JP2006028059A - Dna immobilized body, method for producing the same and collection system using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a DNA immobilized body that firmly immobilizes a DNA, highly controls elution of a DNA in water and sufficiently exhibits function of selectively and specifically collecting a substance, which a DNA has and to provide a method for simply producing the DNA immobilized body and a DNA collection system that uses the DNA immobilized body, makes a DNA collect a substance contained in air or water, which is selectively and specifically collected by a DNA, by sufficiently exhibiting collection function of DNA and is used for high-precision detection of the specific substance and environmental clean-up by efficiently removing the specific substance. <P>SOLUTION: The DNA immobilized body comprises a phosphate group-containing polymer, a divalent or polyvalent metal and a DNA. Preferably the phosphate group-containing polymer contains a specific phosphate group and one or more kinds of specific (meth)acrylic acid esters or their salts. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a DNA immobilized body, and more specifically, suppresses DNA from eluting into water even when the DNA comes into contact with water, and selectively recognizes DNA and uses a specific substance for a double helix. The present invention relates to a DNA fixed body capable of sufficiently exhibiting the uptake function, a method for producing such a DNA fixed body, and a collection system using DNA.

  DNA (deoxyribonucleic acid) plays a role in carrying genetic information in vivo, and is one of the most important substances for life phenomena. Double-stranded DNA has a very sophisticated molecular recognition ability because a large number of base pairs are formed with respect to other single-stranded DNA complementary to single-stranded DNA. In particular, in double-stranded DNA, a double helix structure selectively and specifically intercalates (incorporates) an aromatic compound having a planar chemical structure. It is regarded as promising as a compound detection material and an environmental purification material for removing harmful substances (for example, see Non-Patent Document 1). In order to apply such DNA to a detection material or an environmental purification material, it is necessary to immobilize water-soluble DNA on a base material, and development of a technique for immobilizing DNA on the base material is underway. For example, a method comprising a step of bringing DNA into contact with a carrier in a buffer solution containing morpholine, a morpholine derivative or a salt thereof on the surface of the support (see, for example, Patent Document 1), γ-aminopropyltriethoxysilane A method of immobilizing DNA from a polymer modified with a polymer (for example, see Non-Patent Document 2), a nucleic acid immobilization method for treating a substrate with atomic oxygen plasma (for example, see Patent Document 2), an alkali metal salt of deoxyribonucleic acid and A method for immobilizing deoxyribonucleic acid by coagulating an alkali metal salt of alginic acid with a divalent metal-containing compound (see, for example, Patent Document 3), an aqueous solution of water-soluble DNA on a support or a liquid film thereof, or a support The upper water-soluble DNA thin layer is irradiated with ultraviolet rays having a wavelength in the range of 250 to 270 nm to cure the DNA. A method of immobilizing DNA on the body (for example, see Patent Document 4), a DNA solidified complex in which the DNA immobilization carrier is a calcium-containing substance or an inorganic solid such as silica gel (for example, see Patent Document 5), etc. Has been reported.

In these methods, DNA is fixed to a substrate, or cross-linking between DNAs is brought about, so that DNA is not eluted into water. However, in these methods, the function of the DNA is not completely drawn out, and there remains a problem of how to firmly fix the DNA that has been made non-eluting into water in practical use to a substrate or the like. ing.
Functional materials, Vol. 19, 1999 Chem. Rev., Vol. 96, 1533-1554, 1996 Anal. Chim. Acta, Vol. 365, 19-25, 1998 WO00 / 34456 JP 2002-218976 A JP 7-41494 A JP 2001-81098 A Japanese Patent Laid-Open No. 10-175994

  An object of the present invention is to provide a DNA fixed body capable of suppressing the elution of DNA into water and sufficiently exhibiting the function of selectively and specifically collecting DNA, and a method for producing the same. It is to provide. Furthermore, by using this, DNA can be firmly fixed on a substrate or the like, and a specific substance capable of selectively and specifically collecting DNA contained in air or water can be collected. To provide a collection system using DNA that can be applied to high-precision detection of specific substances and environmental purification that can efficiently remove specific substances is there.

