JP2003149239A - Synthetic polymer material for detection of living body- related substance and method of manufacturing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material by which a living body-related substance such as DNA or the like is immobilized to a synthetic polymer having a chemically stable structure while its function is being maintained, and to provide a method of manufacturing the material. SOLUTION: The synthetic polymer material for the detection of the living body- related substance comprises a polymer layer affinitive to both layers between a living- body-related-substance immobilized polymer layer and a synthetic polymer base layer used to carry the polymer layer. In the method, the synthetic polymer material for the detection of the living body-related substance comprises the polymer layer affinitive to both layers, and is manufactured between the living-body-related-substance immobilized polymer layer and the synthetic polymer base layer used to carry the polymer layer. In the method of manufacturing the synthetic polymer material layer for the detection of the living body-related substance, the polymer layer affinitive to both layers is coated and formed in advance on the synthetic polymer base layer, and the living-body-related-substance immobilized layer is formed on, and adsorbed to, the coating polymer layer by the polymerization of a polymerizing monomer under the existence of the living body-related substance.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a synthetic polymer material for detecting a biological substance and a method for producing the same. The synthetic polymer material for detecting a bio-related substance of the present invention is useful for a microarray used for electrophoresis, DNA analysis and the like.

[0002]

2. Description of the Related Art Immobilization of a bio-related substance on a carrier has been well known in the past, and immobilization on a carrier such as a gel of microorganisms, enzymes, etc. has been extensively studied. In particular, regarding immobilization of biopolymers such as nucleic acids and enzymes to carriers, various immobilization reagents have been developed so far, and glass, metal oxides such as alumina, inorganic compounds such as graphite, dextran, and cellulose. Materials such as natural organic polymers such as agarose and synthetic polymers having a specific functional group such as nylon, polystyrene, polyacrylamide, polyvinyl alcohol, polyamino acid, and synthetic phospholipids are used as carriers.

On the other hand, in the field of life science, the decoding of the human genome is almost completed at present, and the future focus is how to extract useful information from the information encoded in the genome and elucidate its function. Is directed to. Under these circumstances, a new analysis method called a DNA chip has recently been developed, and has been attracting attention as a key device that plays a part in technological innovation in this field. A DNA chip is one in which various kinds of DNA probes are immobilized on a substrate such as glass at predetermined positions. DN to check beforehand on this board
When a DNA fragment having a base sequence complementary to A is immobilized and the DNA solution extracted from the living body is placed on this substrate, the base sequence complementary to the DNA immobilized in the solution is placed. If it contains a DNA fragment having the above, it hybridizes with the immobilized DNA on the substrate and can be found. Using this analysis method, for example, when normal cells such as cancer cells undergo some kind of stimulation to cause gene mutation, quantification of the expression level of the genes in the cells due to the stimulation is quantified. Can be found out.

There are roughly two immobilization techniques used for this DNA chip. One is a DNA of about 20 to 30 bases by stepwise reacting four types of base units of guanine, cytosine, thymine, and adenine using semiconductor lithography technology and photoreaction on a substrate such as silicon.
Method of synthesizing oligo chains [Science 251: 767-773
(Published February 1991)]. The other is a method in which a DNA solution is spotted at a predetermined position on a substrate such as glass which has been previously coated with an amino group by silane treatment or the like. In either method, DNA can be arrayed in high density at a predetermined position on a chip of several centimeters square. With the birth of this DNA chip, it has become possible to analyze the gene expression pattern faster and more easily than the conventional method using Northern blot or the like. These DNA chips are those in which DNA is immobilized on a flat plate, but in addition to this, for example, those in which DNA is immobilized on the surface of glass beads (20th Capillary Electrophoresis Symposium 2000 Abstract, p60-6)
1) Further, as an example of immobilizing DNA in the hollow portion of the capillary, there is a method described in JP-A-11-75812 in which nucleic acid is immobilized on the inner wall of the capillary by photoreaction. Any of these methods is useful as a method for easily immobilizing nucleic acid on a carrier substrate. In the chip described in WO99 / 43853, a biotin-modified thin agarose layer is laid on the surface of the microchannel, and nucleic acid is immobilized by biotin-avidin bond.

As described above, various immobilization forms have been developed with the recent progress of genome analysis techniques, and they are used for immobilization of biopolymers such as DNA currently in practical use. Most of the supports are inorganic materials such as glass and metal, and few examples use synthetic polymers as carriers. One of the reasons for this is that optical detection methods such as fluorescence or ultraviolet rays are the mainstream in the detection method in this field, so it is necessary to select a material that is optically low in noise as the device material. There is something. Another reason is that, in general, synthetic polymer materials are more difficult to immobilize than inorganic materials such as glass. In the case of glass, etc., it has a silanol group as an activation site on its surface, so it is possible to easily form an activated surface on the substrate by treating with a silane coupling agent treatment or a polylysine solution. it can.

