JP4821444B2 - Polymer compound for bioassay and base material for bioassay using the same - Google Patents

Description

  The present invention relates to a bioassay polymer compound used for parallel detection and analysis of a target substance in a biological sample using a physiologically active substance or the like immobilized on the base material, and a bioassay base material using the same About.

  Attempts to decipher the genetic activity and the molecular processes of drug effects at the molecular level have traditionally focused on genomics, but proteomics is more detailed about the biological functions of cells. Provide information. Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantifying expression at the protein level, rather than at the gene level. It also includes studies of events that are not encoded by genes such as post-translational modifications of proteins and interactions between proteins. On the other hand, in recent years, sugar chains have attracted attention as the third chain in the living body. In particular, research on the relationship with cell differentiation, canceration, immune reaction, fertilization, and the like has continued, and attempts to create new drugs and medical materials are continuing. In addition, the sugar chain is a receptor for many toxins, viruses and bacteria, and has attracted attention as a marker for cancer. Recently, sugar chains also interact with amyloid proteins, which are thought to cause cancer cell metastasis and Alzheimer's disease. It has been reported.

  Now that the structure of the genome, which is the “blueprint of life”, has been clarified and a large amount of genome information has become available, proteomics research and sugar chain research are becoming more and more active. There is a need for rapid and high efficiency (high throughput) detection of substances. Therefore, as a molecular array for this purpose, development and research of a substrate on which a physiologically active substance having a sugar such as an antibody, a glycopeptide, or a sugar chain is immobilized has been advanced. Good examples are protein chips including antibody chips and sugar chip chips.

  However, since protein chips and the like are generally developed on the extension line of a DNA chip, studies have been made to immobilize a protein or a molecule that captures it on a glass substrate on a chip surface (for example, patents). Reference 1).

  There is a sandwich method as a method generally used in detection and quantification of specimens such as proteins. In this method, an antibody (primary antibody) is bound to a solid phase, a target protein is trapped by an insolubilized antibody, and then a labeled antibody (secondary antibody) that recognizes and binds to another epitope of the target protein is reacted. Thus, the protein is quantitatively measured by measuring the label (see, for example, Non-Patent Document 1). However, this method requires multiple types of antibodies that do not compete with each other for each target protein when detecting a large amount of proteins at once, such as a protein chip. It contains many problems to be solved.

  In addition, after immobilizing a protein-capturing molecule (hereinafter abbreviated as a capturing molecule) on a substrate, it reacts with another protein (corresponding to an antigen in the case of an antigen-antibody reaction) on the surface as in the sandwich method, for example. In addition, when a labeled protein is reacted and finally detected by a detector or the like, a protein other than the molecule, that is, an antigen or a labeled protein is immobilized on a portion where the capture molecule is not immobilized. , It becomes noise at the time of detection, causing a reduction in the signal-to-noise ratio (S / N ratio), and lowering the detection accuracy (see Non-Patent Document 2, for example). In particular, in experiments conducted by the present inventors, serum and plasma used in clinical diagnosis and the like tend to have high non-specific adsorption of contaminating proteins, resulting in high noise and a low S / N ratio. It was.

  For this reason, in the normal sandwich method, after the primary antibody is immobilized, non-specific adsorption of the antigen and the secondary antibody is carried out to prevent the non-specific adsorption of the antigen and the secondary antibody. Not. In addition, since the adsorption inhibitor is coated after immobilizing the primary antibody, it may be coated on the immobilized protein, and there is a problem that it cannot react with the secondary antibody. Therefore, there is a demand for a biochip that does not have an adsorption inhibitor coating step after immobilization of the primary antibody and has a small amount of nonspecific adsorption of a physiologically active substance.

On the other hand, when immobilizing the primary antibody, it is important to immobilize so that the antigen-binding site of the antibody functions easily. For example, Patent Document 2 describes a method in which a specific immobilizing substance is bonded to a fine hole made on a substrate. However, in this method, when the immobilization substance is an antibody, the amino acid terminal (N terminal) is used to bond the peptide to the substrate, and the antigen binding site may not function sufficiently.
JP 2001-116750 A JP 2005-214889 A "Enzyme immunoassay", Yuji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, Medical School, 1987, p.44-45 “DNA Microarray Practice Manual”, Yoshihide Hayashizaki, Koji Okazaki, Yodosha, 2000, p.57

  The present invention immobilizes saccharides, sugar chains, glycopeptides, antibodies, and / or physiologically active substances having these by suppressing non-specific adsorption / binding of the substance to be detected without coating an adsorption inhibitor. It is an object of the present invention to provide a bioassay polymer compound that can be used and a substrate using the same. The term “substrate” as used herein includes all materials that can be used in bioassays, such as various chips, plates, particles, and containers.