  The present inventors have already developed a DNA hybrid in which DNA is supported on an oxide matrix by removing a dispersion medium from a dispersion containing an oxide colloid and DNA. As a result of further intensive studies, the present inventor can highly suppress the elution of DNA into water by fixing the DNA with a polymer having a phosphate group and a metal ion having a valence of 2 or more, Obtaining the knowledge that DNA can be firmly fixed on a substrate by preparing a coating solution using such a polymer and applying it to the surface of the substrate to complete the present invention It came.

That is, the present invention includes each aspect described below.
(1) A DNA carrier comprising a polymer having a phosphate group, a metal having a valence of 2 or more, and DNA.

  In the DNA fixed body of the present invention, DNA is firmly fixed, elution of DNA into water is highly suppressed, and the function of selectively and specifically collecting substances possessed by DNA is sufficiently exerted. be able to. In addition, the method for producing a DNA fixed body of the present invention can easily produce such a DNA fixed body. Furthermore, the collection system using the DNA immobilized body of the present invention can firmly fix DNA on a substrate or the like, and the DNA is contained in air or water that can be selectively and specifically collected. A substance can be collected by DNA by fully exerting the function of collecting DNA, and can be applied to high-accuracy detection of the specific substance, environmental purification by efficiently removing the specific substance.

  The DNA fixed body of the present invention is not particularly limited as long as it contains a polymer having a phosphate group, a metal ion having a valence of 2 or more, and DNA.

  The polymer having a phosphate group in the DNA immobilized body of the present invention is insoluble in water, and the phosphate group retains the function of collecting DNA and its specific substance via a metal having a valence of 2 or more, In the case of double-stranded DNA, the double-stranded structure is retained, and it has a function of binding tightly and suppressing the DNA from eluting into water. The phosphate group in the polymer having a phosphate group may be any one as long as it reacts with a divalent or higher metal ion and the dispersibility of the polymer having a phosphate group in water decreases. As the phosphate group capable of reacting with a divalent or higher-valent metal ion, specifically, formula (1)

The phosphoric acid group represented by (1) can be exemplified, and any one or two or more of these phosphoric acid groups can be mentioned as preferred, and among these, those represented by (a) are particularly preferred. .

  Examples of the polymer having a phosphoric acid group include those represented by the formula (2)

Those containing one or more of (meth) acrylic acid esters represented by the formula (1) or a salt thereof as a unit are preferable. In the formula, R ¹ represents a hydrogen atom or a methyl group, R ² represents a hydrogen atom, a halogen atom, or an optionally substituted C1-C3 alkyl group, and n represents 1-8. Indicates one of the integers. Examples of the C1-C3 alkyl group represented by R ² in the formula (2) include linear and branched alkyl groups such as a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. Examples of the substituent for the C1-C3 alkyl group include halogen atoms such as chlorine atom, bromine atom and iodine atom. Specific examples of the (meth) acrylic acid esters represented by the formula (2) include the formula (3)

The compound represented by can be mentioned. As the polymer having a phosphoric acid group having such a (meth) acrylic acid ester as a unit, a commercially available polymer can be used. Specifically, a polymer manufactured by Unichemical Co., Ltd. (trade name) : Phosmer).

  The polymer having a phosphoric acid group used in the present invention may contain other vinyl monomer as a unit in addition to the above (meth) acrylic acid ester having a phosphoric acid group. For example, acrylic acid, acrylic acid esters, methacrylic acid, methacrylic acid esters, acrylonitrile, methacrylonitrile, styrene, nucleus-substituted styrenes, alkyl vinyl ethers, alkyl vinyl esters, perfluoroalkyl vinyl ethers, perfluoroalkyl ethers, Fluoro-alkyl vinyl esters, maleic acid, maleic anhydride, fumaric acid, itaconic acid, maleimide, phenylmaleimide and the like can be mentioned. Among these vinyl monomers, methacrylic acid esters, acrylonitrile, styrenes, maleimides, and phenylmaleimides are preferred as units contained in the polymer having a phosphate group used in the present invention.

