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WO2005010525A1 - Serie de supports solides emballes sous vide, et procede d'elaboration - Google Patents

Serie de supports solides emballes sous vide, et procede d'elaboration Download PDF

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Publication number
WO2005010525A1
WO2005010525A1 PCT/JP2004/000595 JP2004000595W WO2005010525A1 WO 2005010525 A1 WO2005010525 A1 WO 2005010525A1 JP 2004000595 W JP2004000595 W JP 2004000595W WO 2005010525 A1 WO2005010525 A1 WO 2005010525A1
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WO
WIPO (PCT)
Prior art keywords
solid support
vacuum
substrate
packed
nucleic acid
Prior art date
Application number
PCT/JP2004/000595
Other languages
English (en)
Japanese (ja)
Inventor
Michifumi Tanga
Hiroshi Okamura
Hirofumi Yamano
Original Assignee
Toyo Kohan Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toyo Kohan Co., Ltd. filed Critical Toyo Kohan Co., Ltd.
Publication of WO2005010525A1 publication Critical patent/WO2005010525A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00608—DNA chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above

Definitions

  • the present invention relates to a vacuum-packed solid support array, in which a plurality of solid supports are aligned and fixed on a substrate, and are vacuum-packed.
  • PCR Polymerase Chain Reaction
  • PCR it is possible to amplify a large number of target sequences with high accuracy, and to amplify efficiently in a short period of time. Widely used for inspection and inspection.
  • the principle of PCR is based on temperature control, and the reaction is carried out by repeating heating and cooling (thermal cycle). That is, after the denatured double-stranded DNA molecule to be amplified is denatured to a single-stranded high-temperature, it is cooled and the primer selected to complement a part of the DNA is annealed, and then heated again. Then, the DNA polymerase is used to extend the DNA behind the primer. By repeating the denaturation, anneal, and elongation processes in multiple cycles, multiple double-stranded DNAs can be amplified.
  • WO 00/22108, WO 02/12891, or JP-A-2002-82116 discloses that DNA can be easily immobilized and DNA is replicated by a DNA amplification reaction.
  • a suitable support a solid support in which a surface treatment layer and a chemically modified layer having a functional group capable of covalently binding to a nucleic acid molecule are sequentially provided on the surface of a substrate is disclosed.
  • Japanese Patent Application Publication No. 2000-516727 also discloses a solid support for immobilizing a protein for the purpose of comprehensively analyzing the protein.
  • Such a solid support is usually commercially available in a container and sealed. Further, such solid supports are taken out one by one from a container and spotted separately, or detection by a hybridization reaction or the like is performed. Therefore, there is a problem in the analysis efficiency and convenience of the sample. Disclosure of the invention
  • the present inventors have developed a solid having a functional group capable of covalently binding to a nucleic acid molecule or protein. After extensive studies on the storage stability of the support, solid supports having an active ester group as a functional group capable of covalently binding to nucleic acid molecules can be left in the air or simply sealed in a container. It has been found that storage alone significantly reduces the amount of nucleic acid molecules or proteins that can be immobilized.
  • An object of the present invention is to improve the storage stability of a solid support having an active ester group as a functional group capable of covalently binding to a nucleic acid molecule or a protein on a substrate, and further improve the storage stability of such a solid support. It is to improve efficiency and convenience.
  • the present inventors have found that by vacuum-packing the solid support, a decrease in the storage stability of the solid support can be significantly prevented. Furthermore, it has been found that the efficiency and convenience in handling the solid support can be improved by aligning and fixing a plurality of the solid supports on a substrate and vacuum-packing the solid support.
  • the present invention includes the following inventions.
  • a solid support array in which a plurality of solid supports having an active ester group as a functional group capable of covalently binding to a nucleic acid molecule or protein are aligned and immobilized on a substrate, and are vacuum-packed.
  • Vacuum-packed solid support array characterized by:
  • the storage stability of a solid support having an active ester group as a functional group capable of covalently binding to a nucleic acid molecule or protein is improved, and a plurality of solid supports can be simultaneously treated.