When immobilizing DNA or the like on a synthetic polymer, it is necessary to activate the substrate by some method like glass. For example, a method of immobilizing terminal aminated DNA by activating the surface of beads of poly-2-hydroxyethyl-methyl methacrylate (PHEMA) with bromine cyanide [Journal of Chromatography B, 666 (1995) 215.
-222] or a method of adsorbing a nucleic acid introduced with an aliphatic hydrocarbon having an amino group at the end onto a microplate (JP-A-7-87999). The former method can be easily immobilized on any material as long as it is a carrier having a hydroxyl group as an active group, but the activating reagent used, bromine cyanide, is highly toxic and poses a difficult factor in handling. It cannot be denied that it contains it. On the other hand, the latter method is a non-covalent immobilization method, but by introducing an aliphatic hydrocarbon as an immobilization site, the adsorptivity to polystyrene-based microplates is improved, and even a short oligonucleic acid can function. Can be fixed while holding.

[0007]

If this phenomenon of adsorption is adopted as a method, it is highly utilized as an optical material such as polymethylmethacrylate, but its structure is chemically stable, so the surface of the base plate is There is a possibility that immobilization of a biological substance such as a nucleic acid on a material that is difficult to activate can be facilitated. As the function of the genome is elucidated in the future, the morphology of analysis equipment will be diversified, and it is expected that the development of immobilization methods according to the equipment morphology will become even more important.

[0008] Therefore, the object of the present invention is to make use of the above-mentioned adsorption ability, and to provide a synthetic polymer excellent in material properties in view of processability and handleability than glass, and particularly to a synthetic polymer whose structure is chemically stable. It is intended to provide a material supporting a biological substance such as a nucleic acid while maintaining its function and a method for producing the material.

[0009]

The inventors of the present invention have focused on the high mutual affinity of the same polymers, and have made synthetic high-performance coatings whose surface is coated with a polymer having the same or similar affinity as the polymer for immobilizing a biological substance in advance. By copolymerizing a polymerizable monomer in the presence of a bio-related substance on a molecular base, it was found that the polymer on which the obtained bio-related substance is immobilized can be easily supported on the synthetic polymer base. Invented.

That is, the present invention is a synthetic polymer material for detecting a bio-related substance, which has a polymer layer on which a bio-related substance-immobilized polymer layer and a polymer layer supporting the polymer layer and having both affinity polymers. .

The synthetic polymer base layer is, for example, an inner wall of a hollow fiber. Further, the constituent monomer of the both-layer affinity polymer contains, for example, the constituent monomer of the biomaterial-immobilized polymer in a ratio of 60% (w / w) or more. Further, the synthetic polymer base layer is polymethylmethacrylate, and a part or all of the constituent monomers of the both-layer affinity polymer and the biomaterial immobilizing polymer are, for example, 2-hydroxyethylmethacrylate. The biological substance is, for example, nucleic acid.

Further, the present invention produces a synthetic polymer material for detecting a bio-related substance, which has a polymer layer on which a bio-related substance is immobilized and a polymer layer supporting the polymer layer and which has both affinity polymer layers. Which is a method of forming a bi-affinity polymer layer by coating on the base layer in advance,
This is a method for producing a synthetic polymer material for detecting a bio-related substance, which comprises forming and adsorbing a bio-related substance-immobilized polymer layer on the coated polymer layer by polymerizing a polymerizable monomer in the presence of the bio-related substance.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described below including preferred embodiments. Examples of the biological substance of the present invention include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), oxypeptide nucleic acid (OPNA)
Examples thereof include nucleic acids, amino acids, proteins, sugars (polysaccharides), lipids, and the like.

The synthetic polymer substrate of the present invention is nylon 6,
Nylon 66, polyamide-based materials such as aromatic polyamide, polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid, polyester-based materials such as polycarbonate, acrylic-based materials such as polyacrylonitrile, polyolefin-based materials such as polyethylene and polypropylene, Polymethacrylate-based materials such as polymethylmethacrylate, polyvinyl alcohol-based materials, polyvinylidene chloride-based materials, polyvinyl chloride-based materials, polyurethane-based materials, phenol-based materials, fluorine-based materials such as polyvinylidene fluoride and polytetrafluoroethylene , Polyalkylene paraoxybenzoate-based materials and the like.

