FR2879483A1 - PROCESS FOR INCREASING THE HYDROPHOBICITY OF AN EXTERNAL COATING OF AN ANALYTICAL DEVICE, SUCH AS A BIOPUCE, SUCH A DEVICE AND METHODS OF MAKING SAME - Google Patents

Abstract

The present invention relates to a method for increasing the hydrophobicity of a perforated outer coating (3) of a chemical and / or biological analysis device (1), such as a biochip, such a device comprising a substrate (2). and this coating which covers the substrate by surrounding a plurality of cuvettes (4) for fixing chemical and / or biological reagents to form sites of analysis. The invention also relates to methods for manufacturing this analysis device.The method according to the invention for increasing the hydrophobicity of this outer coating (3) comprises a treatment of said coating by means of an organic plasma selected from the group constituted by the fluorocarbon plasmas and the siloxane-containing plasmas for depositing a hydrophobic film (7) on said coating, so as to optimize the volume of said reagent located around said film in each cuvette.

Description

i

  The present invention relates to a method for increasing the hydrophobicity of an outer coating of a chemical and / or biological analysis device, such as a biochip, such a device and methods for its manufacture.

  Chemical and / or biological analysis devices, such as microreactor chips, are especially designed to provide a high density of sites for this analysis. These devices typically comprise a substrate provided with microcuvettes, each of which is intended to contain and to locate a determined volume of a specific reagent, 1.0 typically delivered in the form of drops of an oligonucleotide-based reagent, in order to a chemical and / or biological reaction carried out during the analysis. For example, patent document FRA-2 781 886 may be cited in the name of the Applicant for the description of such a device.

  However, these chemical and / or biological reactions, for example PCR type (Polymerase Chain Reaction), require to have a minimum drop volume in each microcuvette so that the polymerase can act. It is known that the fixing of the oligonucleotides is all the more important that the volume of reagent deposited in each cuvette is higher, which led to increase this volume without modifying the surface density of sites on the biochip.

  For this purpose, it was sought to increase the height of each microcuvette, for example by depositing thick photosensitive polymers on the biochip, or by fixing on the latter etched plates of glass, silicon or plastic.

  This increase in the height of the microcuvettes has the disadvantage of potentially generating there air bubbles and unwanted capillary effects during their filling, which penalizes the aforementioned fixing and also the rinsing and emptying of these microcuvettes .

  In addition, this increase in height contributes to slowing down the diffusion of the target species reacting specifically during the analysis with the reagents fixed in the bottom of the microcuvettes.

  In order to increase the volume of reagent introduced into the microcups without modifying the height thereof, hydrophobic materials and hydrophilic materials have been deposited in the past on the edges of the microcuvettes, on the one hand, and on their side walls and their bottoms, on the other hand, as indicated in the patent document FRA-2 783 179 in the name of the Applicant. These hydrophobic materials are obtained by a silanization process using various solvents for a period of several hours and a chemical treatment of the surfaces to be treated, for example by means of soda or boiling water.

  A potential disadvantage of this silanization process lies in the fact that these solvents and this chemical treatment are likely to damage the structure of the bio-chip, in particular the metal electrical contacts and any photosensitive polymers that it contains. In addition, the silane fixation inherent in this process requires the prior addition of an intermediate layer of silica or silicon nitride on the edges of the microcuvettes, when these edges are of polymeric type.

  An object of the present invention is to overcome these disadvantages, and this object is achieved in that the Applicant has surprisingly discovered that, if subjected to treatment with an organic plasma selected from the group consisting of fluorocarbon plasmas and plasmas with siloxane groups, a biocompatible and perforated external coating of a chemical and / or biological analysis device, such as a biochip, comprising a substrate covered by this coating, which surrounds a plurality of cuvettes, for the determination of chemical and / or biological reagents to form test sites, it is possible to significantly increase the hydrophobicity of this coating by depositing a hydrophobic film thereon, so that the volume of the reagent located around said film in each cuvette is optimized.

