JPH07316244A - (poly)oxyalkylene derivative and microsphere - Google Patents

Abstract

PURPOSE:To obtain microspheres for immobolizing a functional substance, which can immobilize the substance stably by covalent bonds on their surfaces, are excellent in dispersion stability in water, and hardly allow the nonspecific adsorption of protein, etc., to occur. CONSTITUTION:Microspheres each having a structure comprising a core consisting of accumulated polystyrene and a shell consisting of accumulated polyoxyalkylene chains are obtd. by polymerizing styrene and a polyoxyalkylene deriv. represented by CH2=C(R)C(=O)(AO)nC(=O)CH2CH2COOH [wherein R is H or CH3; AO is 2-4C oxyalkylene; and n is 1-1,000], e.g. in a solvent mixture comprising an alcohol and water.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is novel and useful (poly).
The present invention relates to an oxyalkylene derivative, microspheres (microspheres) containing the compound as a macromonomer component, functional substance-immobilized microspheres having a functional substance immobilized on the microspheres, and methods for producing the same. More specifically, a (poly) oxyalkylene derivative useful as a raw material for a diagnostic agent, an immobilized enzyme, a drug carrier, a medical material or a polymer for a coating, and a raw material for a drug, an agricultural chemical, a dye, a surfactant, or the like, The present invention relates to microspheres and microspheres on which functional substances are immobilized, which are composed of this derivative and styrene and can be used as diagnostic agents, drug carriers, functional paints, functional dyes, functional inks, etc., and methods for producing these. .

[0002]

2. Description of the Related Art JP-A-57-92332 discloses a polyoxyalkylene derivative having a polymerizable double bond at one end and a functional group at the other end, which is used as a monomer component of a photosensitive resin composition. We are using. However, it has not been suggested to utilize this compound as a macromonomer for producing a polymer for immobilizing a functional substance such as a protein.

By the way, polystyrene latex or microspheres are widely used in the field of paints or diagnostic agents. However, latex or microspheres composed only of polystyrene are poor in dispersion stability in a solvent, especially in water and easily settle, so that it is difficult to use them alone in an aqueous medium, and it is difficult to use them with a surfactant or the like. Combination is required. Further, particularly when polystyrene latex is used as a carrier for a diagnostic agent, since a functional group that reacts with a protein does not exist in the polystyrene latex, the method for carrying the antibody is by adsorption equilibrium, and therefore the antibody carried by adsorption is carried out. There is a problem that the amount is limited or the adsorbed and supported antibody is easily desorbed. In addition, there is a problem that other proteins coexisting in the sample are non-specifically adsorbed and the detection sensitivity and reliability are lowered.

Therefore, by copolymerizing it with styrene or the like, the obtained polystyrene latex or microspheres can be provided with water dispersion stability as a monomer, or by being used as a monomer of a polymer for diagnostics, proteins and the like can be obtained. There is a demand for a monomer having a functional group capable of easily covalently bonding with the functional substance (1), but a compound that sufficiently satisfies such a demand is not known. Therefore, microspheres having excellent dispersion stability in water that sufficiently satisfy the needs in the field of paints or diagnostic agents have not been obtained. Further, microspheres that can easily immobilize a functional substance such as a protein on the surface of the microspheres by a chemical bond have not been obtained.

[0005]

An object of the present invention is to provide a polymerizable double bond at one end of a (poly) oxyalkylene chain,
Another object is to provide a new and useful (poly) oxyalkylene derivative represented by the following general formula (1) having a carboxyl group at the other end. Another object of the present invention is a copolymer of a (poly) oxyalkylene derivative represented by the following general formula (1) and styrene, which is excellent in dispersion stability in water or an organic medium, and is a protein or the like. (EN) Provided are a microsphere capable of easily and stably immobilizing a functional substance of (3) by a chemical bond and less likely to cause non-specific adsorption of a protein and the like, and a method for producing the same. Another object of the present invention is a functional substance-immobilized microsphere in which a functional substance such as a protein is immobilized on the above-mentioned microsphere by a chemical bond, and the activity of the immobilized substance is sufficiently exerted, and a method for producing the same. Is to provide.