  The polymer having a phosphoric acid group is a monomer having a phosphoric acid group such as one or two or more of (meth) acrylic acid esters having a phosphoric acid group or a salt thereof. A vinyl monomer can be added as a monomer and obtained by a polymerization reaction. Such polymerization reaction may be solution polymerization, polymerization by irradiation with ultraviolet rays or electron beams, etc., but solution polymerization is preferred. In the case of solution polymerization, these monomers can be dissolved in a solvent and polymerized using a polymerization initiator. The solvent to be used may be any solvent as long as these raw materials are soluble, such as water, alcohols such as methanol, ketones such as acetone, ethers such as ethylene glycol monomethyl ether, etc. Two or more of these may be mixed and used. As polymerization initiators used, 2,2-azobisisobutyronitrile, 2,2-azobis (2,4-dimethylvaleronitrile), dimethyl 2,2-azobis (2-methylpropionate), dimethyl Examples thereof include azo initiators such as 2,2-azobisisobutyrate, peroxide initiators such as lauryl peroxide, benzoyl peroxide, and tert-butyl peroctoate. When the raw material component is 1, the amount of the solvent used is preferably about 1.0 to 20.0, more preferably 1.5 to 10 by weight. When the raw material component is 1, the amount of the polymerization initiator used is preferably about 0.005 to 0.05 by weight ratio, more preferably around 0.01. When the amount of the solvent and polymerization initiator used is in the above ranges, the polymer of the product does not gel, and these polymers are dissolved in various solvents to easily adjust the coating solution described later. This is preferable in handling. Further, when (meth) acrylic acid ester having a phosphoric acid group is used for preparation of a polymer having a phosphoric acid group, the weight ratio in the raw material of the (meth) acrylic acid ester having a phosphoric acid group is 5% by weight. % Or more, more preferably 10% by weight or more. If the weight ratio of the (meth) acrylic acid ester having a phosphate group in the raw material is 5% by weight or more, crosslinking with DNA by a phosphate group is possible, and if it is 10% by weight or more, it is divalent or more. A number of crosslinks with the DNA of the polymer are formed in combination with the other metals. The polymerization conditions may be a temperature of 40 ° C. or higher, more preferably 60 ° C. or higher and lower than the boiling point of the solvent while stirring the solution.

  The type and size of the DNA used in the DNA fixed body of the present invention are not limited as long as the object of the present invention can be achieved in the DNA fixed body. That is, it may be either single-stranded DNA or double-stranded DNA, the molecular weight is not limited, and may be a DNA precursor oligonucleotide or the like. As such DNA, for example, DNA obtained from animal testis or thymus can be used. Specifically, DNA obtained from salmon, herring or cod testis (white egg), mammals or birds, for example, DNA obtained from the thymus of cows, pigs, chickens and the like can be used. In addition, a DNA sequence having a base pair of (dA)-(dT), specifically, a synthetic DNA having a poly (dA) -poly (dT) type sequence or the like can also be used. Among these, double-stranded DNA is preferable because it has a high effect of collecting a specific substance by DNA, that is, an intercalation function. The molecular weight of such DNA is preferably 100,000 or more, more preferably 500,000 or more.

  The DNA content in the DNA fixed body of the present invention is preferably from 0.01 to 50% by weight, more preferably from 0.1 to 20% by weight. When the content of DNA is 0.01 to 50% by weight, the effect of capturing a specific substance derived from DNA can be sufficiently obtained, the economic efficiency is good, and the content of DNA is 0.1 to 20% by weight. If it is%, such an effect can be obtained more remarkably.

