use a layer of material that is partially immersed, for example the property remains wet against the anterior teeth, while having a strong absorbency or having an immersion area for functionalization.

Applicable in certain cases where the material under consideration is hydrophobic, two conventional photos that make part of the hydrophobic layer mainly by forming openings in the hydrophobic material and forming openings that fit the absorption regions distributed in the hydrophobic layer. Lithography techniques are mainly known. In addition, in the first technique used by Cho et al. (FIG. 5) in " particle separation and concentration digital control of microfluidic systems ", after the deposition of a hydrophobic layer of material on a substrate, a photosensitive resin layer comprising a surfactant The deposition makes it possible to chemically increase the absorbance of the surface with respect to the liquid. In particular, this technique raises the issue of final contamination of hydrophobic materials and thus loses their ability to support the dislocation of liquids. In this technique (FIG. 6), after deposition of a hydrophobic layer of material on a substrate and before deposition of a photosensitive resin, the hydrophobic layer of material is first modified using a plasma to change its hydrophobic properties. It becomes less hydrophobic. This technique also raises the issue of the final change in the surface properties of hydrophobic materials.

With this technique, not only are the openings that become absorbing regions not sufficiently clear and accurate due to possible hydrophobic deposition and consequent formation of chemically functionalized regions, but also hydrophobic regions are the result of disproportionation of the transposition of the liquid. Modified and reduced hydrophobic properties. The same observation can be obtained in the case of the application of these techniques to realize the immersion area in the non-immersion layer for the transferred liquid.

Thus, the ability to partially absorb and thus move the droplets of the liquid is maintained, especially when the liquid is a solution comprising water, while partially moving against the transferred liquid, which allows chemical treatment or interaction with this droplet between its movements. What is needed is a process that makes it possible to make a track of immersed non-immersed movements.

More generally, there is a need for a reliable solution that can solve the above-mentioned problems, in particular for the optimization of anterior teeth and for the manufacture of an optimized anterior track.

It is therefore an object of the present invention to solve the above mentioned problems. To this end, the present invention comprises at least one electrowetting pretreatment track for manipulating a drop on a plan of electrowetting translocation that enables it to be interacted with or chemically treated with the droplet simultaneously with its movement. The first aspect is the device.

This pretreatment track comprises at least two interleaved electrodes that are electrically covered behind the insulated substrate and by the insulating dielectric layer. These insulating substrate units, electrodes and insulating dielectric layers are covered with a layer partially immersed in the droplet being manipulated.

As an alternative to the realization relating to the manipulation of droplets comprising water, the immersion layer is thus partly an absorbent layer.

In the following detailed description, to simplify the proposal, reference is made to the immersed or wetted material or layer, respectively, or partially, to the manipulated droplets.

In another alternative, the device of the invention comprises at least one counter-electrode spaced from the first electrode. This counter-electrode can then be part of an earth line to be placed on, under or inside the immersion layer.

As another alternative, which may be combined with the above, the device comprises a second position track in a manner opposite to the first track, so that the space is to be filled by an insulating fluid which is not electrically mixed with the moved droplet. And a second track formed between the second tracks so that the second tracks are in direct contact with the formed space. The immersion layer of this J-track is possibly partially immersed. This non-immersed layer is also possibly covered with an electrically insulating, semiconductor or conductive top layer.

In another alternative, the J track includes one or more counter-electrodes positioned between the non-immersion layer and the top surface. This possibly includes an insulating dielectric layer located between the non-immersion layer and the counter-electrode.

Preferably, in each combination of these alternatives for the realization of the device, the partial immersion layer of the first track and / or the second track comprises a non-immersion area and an immersion area, the immersion area being a reactive functionalization area.

In another alternative, the device for manipulating droplets in the scheme of the present invention comprises two tracks electrically separated by a space that will be filled by an insulated fluid that is incompatible with the moved droplet. The first track includes an electrically insulating substrate or layer behind at least two interleaved electrodes. There is a non-immersion layer on this unit. The second track partially includes an immersion layer. The partially immersed layer of the first track and / or the second track comprises a non-immersed area and an immersion area, the immersion area being a reactive functionalization area.