[0006]

The present invention provides the following (poly) oxyalkylene derivative, microspheres containing the derivative as a macromonomer component, and functional substance-immobilized microspheres having a functional substance immobilized on the microspheres. Fair,
And the manufacturing methods thereof. (1) A (poly) oxyalkylene derivative represented by the following general formula (1).

[Chemical 4] [In formula, AO is a C2-C4 oxyalkylene group, n
Is the number of repeating units of the oxyalkylene group, and is 1 to 1000
Represents a positive number. R represents a hydrogen atom or a methyl group. (2) A microsphere comprising a copolymer of a (poly) oxyalkylene derivative represented by the general formula (1) and styrene. (3) A method for producing microspheres, which comprises polymerizing the (poly) oxyalkylene derivative represented by the general formula (1) and styrene in a mixed solvent of alcohol and water. (4) A functional substance-immobilized microsphere, wherein the functional substance is immobilized on the microsphere according to (2) above. (5) A method for producing a functional substance-immobilized microsphere, which comprises contacting the microsphere with the functional substance described in (2) above in the presence of a dehydration condensation agent.

In the present invention, "(poly) oxyalkylene" means oxyalkylene in which n is 1 or polyoxyalkylene in which n is 2 or more. Similarly, "(poly) alkylene" means alkylene in which n is 1 or n is 2.
The above-mentioned polyalkylene is meant.

The oxyalkylene group represented by AO in the general formula (1) is an oxyalkylene group having 2 to 4 carbon atoms, and is an oxyethylene group, an oxypropylene group, an oxytrimethylene group or an oxy-1-ethylethylene group. , Oxy-1,2-dimethylethylene group, oxytetramethylene group and the like. These oxyalkylene groups are
Ethylene oxide, propylene oxide, oxetane,
It is a group obtained by addition-polymerizing an alkylene oxide such as 1-butene oxide, 2-butene oxide or tetrahydrofuran.

In the general formula (1), n is a positive number of 1-1000, preferably 2-500, more preferably 5-300. When n is 2 or more, the oxyalkylene groups may be the same or different. In the latter case, they may be added randomly or in blocks.

Examples of different oxyalkylene groups include, for example, random addition of oxyethylene groups and oxypropylene groups, block addition such as polyoxyethylene / polyoxypropylene, or polyoxy. Examples thereof include those added in blocks such as ethylene / polyoxypropylene / polyoxyethylene.

The (poly) oxyalkylene derivative represented by the general formula (1) is, for example, a monomethacrylic acid ester or a monoacrylic acid ester of (poly) alkylene glycol, specifically α-methacryloyl-ω-hydroxypoly (oxy). Ethylene) and succinic anhydride in the absence of solvent or in an organic solvent such as benzene, toluene, chloroform and acetonitrile, using pyridine, triethylamine, sodium methoxide and the like as a catalyst, at 0 to 150 ° C., preferably 40-100 ° C
Then, it can be easily produced by reacting for 30 minutes to 100 hours, preferably for 1 to 24 hours.

The reaction formula when α-methacryloyl-ω-hydroxypoly (oxyethylene) is used as the monomethacrylic acid ester of (poly) alkylene glycol is shown in the following formula (2). In the formula, n represents the same as above.

[Chemical 5]

After completion of the reaction, extraction, distillation, recrystallization, reprecipitation, dialysis, ultrafiltration, gel filtration, ion exchange resin treatment,
It can be easily isolated and purified by a method such as treatment with an adsorbent. The obtained (poly) oxyalkylene derivative is used as a raw material for diagnostic agents, immobilized enzymes, drug carriers, medical materials or polymers for paints, or as raw materials for drugs, agricultural chemicals, dyes or surfactants. can do.

The microspheres of the present invention are composed of a copolymer of the above (poly) oxyalkylene derivative and styrene, and polystyrene is accumulated in the core,
It is a particle having a structure in which a (poly) oxyalkylene chain is accumulated in a shell. The particle size is not particularly limited, but an average particle size of 1 to 1000 nm, preferably 100 to 500 nm is desirable.

The microspheres of the present invention can be schematically represented by the following formula (3).