The divalent or higher metal used in the DNA immobilization body of the present invention binds to both the phosphate group in the polymer having a phosphate group and DNA to form a more stable phosphate bond. Any of them may be used. Examples of the metal ions having a valence of 2 or more include Mg ²⁺ , Ca ²⁺ , Sr ²⁺ , Ba ²⁺ , Mn ²⁺ , Cu ²⁺ , Zn ²⁺ , Al ³⁺ , Fe ³⁺ , Y ³⁺ , ^Examples include Co ³⁺ , In ³⁺ , La ³⁺ , Ti ⁴⁺ , Zr ⁴⁺ , Sn ⁴⁺ . The content of the bivalent or higher metal in the DNA fixed body is, for example, 0.05 to 50 when expressed in terms of the content of the metal oxide in the DNA fixed body when the metal ion is added as a metal oxide. It is preferable that it is weight%, More preferably, it is 0.1-30 weight%. When the metal content is 0.05 to 50% by weight, elution of DNA into water is suppressed, and the function of collecting a specific substance of DNA is maintained. A more remarkable effect can be obtained.

  The DNA fixed body of the present invention contains other components such as other polymers, inorganic powders, surfactants and the like in addition to the above-mentioned polymer having a phosphate group and a metal having a valence of 2 or more. You may contain.

  The method for producing a DNA fixed body of the present invention includes a first step of preparing a dispersion / dissolution solution containing a polymer having a phosphate group and DNA, and the dispersion / solution contains a divalent or higher-valent metal ion. Examples thereof include a second step of adding a dispersion / dissolution solution to prepare a DNA fixing solution and a third step of removing the dispersion medium / solvent of the DNA fixing solution.

  Here, in the present invention, the dispersion / dissolution liquid refers to a liquid containing a substance in a dispersed state, a liquid contained in a dissolved state, or a liquid contained in a dispersed state and a dissolved state.

  In the first step of preparing a dispersion / solution containing a polymer having a phosphate group and DNA, the preparation of the dispersion / solution containing a polymer having a phosphate group and DNA is carried out by using water as a dispersion medium / solvent. In addition, an organic solvent such as alcohol and a mixed solvent thereof can be appropriately selected and used, and a polymer having a phosphate group and each component of DNA can be added to these dispersion medium / solvent. A crosslinking reaction may occur abruptly between a divalent or higher metal ion and a polymer having a phosphate group. In such a case, a dispersion / dissolution solution of a polymer having a phosphate group and DNA is prepared in advance. It is preferable to do. As the DNA to be added, water-soluble DNA alkali salt or lipid-exchanged alcohol-soluble DNA can be used.

  Further, when an aqueous dispersion medium / solvent is used, it is preferable that the phosphate group of the polymer having a phosphate group is partially in the form of an alkali phosphate base. Preparation of a dispersion / solution containing such a polymer having an alkali phosphate base is obtained by dissolving an aqueous solution of an alkali metal hydroxide or an organic alkali metal salt such as an alkali metal alkoxide in an organic solvent in a polymer having a phosphate group. Etc. to add the phosphate group to the formula (4)

(In the formula, M represents an alkali metal) The method of converting to an alkali phosphate is preferable. The DNA is preferably a water-soluble DNA alkali salt, preferably an animal gonad-derived DNA alkali salt. Thereby, DNA and a polymer having a phosphate group are uniformly dispersed and dissolved in an aqueous dispersion / solvent, and an excellent DNA fixed body can be obtained.

  Preparation of a dispersion / dissolution solution containing a divalent or higher metal ion used in the second step of the method for producing a DNA fixed body of the present invention is a metal oxide or metal that generates a bivalent or higher metal ion in the solution. Stir and mix inorganic metal compounds such as halides and metal sulfates, organic acid salts of metal such as metal alkoxides and metal chelate compounds, organic solvents such as water and alcohol, and mixed solvents thereof, as necessary. Depending on the method, heating can be performed. The dispersion / dissolution containing divalent or higher metal ions prepared by such a method is added to the dispersion / solution containing the phosphate group-containing polymer and DNA obtained in the first step, A metal having a bivalent or higher valence by heating, if necessary, such as a hydrogen of a phosphate group of a polymer having a phosphate group, an alkali metal of an alkali metal phosphate, and an alkali metal of an alkali metal salt of DNA. And a cross-link by a divalent or higher metal ion is formed between the polymer having a phosphate group and DNA. Thereby, a DNA fixing solution in which a polymer in which DNA is fixed to a phosphate group via metal ions is dispersed and dissolved can be obtained.