The synthetic polymer substrate may have any shape such as a flat plate shape, a disk shape, a cylindrical shape, and a linear shape. Examples of the linear or tubular body include hollow fibers and solid fibers. The surface morphology may be porous or non-porous. When used for capillary electrophoresis or the like, the solute component to be migrated is irradiated with detection light through the material of the capillary for detection, and thus the material used is required to be optically transparent. Therefore, the material form selected in this case is preferably a non-porous material, and as the organic material, it is preferable to use a material having excellent transparency such as a methacrylate resin represented by polymethylmethacrylate, polystyrene, or polycarbonate. When the hollow fiber is selected as the material, the outer diameter of the hollow fiber used is 2 mm or less, preferably 1 mm or less. The inner diameter is preferably 0.02 mm or more. For capillary electrophoresis, relatively thick hollow fibers are preferable because they are easy to handle.

The polymer having an affinity for both layers between the biomaterial-immobilized polymer layer and the synthetic polymer base layer supporting the polymer layer may be any polymer having an affinity for both layers. In order to enhance the adsorption ability to the biomaterial-immobilized polymer layer, in particular, 60% (w / w) or more of the same constituent monomer as the biomaterial-immobilized polymer,
More preferably, it is desirable to contain 90% (w / w) or more. The biomaterial-immobilized polymer is appropriately selected depending on the type of biomaterial.

For example, when polymethylmethacrylate (PMMA) is selected as the synthetic polymer base and nucleic acid is selected as the bio-related substance, the polymerization of the bio-related substance-immobilizing monomer is carried out in an aqueous system as described later. From the viewpoint of a suitable monomer, it is preferable to use a highly water-soluble monomer, for example, poly- (2-hydroxyethyl-) methacrylate (PHEMA).

When the biocompatible substance is DNA, the synthetic polymer substrate is PMMA hollow fiber, and the biocompatible substance (DNA) -immobilized polymer is PHEMA, for example, the synthetic polymer substrate is coated with a polymer having both affinity layers. By pre-coating the hollow fiber inner wall with a copolymer containing PHEMA or HEMA in an amount of 60% (w / w) or more, the ability to adsorb the DNA-immobilized polymer (PHEMA) to the inner wall can be enhanced. The coating treatment is specifically a synthetic polymer base, DNA
It can be carried out by dissolving PHEMA in a solvent having an affinity for both of the immobilized polymers, for example, ethanol, applying the solution to the inner wall of the hollow fiber, and then evaporating the solvent. Further, a PMMA graft polymer grafted with PHEMA chains can be coated by the same method. On the other hand, if this treatment technique is used, any material can be used as well as PMMA as long as the synthetic polymer substrate can be coated with PHEMA. Further, this coating treatment can also be performed by multi-layer spinning in which the inner layer is PHEMA.

PHEMA inner wall coating P obtained by the above treatment
The adsorption treatment of the DNA-immobilized polymer on the MMA hollow fiber coating layer can be carried out by aqueous polymerization of the polymerizable monomer in the presence of DNA. At that time, DNA having a polymerizable functional group, for example, a vinyl group modified at its end, is previously added to DNA
It is preferable to use A and copolymerize a polymerizable monomer copolymerizable with A from the viewpoint that DNA can be more strongly immobilized.

The introduction of a vinyl group into DNA is described, for example, in JP 2001-122892 A and JP 3-4709 A.
The method described in No. 7 or WO98 / 39351 can be adopted.

Polymerization is carried out, for example, by vinylated DNA, HEM
It can be carried out by filling a polymerization solution obtained by mixing A, water and an initiator into the hollow portion of the hollow fiber by suction introduction, and conducting aqueous polymerization in the hollow portion. As the initiator used here, an azo-based, peroxide-based or redox-based initiator which can be dissolved in the solvent used, for example, 2,2′-azobisisobutyronitrile, 2,2 ′ -Azobis (2-
Methyl butyronitrile) isobutyronitrile, benzoyl peroxide, benzoyl peroxide-dimethylaniline system, and the like. After the completion of the polymerization reaction, water in the hollow portion is removed to obtain a PMMA hollow fiber in which the PHEMA copolymer having the DNA immobilized thereon is adsorbed on the inner wall of the hollow portion.

The synthetic polymeric material for detecting a biological substance of the present invention is a device for assaying a nucleic acid, for example, when the biological substance is a nucleic acid, by hybridizing a base chain complementary to its base sequence. Can be used as

Hereinafter, the present invention will be described more specifically with reference to examples.