  It will be noted that this significantly increased hydrophobicity makes it possible to increase very significantly the volume of reagent actually located in each cuvette, by accentuating the upper curvature of the reagent drop anchored in each cuvette because of the strong hydrophobicity of the external edges. of the latter, in comparison with the volume of the same reagent which would have been located in identical cuvettes surrounded by the same untreated coating.

  It will also be noted that this hydrophobic film forming the outer edges of the cuvettes makes it possible to ensure simultaneous and optimized filling of a multitude of micro-cuvettes (typically several thousand), by soaking and removal of the analysis device in a single reagent, for example based on oligonucleotides for a Polymerase Chain Reaction (PCR) type reaction.

  Indeed, the strong hydrophobicity of the outer edges of the cuvettes results in the reagent practically wetting these edges, which has the effect of pushing it immediately to the inside of the adjacent cuvettes, avoiding the presence of any residual drop. on these edges which could contaminate neighboring bowls by bridging. One can thus do without a micro-dispenser type robot for a controlled payment of the reagent in each bowl.

  It will be further noted that this plasma treatment according to the invention makes it possible to directly deposit said hydrophobic film on said coating, without prior deposition of an intermediate layer based on silica or silicon nitride, for example, such as that mentioned above. relationship with a hydrophobic silanization treatment.

  As a substrate that can be used in said analysis device, any mineral or organic, biocompatible and typically hydrophilic substrate that may be of the active or passive type (i.e. with or without an integrated electronic system, respectively) may be mentioned.

  As a material that can be used to form said coating partially covering said substrate, mention may also be made of any mineral or organic material that is biocompatible and typically hydrophilic.

  As materials that can be used to independently form said substrate and said coating, mention may for example be made of materials based on glass, silicon or a polymer such as a thermoplastic polymer, thermosetting polymer or an elastomer.

  According to a particularly advantageous embodiment of the invention, the hydrophilic material which is used to constitute said coating is based on at least one crosslinked photosensitive polymer which can

to be for example:

  a thermoplastic polymer, preferably a polyimide, such as a crosslinked polyimide of probimide denomination, or a thermosetting polymer, such as a positive or negative photoresist which is sensitive to ultraviolet radiation, advantageously an epoxy resin of denomination Su8.

  According to one embodiment of the invention, said organic plasma used for the surface treatment is advantageously chosen from the group consisting of plasmas of C4F8, CF4, C2F6, C3F8 and C5F8, for obtaining said film. hydrophobic which then comprises CF2 groups.

  According to another embodiment of the invention, said organic plasma is an octamethyltetrasiloxane plasma (OMCTS), for obtaining said hydrophobic film which then comprises SiOxCy groups.

  According to another characteristic of the invention, said organic plasma is used cold in the radiofrequency domain, and it is used for a duration that may for example vary from 30 seconds to about 1 minute in a plasma reactor preferably operating in the field of radio frequencies.

  Said hydrophobic film obtained by this plasma treatment may consist of one or more monolayers having a total thickness ranging from 50 nm to 1 μm.

  Another aspect of the present invention is to provide a chemical and / or biological analysis device, such as a biochip, that is obtainable by the method mentioned above for increasing the hydrophobicity of the outer coating of the invention. analysis device.

  According to this aspect of the invention, said device comprises: an organic or inorganic substrate, an organic or inorganic biocompatible coating, such as a plate, which covers said substrate with perforations, each perforation defining a bowl having a wall; and a bottom are respectively formed by said coating and said substrate, each cuvette being intended to fix a chemical and / or biological reagent to form a site of analysis, and - a hydrophobic film which covers said coating, to the except for said side walls of cuvettes, and which has CF 2 groups or SiOXCy groups, so as to optimize the volume of said reagent located around said film in each cuvette.

