FR2897688A1 - METHOD FOR IMMOBILIZATION OF HYDROGELS ON NON-MODIFIED POLYMERIC MATERIALS, BIOPUCE BASED ON NON-MODIFIED POLYMERIC MATERIALS AND METHOD OF MANUFACTURING THE SAME - Google Patents

Abstract

The invention proposes the use of a number of unmodified polymer materials, used without preliminary modification, for the manufacture of supports for biochips, a modification which is intended to immobilize hydrogels on the surface of the support. The invention also provides a biochip fabricated on the support of unmodified polymeric materials, as well as a method for manufacturing a biochip and a method of immobilizing hydrogel on the supports of unmodified polymeric materials.

Description

METHOD FOR IMMOBILIZING HYDROGELS ON NON-POLYMERIC MATERIALS

  MODIFIED BIOPUCE BASED ON NON-MODIFIED POLYMERIC MATERIALS AND METHOD OF MANUFACTURING THE SAME

  FIELD OF THE INVENTION The invention relates to the field of molecular biology and bio-organic chemistry and relates to the use of polymeric materials that can be used without preliminary chemical modification for the manufacture of a biochip (biochip ) carrying oligonucleotides, proteins, nucleic acids or any other biologically active compound, immobilized in a gel. The invention also relates to a method for manufacturing gel biochips in molecular biology, while sequencing and mapping DNA, detecting mutations, as well as in many medical applications.

  BACKGROUND OF THE INVENTION At present, for the manufacture of biochips, supports made of glass (ceramic), metals and polymeric materials are used. Prior to manufacturing a microchip biochip, glass, metal and, for the most part, the polymers are chemically modified to form on the surface thereof certain active groups that are capable of binding biologically active compounds. The most common is a surface carrying, inter alia, carboxy [1], amino, mercapto [2], aldehyde [3], isocyanate [4], methacrylic [5]. In a number of cases, the immobilization of biologically active compounds is carried out on 2

  nylon membranes [4] and polystyrene (6) without any further modification [1] Nathalie Zammatteo, Laurent Jeanmart, Sandrine Hamels, Stéphane Courtois, Pierre Louette, Laslo Hevesi, José Remacle, Analytical Biochemistry, 2000, 5 280, pages 143-150. [2] Celine Adessi, Gilles Matton, Ayala Handle, Gerardo Turcatti, Jean-Jacques Mermod, Pascal Mayer, Eric Kawashima, Nucleic Acids Research, 2000, Volume 28, No. 20, e87. [3] Edward N Timofeev, Svetlana V. Kochetkova, Andrei D. Mirzabekov, Vladimir L. Florentiev, Nucleic Acids Research, 1996, Volume 24, No. 16, 3142, 3148. [4] Markus Beier, Jorg D. Hoheisel, Nucleic Acids Research, 1999, Volume 27, No. 9, pp. 1970, 1977. [5] Anil Kumar, Zicai Liang, Nucleic Acids Research, 2001, Vol 29, Na 2, 15 e 2. [6] Farah N. Rehman, Mark Audeh, Ezra S. Abrams, Philip W. Hammond, Mary Kenney, T. Christian Boles, Nucleic Acids Research, 1999, Volume 27, No. 2, pp. 649-655. hydrogel-based processes, wherein the process cycle comprises the following steps: (1) chemical modification of the glass support, (2) formation thereon of a gel cell matrix, (3) application of biological macromolecule solutions to the cells according to a predetermined biochip circuit; (4) chemical activation of the cells to immobilize the probe molecules; (5) washing-cleaning and drying of the biochips thus obtained. In order to form the gel cell matrix, there is known a method of laser ablating a specific light absorbing layer underlying the continuous gel layer and having a geometry that is complementary to the cell beam geometry. defined [7]. [7] Ershov et al., U.S. Patent No. 5,570,721. Methods for preparing gel-based biochips in which the step of forming a cell array and step 3 are also known.

  of immobilization of the probe molecules are combined in a single step by the use of a photo- or chemically-induced copolymerization technique [8, 9]. [8] Vasiliskov, A.V. et al., Bio Techniques, 1999, Volume 27, pp. 592-606. [9] RU2216547 C2.

  Essentially, these methods involve the use of compositions that comprise, in addition to a monomer and a crosslinking agent, immobilizable macromolecules, comprising an active group that provides an insertion of these molecules into the network. of polymers in the hydrogel. The materials currently used to manufacture biochip carriers, as well as the biochip fabrication methods thereon, have a number of fundamental disadvantages. The main disadvantages of glass substrates may include the following: o Insufficient chemical homogeneity of the glass surface. This property of the glass leads, after a chemical modification, to the formation of a surface consisting of sections having different hydrophobic (hydrophilic) qualities, and this strongly affects the reproducible nature of the physical properties of biochips, including volume and shape. cells, as well as their arrangement relative to each other. o Relative complexity of implementation and manufacture of biochips having a predetermined surface configuration. o Insufficient mechanical strength. o A comparatively high cost of the substrate with the necessary surface quality. o Preliminary chemical treatment of the glass surface for efficient immobilization of biologically active molecules. The main disadvantages of the polymeric materials used today for the manufacture of carriers may include the following: The surface of the polymer requires a preliminary chemical modification. • The porous structure of nylon filters, used today, actually imposes limitations on the number of biochip elements per unit area. • Gel biochip elements, made on polystyrene without prior chemical modification of its surface, are weakly attached to the surface and, although washed and hybridized, are often subject to destruction. At the present time, all known methods for making gel biochips are based on the use of a chemically modified glass as a support, and with all the disadvantages involved, which affect the productivity of the manufacture, as well as the quality of gel biochips.

