JP4534818B2 - Polymer compound for biomaterial and polymer solution using the same - Google Patents

Description

  The present invention relates to a polymer compound for medical materials used for detection and analysis of a large number of nucleic acids in a biological sample. More specifically, the present invention relates to a polymer compound for biomaterial used for measurement of gene diagnosis and gene expression analysis.

A technique for detecting a target molecule such as a complementary strand of a DNA fragment by immobilizing a nucleic acid fragment such as DNA on a solid substrate such as a plastic substrate, a slide glass, or a magnetic particle is useful for gene diagnosis, gene expression analysis, etc. Provide a simple means.
Means for immobilizing a nucleic acid fragment such as DNA on the surface of the solid phase carrier include (1) a method of synthesizing a nucleic acid fragment on the surface of the solid phase carrier, (2) a covalent bond with a functional group introduced on the surface of the solid phase carrier, Or it is divided roughly into two of the method of forming a non-covalent bond.
In the method (1), a base constituting a target nucleic acid is repeatedly bonded to the surface of a solid phase carrier one by one using a photolithography technique. According to this technique, nucleic acid fragments can be immobilized at a high density on the surface of a solid phase carrier, but it is not suitable for immobilizing long-chain nucleic acids and is not necessarily a widely available technique.
In (2), a method of introducing a functional group such as an aldehyde group or an amino group on the surface of the solid support is common. In the case of introducing an aldehyde group, the nucleic acid can be immobilized on the surface of the solid phase carrier by forming a covalent bond with the amino group previously introduced into the nucleic acid, but as in the case of (1), the long chain It is not suitable as a method for immobilizing nucleic acids.
When introducing an amino group, the surface of the solid phase carrier is treated with poly-L-lysine or a silane coupling agent having an amino group, and the electrostatic charge between the positive charge on the surface and the negative charge on the phosphate group of the nucleic acid is detected. A method of immobilizing using interaction is known. In this case, it is possible to immobilize a long-chain nucleic acid, but nonspecific adsorption to the surface of the detection target substance cannot be sufficiently suppressed, and a high S / N ratio (a signal based on the sample specimen and other signals) The ratio of the signal to the region) cannot be obtained.

In the method of introducing an amino group on the surface of a solid phase carrier, a method of treating the surface of the solid phase carrier with a polymer compound having an amino group in the side chain as a means for avoiding nonspecific adsorption of the detection target substance to the surface (For example, Patent Document 1). Patent Document 1 discloses a technique in which a substrate surface is treated with a polymer compound obtained by copolymerizing a biocompatible monomer and a monomer having an amino group, and DNA is immobilized thereon. However, in this case, the side chain of the monomer having an amino group is short, and the amino group cannot protrude from the high-order structure of the polymer compound, so that it cannot sufficiently interact with the nucleic acid, and the signal based on this is low. It will stop at.
For the purpose of immobilizing a long-chain nucleic acid on the surface of a solid phase carrier, the method of introducing an amino group onto the solid phase surface is most suitable. Since there is no signal that is high based on the sample analyte and that can sufficiently suppress non-specific adsorption of the detection target substance to the solid phase surface, the development thereof has been demanded.
WO2003-018841

An object of the present invention is to provide a polymer material for biomaterials that has a small amount of non-specific adsorption / binding of a substance to be detected and has a high ability to immobilize a physiologically active substance.
Biomaterials mentioned here include, in addition to biodevices such as DNA chips and biochips, gels, porous bodies, membranes, particles, and other biomatrixes that have various physical properties and shapes in the polymer compound itself. .