The present invention
(1) A polymer compound for bioassay comprising at least a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group ,
The polymer compound for bioassay is represented by the following general formula [1] (wherein R1R2 and R3 are hydrogen atoms).
Or a methyl group, R4 shows a hydrophobic group. X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20. When p is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different. Y is a spacer containing an alkylene glycol residue, and Z is an oxygen atom or NH. l, m, and n are natural numbers. )so
Represented,
Prior following general formula [1], Y is represented by the following general formula [2] or [3] (wherein q, r is an integer of 1 to 20.), The hydrophobic group is an alkyl group <br/> characterized high molecular compounds for bioassay,




(2) The polymer compound for bioassay according to (1), wherein the primary amino group is an oxylamino group and / or a hydrazide group,
(3) The polymer compound for bioassay according to (1) or (2), wherein the main chain of the polymer compound is a (meth) acryl skeleton,
( 4 ) The polymer compound for bioassay according to ( 1 ), wherein, in the general formula [1], X is an ethyleneoxy group,
( 5 ) The polymer compound for bioassay according to ( 1 ) or ( 4 ), wherein in the general formula [1], R4 is an alkyl group,
( 6 ) The polymer compound for bioassay according to ( 5 ), wherein the alkyl group has 2 to 10 carbon atoms,
( 7 ) A method for producing a polymer compound for bioassay according to any one of (1) to ( 6 ), wherein at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a primary amino group are previously protected. A method of producing a polymer compound for bioassay comprising the steps of radical copolymerization with a monomer protected in step, and the step of removing the protecting group from the polymer compound obtained by the step,
( 8 ) A method for producing a polymer compound for bioassay according to any one of (1) to ( 7 ), wherein at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a primary amino group can be introduced. A method for producing a polymer compound for bioassay comprising a step of radical copolymerizing a monomer having a functional group, and a step of introducing a primary amino group into the functional group of the polymer compound obtained by the step;
( 9 ) A surface coating material containing the polymer compound for bioassay according to any one of (1) to ( 5 ),
( 10 ) A base material for bioassay, wherein a layer containing the surface coating material according to ( 9 ) is formed on the surface of the base material,
( 11 ) The bioassay substrate according to ( 10 ), wherein the shape of the substrate is any one of a slide-shaped substrate, a 96-well plate, a container, and a microfluidic substrate.
( 12 ) The bioassay substrate according to ( 10 ) or ( 11 ), wherein the substrate is made of plastic.
( 13 ) The bioassay substrate according to ( 12 ), wherein the plastic contains a saturated cyclic polyolefin or polystyrene.
( 14 ) A bioassay substrate in which a physiologically active substance is immobilized on the bioassay substrate according to any one of ( 10 ) to ( 13 ),
( 15 ) The bioassay substrate according to ( 14 ), wherein the physiologically active substance is at least one selected from sugars, sugar chains, and physiologically active substances having these sugars,
( 16 ) The bioassay substrate according to ( 15 ), wherein the physiologically active substance is a glycopeptide or an antibody,
It is.

    By using the polymer compound of the present invention, non-specific adsorption / binding of the detection target substance can be suppressed and the physiologically active substance can be immobilized without coating with an adsorption inhibitor.

    The polymer compound of the present invention includes at least a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group. This polymer compound has a property of suppressing physical adsorption (nonspecific adsorption) of the detection target substance to the base material. Furthermore, it also has the property of immobilizing physiologically active substances. The phosphorylcholine group plays a role in suppressing nonspecific adsorption, and the primary amino group plays a role in immobilizing a physiologically active substance. In particular, when an antibody is immobilized, since the sugar chain of the Fc site of the antibody reacts with the amino group of the polymer compound, the antigen-binding site tends to be sufficiently functional.