  It will be noted that the presence of these CF 2 or SiOXC groups in the hydrophobic film covering the coating alone makes it possible to significantly increase the hydrophobicity of this coating, which is preferably hydrophilic, like the substrate. , so that one can optimize the volume of said reagent located in each cuvette.

  It will also be noted that said lateral wall of each cuvette may have, in a nonlimiting manner, a shape, for example cylindrical, frustoconical or prismatic, the cylindrical geometry being preferred. 30

  According to other characteristics of the device according to the invention: said coating is advantageously based on at least one crosslinked photosensitive polymer of thermoplastic or thermosetting type, such as a polyimide or an epoxy resin, and said hydrophobic film advantageously has a thickness ranging from 50 nm to 1 dam.

  According to a first example of implementation of the invention, a manufacturing method according to the invention of said analysis device comprises the following successive steps: (i) covering said substrate with said plate, (ii) depositing on said bottom of each cuvette a protective layer which is intended to protect said substrate against an organic plasma selected from the group consisting of fluorocarbon plasmas and plasmas with siloxane groups, (iii) depositing said hydrophobic film both on said plate and on said protective layer covering each bottom of the cuvette, by a surface treatment by means of said organic plasma, then (iv) said protective layer coated with said film is removed, to obtain said device comprising said film only on an outer face of said plate.

  According to this first example according to the invention: - said protective layer may be deposited in step (ii) by spoting, and it is preferably based on at least one compound selected from the group consisting of alcohol polyvinyl, photosensitive resins, such as epoxy resins, and sugars, such as sucrose or trehalose and said protective layer is removed in step (iv) by the lift off technique, by means of a bath acetone, alcohol or water. 30

  According to a second example of implementation of the invention, a manufacturing method according to the invention of said analysis device comprises the following successive steps: (i) covering said substrate by said plate, (ii) depositing said film hydrophobic both on said plate and on said bottom of each cuvette, by a surface treatment implemented using an organic plasma selected from the group consisting of fluorocarbon plasmas and plasmas with siloxane groups, (iii) insole then develops, on a part of said hydrophobic film which covers said plate, a protective layer which is intended to protect this film vis-à-vis an oxygen plasma and which is based on at least one resin light-sensitive, such as an epoxy resin, (iv) the other part of said hydrophobic film which covers the bottom of each cuvette is removed by treatment of the assembly obtained in (iii) by means of said oxygen plasma, then (v) eliminating said neck protective guard deposited in (iii), to obtain said device comprising said film only on an outer face of said plate.

  According to this second example according to the invention, said protective layer is removed by means of a bath of acetone, alcohol or water.

  The aforementioned characteristics of the present invention, as well as others, will be better understood on reading the following description of several exemplary embodiments of the invention, given by way of illustration and not limitation, said description being made in relation to the attached drawings, among which: Figure 1 is a schematic cross-sectional view of an analysis device according to the invention in a first phase of its manufacturing method according to a first embodiment of the invention, Figure 2 is a schematic cross-sectional view of the analysis device of FIG. 1 in a second phase of its manufacturing method according to said first example, FIG. 3 is a diagrammatic cross-sectional view of the analysis device of FIG. a third phase of its manufacturing method according to said first example, FIG. 4 is a schematic cross-sectional view of a device In a first phase of its manufacturing method according to a second embodiment of the invention, FIG. 5 is a schematic cross-sectional view of the analysis device of FIG. phase of its manufacturing method according to said second example, FIG. 6 is a schematic cross-sectional view of the analysis device of FIG. 4 in a third phase of its manufacturing method according to said second example, FIG. schematic in cross section 20 of the analysis device of Figure 4 in a fourth phase of its manufacturing method according to said second example, Figure 8 is a bar graph illustrating the contact angles (in degrees) obtained for drops of d water deposited on two untreated hydrophilic coatings and on these same two hydrophobized coatings according to the invention, and FIG. illustrating the volumes of water deposited before overflow (in pL) in three micro-cuvettes of different capacities (in pL), for an analysis device not in accordance with the invention and for another analysis device according to the invention. 'invention.