  • FIG. 1 is a plan view illustrating an embodiment of the solid support array of the present invention.
  • FIG. 2A is a plan view illustrating an embodiment of the solid support array of the present invention.
  • FIGS. (B) to (e) are perspective views showing a manufacturing process until a solid support array is formed from the substrate of the present invention.
  • FIG. 3 is a diagram showing the results of Test Example 1. BEST MODE FOR CARRYING OUT THE INVENTION
  • the solid support in the solid support array of the present invention has a structure having a surface treatment layer and / or an electrostatic layer on a substrate, if necessary.
  • Examples of the material of the substrate used in the present invention include silicon, glass, fiber, wood, paper, ceramics, and plastics (eg, polyester resin, polyethylene Resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin), Metals (eg, stainless steel, nickel, titanium, aluminum).
  • plastics eg, polyester resin, polyethylene Resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin
  • Metals eg, stainless steel, nickel, titanium, aluminum).
  • carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used.
  • soft diamond is a general term for an incomplete diamond structure, which is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited.
  • the surface-treated substrate there is a substrate in which soft diamond is formed on a slide glass.
  • Such substrates may be diamond-like carbon, hydrogen gas 0 9 9 volume 0/0, the remaining methane 1 0 0 containing 1% by volume in the gas mixture, which was developed by ionization deposition Is preferred.
  • the thickness of the surface treatment layer is preferably from 1 nm to 100 ⁇ m.
  • the surface treatment layer of the substrate can be formed by a known method, for example, microwave plasma CVD (Chemical Vapor Deposit, method, ECRCVD (Electric Cvclotron Resonance Chemical Vapor Deposit) method, ICP (Inductive Coupled Plasma) method, DC sputtering method, ECR (Electric Cyclotron Resonance) sputtering method, ion plating method, arc ion plating method, EB (Electron Beam) evaporation method, resistance heating evaporation method It can be performed by ionization evaporation, arc evaporation, laser evaporation, or the like.
  • microwave plasma CVD Chemical Vapor Deposit, method, ECRCVD (Electric Cvclotron Resonance Chemical Vapor Deposit) method, ICP (Inductive Coupled Plasma) method, DC sputtering method, ECR (Electric Cyclotron Resonance) sputtering method, ion plating method, arc ion plating method,
  • the substrate used in the present invention not only the structure having the surface treatment layer formed as described above, but also synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon Metals such as gold, silver, copper, aluminum, tungsten, and molybdenum; plastics (eg, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methyl) Pentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin); a mixture of the above-mentioned metal powder, ceramic powder, etc., with the above-mentioned resin used as a binder, and bonding; raw materials of the above-mentioned metal powder, ceramic powder, etc.
  • the And a sintered body at a high temperature, and a laminate or a composite of the above materials for example, a composite of diamond and another substance, (for example, a two-phase body) It may be
  • the shape and size of the substrate are not particularly limited, examples of the shape include a flat plate, a thread, a sphere, a polygon, and a powder.
  • the width is usually 0.:! ⁇ 10 Omm, length 0.1 ⁇ ; l O Omm, thickness 0.01 ⁇ : about 10mm.
  • a single layer of Ti, Au, Pt, Nb, Cr, TiC, TiN, or the like or a composite film thereof may be formed as a reflective layer on the front or back surface of the substrate.
  • the thickness of the reflective layer is preferably 10 nm or more, more preferably 100 nm or more, since it is necessary that the thickness of the reflective layer be uniform throughout.
  • the surface is intentionally roughened in Ra (JISB 0601) in the range of 1 nm to 1000 nm. This Such a roughened surface is advantageous in that the surface area of the substrate increases and a large amount of DNA probes and the like can be immobilized at a high density.
  • the solid support of the present invention may be provided with an electrostatic layer, if necessary, for electrostatically attracting nucleic acid molecules or proteins.
  • Proteins have the property of being attracted to the cathode side on the acidic side and to the anode side on the basic side in an aqueous solution. Utilizing this property, various proteins can be selected by appropriately selecting the pH of the aqueous solution and the type of the electrostatic layer (either positively charged or negatively charged) according to the isoelectric point of the target protein. It can be attracted electrostatically.
  • 3-lactoglobulin B isoelectric point 5 1
  • calcium carbonate anhydrase isoelectric point 6.0