[Chemical 6]

When imparting hydrophilicity to the microspheres of the present invention, it is preferable to use, as the (poly) oxyalkylene derivative represented by the general formula (1), one in which AO is a single addition of ethylene oxide, and particularly It is preferable to use one in which n is 3 or more. When the types of oxyalkylene groups are different, ethylene oxide is 20 mol% or more,
It is preferable to use a polyoxyalkylene derivative added with 50 mol% or more. By using such a polyoxyalkylene derivative, as shown in the above formula (3), hydrophilic polyoxyalkylene chains are accumulated on the surface of the microspheres, so that microspheres excellent in water dispersion stability can be obtained. can get. In addition, since such a microsphere has amphiphilicity in a polyoxyethylene chain or a polyoxyalkylene chain having a large number of added moles of ethylene oxide, it is dispersed even in an organic solvent such as ethyl acetate, ethanol, methanol, propanol, butanol. It has excellent stability. When imparting lipophilicity to the microspheres, a (poly) oxyalkylene derivative having a large number of added alkylene oxides other than ethylene oxide is used.

The microspheres of the present invention have the general formula (1)
It can be produced by polymerizing a (poly) oxyalkylene derivative represented by and styrene in a mixed solvent of alcohol and water. As the alcohol, a lower alcohol can be used, and an alcohol having 1 to 3 carbon atoms such as methanol, ethanol and propanol can be preferably used. Of these, ethanol is particularly preferable. The mixing ratio of alcohol and water is 5:95 to 99: 1 (5 to 99% by volume), preferably 50:50 to 95: 5 (50 to 95) by volume ratio of alcohol: water.
Volume%) is desirable. No microspheres are formed when water alone or alcohol alone is used as the reaction solvent.

The mixing ratio of the (poly) oxyalkylene derivative and styrene is as follows:
0.01: 99.9 to 50:50 in a molar ratio of styrene,
It is desirable to set it to 0.5: 99.5 to 30:70. If the amount is out of the above range, microspheres tend not to be easily formed. The total monomer concentration in the mixed solvent is 0.001 to 100 mol / l, preferably 0.1 to 1
It is preferably 0 mol / l. Outside this range, microspheres tend to be less likely to be formed.

The reaction is preferably carried out in the presence of a radical polymerization initiator. The radical polymerization initiator is preferably a peroxide or an azo compound having a 10-hour half-life temperature of 10 to 150 ° C. For example, inorganic peroxides such as ammonium persulfate; benzoyl peroxide, t-butyl hydroperoxide, diisopropyl peroxydicarbonate, t
-Organic peroxides such as butylperoxy (2-ethylhexanoate); 2,2'-azobis [2- (5-methyl-2-imidazolin-2-yl) propane] dihydrochloride,
2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2'-azobis [2-
(2-Imidazolin-2-yl) propane], 2,2 '
-Azobisisobutyronitrile, 2,2'-azobis (2-amidinopropane) dihydrochloride, 2,2'-azobis (2-methylbutyronitrile), 4,4'-azobis (4-cyanokyl) Examples thereof include azo compounds such as herb acid). The proportion of the radical polymerization initiator used is 0.001 to 10 mol%, preferably 0.
It is desirable to be 1 to 5 mol%.

The polymerization reaction is carried out in a degassed sealed tube or with nitrogen,
The reaction can be performed under an atmosphere of an inert gas such as argon or carbon dioxide at a reaction temperature of 0 to 150 ° C., preferably 30 to 80 ° C., and a reaction time of 30 minutes to 100 hours, preferably 1 to 24 hours.

As another method, ultraviolet rays, electron beams, γ
Microspheres can be similarly obtained by a method such as a photo-radiation polymerization method by irradiation with rays. In this way, by selecting various conditions such as the monomer concentration at the time of polymerization, the solvent composition, and the monomer composition, the average particle size of 1 to
It is possible to obtain microspheres having a monodispersity of 1000 nm and a CV value of about 5 to 30%. The obtained microspheres can be used as they are, or after being purified by a method such as centrifugation, dialysis, ultrafiltration, gel filtration, treatment with an ion exchange resin, treatment with an adsorbent.

The microspheres of the present invention have a structure in which polystyrene is accumulated in the core and (poly) oxyalkylene chains are accumulated in the shell, as shown in the above formula (3). Excellent dispersion stability. Furthermore, a highly reactive carboxyl group exists at the tip of the (poly) oxyalkylene chain, and this serves as a ligand for immobilizing functional substances, so various functional substances can be immobilized by chemical bonding. Is. Therefore, it can be suitably used as a base material (carrier) for fixing a functional substance. Other than that, it can be used as a functional dye by allowing dyes and pigments to soak into the core part, as well as functional ink,
It can also be used as a functional paint.