  SUMMARY OF THE INVENTION According to a first aspect, the present invention relates to the use of polymeric materials, generally unmodified, used without preliminary modification, for the manufacture of a biochip support, for immobilizing hydrogels on its surface. area. The immobilization of the hydrogels on the surface of the support is carried out during its formation by a polymerization process. With regard to the manufacture of a biochip support, the polymeric materials which are used, generally without preliminary modification, are selected from the following materials: ABS (acrylonitrile-butadiene-styrene copolymer), ABS + PA (a mixture of ABS and polyamide), ABS + PBT (a mixture of ABS and polybutylene terephthalate), ABS + PC (a mixture of ABS and polycarbonate), ABS + PMMA (a mixture of ABS and poly (methyl methacrylate)), ABS + PVC (a mixture of ABS and polyvinyl chloride), ACS (a copolymer of acrylonitrile, chlorinated ethylene and styrene), COC ( copolymers of cycloolefins), MABS (a copolymer of methyl methacrylate, acrylonitrile, butadiene and styrene), PA 6 (polyamide 6), PA 6-3-T (polyamide 6-3-T), PA 11 (polyamide 11), PA 12 (polyamide 12), PA 46 (polyamide 46), PA 66 (polyamide 66), PA 610 (polyamide 610), PA 612 (polyamide 612), PBT (polybutylene terephthalate) ) , PBT + PC (a blend of poly (butylene terephthalate) and polycarbonate), PC + PET (a blend of polycarbonate and poly (ethylene terephthalate)), PC + PMMA (a blend of polycarbonate and poly ( methyl methacrylate), PET or PETP (polyethylene terephthalate), PETG (polyethylene glycol terephthalate), PMMA (poly (methyl methacrylate)), PPA (polyphthalamide, polyamide at elevated temperature), PVC (polyvinyl chloride) and / or mixtures thereof. The invention also proposes the use of polymeric materials and / or blends of such materials, optionally in combination with fillers. Mineral fillers, such as asbestos, fiberglass and / or talc, can be used as fillers. According to a second aspect, the present invention provides a biochip made on a support selected from the polymeric materials cited above and / or mixtures thereof, generally unmodified, a gel layer being immobilized on the surface of the support . The specified polymeric materials and / or mixtures thereof can also be used in combination with fillers. Mineral fillers, including asbestos, fiberglass and / or talc, may be used as fillers. The gel immobilized on the polymer support can also be arranged as separate cells from each other. These cells can also constitute a regular one-dimensional or two-dimensional (beam) structure. The gel cells may comprise immobilized biologically active compounds, and then various biologically active compounds may be immobilized within the different gel cells. Each gel cell of a biochip may further comprise an immobilized fluorescent dye, for example Texas Red, fluorescein, and Cy 5, Cy 3, BODIPY. The immobilization of the compounds is carried out during the formation of the gel by polymerization initiated by thermal, chemical and photochemical. According to a third aspect, the present invention relates to a method for manufacturing gel biochips, the method comprising immobilizing a hydrogel on a support made from unmodified polymeric materials, used without the use of a gel.

  preliminary chemical modification, of the aforementioned type and / or from mixtures thereof. These polymeric materials and / or their mixtures can also be used in combination with fillers. Mineral fillers, including asbestos, fiberglass and / or talc, may be used as fillers.

  Thermally initiated, chemically initiated, or photoinitiated polymerization can be used to form the gel. In the case of the photoinitiated polymerization, it is possible to use a photo-initiated polymerization carried out by a light in the ultraviolet or visible spectrum domains. For gel formation, compositions comprising a monomer, a crosslinking agent and a solvent may be used, which compositions may additionally contain an immobilizable biologically active compound and / or a fluorescent dye, for example Texas Red, fluorescein, Cy 5, Cy 3, BODIPY. In addition, the specified compositions may contain an initiator or a polymerization promoter.

  For the formation of the gel, it is also possible to use compositions comprising a reactive oligomer and a solvent. This type of composition may additionally contain an immobilizable biologically active compound and / or a fluorescent dye, for example Texas Red, fluorescein, Cy 5, Cy 3, BODIPY. In one embodiment, the specified reactive oligomer may also contain, in addition, a biologically active compound in its structure. In addition, the aforementioned compositions may also contain an initiator or a polymerization promoter. To carry out a transfer of specified compositions on the polymer support, it is possible to use a micro-dispenser, for example a micro-distributor rod (needle), pen or jet type.

  Carriers comprising microdrops of incorporated compositions may be placed in an airtight container in an oxygen free atmosphere. The oxygen free atmosphere can be performed with nitrogen, argon and gaseous carbon dioxide. The immobilization of the biologically active compounds is carried out during the formation of the hydrogel. Supports made from polymeric materials are not subject to any chemical modification prior to fabrication of the biochip. 7

  After polymerization, the biochips can be washed with buffer solutions and then with distilled water. The quality of the biochips obtained can be controlled by the diameters of the biochip elements and / or by the fluorescent signal of the dye immobilized in each gel cell of the biochip. According to a fourth aspect, the present invention relates to a method of immobilizing hydrogels on supports made from unmodified polymeric materials of the aforementioned type and / or mixtures thereof, without preliminary chemical modification. The specified polymeric materials and / or mixtures thereof can also be used in combination with fillers. Mineral fillers, including asbestos, fiberglass and / or talc, may be used as fillers. For the formation of the gel, it is possible to use a polymerization initiated thermally, chemically, or photoinitiated. In the case of the photoinitiated polymerization, photoinitiated polymerization carried out by light in the ultraviolet or visible range of the spectrum can be used. Supports made from polymeric materials do not undergo any chemical modification of their surface. For the formation of the gel, compositions comprising a monomer, a crosslinking agent and a solvent may be used, which compositions may additionally contain an immobilizable biologically active compound and / or a fluorescent dye, for example Texas Red, fluorescein, Cy 5, Cy 3, BODIPY. In addition, the aforementioned compositions may also contain an initiator or a polymerization promoter. For the formation of the gel, compositions comprising a reactive oligomer and a solvent can also be used. Such specified compositions may further contain an immobilizable biologically active compound and / or a fluorescent dye, for example Texas Red, fluorescein, Cy 5, Cy 3, BODIPY. In one embodiment, the specified reactive oligomer may also contain, in its structure, a biologically active compound. In addition, the aforementioned compositions may also contain an initiator or a polymerization promoter. 8

  The hydrogels can be produced on the polymer supports in the form of a continuous layer of varying thickness and configuration. The hydrogels can also be arranged on polymer supports in the form of gel cells separated from one another.

  After a transfer of the specified compositions to the polymer support, the latter can be placed in an airtight container in an oxygen-free atmosphere. The oxygen free atmosphere can be performed with nitrogen, argon and gaseous carbon dioxide. After the polymerization, the biochips can be washed with buffer solutions and then with distilled water.