    The unit having a phosphorylcholine group contained in the polymer compound of the present invention is not particularly limited in structure, but as shown in the structural unit on the left side of the structural unit of the following general formula [1], a (meth) acrylic residue And a phosphorylcholine group are preferably bonded via a chain of an alkyleneoxy group X having 1 to 10 carbon atoms. Of these, X is most preferably an ethyleneoxy group. 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. Although l is naturally a natural number, it may be expressed as a composition ratio of each component.

  The composition ratio of the unit having a phosphorylcholine group contained in the polymer compound of the present invention (ratio of l to the sum of l, m, and n) is not particularly limited, but is 5 for all units of the polymer compound. -98 mol% is preferable, More preferably, it is 10-90 mol%, Most preferably, it is 10-80 mol%. When the composition ratio falls below the lower limit, nonspecific adsorption increases. On the other hand, when the upper limit is exceeded, water solubility increases, and the polymer compound may be eluted during the assay. However, this is not the case when a functional group capable of covalently bonding to the substrate is introduced into any part of the polymer compound and chemically bonded to the substrate. For example, a method of introducing a silane coupling agent into the polymer compound is simple and preferable.

    The unit having a hydrophobic group contained in the polymer compound of the present invention is not particularly limited in structure. However, as represented by the structural unit in the center of the structural unit of the general formula [1], (meth) acrylic A structure in which a hydrophobic group is bonded to a group residue is preferable. Although a hydrophobic group is not specifically limited, An alkyl group and aromatics are mentioned. More preferably, the alkyl group is an alkyl group having 2 to 20 carbon atoms. The structure of the alkyl group is not particularly limited, and may be linear, branched, or cyclic. By including a unit having a hydrophobic group in the polymer compound, the wettability is improved even on a hydrophobic base material such as plastic, and coating can be performed without unevenness. In addition, since the hydrophobicity increases, it is possible to prevent the polymer compound from eluting during the assay. In the formula, m is naturally a natural number, but may be expressed as a composition ratio of each component.

  The composition ratio of the unit having a hydrophobic group contained in the polymer compound of the present invention (the ratio of m to the sum of l, m, and n) is not particularly limited, but is based on all units of the polymer compound. 1 to 90 mol% is preferable, more preferably 10 to 80 mol%, and most preferably 20 to 80 mol%. If the upper limit is exceeded, nonspecific adsorption may increase.

  The unit having a primary amino group contained in the polymer compound of the present invention is not particularly limited in structure, but as represented by the structural unit on the right side of the structural unit of the general formula [1], It is preferable that the (meth) acryl group residue and the oxylamino group or hydrazide group have a structure via a spacer Y containing an alkylene glycol residue. In the case of an oxylamino group, Z represents an oxygen atom, and in the case of a hydrazide group, Z represents NH. n is originally a natural number, but may be expressed as a composition ratio of each component. The structure of the spacer Y containing an alkylene glycol residue is not particularly limited, but may be the following general formula [2] or [3] (wherein q and r are integers of 1 to 20). preferable.

  The oxylamino group or hydrazide group can immobilize sugars, sugar chains, glycopeptides, antibodies, and / or physiologically active substances having these. Further, since the amino group is separated from the main chain portion due to the presence of the spacer, the physiologically active substance is easily immobilized. Furthermore, since an alkylene glycol residue is contained in the spacer, non-specific adsorptivity can be expressed. The number of carbon atoms of the alkylene glycol residue is not limited, but an ethylene glycol residue having 2 carbon atoms is most preferable.

  The composition ratio of the unit having a primary amino group contained in the polymer compound of the present invention (ratio of n to the sum of l, m, and n) is not particularly limited, but for all units of the polymer compound 1-94 mol%, more preferably 2-90 mol%, most preferably 5-80 mol%. When the composition ratio falls below the lower limit, the sugar, sugar chain and / or physiologically active substance having these cannot be sufficiently immobilized. On the other hand, if the upper limit is exceeded, non-specific adsorption may increase.

    As long as the chemical structure of the polymer compound of the present invention is a structure including at least a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group, the bonding method is any of random, block, graft, etc. It may be in a form.

The method for synthesizing the polymer compound of the present invention is not particularly limited, but at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a primary amino group are previously protected with a protecting group for ease of synthesis. A production method comprising a step of radical copolymerization of the monomer obtained and a step of removing a protecting group from the polymer compound obtained by the step is preferred. Alternatively, a step of radical copolymerizing a monomer having at least a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer having a functional group capable of introducing a primary amino group, and the polymer compound obtained by the step having a primary amino group The manufacturing method including the step of introducing is preferable.

