WO2005010525A1

WO2005010525A1 - Vacuum-packed solid support array and process for producing the same

Info

Publication number: WO2005010525A1
Application number: PCT/JP2004/000595
Authority: WO
Inventors: Michifumi Tanga; Hiroshi Okamura; Hirofumi Yamano
Original assignee: Toyo Kohan Co., Ltd.
Priority date: 2003-07-25
Filing date: 2004-01-23
Publication date: 2005-02-03
Also published as: JP2005043312A; JP4280124B2

Abstract

It is intended to improve the storage safety of a solid support which has an active ester group as a functional group capable of forming a covalent bond with a nucleic acid molecule or a protein on a substrate. It is also intended to improve the efficiency and convenience in using the above solid support. A vacuum-packed solid support array (6) wherein a plural number of solid supports (2) having an active ester group as a functional group capable of forming a covalent bond with a nucleic acid molecule or a protein are aligned and immobilized on a substrate, characterized by being vacuum-packed.

Description

Description Vacuum-packed solid support array and method for producing the same

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. Background art

Conventionally, polymerase chain reaction has been used to synthesize nucleic acid sequences from existing sequences.

(PCR: Polymerase Chain Reaction). The polymerase chain reaction (PCR) is a method in which the target DNA is sandwiched between a set of primers, and DNA polymerase is repeatedly used to infinitely amplify the region sandwiched by the primers.

According to 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.

Conventionally, 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.

Specifically, 1) raising the temperature of the sample to 95 ° C to dissociate the hydrogen bonds of the double-stranded DNA, and 2) re-bonding with the primers for replicating the DNA. 3) Raise the temperature of the sample to 74 ° C in order to extend the primer with a thermostable polymerase and replicate the DNA. Had to be cycled many times. In such a DNA amplification reaction, a sample is placed in a container of a synthetic resin or the like, and the container is accommodated in an aluminum block to perform the thermal cycle.

However, the thermal cycling took a long time, and it took several hours to obtain a desired amount of DNA. In addition, if the reaction is advanced by controlling the temperature by heating and cooling, there is a limit to changing the temperature instantaneously, and switching to each step cannot be performed smoothly, affecting the accuracy of the amplified nucleic acid sequence. In some cases, DNA other than the target DNA was replicated. In addition, special equipment and technology are required to achieve rapid temperature changes, and there were economic and technical problems such as capital investment.

In view of such a problem, for example, 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. As 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.

Furthermore, 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.

That is, the present invention includes the following inventions.

(1) 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:

(2) The vacuum-packed solid support array according to (1), wherein the substrate has a concave portion, and the solid support is fixed to the concave portion.

(3) The vacuum-packed solid support array according to (1) or (2), wherein the solid support further has an electrostatic layer for electrostatically attracting nucleic acid molecules or proteins.

(4) The vacuum-packed solid support array according to any one of (1) to (3), wherein the nucleic acid molecule is DNA.

(5) The vacuum-packed solid support array according to any one of (1) to (4), wherein the active ester group is an active ester group obtained by succinimidylating a carboxyl group.

(6) An active ester group as a functional group capable of covalently binding to a nucleic acid molecule or protein. The vacuum-packed solid according to any one of (1) to (5), wherein a plurality of the solid supports having the same are aligned and fixed on a substrate, and the solid support is placed in a vacuum-packing bag and vacuum-packed. A method for producing a support array. '

(7) The production method according to (6), wherein the solid support array is dried under reduced pressure at 50 to 200 ° C. and vacuum-packed.

(8) Put the solid support array in a bag for vacuum packing, evacuate, depressurize with inert gas, and evacuate again at least once, then vacuum pack

The production method according to (6) or (7).

According to the present invention, 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.

Thus, efficiency and convenience in using a solid support can be improved. Also, since a plurality of solid supports are vacuum-packed in a single form, they are suitable as a shipping form. Brief Description of Drawings

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).

When the above-mentioned material is used as the substrate material, it is not necessary to apply a surface treatment layer. It is more preferable to perform a surface treatment in order to immobilize on the surface. Synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, It is preferable to use any one of graphite, fullerene, and carbon nanotube), a mixture thereof, or a laminate thereof. Further, 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. Here, 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.