  DESCRIPTION OF THE DRAWINGS The invention is illustrated by the following figures: FIG. 1 shows photographs of biochips produced on poly (methyl methacrylate) (PMMA) during a thermally initiated polymerization (A, D), chemically (B , E) and initiated photoinitiated (C, F). The compositions presented in Example 1 are distributed in the form of drops on a PMMA plate without preliminary modification, and they are then irradiated with UV (2 = 350 nm). PMMA plates carrying immobilized gel elements are washed with buffer solution, water and then dried. To control the quality of the biochip elements, the image of the biochips in terms of transmitted light (A, B, C) and luminescent light (D, E, F) is recorded. Figure 2 shows the results of oligonucleotide hybridization, labeled with a fluorescent dye, obtained on the oligonucleotide biochip, made from various polymeric and glass supports. Oligonucleotides 5'-AATTGGCTCAGCTGGCT-OCH2CH (CH2OH) (CH2) 4 -NH2 (A) and 5'-AATTGGCTCGGCTGGCT-OCH2CH (CH2OH) (CH2) 4 -NH2 (B) are immobilized in a hydrogel according to Example 1 -I, on supports made from different polymeric materials: PMMA (1); PETP (2); PA6 (3); ABS (4); ABS + PBT (5); ACS (6); COC (7); MABS (8); PETG (9); ABS + PA (10); PPP (11); PVC (12); ABS + PMMA (13); 9

  PBT + PC (14); ABS + PC (15); ABS + PVC (16); PBT (17); PC + PET (18); PC + PMMA (19); glass (20). According to Example 2, the biochips obtained are hybridized with an oligonucleotide labeled with a fluorescent dye 3'-TTAACCGAGTCGACCGA-Cy5. After hybridization, the strongest fluorescent signal is observed in biochip cells that contain the immobilized oligonucleotide A, which is fully complementary to the fluorescently labeled one.

  DETAILED DESCRIPTION The present invention relates to the use of polymeric materials for the manufacture of a biochip support for immobilizing hydrogels on its surface, wherein the polymeric materials are selected from the following materials: ABS (acrylonitrile copolymer -butadiene-styrene), ABS + PA (a blend of ABS and polyamide), ABS + PBT (a blend of ABS and polybutylene terephthalate), ABS + PC (a blend of ABS and polycarbonate), ABS + PMMA (a blend of ABS and poly (methyl methacrylate)), ABS + PVC (a blend of ABS and polyvinyl chloride), ACS (a copolymer of acrylonitrile, chlorinated ethylene and styrene), COC (cycloolefin copolymers), MABS (a copolymer of methyl methacrylate, acrylonitrile, butadiene and styrene), PA 6 (polyamide 6), PA 6-3- T (polyamide 6-3-T), PA 11 (polyamide 11), PA 12 (polyamide 12), PA 46 (polyamide 46), PA 66 (polyamide 66), PA 610 ( polyamide 610), PA 612 (polyamide 612), PBT (poly (butylene terephthalate)), PBT + PC (a blend of poly (butylene terephthalate) and polycarbonate), PC + PET (a blend of polycarbonate and poly (ethylene terephthalate)), PC + PMMA (a blend of polycarbonate and poly (methyl methacrylate)), PET or PETP (polyethylene terephthalate), PETG (polyethylene glycol terephthalate), PMMA (poly (methyl methacrylate)), PPA (polyphthalamide, high temperature polyamide), PVC (polyvinyl chloride) and / or mixtures thereof. The polymeric materials used are most often, and preferably, unmodified polymers used without preliminary modification. 10

  On the other hand, the polymeric materials and / or mixtures thereof can be used in combination with fillers, especially mineral fillers, including asbestos, fiberglass and / or talc. Furthermore, in the subject matter of the invention, the immobilization of a hydrogel on the support surface is advantageously carried out during its formation by a polymerization process. The invention also relates to a biochip made on a support made from polymeric materials chosen from the following materials: ABS (acrylonitrile-butadiene-styrene copolymer), ABS + PA (a mixture of ABS and polyamide), ABS + PBT (a mixture of ABS and poly (butylene terephthalate)), ABS + PC (a mixture of ABS and polycarbonate), ABS + PMMA (a mixture of ABS and poly (methyl methacrylate) ), ABS + PVC (a mixture of ABS and polyvinyl chloride), ACS (a copolymer of acrylonitrile, chlorinated ethylene and styrene), COC (cycloolefin copolymers), MABS ( a copolymer of methyl methacrylate, acrylonitrile, butadiene and styrene), PA 6 (polyamide 6), PA 6-3T (polyamide 6-3-T), PA 11 (polyamide 11), PA 12 (polyamide 12), PA 46 (polyamide 46), PA 66 (polyamide 66), PA 610 (polyamide 610), PA 612 (polyamide 612), PBT (poly (butylene terephthalate)), PBT + PC (a mixture of poly ( téréphtala butylene) and polycarbonate), PC + PET (a blend of polycarbonate and polyethylene terephthalate), PC + PMMA (a blend of polycarbonate and poly (methyl methacrylate)), PET or PETP (polyethylene terephthalate), PETG (polyethylene glycol terephthalate), PMMA (poly (methyl methacrylate)), PPA (polyphthalamide, high temperature polyamide), PVC (polyvinyl chloride) and or mixtures thereof, a gel layer being immobilized on the surface of the support. In this biochip, the polymeric materials are most often, and preferably, unmodified materials. The biochips subject of the invention may have one or more of the following characteristics, in any technically feasible combination: the polymeric materials and / or the mixtures thereof are used in combination with fillers, for example mineral fillers, including asbestos, fiberglass and / or talc; 11

  the gel layer formed on the polymer support is arranged in the form of cells separated from each other; the cells of the biochip form a regular one-dimensional or two-dimensional structure (of the beam type in particular); the gel cells also contain the immobilized biologically active compounds and / or the immobilized fluorescent dyes, where, preferably, the different biologically active compounds are immobilized inside the gel cells and in which the immobilized fluorescent dye is advantageously chosen among Texas Red, fluorescein, and Cy 5, Cy 3, BODIPY; the immobilization of the biologically active compounds in the gel is carried out during the formation of the gel by a polymerization initiated by thermal, chemical and photochemical way.