    The monomer having a phosphorylcholine group is not particularly limited in structure. For example, 2- (meth) acryloyloxyethyl phosphorylcholine, 2- (meth) acryloyloxyethoxyethyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine, 10 -(Meth) acryloyloxyethoxynonyl phosphorylcholine, 2- (meth) acryloyloxypropyl phosphorylcholine and the like can be mentioned, and 2-methacryloyloxyethyl phosphorylcholine is preferable from the viewpoint of availability.

    Specific examples of the monomer having a hydrophobic group include n-butyl (meth) acrylate, iso-butyl (meth) acrylate, sec-butyl (meth) acrylate, t-butyl (meth) acrylate, n-neopentyl ( (Meth) acrylate, iso-neopentyl (meth) acrylate, sec-neopentyl (meth) acrylate, neopentyl (meth) acrylate, n-hexyl (meth) acrylate, iso-hexyl (meth) acrylate, heptyl (meth) acrylate, n- Octyl (meth) acrylate, iso-octyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, n-nonyl (meth) acrylate, iso-nonyl (meth) acrylate, n-decyl (meth) acrylate, iso-decyl ( (Meth) acrylate, n-dodecyl (Meth) acrylate, iso-dodecyl (meth) acrylate, n-tridecyl (meth) acrylate, iso-tridecyl (meth) acrylate, n-tetradecyl (meth) acrylate, iso-tetradecyl (meth) acrylate, n-pentadecyl (meta ) Acrylate, iso-pentadecyl (meth) acrylate, n-hexadecyl (meth) acrylate, iso-hexadecyl (meth) acrylate, n-octadecyl (meth) acrylate, iso-octadecyl (meth) acrylate, cyclohexyl (meth) acrylate, isobonyl Examples include (meth) acrylate. Of these, n-butyl methacrylate, n-dodecyl methacrylate, and n-octyl methacrylate are most preferable.

The monomer in which the primary amino group is previously protected with a protecting group is not particularly limited in structure, but the following general formula [4] (wherein R ₃ is a hydrogen atom or a methyl group, Y is a spacer containing an alkylene glycol residue, Z represents an oxygen atom or NH, and W represents a protecting group.) (Meth) acrylic group and oxylamino group or hydrazide group have a structure through a spacer Y containing an alkylene glycol residue. Preferably there is.

  The protecting group W is not limited as long as it can protect the amino group, and can be arbitrarily used. Of these, t-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Z group, Cbz group), 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like are preferably used.

Specific examples of the monomer are those represented by the following formula.

  Deprotection can be performed under general conditions using trifluoroacetic acid, hydrochloric acid, or anhydrous hydrogen fluoride.

  On the other hand, the method for introducing a primary amino group after polymerizing a polymer compound is not limited at all, but at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer having an alkoxy group are radicalized. A method of producing a hydrazide group by reacting an alkoxy group introduced into the polymer compound with hydrazine after copolymerization is preferred. As the alkoxy group, a methoxy group, an ethoxy group, a propoxy group, a t-butoxy group and the like are preferable.

Specific examples of the monomer having an alkoxy group are those represented by the following formula.

    As the synthetic solvent for the polymer compound of the present invention, any solvent may be used as long as each monomer can be dissolved. For example, alcohols such as methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanol are used. , Benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, cyclohexanone, N, N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether And ethylene glycol monobutyl ether. These solvents are used alone or in combination of two or more.

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 molecular weight of the polymer compound of the present invention is preferably 5,000 or more and more preferably 10,000 or more because the polymer compound and unreacted monomer can be easily separated and purified.

    By covering the substrate surface with the polymer compound of the present invention, it is possible to easily impart the property of suppressing nonspecific adsorption of the physiologically active substance and the property of immobilizing the physiologically active substance.

    For coating the base material surface with a polymer compound, for example, a polymer solution in which a polymer compound is dissolved in an organic solvent so as to have a concentration of 0.05 to 50% by weight is prepared, and a known method such as immersion or spraying is used. After coating on the surface of the substrate, drying is performed at room temperature or under heating.

    Examples of organic solvents include ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, cyclohexanol, and other alcohols, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl acetate, ethyl acetate, Examples thereof include butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, and cyclohexanone. These solvents are used alone or in combination of two or more. Of these, alcohols such as ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, and cyclohexanol are preferable because they do not denature the plastic substrate and can be easily dried.