  • human carbonic anhydrase isoelectric point 6.5
  • the electrostatic layer is not particularly limited as long as the nucleic acid molecule or the protein is electrostatically attracted and the amount of the nucleic acid molecule or the protein immobilized is not particularly limited.
  • the layer has a positive charge such as an amino group-containing compound. It can be formed using a compound.
  • amino group-containing compound examples include a compound having an unsubstituted amino group (mono NH 2 ) or an amino group monosubstituted by an alkyl group having 1 to 6 carbon atoms (one NHR; R is a substituent);
  • amino acid for example, ethanolamine
  • the electrostatic layer may be formed without being covalently bonded to the substrate or the surface treatment layer, or may be formed to be covalently bonded to the substrate or the surface treatment layer.
  • the amino group-containing compound is introduced into the film forming apparatus when the surface treatment layer is formed, whereby the amino group is formed. Is formed into a carbon-based film.
  • Ammonia gas may be used as the compound to be introduced into the film forming apparatus.
  • the surface treatment layer may be a multilayer in which a film containing an amino group is formed after forming the adhesion layer, and in this case, the surface treatment layer may be formed in an atmosphere containing ammonia gas.
  • the electrostatic layer When the electrostatic layer is formed without being covalently bonded to the substrate or the surface treatment layer, the electrostatic layer is formed on the substrate in order to increase the affinity, that is, the adhesion between the electrostatic layer and the substrate or the surface treatment layer. It is preferable to introduce a functional group capable of covalently bonding to a nucleic acid molecule after depositing the compound having an unsubstituted or monosubstituted amino group and a carbon compound.
  • the carbon compound used here is not particularly limited as long as it can be supplied as a gas. For example, methane, ethane, and propane, which are gases at normal temperature, are preferable.
  • the ionization vapor deposition method As the method of vapor deposition, the ionization vapor deposition method is preferable, and the conditions of the ionization vapor deposition method are an operating pressure of 0.1 to 50 Pa and an acceleration voltage of 200 to 100 V. It is preferable.
  • the electrostatic layer is formed by covalent bonding to a substrate or a surface treatment layer
  • the surface of the substrate or the substrate to which the surface treatment layer has been applied is chlorinated by irradiating ultraviolet rays in chlorine gas to chlorinate the surface.
  • group-containing compounds for example, polyamines such as polyallylamine, polylysine, 4,4 ′, 4 "-triaminotriphenylmethane, and triamterene are reacted to form an amino group at the end not bonded to the substrate. By introducing, an electrostatic layer can be formed.
  • a reaction for introducing a functional group capable of covalently binding to a nucleic acid molecule or a protein for example, introduction of a carboxyl group using a dicarboxylic acid or a polycarboxylic acid
  • introduction of a carboxyl group using a dicarboxylic acid or a polycarboxylic acid into a substrate provided with an electrostatic layer
  • the substrate is immersed in a solution containing the compound having an unsubstituted or monosubstituted amino group, and then a functional group capable of covalently bonding to a nucleic acid molecule or a protein is introduced.
  • the solvent for the solution include water, N-methylpyrrolidone, and ethanol.
  • the substrate When a carboxylic acid group is introduced into a substrate on which an electrostatic layer has been formed using a dicarboxylic acid or a polycarboxylic acid, the substrate is activated in advance with N-hydroxysuccinimide and / or carposimides. Alternatively, the reaction is preferably carried out in the presence of N-hydroxysuccinimide and Z or carposides.
  • the thickness of the electrostatic layer is 1 ⁇ ⁇ ! Preferably it is ⁇ 500 ⁇ m.
  • the surface of the substrate is coated with an electrostatic layer as described above, and then chemically modified to introduce a carboxyl group.
  • the compound used to introduce a carboxyl group for example, the formula: X - R '- COOH (wherein, X is a halogen atom, R 1 represents a divalent hydrocarbon group of from 1 to 1 2 carbon atoms )),
  • X is a halogen atom
  • R 1 represents a divalent hydrocarbon group of from 1 to 1 2 carbon atoms
  • R 2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms.
  • a dicarboxylic acid represented by, for example, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, and phthalic acid Acids; polycarboxylic acids such as polyacrylic acid, polymethacrylic acid, trimellitic acid, butanetracarboxylic acid; formula: R 3 — CO— R 4 — COOH (wherein R 3 represents a hydrogen atom or a divalent hydrocarbon group having 1 to 12 carbon atoms, and R 4 represents a divalent hydrocarbon group having 1 to 12 carbon atoms.