  In the third step, the dispersion medium / dissolving medium of the DNA fixing solution prepared in Step 2 is removed by spraying, drying, It can be performed by heating or the like, and the dispersion medium / dissolution medium can be removed to obtain a DNA-fixed body in the form of powder, bulk or membrane. When gel containing DNA is precipitated in the dispersion medium by crosslinking, the gel containing DNA is removed by a method such as filtration, natural sedimentation or centrifugation, and the DNA gel body is dried to obtain a DNA fixed body. The method can be used.

  Further, as another method for producing a DNA immobilized body of the present invention, a step of preparing a coating solution using a dispersion / dissolution solution containing a polymer having a phosphate group and DNA, and a substrate using the coating solution are provided. It may be a method having a step of forming a film on top and a step of bringing a dispersion / dissolution containing bivalent or higher metal ions into contact with the film. In this method, in the step of preparing a coating solution using a dispersion / dissolution solution containing a polymer having a phosphate group and DNA, the coating solution is a polymer having a phosphate group and DNA prepared by the same method as described above. For example, the concentration of a dispersion / solution containing a polymer having an alkali metal phosphate and a DNA alkali salt can be adjusted, or a surfactant or the like can be added. Thus, a coating solution can be prepared. In the step of forming a film on a substrate using the obtained coating solution, any of a plate, a tubular body, a fiber, a woven fabric, or a nonwoven fabric can be used as the substrate. The above-mentioned coating solution can be applied and immersed on the surface of such a substrate and temporarily dried to form a film. As a method of application and immersion, an application method such as blade coating or spray coating, or a method such as immersion may be used. In the step of bringing the dispersion / dissolution containing divalent or higher metal ions into contact with the film on the molded substrate, the dispersion / solution containing divalent or higher metal ions prepared by the same method as described above is used. Immerse the film on the material, or contact the film on the base material with a dispersion / dissolution solution containing divalent or higher metal ions by a method such as coating. Can be mentioned. As a result, a cross-link between a DNA having a phosphate group with a metal ion having a valence of 2 or more and DNA is formed, and a DNA immobilization body integrated with a substrate can be formed together with DNA immobilization. In this case, when the substrate is an organic substrate, it is preferable to activate the surface in advance by imparting a phosphate group to the substrate surface in order to enhance adhesion. As a method for imparting a phosphate group to the substrate surface, a method of applying (meth) acrylic acid ester having a phosphate group represented by the above formula (2), an oligomer obtained by vinyl polymerization of this, or the like, And a method of irradiating ultraviolet rays, electron beams or the like after applying these. In the case where the substrate is an inorganic substrate, a component of a (meth) acrylic silane coupling agent can be introduced into the polymer having a phosphate group to improve the affinity with the substrate.

  Examples of the form of the DNA fixed body of the present invention include a coating film formed by coating and dipping on the surface of a substrate such as powder, bulk, plate, tubular body, fiber, woven fabric and non-woven fabric. For example, a column filled with powder or the like can be used.

  The collection system using the DNA of the present invention is particularly limited as long as it has means for bringing the DNA fixed body of the present invention into contact with water and / or gas containing a substance capable of collecting DNA. Rather, as such means, as a DNA fixed body of the present invention, a module formed by coating a film formed by applying to the surface of a substrate such as powder, bulk, plate, tubular body, fiber, woven fabric and non-woven fabric Specifically, as shown in FIG. 1, the material and shape of the substrate 3 forming the column 3 in which the DNA fixed body 1 such as a fiber is packed between the filters 2 and the coating film are formed. Can be mentioned as appropriate.