  The invention furthermore relates, in another aspect, to a method for manufacturing a gel biochip, comprising immobilizing a hydrogel on a support made from polymeric materials, characterized in that the polymeric materials are chosen among the following materials: ABS (acrylonitrile-butadiene-styrene copolymer), ABS + PA (a mixture of ABS and polyamide), ABS + PBT (a mixture of ABS and polybutylene terephthalate), ABS + PC (a mixture of ABS and polycarbonate), ABS + PMMA (a mixture of ABS and poly (methyl methacrylate)), ABS + PVC (a mixture of ABS and poly (vinyl chloride)), ACS (a copolymer of acrylonitrile, chlorinated ethylene and styrene), COC (cycloolefin copolymers), MABS (a copolymer of methyl methacrylate, acrylonitrile, butadiene and styrene), PA 6 ( polyamide 6), PA 6-3-T (polyamide 6-3-T), PA 11 (polyamide 11), PA 12 (polyamide 12), PA 46 (polyamide 46), PA 66 (polyamide) amide 66), PA 610 (polyamide 610), PA 612 (polyamide 612), PBT (poly (butylene terephthalate)), PBT + PC (a mixture of polybutylene terephthalate and polycarbonate), PC + PET ( a mixture of polycarbonate and poly (ethylene terephthalate)), PC + PMMA (a mixture of polycarbonate and poly (methyl methacrylate)), PET or PETP (polyethylene terephthalate), PETG (Polyterephthalate d) ethylene glycol)), PMMA (poly (methyl methacrylate)), PPA (polyphthalamide, polyamide at 12 ° C)

  high), PVC (polyvinyl chloride) and / or mixtures thereof, the polymeric materials being unmodified materials used without preliminary chemical modification. This process may also comprise one or more of the following preferential characteristics, according to all the technically possible combinations: the polymeric materials and / or their mixtures are used in combination with fillers, in particular mineral fillers, including in particular asbestos, fiberglass and / or talc; a thermally initiated polymerization is used to form the gel; a chemically initiated polymerization is used to form the gel; a photo-initiated polymerization is used to form the gel; the photoinitiated polymerization is used in the ultraviolet or visible spectrum domains; compositions comprising a monomer, a crosslinking agent and a solvent are used to form the gel; said compositions further comprise an immobilizable biologically active compound and / or an immobilizable fluorescent dye and / or a polymerization initiator or promoter, the immobilized fluorescent dye being advantageously selected from Texas Red, fluorescein, Cy 5, Cy 3, and BODIPY; compositions comprising an oligomer and a solvent are used to form the gel, wherein said compositions preferably further comprise an immobilizable biologically active compound and / or an immobilizable fluorescent dye and / or a polymerization initiator or promoter, immobilized fluorescent dye being selected from Texas Red, fluorescein, Cy 5, Cy 3 and BODIPY, and wherein the biologically active compound is advantageously the structure of said reactive oligomer; a micro-dispenser is used for transferring said compositions to the polymer support, said micro-dispenser being preferably selected from a rod-type (needle), pen or jet micro-dispenser, and wherein the supports comprising the microdrops of transferred compositions are preferably placed in an airtight container in an oxygen-free atmosphere, this atmosphere

  oxygen-free being advantageously generated by nitrogen, argon and gaseous carbon dioxide; the immobilization of the biologically active compounds in the gel is carried out during the formation of the hydrogel; the supports of the polymeric materials are not subjected to any chemical modification before the fabrication of the biochip; after the polymerization, the biochips are washed successively with buffer solutions and then with distilled water; the quality of the biochips obtained is controlled by the diameters of the elements of the biochips; the quality of the biochips obtained is controlled by the fluorescent signal of an immobilized dye in each gel cell of a biochip.

  Finally, the invention also relates, according to another specific aspect, to a process for immobilizing hydrogels on polymeric materials selected from the following materials: ABS (acrylonitrile-butadiene-styrene copolymer), ABS + PA (a mixture of ABS and polyamide), ABS + PBT (a blend of ABS and polybutylene terephthalate), ABS + PC (a blend of ABS and polycarbonate), ABS + PMMA (an ABS blend) and poly (methyl methacrylate)), ABS + PVC (a blend of ABS and polyvinyl chloride), ACS (a copolymer of acrylonitrile, chlorinated ethylene and styrene), COC (copolymers of cyclo-olefins), MABS (a copolymer of methyl methacrylate, acrylonitrile, butadiene and styrene), PA 6 (polyamide 6), PA 6-3T (polyamide 6-3-T), PA 11 (polyamide 11), PA 12 (polyamide 12), PA 46 (polyamide 46), PA 66 (polyamide 66), PA 610 (polyamide 610), PA 612 (polyamide 612), PBT (polybutylene terephthalate), PBT + PC (u n mixture of poly (butylene terephthalate) and polycarbonate), PC + PET (a mixture of polycarbonate and poly (ethylene terephthalate)), PC + PMMA (a mixture of polycarbonate and poly (methyl methacrylate)) PET or PETP 30 (polyethylene terephthalate), PETG (polyethylene terephthalate), PMMA (poly (methyl methacrylate)), PPA (polyphthalamide, polyamide 14).

  high), PVC (polyvinyl chloride) and / or mixtures thereof, where the polymeric materials are used without preliminary chemical modification. This immobilization process advantageously comprises one or more of the following preferential characteristics, according to all the possible technical combinations: the polymeric materials and / or their mixtures are used in combination with fillers, for example mineral fillers, including asbestos fiberglass and / or talc; a thermally initiated polymerization is used to form the gel; A chemically initiated polymerization is used to form the gel; a photo-initiated polymerization is used to form the gel; the photoinitiated polymerization is carried out in the ultraviolet or visible spectrum domains; the surface of the supports of polymeric materials is not subjected to a chemical modification; compositions comprising a monomer, a crosslinking agent and a solvent are used to form the gel, said compositions preferably further comprising an immobilizable biologically active compound and / or an immobilizable fluorescent dye and / or an initiator or a promoter wherein said immobilized fluorescent dye is, for example, selected from Texas Red, fluorescein, and Cy 5, Cy 3, BODIPY; compositions comprising a reactive oligomer and a solvent are used to form the gel, said compositions preferably further comprising an immobilizable biologically active compound and / or an immobilizable fluorescent dye and / or a polymerization initiator or promoter, said immobilized fluorescent dye being for example selected from Texas Red, fluorescein, and Cy 5, Cy 3, BODIPY, and said reactive oligomer preferably containing in its structure a biologically active compound; the hydrogels are created on the polymer supports in the form of a continuous layer of varying thickness and configuration; the hydrogels are arranged on the polymer supports in the form of cells separated from one another; 15

  the polymer support, after the transfer of said compositions thereon, is placed in an airtight container in an oxygen-free atmosphere; the oxygen-free atmosphere is generated by nitrogen, argon and gaseous carbon dioxide; after the polymerization, the gel formed is washed successively with buffer solutions and then with distilled water.