Preferably, in this alternative of realization, the first track also includes an insulating dielectric layer located between the electrode and the non-immersion layer. Preferably, the device in this alternative of realization also includes a line of earth located above, below or in the non-immersion layer.

In an alternative embodiment, the Jay track includes an upper surface that is electrically insulated, semiconductor, or conductive.

In combination with each one of these alternatives of device realization, the substrate electrically isolated from the first track is preferably transparent, for example a glass substrate.

Preferably, in one or more of the aforementioned alternatives, the immersion region is biochemically functionalized and reactive.

These immersion regions are preferably openings in the non-immersion region. Preferably, the non-wetting material constituting the non-immersion layer and / or the non-immersion area of the partial immersion layer is a polymer of tetrafluoroethylene.

Thus, the device of the present invention advantageously is on a chemically functionalized region while liquid is transferred onto the plan by electrowetting, with only one track or with two facing tracks, with or without counter-electrodes. It makes it possible to manipulate the droplet of liquid while acting chemically on the droplet at its passage time. The required optimization is thus made: for the concentration, very expensive samples and small quantities that preprocess the later analysis in the microsystem during the movement, avoiding contamination and losses in view of the above limitations in the microflow.

According to a second aspect according to the present invention, there is provided a process for manufacturing the device, wherein the impartment of the first or partially immersed layer of the second track is to the "topspin off" used in micro-electronics to form a metallic basis. It is derived from known techniques. As it is known, this "topspin off" technique, however, does not wet, especially if it is a hydrophobic material, such as a polymer of tetrafluoroethylene, if it allows the deposition of the non-immersion layer in the last step to avoid surface treatment. It does not apply for the same reason, because it is not possible to obtain a clear and accurate immersion area for this wet material. Thus, the invention according to the second aspect refers to the manufacturing process of the above-mentioned device, wherein the formation of the first or partially immersed layer of the second track comprises the following steps: in the deposition of the photosensitive material on the substrate. The formation of photosensitive material masks, photolithography, and then exposure of photosensitive materials; Deposition of a material that is not wet on the mask; At least annealing before dissolution; Dissolution of the mask; At least annealing after dissolution.

As an alternative to implementation, the temperature of the annealing before dissolution is lower than the temperature of the annealing after dissolution.

In another alternative of implementation, the first annealing before dissolution is followed by at least another annealing at a higher temperature than that of the first annealing.

In another alternative, possibly in combination with the one described above, the first annealing after dissolution follows at least another annealing at a higher temperature than that of the first annealing.

Preferably, dissolution of the mask is followed by cleaning.

In another alternative of implementation, the deposited wet material is a polymer of tetrafluoroethylene.

Thus, the process of the present invention advantageously includes the formation of a partially immersed layer that includes a clear and accurate immersion region applied to chemical functionalization and that includes a non-immersion region that preserves the high nature of the non-absorbent necessary for the movement of the droplets. To make it possible. Indeed, the layer of material that is not wet is deposited and not surface treated in the last step, and thus its surface properties are not changed.

Finally, the invention according to the third aspect refers to a system for the analysis of microfluidics of a liquid sample comprising at least a means for preparing a sample, coupled to a minimal device for manipulating the droplets according to the invention, as described above. It is at least connected to the means of analysis by itself.

Preferably, the manufacturing means comprise one or more drip reservoirs.

Preferably the analysis means is also a mass spectrometer, a fluorescence detector, a UV light detector or an IR.

The system according to the invention possibly integrates one or more operations of a laboratory, which is usually manually operated by itself, and is integrated into a microsystem called a micro laboratory.

The system according to the invention is thus advantageously capable of analyzing a sample of liquid after transfer by means of automation of the manufacture and transfer incorporated into the micro lab, after the preparation of the sample and then by prepositioning the microvolume weighed with the analyzer. Do. Thus, advantageously, it is possible to reduce the risk of contamination and loss of the sample and to reduce the reaction time.

Other features and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the realization and process of the device with reference to the accompanying drawings.