  Such collection systems include cigarette filters, drinks, milk filter materials, adsorption / cleaning materials in the digestive tract of mammals including humans, air, various wastewater and wastewater, rivers, lakes, etc. An environmental purification system for removing harmful substances from water. Examples of the environmental purification system include a system in which a column filled with DNA fixed body powder or the like is purified through air containing harmful substances or water.

  Here, the harmful substance means a compound that interacts with DNA by intercalation or adsorption, and threatens the DNA structure or DNA genetic information. Although there are unclear parts regarding substances that can interact, harmful substances having an aromatic functional group that is intercalated with DNA and heavy metal ions that are selectively adsorbed on DNA can be mentioned. Specific examples thereof include dioxins such as polychlorodibenzo-para-dioxin, polychlorodibenzofuran and polychlorobisphenyl (PCB), benz [a] pyrene, dichlorodiphenyltrichloroethane (DDT), diethylstilbestrol (DES). , Ethidium bromide, acridine orange, and derivatives thereof.

The collection system of the present invention can also be applied to a test system for substances capable of collecting DNA in the DNA fixed body of the present invention. For example, the DNA fixed body module of the present invention can be used in blood vessels and digestive tracts. It can also be applied to detection of specific substances.
[Example]

[Synthesis Example 1]
100 parts by weight of the following compound was dissolved in 1000 parts by weight of ethanol. The solution was heated for 5 minutes at 60 ° C. in an oil bath while flowing a small amount of nitrogen gas, and then a solution prepared by dissolving 0.1 part by weight of AIBN (azobisisobutyronitrile) in 10 parts by weight of ethanol was added. For 6 hours. The obtained oligomer ethanol solution was subjected to solvent substitution with water. To this oligomer liquid, 2000 parts by weight of ion-exchanged water was added, and alcohol removal was performed at 60 ° C. using an evaporator to finally obtain 450 parts by weight of an approximately oligomer aqueous solution (solid content: 22%). While monitoring the pH and stirring the oligomer solution, 8M KOH aqueous solution and ion-exchanged water were added. The obtained oligomer solution (1) had a pH of 6.5 and a solid content of about 5% by weight.

[Synthesis Example 2]
100 parts by weight of the following compound and 20 parts by weight of 3-methacryloxypropyltrimethoxysilane (248.4 → 179.4) were dissolved in 1000 parts by weight of ethanol. The solution was heated at 60 ° C. for 5 minutes while flowing a small amount of nitrogen gas, and then a solution prepared by dissolving 0.1 part by weight of AIBN in 10 parts by weight of ethanol was added. The reaction was performed at 60 ° C. for 6 hours. The obtained oligomer ethanol solution was subjected to solvent substitution with water. To this oligomer liquid, 2000 parts by weight of ion-exchanged water was added, and alcohol removal was performed at 60 ° C. using an evaporator to finally obtain about 480 parts by weight of an aqueous oligomer solution (solid content: 23%). While stirring this oligomer solution, 8M KOH aqueous solution and ion-exchanged water were added. The obtained oligomer solution (2) had a pH of 6.3 and a solid content of about 4.5% by weight.

[Synthesis Example 3]
100 parts by weight of the following compound was dissolved in 1000 parts by weight of ethanol. The solution was heated at 60 ° C. for 5 minutes while flowing a small amount of nitrogen gas, and then a solution prepared by dissolving 0.1 part by weight of AIBN in 10 parts by weight of ethanol was added. The reaction was performed at 60 ° C. for 6 hours. The obtained oligomer solution was diluted with ethanol to obtain a surface treatment solution having a solid content of 0.5% by weight.

[Synthesis Example 4]
5 parts by weight of double stranded DNA (DNA sodium salt, molecular weight, 6 × 10 ⁶ ) obtained from salmon roe was dissolved in 1000 parts by weight of ion-exchanged water over a period of 1 day. A sodium salt aqueous solution was obtained. [Synthesis Example 5]
Dissolve 20 parts by weight of double-stranded DNA (DNA sodium salt, molecular weight, 6 × 10 ⁶ ) obtained from salmon roe in 1000 parts by weight of ion-exchanged water over a period of 1 day. An aqueous solution was obtained.