  In this invention, it is proposed to use a number of well-known and commercially available polymeric materials for the manufacture of gel biochip carriers, without any preliminary modification. The polymeric materials are selected from the following materials: ABS (acrylonitrile-butadiene-styrene copolymer), ABS + PA (a blend of ABS and polyamide), ABS + PBT (a blend of ABS and poly (terephthalate) butylene)), ABS + PC (a mixture of ABS and polycarbonate), ABS + PMMA (a mixture of ABS and poly (methyl methacrylate)), ABS + PVC (a mixture of ABS and poly ( vinyl chloride)), ACS (a copolymer of acrylonitrile, chlorinated ethylene and styrene), COC (cycloolefin copolymers), MABS (a copolymer of methyl methacrylate, acrylonitrile, butadiene and styrene), PA 6 (polyamide 6), PA 6-3-T (polyamide 6-3-T), PA 11 (polyamide 11), PA 12 (polyamide 12), PA 46 (polyamide 46), PA 66 ( polyamide 66), PA 610 (polyamide 610), PA 612 (polyamide 612), PBT (polybutylene terephthalate), PBT + PC (a mixture of polybutylene terephthalate and polycarbonate), PC + PET 25 (a mixture of polycarbonate and poly (ethylene terephthalate)), PC + PMMA (a mixture of polycarbonate and poly (methyl methacrylate)), PET or PETP (polyethylene terephthalate), PETG (polyethylene glycol terephthalate), PMMA (poly (methyl methacrylate)), PPA (polyphthalamide, high temperature polyamide), PVC (polyvinyl chloride) and / or mixtures of these polymeric materials. 16

  The aforementioned polymeric materials and / or mixtures thereof can also be used in combination with fillers. Mineral fillers, such as asbestos, fiberglass and / or talc, can be used as fillers. Polymeric materials, used without preliminary modification, are intended for the manufacture of a support on the surface of which hydrogels will be immobilized chemically during their formation by a polymerization which is induced thermally, chemically or photochemically. The abovementioned supports are intended for the manufacture of biochips, where biologically active compounds are immobilized in a gel.

  A biochip is a gel layer formed on the polymeric support and arranged in the form of cells separated from each other, each cell being capable of containing, or not containing, the immobilized biologically active compounds. At the same time, biologically active compounds immobilized in different cells may have different nature and properties. The cells can constitute a regular one-dimensional or two-dimensional (beam) structure. The application of the compositions containing the biologically active compounds on the support can be carried out by various means, including the use of an automatic device (for example a robot) equipped with one or more microdispensers of jet or rod / type. needle.

  The immobilization of oligonucleotides, proteins and nucleic acids or other biologically active compounds in a gel can be carried out: during the formation of the gel, during the carrying out of a polymerization initiated by thermal, chemical or photochemical; After formation of a gel on a polymer support. The biologically active compounds may comprise, within their structure, an active group (for immobilization), including one of the following groups: amino, hydrogen sulfide, methacrylamide, acrylamide, acrylate, methacrylate, hydrazide, etc. Alternatively, the biologically active compounds can be used in their native form without preliminary modification. 17

  In addition to the immobilized biologically active compound, each gel cell of a biochip may contain an immobilized fluorescent dye used to control the quality of the biochip and to interpret the results of hybridization on the pellet.

  A method of manufacturing gel biochips on polymer supports provided comprises the following steps: • the preparation of compositions for forming a gel; The transfer of the compositions onto the support; The polymerization of the compositions in an oxygen-free atmosphere; • the washing-cleaning of the biochip; • the quality control of the elements of the biochip. In preparing the compositions to form a homogeneous solution, all components are intimately mixed and degassed.

  The compositions comprise the following components: a monomer, constituting the base of the gel formed and representing an unsaturated compound; as monomer, use is made of acrylamide, methacrylamide, N- [tris (hydroxymethyl) methyl] acrylamide, 2-hydroxyethyl methacrylate, methyl methacrylate or a different monomer containing several bonds, at least one multiple bond to be reactive during the polymerization; A crosslinking agent representing an unsaturated compound comprising two or more multiple bonds; as the crosslinking agent, N, N'-methylenebisacrylamide, N, N'-methylenebismethacrylamide, N, N '- (1,2-dihydroxyethylene) bisacrylamide or polyethylene glycol diacrylate can be used, alone or as a mixture, or a different symmetrical or unsymmetrical crosslinking agent comprising two or more multiple bonds active in polymerization reactions; • an immobilizable biologically active compound (an optional component); 18

  as a biologically active compound, a different oligonucleotide, nucleic acid, protein or different compound may be used; • a fluorescent dye (an optional component); as the fluorescent dye, Texas Red, fluorescein, Cy5, Cy3, BODIPY and other fluorescent dyes may be used; • a solvent; as a solvent, it is possible to use water, glycerol, N, N-dimethylformamide, dimethylsulfoxide, different polar and non-polar solvents, aqueous buffer solutions, glycerol solutions, sucrose solutions, polyol, saline and non-saline solutions of polar and non-polar solvents; • a polymerization initiator or promoter (an optional component); as the initiator or promoter, it is possible to use compounds which contribute to a photochemical or chemical initiation of the polymerization, which are soluble in water or in organic media, namely ammonium persulfate, potassium persulfate, hydrogen peroxide, , benzoyl peroxide, azoisobutyronitrile (AIBN), ferrous iron salts, methylene blue, fluorescein, N, N, N ', N'-tetramethylethylenediamine, 4- (N, N-) dimethylamino) pyridine, triethylamine, acetone, a different polymerization initiator which is induced photo-chemically or chemically; In preparing the compositions, in place of a monomer and / or a crosslinking agent, reactive oligomers containing or not containing biologically active compounds in their structures may be used. When carrying out a thermally initiated or photoinitiated polymerization, it is also possible to omit the initiator or the polymerization promoter.