  • Monohalides of dicarboxylic acids such as succinic monochloride, malonic monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, anhydrous oxalic acid, maleic anhydride, and butanetetracarboxylic anhydride. are listed.
  • the carboxyl group introduced as described above can be combined with a dehydrating condensing agent such as cyanamide-d-carboimide (eg, 11- [3- (dimethylamino) propyl] -13-ethylcarbodiimide) and N-hydroxy.
  • cyanamide-d-carboimide eg, 11- [3- (dimethylamino) propyl] -13-ethylcarbodiimide
  • Active esterification succinimidylation
  • succinimidylation can be carried out with a compound of succinimide.
  • X—C (R 6 ) H-COOR 7 (where X is a halogen atom, R 6 is a hydrogen atom, a phenyl group or an alkyl group having 1 to 12 carbon atoms, and R 7 is a monovalent carbon atom.
  • the present invention enables simultaneous processing of a plurality of solid supports by aligning and immobilizing the plurality of solid supports on a substrate, thereby improving the efficiency and convenience in using the solid supports. It is to let.
  • the material of the substrate 1 for aligning and fixing the solid support 2 is, for example, silicon, glass, fiber, wood, paper, ceramics, plastic (for example, polyester resin, polyethylene resin, polypropylene resin). , ABS resin ( Acrylonitrile Butadiene Styrene resin), Nylon, Acrylic resin, Fluororesin, Polycarbonate resin, Polyurethane resin, Methylpentene resin, Phenol resin, Melamine resin, Epoxy resin, Chloride chloride resin), Metal (for example, Stainless steel, Nickel, Titanium, Aluminum, aluminum alloys).
  • the substrate 1 of the present invention is usually in the form of a flat plate, and its size is not particularly limited as long as it can fix a plurality of solid supports 2, but it is usually 0.1 to 200 mm in width and 0.2 to 200 mm in length.
  • the thickness is about 0.1 to 20 mm and the thickness is about 0.1 to 10 mm.
  • the solid support 2 may be cut and divided and immobilized on a substrate.
  • the substrate 1 preferably has a tackifier for immobilizing the solid support 2.
  • the tackifier refers to a material having tackiness.
  • the tackifier examples include Tatsukiroll 101 (Taoka Chemical), Hitachil 1501 (Hitachi Kasei), and denatured alkylphenol formaldehyde. Resin (Takikuronore 130), hitanol 501 and the like.
  • the solid support 2, adhesive tape or acrylic as good D adhesive tape be immobilized by Ri substrate 1 in an adhesive, polyiso-butylene, SBR, butyl rubber, chloroprene rubber, chloride bi - Honoré - acetic acid Vinyl copolymer and polybutyral can be used, and benzoic acid resin, polybutene, and bamidothion can be used as the adhesive.
  • a plurality of solid supports 2 are aligned and immobilized on a substrate.
  • To be aligned and fixed means to be arranged in a certain order, for example, to be positionable in a device used in the art such as a spotting device. .
  • a device used in the art such as a spotting device.
  • the pitch between the supports is usually about 0 to 10 mm. Since a plurality of solid supports 2 are aligned and immobilized on the substrate, a plurality of types of solid supports 2 are installed at once in a spotting device or the like, and the positioning of the device is set.
  • Nucleic acid molecules and the like can be spotted on a plurality of solid supports as they are, and the operation of immobilizing nucleic acid molecules or proteins on the solid support 2 can be performed efficiently and quickly.
  • a plurality of solid supports 2 on a solid on which nucleic acid molecules or proteins are immobilized may be used as such for a hybridization reaction, an antigen-antibody reaction or a PCR reaction, or may be removed and used separately. Is also good.
  • the base 1 has the concave portion 3, and the solid support 2 may be fixed in the concave portion 3.
  • the black portion corresponds to the concave portion 3.
  • the size of the concave portion 3 is not particularly limited as long as the solid support 2 can be held inside the solid support 2, but each of the recesses 3 has a width of about 0.1 to 0.5 depending on the width, length and thickness of the solid support 2. It is preferable that the diameter is about mm larger.
  • the width is about 0.2 to: L 00.5 ram, the length is 0.2 to: L 00.5 mm, and the depth is about 0.01 to 10 mm.
  • the base 1 may have a horizontal communication path composed of the recess 3 and the groove connecting the recess 3.
  • the width of the horizontal communication channel is usually about 0.1 to 10 mm. This horizontal communication path facilitates the removal of each solid support 2 from the substrate 1.
  • the horizontal communication path allows the solution used for the high pre-sidation reaction and the like to smoothly spread to the fixed support 2.