（ ４ ） ホスホリルコリン基を有する単量体（ ａ ） が２ − メタクリロイルオキシエチルホスホリルコリンである（ １ ） または（ ２ ） 記載のバイオマテリアル用高分子化合物、 （ ５ ） アミノ基を有する単量体（ ｂ ） が下記の一般式［ ２ ］ （ 式中Ｒ 2 は水素原子またはメチル基を示し、Ｙ は― Ｎ Ｈ ― または酸素原子を示す。Ｚ は炭素数１ 〜 ５ のアルキレンオキシ基またはアルキル基を示し、ｑ は1〜２０の整数を示す。繰り返されるＺ は、同一であっても、異なるアルキレンオキシ基またはアルキル基の連鎖であってもよい。Ｗはアミノ基またはその塩を示す。） で表されるものである（ １ ） 〜 （ ４ ） いずれか記載のバイオマテリアル用高分子化合物、

（ ６ ） 式［ ２ ］ のＺ がエチレンオキシ基である（ ５ ） 記載のバイオマテリアル用高
分子化合物、
（ ７ ） 式［ ２ ］ のＷ が― Ｎ Ｈ ２ またはその塩である（ ５ ） 又は（ ６ ） 記載のバイオマテリアル用高分子化合物、
（ ８ ） アミノ基を有する単量体（ ｂ ） がＮ ― （ ２ ― （ ２ ― （ ２ ― アミノエトキシ）エトキシ） エチル） メタクリルアミドである（ １ ） 〜 （ ４ ） いずれか記載のバイオマテリアル用高分子化合物、
（ ９ ） アルキル基を有する単量体（ ｃ ） がｎ ― ブチルメタクリレートである（ ２ ）〜（ ８ ） いずれか記載のバイオマテリアル用高分子化合物、
（ １ ０ ） 共重合がラジカル重合である（ １ ） 〜 （ ９ ） いずれか記載のバイオマテリアル用高分子化合物、
（ １ １ ） （ １ ） 〜 （ １ ０ ） いずれか記載のバイオマテリアル用高分子化合物を溶媒に溶解させたことを特徴とする高分子溶液、
（ １ ２ ） 溶媒がエタノール、メタノール、水のいずれか１ 種類以上から選ばれる単独または混合溶媒である（ １ １ ） の高分子溶液、である。 The present invention
(1) A polymer compound obtained by copolymerizing a monomer (a) having a phosphorylcholine group and a monomer (b) having an amino group, wherein the monomer (b) having an amino group is A polymer compound for biomaterials, characterized in that it is a compound having 5 to 20 atoms linked in a chain between an amino group and a polymerizable group,
(2) A polymer compound obtained by copolymerizing a monomer (a) having a phosphorylcholine group, a monomer (b) having an amino group, and a monomer (c) having an alkyl group, A polymer compound for biomaterial, wherein the monomer (b) having an amino group is a compound having 5 to 20 atoms linked in a chain between an amino group and a polymerizable group,
(3) The monomer (a) having a phosphorylcholine group has the following general formula [1] (wherein R1 represents a hydrogen atom or a methyl group, and X1 represents an alkyleneoxy group having 1 to 10 carbon atoms. p represents an integer of 1 to 2 0. When p is an integer of 2 or more and 2 0 or less, the repeated X 1 may be the same or a chain of different alkyleneoxy groups. (1) or the polymer compound for biomaterials according to (2),

(4) The polymer compound for biomaterial according to (1) or (2), wherein the monomer (a) having a phosphorylcholine group is 2-methacryloyloxyethyl phosphorylcholine, (5) the monomer having an amino group (b) ) Represents the following general formula [2] (wherein R 2 represents a hydrogen atom or a methyl group, Y represents —NH— or an oxygen atom, Z represents an alkyleneoxy group or alkyl group having 1 to 5 carbon atoms). Q represents an integer of 1 to 20. Repeated Z 1 may be the same or a chain of different alkyleneoxy groups or alkyl groups, and W represents an amino group or a salt thereof. (1) to (4) The polymer compound for biomaterial according to any one of the following:

(6) The polymer compound for biomaterials according to (5), wherein Z in formula [2] is an ethyleneoxy group,
(7) The polymer for biomaterial according to (5) or (6), wherein W in formula [2] is -NH2 or a salt thereof,
(8) The monomer (b) having an amino group is N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) methacrylamide (1) to (4) for biomaterials according to any one of Polymer compounds,
(9) The polymer compound for biomaterial according to any one of (2) to (8), wherein the monomer (c) having an alkyl group is n-butyl methacrylate,
(1 0) The polymer compound for biomaterial according to any one of (1) to (9), wherein the copolymerization is radical polymerization,
(1 1) (1) to (1 0) A polymer solution, wherein the polymer compound for biomaterial according to any one of the above is dissolved in a solvent,
(1 2) The polymer solution according to (1 1), wherein the solvent is a single or mixed solvent selected from one or more of ethanol, methanol, and water.