  To carry out a transfer of the compositions onto the polymer support, it is possible to use robots equipped with micro-dispensers of various types, including microparticles.

  robots equipped with rod-type (needle), pen-type and jet type micro-dispensers. In carrying out the polymerization, the carriers carrying micro-drops of compositions are placed inside hermetically in an oxygen-free atmosphere (nitrogen, argon, gaseous carbon dioxide, etc.). The polymerization initiated thermally in the micro-drops of solution is carried out in an oxygen-free atmosphere at a temperature T of 60 to 80 C. To carry out a polymer-initiated polymerization inside 10 microdrops of solution, these are maintained in an oxygen-free atmosphere at a temperature T of 60 to 80 C, depending on the chosen initiator. The photoinitiated polymerization process is carried out by UV radiation with a 7, 312 min. The biochips obtained are washed first with buffer solutions and then with distilled water and then used. The quality of the biochips obtained is determined by the relative error of the diameters of the elements of the biochip or volumes thereof. The volume of the elements of the biochip is proportional to the fluorescent signal of an immobilized dye within each gel cell of a biochip. A method of immobilizing hydrogels on polymer supports during formation of the hydrogels involves the generation of covalent bonds between the macromolecules of the polymer supports and the hydrogels, the covalent bonds being generated on the surface of the supports. of polymer during the formation of the gel during the thermally, chemically and photochemically initiated polymerization. The covalent bonds between the polymer support and the hydrogel are generated by one of the following possible routes: a) The bringing into play of several bonds, existing in the structure of the polymer molecules, in the polymerization reaction with monomers that form a hydrogel during the polymerization reaction. 20

  b) The use of fragments of the support polymer molecules in chain transfer reactions during the formation of the gel during the initiated polymerization. c) The modification of the polymer surface by bifunctional reagents which carry in their structure an unsaturated fragment and which are part of the compositions for the formation of a hydrogel. According to route a), polymers and compositions based on these, obtained by the polymerization process, can also be reacted: ABS, ABS + PVC, PC + PMMA, ACS, COC, MABS , PMMA, PVC. This is possible because of the nature of the radical polymerization reaction, so that polymers are obtained, i.e. in the chain termination step, a process of forming many terminal bonds takes place during intermolecular disproportionation [10]. [10] A. M. Shur, High-molecular Compounds, M .; The Higher School 15 Publishing House, 1981, pages 100 to 104 (in Russian). According to route a), polymers and compositions based on these, obtained by the polycondensation process, can also be reacted, namely: PBT, PBT + PC, PC + PET, PET or PETP, PETG . The formation of several terminal bonds in macromolecules of these polymers is evidenced by the intramolecular dehydration processes which take place involving alcohol groups under the conditions of the production of polymers [II]. [11] Yu. S. Shabarov, Organic Chemistry V1, M .; Chemistry, 1996, pp 193 - 203 (in Russian). By way of b), all the specified polymeric materials [12] can be reacted, however, the reaction conditions are highly dependent on the nature and structure of the polymers. [12] A. M. Shur, High-molecular Compounds, M .; The Higher School Publishing House, 1981, pages 104-113 (in Russian). The polycondensation polymers containing terminal amino groups, namely: ABS + PA, PA6, PA6-3-T, PA11, PA12, PA46, PA66, PA610, PA612, PPA, can react according to channel c). The necessary condition is the presence of bi-functional derivatives which are active in a nucleophilic addition or in 21

  substitution reactions, for example N, N-methylenebisacrylamide [13] or N-hydroxysuccinamide-6-methacryloylaminohexane ester, respectively, in the composition for generating a hydrogel. [13] General Organic Chemistry, Volume 3, Nitrogen-containing Compounds, under the direction of N.K. Kochetkov, M: Chemistry, 1982, pp. 61-62 (in Russian). The concept of the invention is described in detail with reference to the examples of individual embodiments, which will not be considered by the reader as limiting the scope of the claims of the invention.

  As shown in Figure 2, biochips that are made on various polymer supports, act when hybridized in the same manner as in the case of glass commonly used for the fabrication of a biochip, and the results of the hybridization are practically unaffected by the nature of the surface of the polymeric material.

  Since the nature of the polymer support does not affect the properties of the gel, it is then obvious that biologically active compounds other than oligonucleotides, such as nucleic acids, proteins, sugars, lipids, and the like. , which can be immobilized in a gel, can also be immobilized according to the present invention, in addition, they would not lose their properties when immobilized in a gel on the polymer support. The following nonlimiting examples are illustrative embodiments intended inter alia to attest to the feasibility of the invention. Those skilled in the art will easily discover other embodiments of the invention, covered by the appended claims.

  Example L Fabrication of a Biochip on a Polymer Support, Where Oligonucleotides are Immobilized in a Gel

  1. Photoinitiated polymerization To a mixture of 2-hydroxyethyl methacrylate (m = 0.030 g), N, N'-methylenebisacrylamide (m = 0.007 g) and 2-acryloyloxyethyl methacrylate (m = 0.003 g), poured a solution of N, N, N ', N'-tetramethylethylenediamine in 22

  demineralised water (V = 210 l, 1: 1) containing a Texas Red dye (n = 40 nmol), and they are mixed until complete dissolution of the components, then glycerol is poured into it (V = 650 l). An oligonucleotide solution in water (v = 100 g, C = 2 nmol / well) is added to the resulting solution. The mixture is vigorously stirred. The composition is applied to a polymer support using a QArray robot (Genetix, GB). The droplet beam obtained is irradiated with UV light (1 = 350 nm, t = 60 min, T = 55 ° C.) in a dry argon atmosphere, washed with a phosphate buffer (0.1 M, t = 15 min, T = 30 ° C.) and then with water (t = 15 min, T = 60 ° C.) and air dried (T = 25 ° C.) in a dust-free atmosphere. Using special equipment equipped with a charge-coupled camera (CCD camera) and a computer, photographs of biochips in the field of transmitted visible light (C) and fluorescent light are obtained. The quality of the elements of the biochip is determined using special software based on the relative error of diameters or fluorescent signals of all elements of the biochip. Figure 1 (C, F) shows the photographs of a biochip obtained by the present procedure on poly (methyl methacrylate) (PMMA) in the field of transmitted light and fluorescent light.