1a to 1d schematically illustrate the characteristics of the non-absorbance and the absorbency of the surface with respect to the droplets,

2a to 2r schematically illustrate in accordance with the invention various alternatives for the realization of the device (a transverse cross-section perpendicular to the meaning of transposition of the drop),

3 schematically shows the translocation of a drop onto a track of the device according to the first embodiment,

4 schematically shows the translocation of a drop onto a track of a device according to a second embodiment,

5 schematically illustrates a process for forming an opening in a material that is not wet according to conventional photolithographic techniques using surfactants in the resin,

6 schematically illustrates a process for forming an opening in a material that is not wet according to conventional photolithographic techniques by modification of the surface by plasma,

7 schematically illustrates an embodiment of a process for forming an opening in a material that is not wet according to the present invention,

8 schematically illustrates the chemical functionalization of the immersion region,

9 schematically illustrates the chemical treatment of a droplet of a sample between its translocations,

10 schematically shows an embodiment of a system according to the invention.

2a to 2r schematically illustrate various embodiments of the device of the invention (transverse cross-section perpendicular to the meaning of transposition of the drop).

In these FIGS. 2A-2N, the device comprises a track having at least one substrate 1, which is not necessary but preferably for example Pyrex®. The interleaved electrodes 2 overlie this substrate 1. The concept of interleaved electrodes will be more specific with reference to FIGS.

On these electrodes 2 is a dielectric insulating layer 3 composed of, for example, an oxide or a polymer. There is a non-immersion layer 4 which is partially immersed by the process of forming the immersion opening 5 in the material 4 which is not wet on the electrically insulating layer 3, which process will be described in more detail with reference to FIG.

In the embodiment of FIGS. 2A-2D, the device comprises only one track consisting of layers 1, 2, 3 and 4. The apparatus of FIG. 2A makes it possible to realize a transposition by electrowetting that does not require counter-electrodes, which is described in more detail with reference to FIG. 3. The device of FIG. 2B is presented on a partially immersed layer 4 (FIG. 2B), or is not covered by a partially immersed layer 4 (FIG. 2C), or is covered and fitted by a partially immersed layer 4. (FIG. 2D) One counter-electrode each represents the shape of earth line 6. The apparatus of FIGS. 2B-2D makes it possible to carry out an electrowetting translocation with an earth line to the counter-electrode, which will be described in more detail with reference to FIG. 4.

2E illustrates an embodiment of adding a second forming track of the non-immersion layer 7 itself covered with an upper face 8 which may be electrically insulating, electrically semiconductor or still electrically conductive. This Jay track is positioned with respect to the first with the use of Hold 9 which makes it electrically possible to maintain a space 10 of the transposition that is to be filled with an incompatible insulator against the moved droplet.

In order to perform the electrowetting translocation, the fluid filling space 10 must actually be electrically insulated. In addition, the fluid must be incapable of actually miscible with the liquid so as not to interact with the moved droplet. In the case of drops of aqueous solutions, for example air or oil may be questionable.

In particular, FIGS. 2F-2H show embodiments based on the apparatus of FIGS. 2B-2D, respectively, with the addition of a J-track as described above.

In the embodiment of the apparatus of FIG. 2I, the Jay track further comprises one or more counter-electrodes 11 inserted between the non-immersion layer 7 and the upper face 8. Thus, this does not use an earth line, as opposed to the device of FIGS. 2F-2H since the counter-electrode appears in the J-track. However, the style of this transposition is the same as that of FIGS. 2F-2H.

The embodiments of FIGS. 2J-21 are derived directly and directly from the embodiments of FIGS. 2F-2H, respectively (transverse cross-section perpendicular to the meaning of transposition of the drop) from the embodiments of FIGS. Is partially immersed by the process of forming the immersion opening 5 in the non-wet material 7 which will be described in more detail later with reference to FIG.

FIG. 2M shows an embodiment as already described in FIG. 2E with the following differences: The non-immersion layer 7 of the J. track is the immersion opening 5 in the non-wet material 7 which will be described in more detail later with reference to FIG. Partial dipping is achieved by the forming process.

The embodiment of FIG. 2N is derived from the embodiment of FIG. 2I with the following two differences: The non-immersion layer 7 of the J. track is an immersion layer according to the process to be described in more detail later with reference to FIG. Partial immersion is achieved by formation of the immersion opening 5 in 7; And permitting the biochemical functionalization of these immersion openings 5 without interaction to the insulating dielectric layer 12, electrode 11, similar to the one inserted between the immersion layer 7 and the counter-electrode 11 present in the first track.