  • the recess 3 can be made by a method usually used in the art. For example, it can be formed by forming a tackifier on the first layer and further forming a third layer having the concave portion 3 thereon.
  • a three-layer substrate 1 can be manufactured by a method commonly used in the art, for example, a photolithography. It can be manufactured by methods, press molding, extrusion molding, die molding, three-layer bonding, injection molding, etc.
  • the concave portion 3 can be formed by performing press molding on a metallic base. In this case, it is preferable that a tackifier for fixing the solid support 2 be present on the bottom surface of the concave portion 3 formed by pressing.
  • the substrate 1 preferably has a through hole smaller than the size of the solid support 2 in a portion holding the solid support 2.
  • the through hole facilitates drainage of the activating liquid and facilitates removal of the solid support 2.
  • the solid support array 6 into which the active ester group has been introduced as described above is left alone in the air or simply sealed and stored in a container to reduce the amount of nucleic acid molecules or proteins that can be immobilized. Since the solid support 2 has a plurality of solid supports 2 immobilized on the substrate, the solid support array 6 is placed in a vacuum packing bag and vacuum-packed. By vacuum packing, it is possible to remarkably prevent a decrease in the amount of immobilized nucleic acid molecules and the like.
  • the material of the vacuum-packing bag there is no particular limitation on the material of the vacuum-packing bag as long as it does not allow water and oxygen to pass through.
  • stacked or vapor-deposited the metal is mentioned.
  • the thickness of the film used for the vacuum bag is not particularly limited, and a film having an appropriate mechanical strength can be used according to the weight of the contents. Thick ones are preferred. If it is less than 20 ⁇ , mechanical strength tends to be insufficient, and if it exceeds 300 / m, handleability is poor.
  • a structure in which two films constituting the bag for vacuum packing are overlapped and three sides thereof are laminated is preferable.
  • the solid support array 6 is preferably dried under reduced pressure before the solid support array 6 is vacuum-packed.
  • the temperature during drying under reduced pressure is preferably _
  • the temperature during vacuum packing is preferably 15 to 100 ° C, more preferably 25 to 50 ° C.
  • the pressure at the time of vacuum packing is preferably 1 to 1 ⁇ 1 CT s torr, more preferably 1 ⁇ 10 ⁇ 1 to 1 ⁇ 1 CT 4 torr.
  • the solid support 2 is directly sealed in the bag for vacuum packing so as to minimize the space volume.
  • the vacuum-packed solid support 2 of the present invention can be used for immobilization of any nucleic acid molecule of DNA or RNA.
  • the number of bases of DNA and RNA is usually 1 to 200, preferably 5 to 150.
  • DNA can be immobilized in either single-stranded or double-stranded form. It can also be used for immobilizing various proteins.
  • the solid support immobilized on a plurality of substrates may be directly subjected to the immobilization reaction, or the solid supports 2 may be separately removed from the substrate 1 and then individually subjected to the immobilization reaction. .
  • the terminal base of the oligonucleic acid is immobilized to the active esterified carboxyl group by hydrogen bonding, and the DNA having a base sequence complementary to this oligonucleic acid is further immobilized. It can also be used as a DNA library chip. Also, instead of DNA, nucleotides, oligonucleotides, DNA fragments, proteins and the like can be immobilized to give a library.
  • a plurality of solid supports 2 on which nucleic acid molecules or proteins are immobilized may be immobilized on a substrate and vacuum-packed.
  • Such a solid support array 6 is open After the sealing, the solid support 2 can be used as it is or after removing each one of the solid supports 2 from the substrate and used for an elongation reaction or a hybridization reaction of a nucleic acid molecule or the like.
  • a 0.46 mm deep grating groove (3 mm x 3 mm) was formed on a silicon wafer with a thickness of 0.625 mm and a diameter of 2.5 inch using an Nd-YAG laser.
  • a DLC layer was formed to a thickness of 10 nm on this silicon wafer at an accelerating voltage of 0.5 kV using a gas mixture of 95% by volume of methane gas and 5% by volume of hydrogen as a raw material. Thereafter, ammonia gas was inserted into the first chamber at a rate of 5 cm 3 / min. Amination was performed by treating the DLC surface with ammonia plasma at an operating pressure of 2 Pa.