    By using the polymer compound of the present invention, it is possible to suppress nonspecific adsorption / binding of the substance to be detected without coating with an adsorption inhibitor, and to reduce signal decrease due to substrate washing, further simplifying It is possible to obtain a biomaterial that can detect the binding of a target molecule to a nucleic acid or the like immobilized thereon with high sensitivity.

    The polymer compound of the present invention is a polymer compound obtained by copolymerizing a monomer (a) having a phosphorylcholine group and a monomer (b) having an amino group, the monomer having an amino group The body (b) is characterized by having 5 to 100 atoms connected in a chain between an amino group and a polymerizable group. This polymer compound is a polymer that has both the property of suppressing nonspecific adsorption of physiologically active substances and the property of immobilizing nucleic acids, etc., and the phosphorylcholine group plays a role of suppressing nonspecific adsorption of physiologically active substances. The group plays a role in immobilizing nucleic acids and the like.

The monomer (b) having an amino group used in the present invention is not particularly limited as long as it gives an amino group to the end of the side chain having 5 to 100 atoms connected in a chain in the polymer compound. Although it is not a thing, it is preferable that it is a monomer represented by General formula [2].
In the monomer (b) having an amino group, when the number of atoms connected in a chain between the amino group and the polymerizable group is 4 or less, the amino group protrudes from the higher order structure of the polymer compound. In addition, when the number of atoms is 101 or more, the chain molecular structure forms an irregular high-order structure, and the orientation of the amino group becomes disordered, so that sufficient interaction with nucleic acid or the like is obtained. I can't. Therefore, in the monomer (b) having an amino group, the number of atoms connected in a chain between the amino group and the polymerizable group is limited to 5 to 100, preferably 5 to 50 atoms. More preferably, it has 5 to 20 atoms.

The structural unit Z of the chain molecular structure in the general formula [2] includes an alkyleneoxy group or an alkyl group, and an alkyleneoxy group is preferable in view of appropriate hydrophilicity and flexibility. Examples of the alkyleneoxy group include an ethyleneoxy group, a propyleneoxy group, an isopropyleneoxy group, a butyleneoxy group, and a pentyleneoxy group, and an ethyleneoxy group is preferable from the viewpoint of hydrophilicity and flexibility.
Q in the general formula [2] represents an integer of 1 to 20, and the repeated Zs may be the same or a chain of different alkyleneoxy groups or alkyl groups.
W in the general formula [2] is an amino group or a salt thereof, and examples thereof include a primary amino group, a secondary amino group, a tertiary amino group, a quaternary ammonium group, or a salt thereof. A primary amino group or a salt thereof is preferable in that it can be used for reaction with other low molecular compounds or high molecular compounds at the end of the side chain of the high molecular compound.
Preferable examples of the monomer represented by the general formula [2] include N- (2- (2-aminoethoxy) ethyl) methacrylamide, 2- (2-aminoethoxy) ethyl methacrylate, N- (2 -(2- (2-aminoethoxy) ethoxy) ethyl) methacrylamide, 2- (2- (2-aminoethoxy) ethoxy) ethyl methacrylate, N- (2- (2- (2- (2-aminoethoxy)) Ethoxy) ethoxy) ethyl) methacrylamide, 2- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) ethyl methacrylate, N- (3- (2-aminoethoxy) propyl) methacrylamide, 3- (2 -Aminoethoxy) propyl methacrylate, N- (3- (3- (2-aminoethoxy) propoxy) propyl) methacrylamide, 3- (3- (2- Minoethoxy) propoxy) propyl methacrylate, N- (2- (3- (3- (3-aminoethoxy) propoxy) propoxy) propyl) methacrylamide, 2- (3- (3- (3-aminoethoxy) propoxy) propoxy ) Propyl methacrylate or a salt thereof, but N- (2- (2- (2-aminoethoxy) ethoxy) is excellent in copolymerization with the monomer (a) having a phosphorylcholine group. More preferred is ethyl) methacrylamide.