The embodiment described in FIG. 2O relates to an apparatus having two tracks. The first track differs from the first track in the above embodiment in that the non-immersion layer 4 constituting it is not partially immersed: the immersion opening is not shaped in this non-immersion layer 4. In addition, this embodiment does not require an insulating dielectric layer between the interleaved electrode 2 and the non-immersion layer 4 if this immersion layer 4 is electrically insulated by itself. In particular, the hydrophobic layer is made of a material such as tetrafluoroethylene polymer. In practice, however, these materials are electrically insulated if the layer is large (about a few micrometers in thickness). Also, in the case of FIG. 2O where the thickness of the non-immersion layer 4 is not sufficient, a layer 3 type insulating dielectric layer, as in the other figures, may be inserted between the layer of the interleaved electrode 2 and the side-side non-immersion layer 4. will be.

Earth line 6 on the non-immersion layer 4 acts as the counter-electrode. In this embodiment, the Jay track is the same as that of the embodiment of Figures 2J-2M.

In each embodiment of FIGS. 2P and 2Q, the non-immersion layer 4 does not include the opening 5 and is not partially immersed. These embodiments thus derive from the embodiments of FIGS. 2K and 21 with the above-mentioned differences (the embodiment of FIGS. 2K and 21 is partially immersed but layer 4 is completely non-immersed).

Finally, the embodiment of FIG. 2R retakes the transpose mode of FIGS. 2A, 2E, and 2M. That is, there is no use of the counter-electrode, and as in the embodiment of FIGS. 2O-2P, there is no immersion layer 7 and no immersion in the first track, which is completely immersed, with no openings 7 and no openings in the partially immersed J track. Layer 4 appears. In addition, as in the embodiment of FIG. 2O, this embodiment does not require an insulating dielectric layer between the interleaved electrode 2 and the non-immersion layer 4, in particular if this immersion layer 4 is electrically insulated by itself, in particular In the case of the hydrophobic layer, it is made of a material such as tetrafluoroethylene polymer. In this case, however, still in practice, these materials are electrically insulated if the thickness of the layer is large (about several micrometers in thickness). Also, in the case of FIG. 2R where the thickness of the non-immersion layer 4 is not sufficient, a layer 3 type insulating dielectric layer, as in other figures, may be inserted between the interleaved electrode 2 layer and the side-side non-immersion layer 4. will be.

3 schematically shows the translocation of a drop onto a track of an apparatus according to an embodiment. This figure is divided into two parts. In the simplified upper part (Figs. A, B and C) for ease of explanation, the representation of the device is based on a non-immersed or partially immersed or insulating dielectric layer located between droplet 15 and electrodes 1, 2, 3 and 4. Shown in an unexposed plan view. In the lower part (Figs. A ', B' and C '), the marking of the device shows the side cross section in the direction of the transposition of the drop.

In more detail, the device is of the type in FIG. 2A, ie with only one track. However, the following detailed description of the transposition of the droplets can be applied more generally in the case of FIGS. 2A, 2E, 2M and 2R, ie with interleaved electrodes without counter electrodes, possibly with a higher plane of the second. It can be applied to transpose on a track with.

The device thus requires several interleaved electrodes 1, 2, 3, 4, which are placed behind the substrate 10 and are electrically insulators, possibly transparent. On the layer of interleaved electrodes there is an insulating dielectric layer 11 and an unimmersed layer 12. This non-immersed layer 12 may be partially immersed in accordance with the embodiment in which its shape is shown (see FIG. 2 described above), without changing the following description regarding transposition. Drop 15 is initially on electrode 2 (step A). By relating the potential difference between electrode 3 and electrodes 1, 2 and 4, the drop is moved to electrode 3 (step B). To move this to electrode 4, a potential difference is created between electrode 4 and electrodes 1, 2 and 3.

4 schematically shows the translocation of a drop onto a track of a device according to another embodiment. This figure is also divided into two parts. In the upper parts (Figs. A, B and C), which are easily described and simplified as described with respect to FIG. 3, the markings on the device are immersed or partially immersed between the drop 15 and the electrodes 1, 2, 3 and 4. The layer or insulating dielectric layer is shown in a partial plan view without exposure. In the lower part (Figs. A ', B' and C '), the marking of the device shows the side cross section in the direction of the transposition of the drop.