  • a 5% aqueous solution of polyacrylic acid was applied to the slide glass and dried, and then insolubilized by ultraviolet irradiation for 60 minutes. Then, in 300 ml of 0.1M phosphate buffer (pH6), 0.1M of 1- [3- (dimethylamino) propyl] _3-ethylcarboimide and 1 ⁇ -hydroxysuccinyl of 20111] ⁇ to obtain a solid support having an active ester group by immersing for 30 minutes in the activation solution obtained by dissolving bromide was dried at 100 ° C, 1 X 10 _ 2 torr.
  • polyacrylic acid was added as polyvalent carboxylic acid to the amino group of the surface treatment layer composed of methane and ethylenediamine in the presence of 0.1M 1- [3- (dimethylamino) propyl] -13-ethylcarpo- imide.
  • 0.1 M phosphate buffer (pH 6) in 300 ml of 0.1 M 1- [3- (dimethylamino) propyl] -13-ethylcarbodiimide and 20 mM N-hydroxysuccinimide
  • the solid support having an active ester group was obtained by immersing it in an activating solution in which the solid support was dissolved for 30 minutes, and dried at 100 ° C. and 1 ⁇ 1 O ′ 2 torr.
  • a 10-nm-thick DLC layer was formed at an accelerating voltage of 0.5 kV from a gas mixture of 95% by volume of methane gas and 5% by volume of hydrogen by ionization evaporation. Then, it was chlorinated by irradiating it with ultraviolet light for 30 minutes in chlorine gas. Thereafter, the substrate was immersed in an aqueous solution of polyallylamine (0.1 g / 1) to form an electrostatic layer.
  • polyacrylic acid was polycondensed to the amino group of the electrostatic layer as a polyvalent carboxylic acid in the presence of 0.1 M of 111- [3- (dimethylamino) propyl]-(3-ethylcarbodiimide).
  • 0.1 M phosphate buffer (pH 6) in 300 ml of an activation solution containing 0.11-11- [3- (dimethylamino) propyl] -13-ethylcarbodiimide and 20 mM N-hydroxysuccinimide a solid support having an active ester group by immersing 30 minutes, which was drying at 100 ° C, 1 X 10- 2 torr.
  • a 5% aqueous solution of polyacrylic acid was applied to a slide glass on which DLC was formed to a thickness of 10 nm, dried and then insolubilized by irradiation with ultraviolet light for 60 minutes. Then, in 300 ml of 0.1 M phosphate buffer (pH 6), 0.1 M of 1- [3- (dimethylamino) propyl] -131-ethylcarbodiimide and 20 mM of N-hydroxysuccinimide
  • the solid support having an active ester group was obtained by immersing the substrate in an activation solution in which the medium was dissolved for 30 minutes, and dried at 100 ° C. and 1 ⁇ 1 (T 2 torr).
  • a plurality of holes slightly smaller than the solid support 2 obtained in the production example were punched out in a grid shape using a press machine on a 0.625 mm thick JIS 3004 aluminum alloy plate. In this way, an upper plate 5 having a plurality of through holes in a lattice was prepared.
  • the solid support 2 of 3 mm square obtained in Production Example 1 was subjected to chip transfer equipment SCH (Sanyo High-Technology Co., Ltd.) as shown in FIGS. 2 (b) to 2 (c). It was arranged in the recess 3 of the substrate (FIG. 2 (a)).
  • chip transfer equipment SCH Sanyo High-Technology Co., Ltd.
  • FIGS. 2 (b) to 2 (c) It was arranged in the recess 3 of the substrate (FIG. 2 (a)).
  • an upper plate 5 was placed as a cover on the substrate 1, and four end portions of each solid support 2 were pressed. Since the through-hole of the upper plate 5 is slightly smaller than the solid support 2 fixed on the base, the solid support 2 is fixed by pressing the end of the solid support 2 with the surface of the solid support 2 exposed. be able to. The surface of the solid support 2 is exposed as much as possible.
  • the solid support array 6 shown in FIG. 2 (e) manufactured in Example 2 was individually placed in a polyethylene bag laminated with aluminum, and was evacuated using a vacuum packing device so that there was no space. X1 (packed after evacuation to T 2 torr. The substrate was stored for 30 days in an oven set at 40 ° C., and the amount of immobilized oligonucleotide was measured. The nucleotide immobilization performance was almost the same as the initial performance.
  • the Cy3-labeled oligonucleotide was immobilized as follows.
  • 0.1 ⁇ g / ⁇ 1 was prepared; about 1 n1 of 500 b ⁇ of Cy3-labeled double-stranded DNA amplified by PCR using LDNA as type ⁇ was cut into a substrate (opened) using a microarray maker. On a solid support). Then, after heating in an oven at 80 ° C. for 3 hours, the plate was washed with 2 ⁇ S SCZ0.2% SDS, and the fluorescence intensity of the spotted DNA was measured.
  • the intensity immediately after activation or immediately after purchase is set to 1, and the intensity after 1 day, 10 days, or 30 days is divided by the intensity immediately after activation or immediately after purchase. Rate.
  • the sample that was vacuum-packed directly into the film had the highest residual ratio, and hardly changed even after 30 days.
  • the sample left in the air had the lowest residual rate and a large deterioration with time.
  • the storage stability of the solid support 2 having an active ester group as a functional group capable of covalently binding to a nucleic acid molecule or a protein on a substrate can be improved.