As an example of 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 μm.

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. As 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.

Although 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. When a flat plate is used, the width is usually 0.:! ~ 10 Omm, length 0.1 ~; l O Omm, thickness 0.01 ~: about 10mm.

Further, 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.

When glass is used as the substrate, it is also preferable that 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. For example, when the pH of the aqueous solution is more basic than the isoelectric point of the protein (for example, phycocyanin (isoelectric point 4.45 to 4.75)),] 3-lactoglobulin B (isoelectric point 5 1), calcium carbonate anhydrase (isoelectric point 6.0) and human carbonic anhydrase (isoelectric point 6.5) when reacted in an aqueous solution with a pH of 7.0. What is necessary is just to use what has a positive charge as an electrostatic layer. 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.For example, the layer has a positive charge such as an amino group-containing compound. It can be formed using a compound. Examples of the amino group-containing compound 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); For example, ethylenediamine, hexamethylenediamine, n-propylamine, monomethamine, dimethylamine, monoethylamine, cetamine, arylamine, aminoazobenzene, amino alcohol (for example, ethanolamine), atalinol, aminoaminobenzoic acid, aminoanthraquinone The amino acid

(Glycine, alanine, palin, leucine, serine, threonine, cystine, methionine, fenylalanine, tryptophan, tyrosine, proline, cystine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, histidine, histidine) These polymers (for example, polyallyl , Polylysine) and copolymers; and polyamines (polyvalent amines) such as 4,4 ', 4 "-triaminotriphenylmethane, triamterene, sponolemidine, sponolemin, and putrescine.

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.

In the case where the electrostatic layer is formed without being covalently bonded to the substrate or the surface treatment layer, for example, 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. Further, 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.

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. 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.

When the electrostatic layer is formed by covalent bonding to a substrate or a surface treatment layer, for example, 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. Among the 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. When 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) into a substrate provided with an electrostatic layer is performed in a solution. Preferably, 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. Examples of the solvent for the solution include water, N-methylpyrrolidone, and ethanol.

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.

When the substrate is immersed in a solution containing a compound having an unsubstituted or monosubstituted amino group to form an electrostatic layer, if a polyamine is used as the amino group-containing compound, adhesion to the substrate may occur. Excellent in immobility, and the amount of immobilized nucleic acid molecules is further improved.

The thickness of the electrostatic layer is 1 η π! Preferably it is ~ 500 μm.

If necessary, 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 )), For example, chloroacetic acid, fluoroacetic acid, bromoacetic acid, odoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; Formula: HOOC— R ² — COOH

(In the formula, 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. A) a keto acid or an aldehyde acid represented by the formula: X—OC—R ⁵ —COOH (wherein, X is a halogen atom, and R ⁵ represents a single bond or 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. Active esterification (succinimidylation) can be carried out with a compound of succinimide.

In the formula, 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. by reacting with non Harokarubon acid ester represented by the hydrogen radical), the ^{formula:. -COO-C (R 6} ) H-COOR 7 ( wherein, R ⁶ and R ⁷ are the same as defined above). An active ester group represented by the following formula can be introduced.

In the case where a nucleic acid molecule or protein is immobilized by spotting on the solid support obtained as described above, or in the case where another nucleic acid molecule is hybridized to the nucleic acid molecule immobilized on the solid support, one If one solid support is processed, the processing steps become complicated and it is difficult to analyze a large amount of sample. 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.

In the present invention, 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. In the present invention, after the solid support 2 is prepared, 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. Examples of the tackifier include Tatsukiroll 101 (Taoka Chemical), Hitachil 1501 (Hitachi Kasei), and denatured alkylphenol formaldehyde. Resin (Takikuronore 130), hitanol 501 and the like. Alternatively, 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.

By immobilizing a plurality of solid supports 2 on one substrate, different kinds of solid supports can be used in a required combination as needed. Also, a plurality of solid supports 2 on which different kinds of nucleic acid molecules or proteins are immobilized can be immobilized on one substrate, and various tests can be performed at once.