  II. Thermally Initiated Polymerization to a mixture of 2-hydroxyethyl methacrylate (m = 0.075 g), N, N'-methylenebisacrylamide (m = 0.0175 g) and 2-acryloyloxyethyl methacrylate (m = 0.0075 g), a solution of N, N, N ', N'-tetramethylethylenediamine is poured into demineralised water (V = 200 l, 1: 1) containing a Texas Red dye (n = 40 nmol), and mixing them until the complete dissolution of the components, then glycerol is poured in (V = 600 l). An oligonucleotide solution in water (v = 100 l, C = 2 nmol / g) is added to the resulting solution. The mixture is vigorously stirred. The composition is applied to a polymer support using a QArray robot (Genetix, GB). The droplet beam obtained is maintained at a temperature of 80 ° C. for 60 minutes in a dry argon atmosphere, washed with a phosphate buffer (0.1 M, t = 15 min, T = 30 ° C.) and then with water (t = 15 min, T = 60 C) and on the 23

  air dried (T = 25 C) in an atmosphere free of dust. Using special equipment equipped with a CDD (charge coupled device) camera and a computer, photographs of the biochip are obtained in the field of transmitted visible light and fluorescent light. The quality of the elements of the biochip is determined using special software based on the relative error of diameters or fluorescent signals of all elements of the biochip. Figure 1 (A, D) shows the photographs of a biochip obtained by the present procedure on poly (methyl methacrylate) (PMMA) in the field of transmitted light and fluorescent light.

  III. Chemically initiated polymerization of a mixture of 2-hydroxyethyl methacrylate (m = 0.075 g), N, N'-methylenebisacrylamide (m = 0.0175 g) and 2-acryloyloxyethyl methacrylate (m = 0.0075 g), a phosphate buffer solution (pH 11.6, C = 0.05 M, V = 190 l) containing a Texas Red type dye (n = 40 nmol) and ammonium persulphate (m = 0.010 g) is poured into it. and they are mixed until complete dissolution of the components, then glycerol is poured in (V = 600 l). An oligonucleotide solution in water (v = 100 l, C = 2 nmol / l) is added to the resulting solution. The mixture is vigorously stirred. The composition is applied to a polymer support using a QArray robot (Genetix, GB). The droplet beam obtained is placed in an airtight chamber saturated with N, N, N ', N'-tetramethylethylenediamine vapor in an argon atmosphere and maintained at a temperature of 80 ° C. for 60 minutes. washed with a phosphate buffer (0.1 M, t = 15 min, T = 30 ° C.) and then with water (t = 15 min, T = 60 ° C.) and dried in air ( T = 25 C) in an atmosphere free of dust. Using special equipment equipped with a CCD camera and a computer, photographs of the biochip are obtained in the field of transmitted visible light and fluorescent light. The quality of the elements of the biochip is determined using special software based on the relative error of diameters or fluorescent signals of all elements of the biochip. Figure 1 (B, E) presents the photographs of a 24

  biochip obtained by the present procedure on poly (methyl methacrylate) (PMMA) in the field of transmitted light and fluorescent light. As shown by the results presented in FIG. 1, the thermally, chemically and photo-initiated polymerizations actually provide a degree of immobilization of an identical hydrogel on the support, or else, in other words, the same quality of biochips. .

  Example 2. Hybridization on oligonucleotide biochips. A solution (1 M NaCl, 1 μM EDTA, 1% Tween 20, 5 mM phosphate buffer, pH 7.0, V = 35 μl) was hybridized containing a fluorescently labeled oligonucleotide (C = 10 mM) (t = 12). h, T = 37 C) with an oligonucleotide biochip made according to Example I-1. The biochip is washed with a hybridization solution that does not contain the fluorescently labeled oligonucleotide and is dried. The fluorescent signal is recorded using a fluorescent microscope equipped with a CDD camera and a computer. The hybridization results are shown in FIG. 2. As shown by the results presented in FIG. 2, the strongest fluorescent signal after hybridization is observed in the biochip cells which contain the fully complementary immobilized oligonucleotide A. of the fluorescently labeled oligonucleotide which is not dependent on the nature of the polymeric material used for the manufacture of the support.

  Industrial Field of Application The method of manufacturing a biochip according to the present invention is intended for the manufacture of biochips. The biochips according to the present invention can be used as self-supporting articles for conducting scientific research to study various interactions of biologically active compounds including: oligonucleotide-oligonucleotide, oligonucleotide-nucleic acid, protein-protein, protein-nucleic acid, etc. • can be incorporated into compositions of different medical diagnostics to quickly detect and identify a causative agent (vector) and / or disease. The method of immobilizing hydrogels on polymer supports can be used to make a biochip for various applications; Can be used to manufacture polymer articles having various applications, the surface of which is to be covered with a hydrogel layer, for example, different electrodes and sensors, where the working surface is covered with a hydrogel in which is immobilized a biologically active compound.