The functional substance-immobilized microsphere of the present invention is a microsphere in which a functional substance is immobilized on the microsphere by a chemical bond (covalent bond). The functional substance to be immobilized is not particularly limited, but for example, a labeling substance such as a dye, a dye, a radiation labeling compound, a fluorescent compound, a chemiluminescent compound, an electrode-sensitive compound; a photoresponsive compound,
External stimuli-responsive compounds such as pH-responsive compounds and heat-responsive compounds; enzymes, antibodies, antigens, other proteins, sugars,
Examples include physiologically active substances such as lipids, glycoproteins, glycolipids, and hormones; drugs. Among these, those having an amino group, a hydroxyl group or a thiol group in the molecule are preferable, and those having a primary amino group that easily reacts with a carboxyl group are particularly preferable.

Specific examples of the above-mentioned antibody include a polyclonal antibody, a monoclonal antibody, a part of these units, and the like. These antibodies may be derived from any animal species such as human, goat, sheep, rabbit and chicken, and may be derived from any hybridoma. Specific examples of the above-mentioned antigens include proteins, oligosaccharides, high molecular sugars, low molecular haptens, and cholesterol. However, in the case of a hapten, for immobilization, an amino group in the molecule,
It is necessary to have a functional group, either a hydroxyl group or a thiol group. Microspheres having such an antibody or antigen immobilized thereon are suitably used as a diagnostic agent.

The functional substance is immobilized by covalent bond such as amide bond, ester bond or thiol ester bond between the carboxyl group of the microsphere and the functional group such as amino group, hydroxyl group or thiol group of the functional substance. Is formed by forming.

The functional substance-immobilized microspheres of the present invention can be easily prepared by bringing the microspheres into contact with the functional substance in the presence of a dehydration condensation agent. As the contact method, a method of stirring in a reaction solvent can be adopted. Examples of the dehydration condensing agent include N-cyclohexyl-N '-(2-morpholinoethyl) carbodiimide = meso-p-toluenesulfonate, 1-ethyl-
Examples thereof include water-soluble dehydration condensation agents such as 3- (3-dimethylaminopropyl) carbodiimide hydrochloride; water-insoluble dehydration condensation agents such as dicyclohexylcarbodiimide and 1,1′-carbonyldiimidazole. Among these, it is preferable to use a water-soluble dehydrating condensing agent.

The immobilization of the functional substance on the microspheres can be carried out by reacting the microspheres with the functional substance in a reaction solvent in the presence of the dehydration condensation agent. As the reaction solvent, water, physiological saline, or a phosphate buffer of pH = 3 to 10, preferably 5 to 9,
Buffer solutions such as carbonate buffer solution, Tris buffer solution, acetate buffer solution; mixed solvents of these aqueous solvents and organic solvents such as ethanol, methanol, acetonitrile, tetrahydrofuran, dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, etc. Can be used.

The concentration of microspheres in the reaction solution is
0.001 to 50% by weight, preferably 0.01 to 30% by weight, and the concentration of the functional substance is 1 nmol / l to 1 mol /
1, preferably 1 μmol / l to 100 mmol / l,
The concentration of the dehydration condensing agent is 1 μmol / l to 10 mol / l,
It is preferably 1 to 100 mmol / l.

The reaction temperature is -10 to + 120 ° C, preferably 0 to 50 ° C for the reaction with an amino group, 30 to 80 ° C for the reaction with a hydroxyl group or a thiol group, and the reaction time is 10 minutes to 300 hours. It is desirable that the time is 30 minutes to 24 hours. Outside the range of such conditions, the stability of the microspheres tends to deteriorate. When microspheres having a fixed functional substance are used as a diagnostic agent, the microspheres have an average particle size of 100 to 5
It is preferable to use the one of 00 nm.

The immobilization reaction between the microspheres and the functional substance having an amino group is schematically represented by the following formula (4) or (5). In the formula, PS represents polystyrene and P represents a protein or a functional substance. n shows the same thing as the above.