  As illustrated in FIGS. 3 and 7, a chemical and / or biological analysis device 1 according to the invention, such as a biochip, comprises: a substrate 2; a biocompatible plate 3 which covers the substrate 2 by presenting perforations, each perforation defining a microcuvette 4 including a cylindrical side wall 5 and a bottom 6 are respectively formed by the plate 3 and the substrate 2, each microcuvette 4 being intended to fix a chinnic and / or biological reagent in order to constitute an analysis site, and - a hydrophobic film 7 which covers the plate 3, with the exception of the side walls 5 of the microcuvettes 4.

  As illustrated in FIGS. 1 to 3, a method of manufacturing the analysis device 1 according to a first exemplary implementation of the invention comprises the following successive steps: (i) being deposited by a spot-ing apparatus 8, on the bottom 6 of each microcuvette 4, a protective layer 9 (see FIG. 1) which is intended to protect the substrate 2 from the plasma used in step (ii) and which is preferably based on of polyvinyl alcohol, a photoresist or a sugar, (ii) the hydrophobic film 7, 7 'is deposited on both the plate 3 and on the protective layer 9 covering the bottoms 6 (see FIG. ) by surface treatment with cold fluorocarbon or siloxane-containing plasma; this treatment is carried out in a NEXTRAL NE110 commercial plasma reactor which uses, in a vacuum chamber maintained at a pressure of 60 mTorr, a radio frequency generator, then (iii) is removed by lift off, by means of a bath of acetone, alcohol or water, the protective layer 9 coated with the film 7 ', to obtain the device 1 illustrated in Figure 3 which comprises the film 7 covering only the plate 3. io

  As illustrated in FIGS. 4 to 7, a method of manufacturing the analysis device 1 according to a second example of implementation of the invention comprises the following successive steps: (i) the hydrophobic film 7, 7 "is deposited at the each time on the plate 3 and on the bottom 6 of each microcuvette 4 (see FIG. 4), by a surface treatment which is carried out as described in the first example above by means of a cold fluorocarbon plasma or with siloxane groups, (ii) is insole then develops, on the part of the film 7 which covers the plate 3, a protective layer 10 (see Figure 5) which is intended to protect the film 7 vis-à-vis an oxygen plasma PO2 and which is based on a photoresist, (iii) the other part of the hydrophobic film 7 "which covers the bottoms 6 is eliminated by treatment of the assembly obtained in (ii) with average oxygen PO2 plasma (see Figure 6) implemented in a NEXTRAL NE110 reactor that uses, in a chamber s or vacuum maintained at a pressure of 10 mTorr, a radiofrequency generator, then (iv) is removed by means of a bath of acetone, alcohol or water the layer 10 deposited in (ii), to obtain the device 1 illustrated in FIG. 7 which comprises the hydrophobic film 7 covering only the plate 3.

  Moisture tests and microcuvette filling tests: 1) Wetting tests: For the tests described below in connection with FIG. 8, wetting measurements were carried out: of two different hydrophilic Ti and T2 control coatings based on photosensitive polymers which are usable in the analysis device 1 according to the invention and which have not been treated (each of these two coatings being devoid of any hydrophobic film on its surface), and - of these two same Coatings having been treated with a plasma according to the invention, so as both to be covered with the hydrophobic film of the invention to give respectively two laminates 11 and 12.

  The first hydrophilic control coating Ti is based on an annealed probimide type polyimide, which is marketed by OLIN MATERIALS under the name Polyimide 7520.

  The second hydrophilic control coating T2 is based on an epoxy resin marketed by MICROCHEM under the name SU-8.

  The first laminate according to the invention 11 consists of said Ti coating having been treated with a cold fluorocarbon CF4 (octafluorocyclobutane) plasma for about 1 minute in said NEXTRAL NE110 plasma reactor which uses, in a vacuum chamber maintained at 60 mTorr, a radio frequency generator set at 512 MHz. This laminate 11 is thus provided with a hydrophobic film according to the invention having CF 2 groups and a thickness of a few hundred nm.