    As the substrate used in the present invention, a slide-shaped substrate, a 96-well plate, a container, and a microfluidic substrate are preferable. Examples thereof include a plastic substrate, a glass substrate, and a substrate having a metal vapor deposition film. Specific examples of the plastic substrate include substrates made of cycloolefin polymer, polypropylene, polyethylene, polysalophone, polyimide, polycarbonate, polymethyl methacrylate, and the like.

    The substrate coated with the polymer compound can immobilize sugars, sugar chains, glycopeptides, antibodies, and / or physiologically active substances having these, and can be suitably used for bioassay applications. Examples of physiologically active substances include glycoproteins, glycolipids, and sugar chain genes. As a method for immobilizing these physiologically active substances on the substrate, a method of spotting a solution in which the physiologically active substance is dissolved using a spotter, or dispensing a solution in which the physiologically active substance is dissolved into a container or the like. There are ways to fix it.

    At the time of immobilization, the physiologically active substance can be more effectively immobilized on the base material by oxidizing the glycoprotein, glycolipid, sugar chain gene, and antibody to an aldehyde with an oxidizing agent. The oxidizing agent to be used is not particularly limited, but periodic acid is used. These concentrations are 0.04 to 0.16M. In addition, a sodium bicarbonate solution (pH 8.1) is usually used as a buffer for the oxidation reaction. The aldehyde group of the physiologically active substance thus oxidized reacts with the primary amino group on the substrate to generate a Schiff base, which can be chemically immobilized.

    Various buffer materials are preferably used as the solution for dissolving the physiologically active substance. Although not particularly limited, for example, sodium carbonate, sodium bicarbonate, potassium phosphate, dipotassium hydrogen phosphate, Tris-HCl buffer, Tris acetate buffer, PBS buffer, sodium citrate, sodium acetate, HEPES (N-2 -Hydroxyethylperazine-N'-ethanesulfonic acid) buffer, MOPS (3- (N-morpholino) propanesulfonic acid) buffer, etc. are used.

    The pH of the solution for dissolving the physiologically active substance is preferably 2 to 8 when the sugar or sugar chain is dissolved. When glycoprotein is dissolved, the pH is preferably 4-9. When the sugar nucleic acid is dissolved, the pH is preferably 2-8. When glycolipid is dissolved, the pH is preferably 2-8.

    The concentration of the physiologically active substance in the solution is not particularly limited, but is preferably 0.0001 mg / ml to 10 mg / ml.

    The temperature for immobilizing the physiologically active substance solution is preferably 0 ° C to 100 ° C.

(Synthesis Example 1 of polymer compound)
2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), N- [2- [2- [2- (t-butoxycarbonylaminooxyacetylamino) ethoxy] ethoxy] ethyl] -methacrylamide (OA) The compound represented by the formula [5] was dissolved in ethanol so as to be 0.25 mol / L, 0.55 mol / L, and 0.20 mol / L, respectively, to prepare a monomer mixed solution. Further AIBN was added to 0.01 mol / L, and the mixture was stirred until uniform. Then, after making it react at 60 degreeC under argon gas atmosphere, the reaction solution was dripped in diethyl ether, and precipitation was collect | recovered. The obtained polymer compound was measured by 1H-NMR, and the composition ratio of the polymer compound was calculated. Table 1 shows the results.

(Deprotection of polymer compounds)
The polymer compound was treated with 2N HCl-dioxane-ethanol solution for 4 hours at room temperature to remove the BOC group. 1H-NMR measurement of the polymer compound after deprotection was performed, and the deprotection was confirmed by the disappearance of the peak due to trimethyl of the BOC group.

(Synthesis Example 2 of Comparative Polymer Compound)
MPC, BMA, and p-nitrophenyloxycarbonyl-4.5-ethylene glycol methacrylate (MEO4.5NP) were added to ethanol so as to be 0.25 mol / L, 0.60 mol / L, and 0.15 mol / L, respectively. It was dissolved to prepare a monomer mixed solution. Further 0.01 mol / L of AIBN was added thereto and stirred until uniform. Then, after making it react at 60 degreeC under argon gas atmosphere for 4 hours, the reaction solution was dripped in the mixed solvent of diethyl ether and chloroform, and precipitation was collect | recovered. The obtained polymer compound was measured by 1H-NMR, and the composition ratio of the polymer compound was calculated. Table 2 shows the results.