Claims (34)

  1. Biochip manufactured on a support made from polymeric materials selected from the following materials: ABS (acrylonitrile-butadiene-styrene copolymer), ABS + PA (a mixture of ABS and polyamide), ABS + PBT (a mixture of ABS and poly (butylene terephthalate)), ABS + PC (a mixture of ABS and polycarbonate), ABS + PMMA (a blend of ABS and poly (methyl methacrylate)), ABS + PVC (a mixture of ABS and polyvinyl chloride), ACS (a copolymer of acrylonitrile, chlorinated ethylene and styrene), COC (cycloolefin copolymers), MABS (a copolymer of methyl methacrylate, acrylonitrile, butadiene and styrene), PA 6 (polyamide 6), PA 6-3T (polyamide 6-3-T), PA 11 (polyamide 11), PA 12 (polyamide 12), PA 46 (polyamide 46 PA 66 (polyamide 66), PA 610 (polyamide 610), PA 612 (polyamide 612), PBT (poly (butylene terephthalate)), PBT + PC (a mixture of polybutylene terephthalate) and polycarbonate. nate), PC + PET (a mixture of polycarbonate and polyethylene terephthalate), PC + PMMA (a mixture of polycarbonate and poly (methyl methacrylate)), PET or PETP (polyethylene terephthalate), PETG (polyethylene terephthalate), PMMA (poly (methyl methacrylate)), PPA (polyphthalamide, high temperature polyamide), PVC (polyvinyl chloride) and / or mixtures thereof, a diaper gel being immobilized on the surface of the support.   The biochip of claim 1, wherein the polymeric materials are unmodified materials.   3. Biochip according to claim 1 or 2, characterized in that the polymeric materials and / or mixtures thereof are used in combination with fillers.   4. Biochip according to claim 3, characterized in that the fillers are mineral fillers, including asbestos, fiberglass and / or talc.   5. Biochip according to claim 1 or 2, characterized in that the gel layer formed on the polymer support is further arranged in the form of cells separated from each other.   6. biochip according to claim 5, characterized in that the cells form a regular one-dimensional or two-dimensional (beam) structure.   7. The biochip according to claim 3, characterized in that the gel cells further contain immobilized biologically active compounds and / or immobilized fluorescent dyes.   8. biochip according to claim 7, characterized in that the different biologically active compounds are immobilized inside the gel cells.   9. Biochip according to claim 7, characterized in that the immobilized fluorescent dye is selected from Texas Red, fluorescein, and Cy 5, Cy 3, BODIPY.   10. Biochip according to claim 5, characterized in that the immobilization of the biologically active compounds in the gel is carried out during the formation of the gel by polymerization initiated by thermal, chemical and photo-chemical.   A method of manufacturing a gel biochip, comprising immobilizing a hydrogel on a support made from polymeric materials, characterized in that the polymeric materials are selected from the following materials: ABS (acrylonitrile copolymer) butadiene-styrene), ABS + PA (a mixture of ABS and polyamide), ABS + PBT (a mixture of ABS and polybutylene terephthalate), ABS + PC (a mixture of ABS and polycarbonate ), ABS + PMMA (a blend of ABS and poly (methyl methacrylate)), ABS + PVC (a blend of ABS and polyvinyl chloride), ACS (a copolymer of acrylonitrile, chlorinated ethylene and styrene), COC (cycloolefin copolymers), MABS (a copolymer of methyl methacrylate, acrylonitrile, butadiene and styrene), PA 6 (polyamide 6), PA 6-3T ( polyamide 6-3-T), PA 11 (polyamide 11), PA 12 (polyamide   12), PA 46 (polyamide 46), PA 66 (polyamide 66), PA 610 (polyamide 610), PA 612 (polyamide 612), PBT (poly (butylene terephthalate)), PBT + PC (a blend of poly (butylene terephthalate) and polycarbonate), PC + PET (a mixture of polycarbonate and poly (ethylene terephthalate)), PC + PMMA (a blend of polycarbonate and poly (methyl methacrylate)), PET or PETP (polyethylene terephthalate), PETG (polyethylene glycol terephthalate), PMMA (poly (methyl methacrylate)), PPA (polyphthalamide, high temperature polyamide), PVC (polyvinyl chloride) and / or mixtures thereof, the polymer materials being unmodified materials used without preliminary chemical modification. Process according to claim 11, characterized in that the polymeric materials and / or mixtures thereof are used in combination with fillers.   13. The method of claim 12, characterized in that the fillers are mineral fillers, including asbestos, fiberglass and / or talc. 10   14. The method of claim 11, characterized in that a thermally initiated polymerization is used to form the gel.   15. The method of claim 11, characterized in that a chemically initiated polymerization is used to form the gel.   16. The method of claim 11, characterized in that a photoinitiated polymerization is used to form the gel.   17. The method according to claim 16, characterized in that the photoinitiated polymerization is used in the ultraviolet and visible spectral domains.   18. The method of claim 11, characterized in that compositions comprising a monomer, a crosslinking agent and a solvent are used to form the gel.   19. The method of claim 18, characterized in that said compositions further comprise an immobilizable biologically active compound and / or an immobilizable fluorescent dye and / or a polymerization initiator or promoter.   20. The method of claim 19, characterized in that the immobilized fluorescent dye is selected from Texas Red, fluorescein, Cy 5, Cy 3 and BODIPY.   21. The method of claim 11, characterized in that compositions comprising an oligomer and a solvent are used to form the gel. 15 25 30   22. The method according to claim 21, characterized in that said compositions further comprise an immobilizable biologically active compound and / or an immobilizable fluorescent dye and / or an initiator or a polymerization promoter.   23. The method of claim 22, characterized in that the immobilized fluorescent dye is selected from Texas Red, fluorescein, Cy 5, Cy 3 and BODIPY.   24. The method of claim 21, characterized in that the biologically active compound constitutes the structure of said reactive oligomer.   25. The method of claim 22, characterized in that the biologically active compound constitutes the structure of said reactive oligomer.   26. The method of claim 11, characterized in that a micro-dispenser is used to transfer said compositions to the polymer support.   27. The method of claim 26, characterized in that said micro-dispenser is selected from a micro-distributor type rod (needle), pen or jet.   28. A method according to claim 26, characterized in that the supports comprising microdroplets of transferred compositions are placed in a sealed container in an oxygen-free atmosphere.   29. The method of claim 27, characterized in that said oxygen-free atmosphere is generated by nitrogen, argon and gaseous carbon dioxide.   30. The method of claim 11, characterized in that the immobilization of the biologically active compounds in the gel is carried out during the formation of the hydrogel. 30   31. The method of claim 11, characterized in that the supports of the polymeric materials are not subjected to any chemical modification before the manufacture of the biochip.   32. The method of claim 11, characterized in that, after the polymerization, the biochips are washed successively with buffer solutions and then with distilled water.   33. Method according to any one of claims 11 to 32, characterized in that the quality of the biochips obtained is controlled by the diameters of the elements of the biochips.   34. Process according to any one of claims 11 to 32, characterized in that the quality of the biochips obtained is controlled by the fluorescent signal of a dye immobilized in each gel cell of a biochip.