  The second laminate according to the invention 12 consists of said coating T2 having been treated under the same conditions by this same cold CF4 plasma, in order to obtain a hydrophobic film also having CF 2 groups and a thickness similar to that of the film. from 11.

  The wetting of the Ti, T2 and laminates 11, 12 coatings was quantified by the so-called water drop method, which is well known to those skilled in the art. This method involves depositing a drop of water on the surface to be tested by means of a micro-syringe and measuring, with a camera incorporated in a DIGIDROP brand device, the contact angle between the base drop on the surface and the outer tangent to this surface. In known manner, a surface is considered hydrophobic if the measured contact angle is greater than 90, and hydrophilic if this contact angle is less than 90.

  The graph of FIG. 8 illustrates the average contact angles θ obtained (in degrees) for each coating T1, T2 and laminate 11, 12. This graph shows that the contact angles relative to the control coatings Ti and T2 are between 60 and 80, testifying to the hydrophilic nature of these photosensitive polymer-based coatings, while the average contact angles relative to the laminates 11 and 12 incorporating the CF4 plasma-deposited hydrophobic film are greater than 100.

  In other words, the plasma treatment according to the invention makes it possible to make a coating plate 3 very hydrophobic for an analysis device 1, such as a biochip.

  2) Filling tests: We quantified the optimization of the volume of water Ve actually located before overflow in three types of microcuvettes 4 of different volumes Vc which are formed on the surface of biochips B11 according to the invention, which are manufactured according to the first or second examples above and thus each incorporate a hydrophobic film 7 on the edges of the microcuvettes 4 formed on the coating plate 3. The plate 3, of the probimide type, and the film 7 with CF2 groups are identical to those of said laminate 11 according to the invention.

  The results of volumes Ve = f (Vc) obtained with these three biochips B11 were compared with the corresponding results obtained with three biochips control BT1, which differ only from the biochips B11 in that they do not comprise a hydrophobic film 7 to the hydrophilic surface of the plate 3, like the control coating Ti based on probimide.

  The graph of FIG. 9 illustrates the mean water volumes Ve (in pL) located before overflow in each microcuvette 4 of the control biochip BT1 and according to the invention B11, when the microcuvettes 4 each have a capacitance Vc of 0.1 μL, 0.5 μL and 1 μL.

  This graph shows that the plasma treatment according to the invention makes it possible to multiply by at least two or even three the volume of water Ve located in each microcuvette 4 (Ve increases from 5 to approximately 15 μL when Vc = 1 pL), due to the accentuation of the upper curvature of the dome-shaped drops of water which are respectively anchored in the microcuvettes 4 because of the strong hydrophobicity of the external edges 7 of the latter, in comparison with the volume of Ve water located in each microcuvette 4 surrounded by the untreated plate 3.

  It will further be noted that the hydrophobic film 7 deposited according to the invention makes it possible to ensure simultaneous and optimized filling of thousands of microcuvettes 4, by partial soaking and removal of the biochip 1 in a reagent, because of this high hydrophobicity. aforementioned edges 7. It is thus possible to dispense with a micro-dispenser-type robot for controlled delivery of the reagent into the microcuvettes 4, and the presence of residual drops of reagent on these edges 7 which is capable of contaminating micro-organisms is avoided. 4 adjacent bowls by bridging.