(Example)
Saturated cyclic polyolefin resin (hydrogenated ring-opening polymer of 5-methyl-2-norbornene, MFR (Melt flow rate): 21 g / 10 min, hydrogenation rate: substantially 100%, heat distortion temperature 123 ° C.) A solid-phase substrate was prepared by processing into a slide glass shape (size: 76 mm × 26 mm × 1 mm). This solid phase substrate was immersed in a 0.3 wt% ethanol solution of the polymer compound obtained in Polymer Compound Synthesis Example 1 and dried to introduce a layer containing the polymer of Synthesis Example 1 on the substrate surface. .

(Comparative example)
Saturated cyclic polyolefin resin (hydrogenated ring-opening polymer of 5-methyl-2-norbornene, MFR (Melt flow rate): 21 g / 10 min, hydrogenation rate: substantially 100%, heat distortion temperature 123 ° C.) A solid-phase substrate was prepared by processing into a slide glass shape (size: 76 mm × 26 mm × 1 mm). This solid phase substrate was immersed in a 0.3 wt% ethanol solution of the polymer compound obtained in Polymer Compound Synthesis Example 2 and dried to introduce a layer containing the polymer of Synthesis Example 2 on the substrate surface. .

<Experiment 1>
Step 1 (immobilization of primary antibody)
The sandwich method was performed on the substrate obtained in the example. In detail, first, anti-mouse IgG2a (5 mg), which is a primary antibody, was added to an aqueous solution (pH 8.1) containing 0.1 M sodium periodate (manufactured by Wako Pure Chemical: 199-08062) and 0.1 M sodium bicarbonate. The product was dissolved in 1 ml of Kojun Pure Chemical Co., Ltd .: 197-01302) and allowed to stand for 30 minutes. Subsequently, the salt was removed by applying the reaction solution to a column made of PEARCE, a distant plastic column (product code: 20439). The substrate was adjusted to 1 mg / ml with PBS buffer (Nissui Pharmaceutical: Dulbecco PBS (-) for tissue culture dissolved in 9.6 g in pure water) pH 7.4) by an automatic spotter. After spotting the oxidized anti-mouse IgG2a, the primary antibody was immobilized by allowing it to stand in a room temperature environment for 24 hours.
Next, the sandwich method was performed on the substrate obtained in the comparative example. For details, first spot anti-mouse IgG2a, which is a primary antibody prepared to 1 mg / ml with carbonate buffer (pH 9.5 manufactured by Wako Pure Chemical Industries, Ltd.) with an automatic spotter on the substrate, and then let stand in a room temperature environment for 24 hours. By doing so, the primary antibody was immobilized.

Process 2 (Suction prevention treatment)
Thereafter, the substrate of the examples was treated with an aqueous solution of 10 mg / ml succinic anhydride (manufactured by Wako Pure Chemicals: 194-04352) for 1 hour to inactivate the remaining oxylamine.
The substrate of the comparative example was immersed in 0.1 mol / liter ethanolamine (manufactured by Wako Pure Chemicals, deer special grade), 0.1 mol / liter tris buffer (manufactured by SIGMA) aqueous solution (pH 9.5) for 1 hour, and the rest The active ester part was deactivated.

Step 3 (antigen-antibody reaction 1)
Thereafter, an FBS (calf serum) solution was prepared by diluting to 10% with PBS buffer (Nissui Pharmaceutical: Dulbecco PBS (-) for tissue culture dissolved in 1 liter of pure water). Mouse IgG2a which is an antigen was added to this solution to prepare a solution to 20 nmol / liter. The antigen-antibody reaction was carried out by bringing this solution into contact with each substrate at 37 ° C. for 2 hours. 1 × SSC buffer (diluted SSC20 × Buffer manufactured by Zymed Laboratories, Inc.) supplemented with 0.05 wt% nonionic surfactant Tween20 (manufactured by Roche Diagnostics) after antigen-antibody reaction For 5 minutes at room temperature.