50 parts by weight of the oligomer solution (1) obtained in Synthesis Example 1 was added to 100 parts by weight of the DNA sodium salt aqueous solution obtained in Synthesis Example 4 and stirred. 5 parts by weight of 5% by weight AlCl ₃ aqueous solution was added to the obtained transparent liquid. A white precipitate was observed from the mixed solution. After standing at room temperature for 4 hours, the precipitate was taken out with a centrifuge, rinsed with ion-exchanged water, dried for 1 hour in a drier set at 60 ° C., and DNA fixed body 1 (DNA: 16.15 wt. %, Polymer: 80.07% by weight, Al ₂ O ₃ : 3.09% by weight), about 3.0 parts by weight were obtained.

  The degree of DNA immobilization in DNA immobilization body 1 was evaluated. 0.015 parts by weight of DNA immobilized body 1 was added to 50 parts by weight of ion-exchanged water, and the mixture was prepared at room temperature for 1 day. The DNA concentration in the supernatant was evaluated with a spectrophotometer. Absorbance at 260 nm was 0.05. The amount of DNA eluted was about 5% of the theoretical charged DNA.

  The DNA double helix structure in DNA fixed body 1 was evaluated. When 0.5 part by weight of DNA complex 1 was immersed in 10 parts by weight of 50 ppm ethidium bromide aqueous solution, DNA fixed body 1 became pink and the color of the solution lightened. When the ultraviolet light was irradiated with a 366 nm ultraviolet lamp, the DNA fixed body 1 showed a strong orange fluorescent color. It was found that the intercalation characteristics of DNA were retained.

50 parts by weight of the oligomer solution (1) obtained in Synthesis Example 1 was added to 100 parts by weight of the DNA sodium salt aqueous solution obtained in Synthesis Example 4 and stirred. 5 parts by weight of a 3% by weight aqueous CaCl ₂ solution was added to the obtained transparent liquid. A white precipitate was observed from the mixed solution. After standing at room temperature for 4 hours, the precipitate was taken out with a centrifuge, rinsed with ion-exchanged water, dried for 1 hour in a drier set at 60 ° C., and DNA immobilized body 2 (DNA: 17.7 wt. %, Polymer: 79.6% by weight, CaO: 2.7% by weight) to obtain about 2.9 parts by weight.

  The degree of DNA immobilization in the DNA immobilization body 2 was evaluated. The same procedure as in Example 1 was conducted, except that 0.014 part by weight of DNA immobilized body 2 was added to 50 parts by weight of ion-exchanged water. Absorbance at 260 nm was 0.045. The amount of DNA eluted was about 4.5% of the theoretical charged DNA.

  The DNA double helix structure in the DNA fixed body 2 was evaluated in the same manner as in Example 1. It was found that the DNA fixed body 2 showed a strong orange fluorescent color, and the DNA intercalation characteristics were maintained.

To 100 parts by weight of the aqueous DNA sodium salt solution obtained in Synthesis Example 4, 80 parts by weight of the oligomer solution (2) obtained in Synthesis Example 2 was added and stirred. 10 parts by weight of a 5% by weight AlCl ₃ aqueous solution was added to the obtained transparent liquid. A white precipitate was observed from the mixed solution. After standing at room temperature for 4 hours, the precipitate was taken out with a centrifuge, rinsed with ion exchange water, dried in a drier set at 60 ° C. for 1 hour, and DNA fixed body 3 (DNA: 10.7 wt. %, Polymer: 85.3% by weight, Al ₂ O ₃ : 4.1% by weight), about 3.0 parts by weight were obtained.

  The degree of DNA immobilization in the DNA immobilization body 3 was evaluated. The same procedure as in Example 1 was carried out except that 0.023 part by weight of DNA immobilized body 3 was added to 50 parts by weight of ion-exchanged water. Absorbance at 260 nm was 0.035. The amount of DNA eluted was about 3.5% of the theoretical charged DNA.