More specifically, the device corresponds to a device having an earth line as only one track and the counter-electrode as in FIG. 2B above. However, the following detailed description of the transposition of the drop for this device is also generally applicable in the case of FIGS. 2C, 2D, 2F, 2G, 2H, 2J, 2K, 2I1 2O, 2P, 2Q.

The device comprises a layer of interleaved electrodes (1, 2, 3, 4), possibly transparent, placed behind the substrate 10 and electrically insulated. Above this electrode layer is an insulating dielectric layer 11. Above this insulating dielectric layer 11 is a non-immersion layer 12. This layer is possibly partially immersed, depending on its shape (see FIG. 2). At the top of this non-immersion layer 12 (possibly partially immersed) is the electrode of the earth or a line of earth.

Drop 15 is initially on electrode 2 (step A). By relating the potential difference between electrode 3 and electrodes 1, 2 and 4 and the earth's electrode, the drop is moved to electrode 3 (step B). In order to move this to the electrode 4, a potential difference is made between the electrode 4 and the electrodes 1, 2 and 3, and the earth electrode.

If the earth's electrode or the earth's line is replaced by a higher-positioned counter-electrode (in the case of FIGS. 4I and 4N), the above description with respect to FIG. 4 still applies.

The process of making a non-immersion layer partially immersed in one of the tracks of the device of the invention is described in detail below with reference to FIG. 7, recalling the steps of the technique with reference to FIGS. 5 and 6.

FIG. 5 schematically illustrates a process for forming an opening that becomes partially submerged in a material that is not wet according to conventional photolithographic techniques using a surfactant. In step (A), two layers of non-wetting material are deposited on the substrate 1. In step (B), a resin layer 3 comprising a surfactant is deposited on the non-immersion layer 2. The surfactant increases the absorbency of the non-immersed layer with respect to the resin, thus fixing the resin on this layer. Step (C) is a photolithographic step, strictly speaking UV is emitted to layer 3. If layer 3 is not a positively known resin, ultraviolet radiation includes the fracture of macromolecules in the exposed areas, which provides increased solubility to these areas with the exposure solvent to be used in step (D), while this part Not exposed and compounded. Thus, this is what the result of the exposure step (D) is. The exposure of the resin is accompanied by an attack by the exposed wet material and thus an area or opening appears in the non-immersion layer 2 (step (E)). This technique involves the risk of a final change in the surface properties of the material that is not wet due to the use of surfactants in the resin.

Figure 6 schematically illustrates the steps of a process for forming openings in a material that is not wet by plasma according to conventional photolithographic techniques. This technique differs from the above in that it comprises an additional step consisting of plasma-argon radiation (step (B)) for the non-immersion layer 2 before deposition of the resin layer. This is spinning which modifies the surface properties of the non-immersed layer 2, whereas in the above technique (Fig. 5), the presence of surfactants in the resin acting in this role. The following steps ((C), (D), (E), (F)) are the same as those of steps (B), (C), (D) and (E) of FIG. 5, respectively. The results are the same as for photolithographic techniques using surfactants. That is, there is a risk of the final change of the surface properties of the non-immersed layer 2.

Therefore, the process of the present invention shown in FIG. 7 first of all imparts a partial immersion layer by imparting a photosensitive material mask by the deposition of this material 2 on the substrate 1 (step (A)), photolithography (step (B)), and the manufacturing process of one or more tracks of the device described above, including exposure of the photosensitive material (step (C)). In the embodiment shown in FIG. 7, negative resins, such as photosensitive materials, ie UV radiation, were used to include polymerization of the exposed areas from the increased areas of the unexposed areas by exposure. Thus, this is the non-exposed area in step (B) that disappears in step (C), while the area exposed in step (B) is present in step (C) and is located by line 2. The choice of negative resin is of course not limited in the present invention. Considering the process of the present invention, it is precisely the same as in the case of the use of a positive resin.

Step (C) is followed by step (D) of deposition of the non-wet material layer 3.

For example, a photolithographic step may be used with a resin having the following parameters:

Resin AZ 4562,

-Exposure at AZ 351 B.