    The monomer (a) having a phosphorylcholine group used in the present invention is not particularly limited in structure, but the (meth) acryl group and phosphorylcholine group represented by the general formula [1] are alkyleneoxy having 1 to 10 carbon atoms. Preference is given to compounds bonded via a chain of group X. The repeating number of the alkyleneoxy group X in the formula is an integer of 1 to 20, and when the repeating number is 2 or more and 20 or less, the carbon number of the repeated alkyleneoxy group may be the same or different. Examples of the monomer (a) having a phosphorylcholine group include 2- (meth) acryloyloxyethyl phosphorylcholine, 2- (meth) acryloyloxyethoxyethyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine, 10- (meth). Examples thereof include acryloyloxyethoxynonyl phosphorylcholine, 2- (meth) acryloyloxypropyl phosphorylcholine, and 2-methacryloyloxyethyl phosphorylcholine is preferable from the viewpoint of availability.

    The polymer compound used in the present invention may contain other groups in addition to the phosphorylcholine group and amino group. For example, an alkyl group-containing monomer (c) may be copolymerized, and the alkyl group-containing monomer (c) is preferably n-butyl methacrylate.

    The method for synthesizing the polymer compound of the present invention is not particularly limited, but includes at least a monomer (a) having a phosphorylcholine group and a monomer (b) having an amino group for ease of synthesis. The mixture is preferably radically polymerized in a solvent in the presence of a polymerization initiator.

    Any solvent may be used as long as each monomer can be dissolved, and examples thereof include methanol, ethanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, and chloroform. These solvents are used alone or in combination of two or more. Generally, when the polymer compound is applied to a plastic substrate, ethanol and methanol are preferable because they do not denature the substrate.

The polymerization initiator may be any ordinary radical initiator, for example, 2,
Examples include azo compounds such as 2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), organic peroxides such as benzoyl peroxide and lauryl peroxide. be able to.

    The chemical structure of the polymer compound of the present invention may be in any form such as random, block, graft, etc., as long as at least a phosphorylcholine group and an amino group are copolymerized.

Example 1
2-Methacryloyloxyethyl phosphorylcholine (MPC), N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) methacrylamide (NAEMA), n-butyl methacrylate (BMA), respectively, in this order 0.25 mol / L , 0.05 mol / L and 0.70 mol / L were dissolved in ethanol to prepare a monomer mixed solution. Thereto was further added 0.002 mol / L AIBN and stirred until uniform. Then, after reacting at 60 ° C. for 4 hours under an argon gas atmosphere, the reaction solution was dropped into a mixed solvent of diethyl ether and chloroform, and a precipitate was collected.
The collected polymer substance was dissolved in an ethanol solvent so as to be 0.3% by weight. A plastic substrate obtained by processing a saturated cyclic polyolefin resin into a slide glass shape (dimensions: 76 mm × 26 mm, 1 mm) is immersed in an ethanol solution in which the polymer substance is dissolved, and has a phosphorylcholine group and an amino group on the plastic substrate surface Polymeric material was introduced. At this time, the number of atoms between the amino group and the polymerizable group is ten.

(Example 2)
2-Methacryloyloxyethyl phosphorylcholine (MPC), N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) methacrylamide (NAEMA), n-butyl methacrylate (BMA), respectively, in this order 0.25 mol / L , 0.10 mol / L and 0.65 mol / L were dissolved in ethanol to prepare a monomer mixed solution. Thereto was further added 0.002 mol / L AIBN and stirred until uniform. Then, after reacting at 60 ° C. for 4 hours under an argon gas atmosphere, the reaction solution was dropped into a mixed solvent of diethyl ether and chloroform, and a precipitate was collected.
After that, it was dissolved in an ethanol solvent by the same method as in Example 1, and a plastic substrate was further immersed therein to introduce a polymer substance having a phosphorylcholine group and an amino group on the surface of the plastic substrate. At this time, the number of atoms between the amino group and the polymerizable group is ten.