Step 4 (antigen-antibody reaction 2)
After washing, biotin-labeled anti-mouse IgG2a as a secondary antibody is added to a PBS buffer (manufactured by Nissui Pharmaceutical: Dulbecco PBS (-) for tissue culture dissolved in 9.6 g in pure water). A 20 nmol / liter solution was prepared. This solution and each substrate were subjected to antigen-antibody reaction at 37 ° C. for 2 hours. 1 × SSC buffer (diluted SSC20 × Buffer manufactured by Zymed Laboratories, Inc.) supplemented with 0.05 wt% nonionic surfactant Tween20 (manufactured by Roche Diagnostics) after antigen-antibody reaction For 5 minutes at room temperature.

Step 5 (labeling)
Finally, Cy5-labeled streptavidin is added to a PBS buffer (manufactured by Nissui Pharmaceutical: Dulbecco PBS (-) for tissue culture in which 9.6 g is dissolved in 1 liter of pure water) to give a 20 nmol / liter solution. Was made. After this solution and the substrate were reacted at 37 ° C. for 30 minutes, 1 × SSC buffer (Zymed Laboratories) to which 0.05 wt% of a nonionic surfactant Tween 20 (manufactured by Roche Diagnostics) was added. , Inc. SSC20 × Buffer was used for dilution) and washed at room temperature for 5 minutes for labeling.

    The amount of fluorescence was measured for each substrate, and the spot signal intensity value and the background value were evaluated. The results of the background value are shown in Table 3.

A microarray scanner “ScanArray” manufactured by Packard BioChip Technologies was used for the measurement of the amount of fluorescence in Examples and Comparative Examples. The measurement conditions were laser output 90%, PMT sensitivity 50%, excitation wavelength 649 nm, measurement wavelength 670 nm, and resolution 50 μm.
In Examples, the spot signal value was stronger than in any of the comparative examples, and the S / N ratio was large.

Claims (16)

A polymer compound for bioassay comprising at least a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group ,
The polymer compound for bioassay is represented by the following general formula [1] (wherein R1R2 and R3 are hydrogen atoms).
Or a methyl group, R4 shows a hydrophobic group. X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20. When p is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different. Y is a spacer containing an alkylene glycol residue, and Z is an oxygen atom or NH. l, m, and n are natural numbers. )so
Represented,
Prior following general formula [1], Y is represented by the following general formula [2] or [3] (wherein q, r is an integer of 1 to 20.), The hydrophobic group is an alkyl group <br/> A characteristic high molecular compound for bioassay.



The polymer compound for bioassay according to claim 1, wherein the primary amino group is an oxylamino group and / or a hydrazide group. The polymer compound for bioassay according to claim 1 or 2, wherein the main chain of the polymer compound is a (meth) acryl skeleton. In Formula [1], X bioassay polymeric compound of claim 1 wherein the ethyleneoxy group. The polymer compound for bioassay according to claim 1 or 4 , wherein, in the general formula [1], R4 is an alkyl group. The polymer compound for bioassay according to claim 5 , wherein the alkyl group has 2 to 10 carbon atoms. The method for producing a polymer compound for bioassay according to any one of claims 1 to 6 , wherein at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer in which a primary amino group is previously protected with a protecting group A method for producing a polymer compound for bioassay, comprising the step of radical copolymerization with a polymer compound, and a step of removing the protecting group from the polymer compound obtained by the step. A method according to claim 1-7 bioassay polymer compound according to any monomer having at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a functional group capable of introducing a primary amino group A method for producing a polymer compound for bioassay comprising the step of radical copolymerization of a polymer, and a step of introducing a primary amino group into the functional group of the polymer compound obtained by the step. Surface coating material including the claims 1-5 bioassay polymer compound according to any. A bioassay substrate, wherein a layer containing the surface coating material according to claim 9 is formed on the surface of the substrate. The bioassay substrate according to claim 10, wherein the shape of the substrate is any one of a slide-shaped substrate, a 96-well plate, a container, and a microfluidic substrate. The bioassay base material according to claim 10 or 11, wherein the base material is made of plastic. The bioassay substrate according to claim 12 , wherein the plastic contains a saturated cyclic polyolefin or polystyrene. A bioassay substrate in which a physiologically active substance is immobilized on the bioassay substrate according to any one of claims 10 to 13 . The bioassay substrate according to claim 14 , wherein the physiologically active substance is at least one selected from sugars, sugar chains, and physiologically active substances having these. The bioassay substrate according to claim 15 , wherein the physiologically active substance is a glycopeptide or an antibody.