Deposition step (D) of the non-wetting material 3 is followed by the first step of annealing. According to the selected material (eg polymer of tetrafluoroethylene) it can be annealed at 50 ° C. for 5 minutes. Although not necessary, preferably, this annealing is followed by other supplementary annealing. This other annealing can also be performed at 110 ° C. for 5 minutes.

In certain cases of hydrophobic materials, such as tetrafluoroethylene polymers, in this step, little solvent remains in the materials. However, after dissolution of the resin mask 2 (step (E), annealing of the second step is necessary. In practice, the resin polymerizes at the temperature of the annealing of the hydrophobic material, which is difficult to remove. This results in removing traces of the resin on the substrate. These traces tend to make it difficult to be almost impossible to remove at the time of dissolution step, and are easy to partially modify the properties of the surface of the immersion layer (partially absorbent in the case of water absorption). : The opening may not be fully immersed (or hydrophobic to water absorbency) and the non-opening region may not be fully immersed (hydrophobic). Before, first, the resin is dissolved in, for example, acetone, for example for 30 to 40 seconds, although not necessarily, but preferably, Step of dissolving, for example followed by a step of cleaning in alcohol.

Finally, in the annealing of the second step, for example (depending on the material selected), it can proceed for 5 minutes at 170 ° C. to completely remove the solvents that may be present in the hydrophobic material. If possible, another annealing can be performed supplementally for 15 minutes at 330 ° C., for example, to obtain maximum bonding with a constant surface of the material that is not wet on the substrate.

On the other hand, the process according to the invention makes it possible to form a partially immersed layer in a material which is advantageously not wet. This result is obtained by the formation of openings in the non-wetting material, which results in immersion regions that are subjected to chemical and biochemical functionalization. The non-opening area remains completely unimmersed and thus preserves the highly absorbent, high nature required for the movement of the droplets. In particular, the fact that a layer of non-wetting material is deposited at the end of the process, contrary to the state of the art, makes it impossible to surface-treat the material (technology using surfactants or plasma-argon).

Thus, the apparatus of the present invention includes at least a layer that is partially immersed by forming an immersion opening in the non-immersion layer as described above. These immersion regions can be activated and chemically functionalized for the droplet being manipulated (FIG. 8) and then reacted with the droplet being manipulated (FIG. 9). Thus, as already described, it is mainly used as a translocation of the droplets to activate regions that have not yet been functionalized based on the droplets containing agents that allow functionalization.

In particular, FIG. 8 (i.e., the same representation format as the top for the top views of FIGS. 3 and 4 without insulation and non-soaked dielectric layers between interleaved electrodes and droplets) allows functionalization on the base of electrode 1. It is shown that droplet 15 containing the agent moves to electrode 2 with region 5 capable of functionalization thereon and then reaches electrode 3 after chemically activating and functionalizing region 5.

In FIG. 9 (i.e., in the same representation format as the top for the top views of FIGS. 3 and 4 without each dielectric insulating layer immersed between the interleaved electrodes and the droplets), 15 first moves on electrode 1 above the track and It then moves to electrode 2 with functionalized region 5 above it and shows that after reaction with the functionalized region it reaches and changes 3.

10 schematically shows an embodiment of a system according to the invention. The system comprises one or more preparation means 1 for analyzing a sample of liquid, one or more devices 2 for manipulating the droplets according to the invention and one or more means 3 for analyzing the output. Formulation means 1 comprises one or more drip reservoirs. Analysis means 3 may for example be a mass spectrometer, a fluorescence detector or a UV light detector. Device 2 according to the invention in the middle of this system is connected upstream with preparation 1 and downstream with analysis means 3.

Thus, the system according to the invention can be incorporated into one or more instruments of the laboratory, which are generally manually realized, and microsystems which are themselves incorporated. This system is called a micro laboratory.

Next, two examples of functionalization of the present invention include a substrate of Pyrex®, an intercalated electrode of nickel, hundreds of nanometers thick, a micrometer resin SU8 layer deposited by centrifugation, and a dielectric insulating layer. A description will be given based on an example of the implementation of the device. Finally the device comprises a hydrophobic layer deposited on the above-mentioned resin layer with tetrafluoroethylene polymer and also by centrifugation.