(Comparative Example 1)
2-Methacryloyloxyethyl phosphorylcholine (MPC), 2-aminoethyl methacrylate hydrochloride (AEMHCl), and n-butyl methacrylate (BMA) were sequentially added to 0.25 mol / L, 0.05 mol / L, and 0.70 mol / L, respectively. In this way, it was dissolved in ethanol to prepare a monomer mixed solution. Thereto was further added 0.002 mol / L AIBN and stirred until uniform. Then, after reacting at 60 ° C. for 4 hours under an argon gas atmosphere, the reaction solution was dropped into a mixed solvent of diethyl ether and chloroform, and a precipitate was collected.
After that, it was dissolved in an ethanol solvent by the same method as in Example 1, and a plastic substrate was further immersed therein to introduce a polymer substance having a phosphorylcholine group and an amino group on the surface of the plastic substrate. At this time, the number of atoms between the amino group and the polymerizable group is four.

(Comparative Example 2)
2-Methacryloyloxyethyl phosphorylcholine (MPC), 2-aminoethyl methacrylate hydrochloride (AEMHCl), and n-butyl methacrylate (BMA) were sequentially added to 0.25 mol / L, 0.10 mol / L, and 0.65 mol / L, respectively. In this way, it was dissolved in ethanol to prepare a monomer mixed solution. Thereto was further added 0.002 mol / L AIBN and stirred until uniform. Then, after reacting at 60 ° C. for 4 hours under an argon gas atmosphere, the reaction solution was dropped into a mixed solvent of diethyl ether and chloroform, and a precipitate was collected.
After that, it was dissolved in an ethanol solvent by the same method as in Example 1, and a plastic substrate was further immersed therein to introduce a polymer substance having a phosphorylcholine group and an amino group on the surface of the plastic substrate. At this time, the number of atoms between the amino group and the polymerizable group is four.

(Preparation of PCR product)
The DNA amplified by PCR using PCR Thermal Cycler 480 (manufactured by Takara Bio Inc.) was used for the preparation of long DNA and fluorescently labeled long DNA. As a primer, oligonucleotide (manufactured by Sigma Genosys), sequence 1: TCGTGCCGTGGACATTAAGGAGAAGC (SEQ ID NO: 1), sequence 2: CTAGAAAGCATTTGCGGTGGACGAT (SEQ ID NO: 2) was used, and cDNA library (Humanliver) (manufactured by Takara Bio Inc.) was used as a template. A DNA fragment (502 bp) of the β-actin gene was amplified.
The composition of the PCR reaction solution was 200 μM dNTP, TakaraExTaq (manufactured by Takara Bio Inc.), ₂ mM MgCl ₂ , Ex Taq Buffer. The PCR reaction was pre-denaturation: 90 ° C., 30 seconds; Second, annealing: 60 ° C., 30 seconds, elongation: 72 ° C., 45 seconds; 30 cycles, elongation: 72 ° C., 3 minutes; 1 cycle. After confirming that this fragment was amplified by agarose gel electrophoresis, the PCR amplification product was purified and concentrated using Microcon 100 (Millipore).
Preparation of fluorescently labeled long-chain DNA was performed in the same manner as described above using Cy3 labeled oligonucleotide.

(DNA immobilization, hybridization)
The long-chain DNA obtained above was dissolved in a predetermined concentration using a predetermined aqueous solution, dispensed into a 96-well plate, and a substrate prepared in Examples and Comparative Examples using a micropin type microarray spotter. Each was spotted.
After spotting, the long DNA was immobilized by heat treatment at 80 ° C. for 1 hour and irradiation with UV (120 mJ), followed by washing with 0.1% SDS. Thereafter, an aqueous solution of 2 × SSC (sodium citrate buffer) and 0.1% SDS was prepared and boiled on a hot plate, and then the substrate was immersed for 1 minute and then immersed in pure water for 1 minute. .
The substrate was immersed in 5 × SSC, 0.3% SDS, 0.1 mg / ml BSA aqueous solution at 50 ° C. for 1 hour to block the portion where DNA was not spotted. Subsequently, after washing with water, a hybridization reaction was performed at 50 ° C. for 16 hours. As the hybridization solution, 5 × SSC, 0.3% SDS in which the fluorescently labeled long-chain DNA obtained above was dissolved at a predetermined concentration was used.
After completion of hybridization, the cells were immersed in 0.2 × SSC, 0.1% SDS at 42 ° C. for 10 minutes. Then, it was immersed in 0.2 × SSC and 0.1% SDS at room temperature for 10 minutes and washed in the order of 2 × SSC, 1 × SSC, and 0.1 × SSC. The substrate was then dried by centrifuging.