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. . For example, when arranged in a line, in a grid, or in a radial pattern. If the solid support 2 is fixed in a grid, 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.

In one embodiment of the present invention, the base 1 has the concave portion 3, and the solid support 2 may be fixed in the concave portion 3. One example of this embodiment is shown in FIGS. 1 and 2 (a) to (e). In FIGS. 1 and 2 (b) to (e), the black portion corresponds to the concave portion 3. By fixing the solid support 2 in such a concave portion 3, the solid support 2 becomes difficult to be separated from the base 1. 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. More specifically, it is preferable that 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. Further, in this embodiment, as shown in FIG. 1, 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. In addition, 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. Such 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. In addition to the three-layer structure, 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.

Further, 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.

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.For example, a single-layer film of polyethylene or polyester, a laminated film of polyethylene and polyester, or a resin such as aluminum What laminated | 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 μηι, mechanical strength tends to be insufficient, and if it exceeds 300 / m, handleability is poor. As the form of the bag for vacuum packing, a structure in which two films constituting the bag for vacuum packing are overlapped and three sides thereof are laminated is preferable.

In the present invention, 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 _

14

Is 50 to 200 ° C, more preferably 70 to 120 ° C. Vacuum drying pressure during preferably 1~ 1 X 10- ^s torr, more preferably 1 X 10 - Ru ¹ ~ ¹ X ¹ O_ ⁴ torr der.

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.

It is preferable to perform at least one operation of evacuating, depressurizing with an inert gas, and evacuating again before vacuum packing. As the inert gas used here, for example, nitrogen gas, argon gas, neon gas or a mixture thereof can be mentioned. It is preferable that the solid support 2 is directly sealed in the bag for vacuum packing so as to minimize the space volume.

After being opened, 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. In addition, DNA can be immobilized in either single-stranded or double-stranded form. It can also be used for immobilizing various proteins. After opening, 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. .

Using the solid support array 6, 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. Example

Hereinafter, the present invention will be described with reference to Production Examples and Examples, but the present invention is not limited thereto.

(Production Example 1)

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.

Then, after condensing butanetetracarbonic anhydride as a polyvalent carboxylic acid to the amino group of the surface treatment layer, 0.1M of 1.1M in 300 ml of 0.1 M phosphate buffer (pH 6) was added. [Propyl] -31-ethylcarbodiimide and 20 mM N-hydroxysuccinimide were immersed in an activation solution for 30 minutes to obtain a solid support having active ester groups. C, dried at 1 X 10 " ² torr. The solid support thus obtained was stuck on a Daishinda film, and each solid support of 3 mm square was mounted on a CHIP MATRIX E PANDER (TECHNOVISION. INC.). Divided into

(Production Example 2)

After immersing the Suraido glass 10 min 2 Weight ^_0/0 3 § amino propyltriethoxysilane ethanol solution was taken out, washed with ethanol, and dried for 10 minutes at 1 10 ° C. Next, succinic anhydride was condensed on the substrate having the amino group introduced. Later, 0.1 M of 1- [3- (dimethinoleamino) propyl] -13-ethylcarbodiimid and 2 OmM of N-hydroxylsk in 300 ml of 0.1 M phosphate buffer (pH 6) The solid support having an active ester group was obtained by immersing in a activating solution in which synimide was dissolved for 30 minutes, and dried at 100 ° C. and 1 × 10-orr.

(Production Example 3)

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.

(Production Example 4)

3. 15 mm (width) x 3.5 mm (length) x 1 mm (thickness) glass slides, using methane gas as a carrier gas, at a rate of 15 cm ³ Z by ionization deposition. C was introduced into the chamber through ethylenediamine kept warm. At an operating pressure of 2 Pa and an accelerating voltage of 0.5 kV, a layer composed of C, N and H was formed to a thickness of 20 nm using methane and ethylenediamine as raw materials.

Then, 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. After condensing with 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.

(Production Example 5)

3.5 mm (width) x 3.5 mm (length) x 1 mm (thickness) slide glass 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.

After that, 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.

(Production Example 6)

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).

(Example 1) Manufacturing method of substrate

1) 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.