Affinity  Example engine:

Areas not covered by the hydrophobic layer are, for example, support-NH ₂ graft streptavidin and are surface treated to convert them to reactive surfaces.

Thus, with devices comprising such functionalized regions, for example, a droplet of liquid containing protein and moving into an electrode on the functionalized region is itself directed against a surface grafted during the functionalization to be immobilized on these surfaces. You know the molecule of interest (for example, some protein, such as biotin) that has affinity. When the chemical reaction ends, this droplet travels on its path in the device. It is then possible to remove (eg, by disruption of non-covalent interactions) and to have the molecules of interest through the passage of certain mixtures (e.g., to break up the charge mixture) on these regions. . Thus, such a device makes it possible to separate from the molecule of interest.

Example of isolation engine:

In this device, the area not covered by the hydrophobic layer is surface treated for the purpose of converting it to a reactive surface, for example, a support-NH ₂ graft trypsin.

Thus, in a device comprising such a functionalized region, a drop of liquid that travels to the path of the electrode will be fixed to the functionalized region, and certain target molecules (eg proteins) will react with the grafted surface. The result of this reaction is to isolate the molecule (e.g., a peptide obtained by tript isolation). The drop then moves on its path in the device. Thus, such a device is capable of analyzing long chain molecules, for example by analysis by mass spectrometer, by isolating in advance by means of specific enzymes.

Thus, the devices, processes and systems of the present invention provide a basic component of a microsystem for moving fine amounts to other functionalized areas in an architecture that completes itself with other auxiliary functions and upstream or downstream integration. Enable implementation Thus, it is possible to design specific microsystems that are distinguished from each other by the continuity and nature of the biochemical manipulations being performed.

The foregoing detailed description is given by way of example and not by way of limitation. In particular, the selection of the tetrafluoroethylene polymer as an immersed or partially immersed layer is a choice that has been adopted in the sense that it is actually, but not limited to, non-immersed in water and therefore hydrophobic. More generally, biocompatible, non-wetting materials (which do not absorb migrated materials, do not mix with migrated materials, do not cause chemical reactions, and do not precipitate) will always be the subject. Therefore, the above description should be taken in a neutral posture, and also the equality of its properties should appear on the surface.

In the same way, the choice of silicon or Pyrex® as the substrate is of course not limited in the present invention. Moreover, a positive or negative resin may be selected in the framework of the manufacturing process of the apparatus of this invention. It is also to be appreciated that the temperature and time of the annealing step within the framework of the manufacturing process of the device of the invention are not limited and depend primarily on the function of the selected wet material. In addition, the use of acetone for dissolution and the use of alcohol for cleaning is also not limiting. All other things that apply to dissolution and washing can be used.

Moreover, the examples of transposition in the certain directions mentioned in this description are not limiting inventions. Of course it is possible to consider a transpose matrix that allows the droplet to move anywhere on the track. The transposition option is essentially dependent on the geometry of the electrode. The matrix of electrodes can in fact be used to achieve transpose of the matrix type. In addition, the shape of the electrode in the example of the present description is by no means limited. Any shape that allows insertion of the electrode is suitable.

 In addition, in systems incorporated such as the system of the present invention, the list of examples of manufacturing means of the pre-device upstream is not exhaustive and thus not limiting. This also applies to the list of examples of manufacturing means downstream of the transposition device.

Finally, examples of functionalization of the partially immersed regions of the immersion layer and examples of drip treatment by these functionalized regions are not limited to the present invention. In general, whatever you do, you will actually be interested in the separation, classification, or isolation of molecules. Other manipulations by chemical and / or biochemical reactions may also be considered.

Claims (26)