EXAMPLES <Example 1> Polymethylmethacrylate (PM
MA) Immobilization of DNA probe on inner wall of hollow fiber (1): 1) Coating of PHEMA on inner wall of hollow part PMMA hollow fiber (manufactured by Mitsubishi Rayon Co., Ltd., outer diameter 25)
After cutting 0 μm, inner diameter 130 μm) into 50 cm, ethanol was sucked through the hollow portion to wash. Next, an ethanol solution of poly- (2-hydroxyethyl-) methacrylate (PHEMA) was passed in the same manner to attach the ethanol solution to the inner wall surface of the hollow portion, and then the ethanol solvent was evaporated to remove PHEMA from the hollow portion. A hollow fiber (A) whose inner wall was coated was obtained. 2) Adsorption of the DNA probe-immobilized polymer onto the coated surface A 200 μl reaction tube was prepared with a polymerization solution (B) having the following composition. HEMA 5 μl Distilled water (Otsuka Pharmaceutical Co., Ltd.) 43 μl 50 pmol / μl DNA fragment (AGCT) aqueous solution 1 μl 10% VA044 aqueous solution (Wako Pure Chemical Industries, Ltd.) 1 μl It was filled and polymerized under a nitrogen atmosphere at 55 ° C. for 3 hours. After the completion of the polymerization, it was washed with water several times to obtain a PMMA hollow fiber having the DNA-immobilized polymer adsorbed on the inner wall.

<Example 2> Immobilization of DNA probe on inner wall of PMMA hollow fiber (2): 1) Coating of PHEMA on inner wall of hollow portion PMMA hollow fiber (manufactured by Mitsubishi Rayon Co., Ltd., outer diameter 25)
After cutting 0 μm, inner diameter 130 μm) into 50 cm, ethanol was sucked through the hollow portion to wash. Next, a methyl cellosolv solution of a graft polymer (C) of main chain PMMA / side chain PHEMA manufactured by Soken Chemical Co., Ltd. was passed in the same manner to attach the polymer solution to the inner wall surface of the hollow portion, and then the solvent was added. Evaporation gave a hollow fiber (D) in which the inner wall of the hollow portion was coated with PHEMA. 2) Adsorption of the DNA probe-immobilized polymer on the coated surface In the same manner as in 2) of Example 1, PMMA hollow fibers having the inner wall coated with the DNA-immobilized polymer were obtained.

<Reference Example 1> Hybridization using the capillaries obtained in Examples 1 and 2: (1) Preparation of sample solution 200 μl of a sample solution was prepared with the following composition to prepare a sample solution (E). 20 × SSC 40 μl 10% SDS 10 μl 1.16 pmol / μl DNA fragment (AGCT) solution 8.6 μl (Note) Labeled with Cy5 dye Distilled water (Otsuka Pharmaceutical Co., Ltd.) 141.6 μl (2) Hybridization Obtained in Examples 1 and 2 Prepare 3 capillaries cut to a length of 3 mm, put them in a vinyl chloride sealed container together with 30 μl of the sample solution (E) prepared in (1), and seal for 16 hours at 55 ° C for hybridization. went. After 16 hours, the capillary was taken out from the container, and the side surface of the capillary was observed with a fluorescence microscope at an excitation wavelength of 633 nm. As a result, it was confirmed by fluorescence detection that the sample was hybridized with the inner wall of the capillary.

[0026]

EFFECT OF THE INVENTION By coating a synthetic polymer substrate with a polymer having the same or affinity as the polymer on which the biological substance is immobilized, the material is superior to glass in terms of workability and handleability. It is possible to support a biological polymer such as DNA while retaining its function on a synthetic polymer, especially a synthetic polymer whose structure is chemically stable. The obtained synthetic polymer material for detecting biological substances is used for electrophoresis and DN.
A It becomes possible to use it for manufacturing the array for analysis.

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Claims (6)

[Claims] 1. A synthetic polymer material for detecting a bio-related substance, comprising a bio-affinity-immobilized polymer layer and a polymer layer supporting the polymer layer and having both affinity polymer layers. 2. The synthetic polymer material for detecting a bio-related substance according to claim 1, wherein the synthetic polymer base layer is an inner wall portion of a hollow fiber. 3. The constituent monomer of the both-layer affinity polymer is 60% of the constituent monomer of the biomaterial immobilizing polymer.
The synthetic polymer material for detecting a bio-related substance according to claim 1 or 2, which is contained in a ratio of not less than% (w / w). 4. The synthetic polymer base layer is polymethylmethacrylate, and part or all of the constituent monomers of the bilayer affinity polymer and the biomaterial immobilization polymer are 2-hydroxyethylmethacrylate. 1
4. The synthetic polymer material for detecting a bio-related substance according to any one of 3 to 3. 5. The synthetic polymer material for detecting a bio-related substance according to claim 1, wherein the bio-related substance is a nucleic acid. 6. A method for producing a synthetic polymeric material for detecting a biologically relevant substance, comprising a polymer layer on which a biologically relevant substance is immobilized and a synthetic polymer substrate layer carrying the polymer layer, and having both affinity polymer layers. Then, a polymer layer having affinity for both layers is formed in advance by coating on the synthetic polymer base layer, and then a polymer layer on which a biomaterial is immobilized by polymerizing a polymerizable monomer in the presence of the biomaterial. A method for producing a synthetic polymer material for detecting a biological substance, which is formed and adsorbed.