2) On a 0.4 mm thick JIS 3004 aluminum alloy plate, using a press machine, a plurality of concave portions 3 large enough to accommodate the solid support 2 obtained in Production Example 1 were provided in a grid pattern. The position of the concave portion 3 in the base 1 is aligned with the position of the through hole in the upper plate 5. Further, as shown in FIG. 2A, the size of the recess 3 is smaller than that of the recess 3. A through hole 4 was provided. The through-holes 4 make it easier for the activation liquid to run out, and when the solid support 2 is detached, the solid support 2 can be easily taken out by pushing it out from the through-hole 4 on the back.

3) A double-sided tape was applied to the concave portion 3 of the lower plate to prepare the base 1.

(Example 2) Method for producing solid support array

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)). Next, as shown in FIGS. 2 (d) to 2 (e), 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.

(Example 3)

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.

(Comparative Example 1)

Five activated ester groups obtained in Production Example 1 were placed in a slide case, and then placed in a polyethylene bag on which aluminum was deposited, followed by vacuum packing at room temperature. After this substrate was stored in an oven set at 40 ° C. for 30 days, the amount of immobilized oligonucleotide was measured. As a result, the performance was significantly deteriorated as compared with the initial performance. (Test Example 1) The time-dependent change in the amount of the Cy3-labeled oligonucleotide immobilized on the vacuum-packed solid support array 6 obtained in Example 3 (fluorescence intensity using a fluorescence scanner FLA8000, manufactured by Fuji Photo Film Co., Ltd.) was examined.

The Cy3-labeled oligonucleotide was immobilized as follows.

0.1 μg / μ1 was prepared; about 1 n1 of 500 bρ of Cy3-labeled double-stranded DNA amplified by PCR using LDNA as type 铸 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 results are shown below.

(1) After activation, leave at 40 ° C in air

Immediately after activation: 37,000 → 1 day later: 29,100 → 10 days later: 20,500 → 30 days later: 12,300

(2) After activation, store in a vacuum pack in a case at 40 ° C.

Immediately after activation: 37,200 → 1 day later: 33,000 → 10 days later: 29,400 → 30 days later: 24,500

(3) After activation, vacuum-pack directly into film and store at 40 ° C

Immediately after activation: 33,400 → 1 day later: 31,700 → 10 days later: 30,400 → 30 days later: 31,000 For comparison, a commercially available solid support having an active ester group was left in the air.

Immediately after purchase: 30,000 → After 1 day: 22,600— After 100: 15,100 → After 30 days: 5,700

In Fig. 3, 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. According to Fig. 3, the sample that was vacuum-packed directly into the film had the highest residual ratio, and hardly changed even after 30 days. On the other hand, it was found that the sample left in the air had the lowest residual rate and a large deterioration with time. Industrial applicability

According to the present invention, 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.

Claims

The scope of the claims

1. 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 a protein are aligned and immobilized on a substrate, and are vacuum-packed. Characterized by a vacuum-packed solid support array

2. The vacuum-packed solid support array according to claim 1, wherein the substrate has a concave portion, and the solid support is fixed to the concave portion.

3. The vacuum-packed solid support array according to claim 1, wherein the solid support further has an electrostatic layer for electrostatically attracting nucleic acid molecules or proteins.

4. The vacuum-packed solid support array according to any one of claims 1 to 3, wherein the nucleic acid molecule is DNA.

5. The vacuum-packed solid support array according to any one of claims 1 to 4, wherein the active ester group is an active ester group obtained by succinimidylating a carboxyl group.

6. A plurality of solid supports having an active ester group as a functional group capable of covalently binding to a nucleic acid molecule or a protein are aligned and immobilized on a substrate, and this is put in a vacuum packing bag and vacuum-packed. The method for producing a vacuum-packed solid support array according to any one of claims 1 to 5.

7. The method according to claim 6, wherein the solid support array is vacuum-dried after drying at 50 to 200 ° C. under reduced pressure.

8. The vacuum packing method according to claim 6 or 7, wherein the solid support array is put in a bag for vacuum packing, and after evacuation, an operation of depressurizing with an inert gas and evacuating again is performed at least once, followed by vacuum packing. Manufacturing method.