  The DNA double helix structure in the DNA fixed body 3 was evaluated in the same manner as in Example 1. It was found that the DNA fixed body 3 showed a strong orange fluorescent color and retained the DNA intercalation characteristics.

To 100 parts by weight of the aqueous DNA sodium salt solution obtained in Synthesis Example 5, 100 parts by weight of the oligomer solution (2) obtained in Synthesis Example 2 was added and stirred. A nonwoven fabric having a thickness of 2 mm was dipped in the obtained transparent coating solution for 10 minutes and then dried for 5 hours in a drier set at 60 ° C. The obtained non-woven fabric was immersed in a 5 wt% AlCl ₃ aqueous solution for 5 minutes, dried at 60 ° C. for 5 hours, and then air-cooled in air. The obtained non-woven fabric was sufficiently rinsed with ion-exchanged water and dried at 60 ° C. for 4 hours to obtain a non-woven fabric coated with the DNA immobilized body 4.

  The DNA double helix structure in the DNA fixed body 4 was evaluated. A 50 ppm ethidium bromide aqueous solution was allowed to flow in the center of the nonwoven fabric coated with the DNA immobilizing body 4 for 1 minute. Thereafter, it was sufficiently washed with ion-exchanged water and dried at 50 ° C. for 20 minutes. When the ultraviolet light was applied with a 366 nm ultraviolet lamp, the DNA fixed body 4 through which the aqueous ethidium bromide solution passed showed a strong orange fluorescent color. It was found that the intercalation characteristics of DNA were retained.

  A non-woven fabric having a thickness of 2 mm was immersed in the surface treatment liquid obtained in Synthesis Example 3 and dried in the air to activate the non-woven fabric. Then, the nonwoven fabric which coat | covered the DNA fixing body 5 was obtained like Example 4 except having immersed the nonwoven fabric which performed surface activation in the transparent coating liquid for 30 second.

  The DNA double helix structure in the DNA fixed body 5 was evaluated in the same manner as in Example 4. It was found that the DNA fixed body 5 showed a strong orange fluorescent color, and the DNA intercalation characteristics were maintained.

It is the schematic which shows the collection system of this invention.

Claims (9)

  A DNA fixed body comprising a polymer having a phosphate group, a metal having a valence of 2 or more, and DNA. The polymer having a phosphate group has the formula (1)

The DNA fixed body according to claim 1, which has any one or two or more phosphate groups represented by the formula (1). The polymer having a phosphate group is represented by the formula (2)

(In the formula, R ¹ represents a hydrogen atom or a methyl group, R ² represents a hydrogen atom, a halogen atom or an optionally substituted C1-C3 alkyl group, and n represents 1 to 1) The DNA fixed body according to claim 1 or 2, wherein one or more of (meth) acrylic acid esters represented by the formula (1) or a salt thereof are included as a unit.   A first step of preparing a dispersion / dissolution containing a polymer having a phosphate group and DNA, and adding a dispersion / dissolution containing a divalent or higher metal ion to the dispersion / dissolution, A method for producing a DNA fixed body, comprising a second step of preparing and a third step of removing a dispersion medium / dissolution medium of the DNA fixing solution.   The method for producing a DNA fixed body according to claim 4, wherein the polymer having a phosphate group is a polymer having an alkali phosphate base, and the DNA is a DNA alkali salt.   A step of preparing a coating solution using a dispersion / dissolution solution of a polymer having a phosphate group and DNA, a step of forming a film on a substrate using the coating solution, and a metal having a valence of 2 or more And a step of bringing a dispersion / dissolution solution containing ions into contact with each other.   7. The method for producing a DNA fixed body according to claim 6, wherein the polymer having a phosphate group in the step of preparing the coating solution is a polymer having an alkali phosphate base, and the DNA is a DNA alkali salt.   A DNA collection body according to any one of claims 1 to 3, further comprising means for bringing water and / or gas containing a substance capable of collecting DNA into contact.   9. The collection system using DNA according to claim 8, which is used for an environmental purification system.

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