(Numericalization of fluorescent intensity of spots by fluorescent scanner)
Using the microarray scanner “ScanArray Lite” (manufactured by Packard Biochip Technology), the fluorescence of the spots of each substrate obtained above was detected. The measurement conditions at this time were laser output 90%, PMT sensitivity 65%, excitation wavelength 550 nm, measurement wavelength 570 nm, and resolution 50 μm.
Quantification of the fluorescence intensity of the spot was performed using analysis software “QuantArray” attached to the scanner.

Table 1 shows the fluorescence signal values and CV values obtained with the substrates of Examples 1 and 2 and Comparative Examples 1 and 2. The CV value is a value obtained by dividing the average value of each fluorescence amount of 96 spots by the standard deviation of each fluorescence amount of 96 spots, and represents a variation in signal.
In Examples 1 and 2, as compared with Comparative Examples 1 and 2, a significant increase in the fluorescence signal value was observed, and the CV value was also good. This result supported the effect of the present invention.

Claims (12)

A polymer compound obtained by copolymerizing a monomer (a) having a phosphorylcholine group and a monomer (b) having an amino group, wherein the monomer (b) having an amino group has an amino group and A polymer compound for biomaterials, which is a compound having 5 to 20 atoms connected in a chain with a polymerizable group. A polymer compound obtained by copolymerizing a monomer (a) having a phosphorylcholine group, a monomer (b) having an amino group, and a monomer (c) having an alkyl group, The polymer compound for biomaterials, wherein the monomer (b) is a compound having 5 to 20 atoms linked in a chain between an amino group and a polymerizable group. The polymer compound for biomaterial according to claim 1 or 2, wherein the monomer (a) having a phosphorylcholine group is represented by the following general formula [1].

(In the formula, R 1 represents a hydrogen atom or a methyl group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, p represents an integer of 1 to 2O, and p is an integer of 2 to 20 inclusive. In some cases, the repeated X 1 may be the same or a chain of different alkyleneoxy groups.) The polymer compound for biomaterial according to claim 1 or 2, wherein the monomer (a) having a phosphorylcholine group is 2-methacryloyloxyethyl phosphorylcholine. The polymer compound for biomaterial according to any one of claims 1 to 4, wherein the monomer (b) having an amino group is represented by the following general formula [2].

(Wherein R 2 represents a hydrogen atom or a methyl group, Y represents —NH— or an oxygen atom, Z represents an alkyleneoxy group or alkyl group having 1 to 5 carbon atoms, and q represents 1 to 20 (Indicates an integer. The repeated Z 1 s may be the same or different chain of alkyleneoxy groups or alkyl groups. W 1 represents an amino group or a salt thereof.) The polymer compound for biomaterial according to claim 5, wherein Z in formula [2] is an ethyleneoxy group. The polymer compound for biomaterial according to claim 5 or 6, wherein W in formula [2] is -NH2 or a salt thereof. The polymer compound for biomaterial according to any one of claims 1 to 4, wherein the monomer (b) having an amino group is N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) methacrylamide. The polymer compound for biomaterials according to any one of claims 2 to 8, wherein the monomer (c) having an alkyl group is n-butyl methacrylate. Copolymerization is radical polymerization, The high molecular compound for biomaterials in any one of Claims 1-9. A polymer solution obtained by dissolving the polymer compound for biomaterial according to claim 1 in a solvent. The polymer solution according to claim 1, wherein the solvent is a single or mixed solvent selected from at least one of ethanol, methanol, and water.