Claims (13)

  A method for increasing the hydrophobicity of a biocompatible and perforated external coating (3) of a chemical and / or biological analysis device (1), such as a biochip, said device comprising a substrate (2) and said coating which covers said substrate by surrounding a plurality of test cuvettes (4) for fixing chemical and / or biological reagents to form test sites, characterized in that it comprises a treatment of said coating using an organic plasma selected from the group consisting of fluorocarbon plasmas and siloxane-containing plasmas, for depositing a hydrophobic film (7) on said coating, so as to optimize the volume of said reagent located around said film in each bowl.   2. Method according to claim 1, characterized in that said organic plasma is chosen from the group constituted by the plasmas of C4F8, CF4, C2F6, C3F8 and C5F8, for obtaining said film (7) which comprises CF2 groups.   3. Process according to claim 1, characterized in that said organic plasma is an octamethyltetrasiloxane plasma, for obtaining said film (7) which comprises SiOXCy groups.   4. Method according to one of the preceding claims, characterized in that said coating (3) is based on at least one crosslinked photosensitive polymer of the thermoplastic or thermosetting type, such as a polyimide or an epoxy resin.   5. Method according to one of the preceding claims, characterized in that said plasma is used cold in the field of radio frequencies.   6. Method according to one of the preceding claims, characterized in that said hydrophobic film (7) has a thickness ranging from 50 nm to 1 μm.   7. Chemical and / or biological analysis device (1), such as a biochip, obtainable by a method according to one of the preceding claims, characterized in that it comprises: a substrate (2), a biocompatible coating (3), such as a plate, which covers said substrate with perforations, each perforation defining a bowl (4) of which a lateral wall (5) and a bottom (6) are respectively formed by said coating; and said substrate, each cuvette being for fixing a chemical and / or biological reagent to form an analysis site, and - a film (7) which has a hydrophobicity greater than that (s) of said substrate and said coating and which covers said coating, with the exception of said side walls of cuvettes, characterized in that said film has CF 2 groups or SiOXC 3 groups, so as to optimize the volume of said reagent located around said film in each cuvette.   8. Analysis device (1) according to claim 7, characterized in that said coating (3) is based on at least one crosslinked photosensitive polymer of the thermoplastic or thermosetting type, such as a polyimide or an epoxy resin.   9. Analysis device (1) according to claim 7 or 8, characterized in that said hydrophobic film (7) has a thickness ranging from 50 nm to 1 .mu.m.   10. A method of manufacturing an analysis device (1) according to one of claims 7 to 9, characterized in that it comprises the following successive steps: (i) covering said substrate (2) by said coating ( 3), (ii) depositing on said bottom (6) of each bowl (4) a protective layer (9) which is intended to protect said substrate against an organic plasma selected from the group consisting of the fluorocarbon plasmas and the plasmas with siloxane groups, (iii) depositing said hydrophobic film (7, 7 ') on both said coating and on said protective layer covering each bottom of the cuvette, by a surface treatment by means of said organic plasma, then (iv) removing said protective layer coated with said film (7 '), to obtain said device comprising said film (7) only on an outer face of said coating.   11. The manufacturing method according to claim 10, characterized in that said protective layer (9) deposited in step (ii) is based on at least one compound selected from the group consisting of polyvinyl alcohol, photosensitive resins and sugars.   12. A method of manufacturing an analysis device (1) according to one of claims 7 to 9, characterized in that it comprises the following successive steps: (i) covering said substrate (2) by said coating (3). ), (ii) depositing said hydrophobic film (7, 7 ") on both said coating and on said bottom (6) of each cuvette (4), by a surface treatment carried out using a plasma organic material selected from the group consisting of fluorocarbon plasmas and siloxane-containing plasmas, and (iii) a portion of said hydrophobic film (7) covering said coating is insoluble and then coated with a protective layer (10) for to protect this film vis-à-vis an oxygen plasma and which is based on at least one photoresist, (iv) the other part of said hydrophobic film (7 ") which covers said bottom of each cuvette, by treatment of the assembly obtained in (iii) by means of said oxygen plasma, then (v) eluting mine said protective layer deposited in (iii), to obtain said device comprising said film (7) only on an outer face of said coating.   13. The manufacturing method according to one of claims 10 to 12, characterized in that said protective layer (9 or 10) is removed by means of a bath of acetone, alcohol or water.