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Abstract

L'invention concerne l'amélioration de la sécurité de stockage de support solide comportant un groupe ester actif comme groupe fonctionnel capable de former une liaison covalente avec une molécule d'acide nucléique ou une protéine sur un substrat. L'invention concerne aussi l'amélioration de l'efficacité et de la commodité d'utilisation d'un tel support solide. On décrit une série de supports solides emballés sous vide (6) qui comporte plusieurs supports solides (2) ayant un groupe ester actif comme groupe fonctionnel capable de former une liaison covalente avec une molécule d'acide nucléique ou une protéine, alignés et immobilisés sur un substrat, et emballés sous vide.
PCT/JP2004/000595 2003-07-25 2004-01-23 Serie de supports solides emballes sous vide, et procede d'elaboration WO2005010525A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2003280064A JP4280124B2 (ja) 2003-07-25 2003-07-25 真空パック固体支持体アレイ及びその製造方法
JP2003-280064 2003-07-25

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WO2005010525A1 true WO2005010525A1 (fr) 2005-02-03

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8455400B2 (en) 2005-05-24 2013-06-04 Hipep Laboratories Substrate for biochip and biochip
EP3581930A4 (fr) * 2017-02-08 2020-12-16 Toyo Seikan Group Holdings, Ltd. Transporteur pour immobilisation de molécules de type biologique

Families Citing this family (8)

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JP5041680B2 (ja) * 2005-06-17 2012-10-03 株式会社ハイペップ研究所 バイオチップ用基板及びバイオチップ
JP2008105973A (ja) * 2006-10-24 2008-05-08 Toyo Kohan Co Ltd ポリペプチド固定化担体の保存方法
KR101414232B1 (ko) * 2007-08-02 2014-08-06 삼성전자 주식회사 바이오 칩 패키지 및 바이오 칩 패키지 기판
JP4999171B2 (ja) * 2007-08-06 2012-08-15 日本電信電話株式会社 タンパク質機能解析装置
JP2018078864A (ja) * 2016-11-18 2018-05-24 東洋製罐グループホールディングス株式会社 生体関連分子固定化用担体
JP6931181B2 (ja) * 2016-12-20 2021-09-01 東洋製罐グループホールディングス株式会社 生体関連分子固定化用担体
WO2018092908A1 (fr) * 2016-11-18 2018-05-24 東洋製罐グループホールディングス株式会社 Support pour immobilisation de molécules biologiquement pertinentes
FR3091703B1 (fr) * 2019-01-11 2021-02-12 Fermentalg Procédé d’extraction de phycocyanines

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JP2000046832A (ja) * 1999-08-05 2000-02-18 Dainabotto Kk クロマトグラフィ免疫分析装置
JP2003344386A (ja) * 2002-05-21 2003-12-03 Advanced Gene Technology Corp 薬草の品質を確認する方法
JP2004020328A (ja) * 2002-06-14 2004-01-22 Toyo Kohan Co Ltd 化学修飾を施した固体支持体およびその用途

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
JP2000046832A (ja) * 1999-08-05 2000-02-18 Dainabotto Kk クロマトグラフィ免疫分析装置
JP2003344386A (ja) * 2002-05-21 2003-12-03 Advanced Gene Technology Corp 薬草の品質を確認する方法
JP2004020328A (ja) * 2002-06-14 2004-01-22 Toyo Kohan Co Ltd 化学修飾を施した固体支持体およびその用途

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8455400B2 (en) 2005-05-24 2013-06-04 Hipep Laboratories Substrate for biochip and biochip
EP3581930A4 (fr) * 2017-02-08 2020-12-16 Toyo Seikan Group Holdings, Ltd. Transporteur pour immobilisation de molécules de type biologique

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