An apparatus for manipulating a droplet on an anterior tooth by an electrowetting surface comprising at least one track, the track comprising: a track; Electrically insulated substrate having an upper surface, At least two primary conductive electrodes having an upper surface and a lower surface, the lower surface of the conductive electrode being placed on an upper surface of the electrically insulated substrate, each of the primary electrodes being at least one other of these primary electrodes; Intertwined, An insulating dielectric layer having a top side and a bottom side, the bottom side of which lies on the top side of the first electrode, A partial immersion layer having a top side and a bottom side, the bottom side of which lies on the top side of the insulating dielectric layer, Characterized in that it comprises a. The device of claim 1, wherein the device comprises at least one counter-electrode spaced from the first electrode. 3. The apparatus of claim 2, wherein the separated counter-electrode is an earth line positioned over or under or partially in the top surface of the immersion layer. 4. 4. The apparatus according to any one of claims 1 to 3, wherein the device comprises a Jay track located opposite the first track, the space being formed between the first and second tracks, the second track being spaced. And a non-immersion layer having a lower side on one side and an upper side on the other side. 5. The apparatus of claim 4, wherein the immersion layer of the Jay track is partially immersed. 6. The apparatus of any of claims 4-5, wherein the second track comprises an upper layer, which is an electrically insulating, semiconductor or conductor located on one side of the upper surface of the non-immersed layer. 7. The apparatus of any one of claims 4 to 6, wherein the second track comprises one or more counter-electrodes positioned between the non-immersion layer and the top surface. 8. The apparatus of claim 7, wherein the second track comprises an insulating dielectric layer positioned between the non-immersion layer and the counter-electrode. The device of claim 1, wherein the partially immersed layer of the first track and / or the second track comprises a region and an immersion region, wherein the immersion region is a reactive functionalization region. Electrically insulated substrate having an upper surface, At least two primary conductive electrodes having an upper surface and a lower surface, the lower surface of the conductive electrode being placed on an upper surface of the electrically insulated substrate, each of the primary electrodes being at least one other of these primary electrodes; Intertwined, An immersion layer having an upper side and a lower side, positioned on one side of the upper side of the first electrode A best track comprising a; Partially immersed layer with top and bottom sides A J track comprising a; An apparatus for manipulating a drop between two translocations by an electrowetting surface, comprising two tracks separated by a space, characterized in that Wherein the partial-immersion layer of the first track and / or the second track comprises a non-immersion area and an immersion area, the immersion area being a reactive functionalization area. 12. The apparatus of claim 10, wherein the second track comprises an insulating dielectric layer positioned between the bottom surface of the non-immersion layer and the top surface of the first electrode. 12. The device of any one of claims 10 or 11, wherein the device comprises an earth line positioned on or below or in the top surface of the non-immersion layer. 13. The apparatus of claim 10 or 12, wherein the second track comprises a layer that is electrically insulating, conductive, or semiconductor located on one side of the top surface of the non-immersed layer. 14. An apparatus according to any one of the preceding claims, wherein the electrically insulating substrate of the first track is transparent. 15. The apparatus of claim 14, wherein the electrically insulating substrate of the first track is glass. 16. The apparatus of any one of claims 9 or 15, wherein the immersion area is an opening in the non-immersion area. The device according to claim 9, wherein the immersion region is biochemically functionalized and reactive. 18. The method according to any one of claims 1 to 17, wherein the non-immersed layer and / or the non-immersed area of the partially immersed layer is immersed in water and therefore hydrophobic, and the immersed region is immersed in water. And therefore hydrophilic. 19. The device of any one of the preceding claims, wherein the non-immersed layer and / or the non-immersed area of the partially immersed layer is a tetrafluoroethylene polymer. Impartation of a partially immersed layer of the first track or the second track; Depositing a photosensitive material on the substrate, then photolithography, and applying a mask to the photosensitive material by photosensitive exposure, Depositing a material that is not wet on the mask; At least one annealing step before dissolution, Dissolving the mask, At least one annealing step after melting Method for manufacturing a device according to any one of claims 1 to 29 comprising a. The method of claim 20, wherein the temperature of the annealing step before the dissolution is lower than the temperature of the annealing step after the dissolution. 22. The method of any one of claims 20 to 21, wherein depositing the wet material on the mask is depositing a tetrafluoroethylene polymer. At least one means for preparing a liquid sample in at least one outlet, At least one drip operating device according to any one of claims 1 to 29, wherein one of the inlets is connected to one outlet of the dispensing means and has at least one outlet; At least one analysis means connected to one outlet of the droplet manipulation device, the one of its inlets. 21. The system of claim 20, wherein said dispensing means comprises one or more reservoirs. 25. The system of any one of claims 23 to 24, wherein said analyzing means is a mass spectrometer, a fluorescence detector, a UV light detector. 26. The system of any one of claims 23 to 25, wherein the system is incorporated into a micro lab.

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