[Chemical 7]

[0031]

[Chemical 8]

The mixture after the reaction can be purified by a method such as centrifugation, gel filtration, ultrafiltration, dialysis, ion exchange resin treatment, adsorbent treatment and the like. Since the functional substance-immobilized microspheres thus obtained are immobilized by a covalent bond, the functional substance is not desorbed and is stably immobilized. In addition, since (poly) oxyalkylene chains are accumulated on the surface, nonspecific adsorption of proteins etc. does not occur easily. Therefore, even when using functional substance-immobilized microspheres as a diagnostic agent, detection sensitivity and reliability are high. Will be high.
Furthermore, the activity of the immobilized functional substance is sufficiently exerted. The functional substance-immobilized microspheres of the present invention can be used as immobilized enzymes, drug carriers, etc., as well as diagnostic agents.

[0033]

According to the present invention, a novel and useful (poly) oxyalkylene derivative having a polymerizable double bond at one end of a (poly) oxyalkylene chain and a carboxyl group at the other end is obtained. To be

The microspheres of the present invention have the general formula (1)
As it is composed of a copolymer of (poly) oxyalkylene derivative and styrene, it has excellent dispersion stability in water or organic solvent, and functional substances can be easily fixed by chemical bond. Can be converted. In addition, nonspecific adsorption of proteins and the like is unlikely to occur on the microspheres. In the method for producing microspheres of the present invention, the monomers are polymerized in a mixed solvent of alcohol and water, so that the above microspheres can be easily and efficiently produced.

In the functional substance-immobilized microsphere of the present invention, the functional substance is fixed to the microsphere by a chemical bond, so that the functional substance is not detached and the activity is sufficiently exhibited. . In the method for producing a functional substance-immobilized microsphere of the present invention, since the microsphere and the functional substance are contacted in the presence of a dehydration condensing agent, the functional substance-immobilized microsphere can be simply and efficiently prepared. It can be manufactured.

[0036]

EXAMPLES The present invention will now be described in more detail by way of examples, which should not be construed as limiting the invention thereto. Example 1-1 10 g (23.8 mmol) of α-methacryloyl-ω-hydroxypoly (oxyethylene) (n≈7.6), 2.6 g (26.2 mmol) of succinic anhydride and a catalytic amount of pyridine were mixed. The reaction was carried out at 80 ° C. for 4 hours with stirring. 20 ml of distilled water was added thereto, washed with diethyl ether, further extracted with chloroform, and chloroform was distilled off under reduced pressure to obtain the target compound of the following formula (6).

[Chemical 9]

The analysis results of ¹ H-NMR and IR are as follows. ¹ H-NMR (270 MHz, CDCl ₃ , TMS (pp
m), J (Hz)) 6.11 (b; s, 1H) 5.56 (a; s, 1H) 4.26 (d, f; m, 4H) 3.63 (e; m, about 28H) ) 2.65 (g; s, 4H) 1.93 (c; s, 3H) h; un detect IR (liquid film method; cm ^-1 ) 2900 (carboxylic acid) 1728 (ester bond) 1655,895 (vinyl) Basis)

Example 1-2 10 g (3.2 mmol) of α-methacryloyl-ω-hydroxypoly (oxyethylene) (n≉68), 0.5 g (5 mmol) of succinic anhydride and a catalytic amount of pyridine were mixed, The reaction was carried out at 80 ° C. for 4 hours with stirring. After completion of the reaction, the target compound of the following formula (7) was obtained in the same manner as in Example 1-1.

[Chemical 10]

The analysis results of ¹ H-NMR and IR are as follows. ¹ H-NMR (270 MHz, CDCl ₃ , TMS (pp
m), J (Hz)) 6.11 (b; s, 1H) 5.56 (a; s, 1H) 3.63 (d; m, about 270H) 2.65 (e; s, 4H) 1 .93 (c; s, 3H) f; un detect IR (liquid film method; cm ^-1 ) 2900 (carboxylic acid) 1728 (ester bond) 1655,895 (vinyl group)

Example 1-3 Polyethylene glycol / polypropylene glycol block copolymer 10 having a methacryloyl group at one end
g (5.9 mmol), 1 g of succinic anhydride (10 mmo
1) and a catalytic amount of pyridine were mixed and reacted at 80 ° C. for 4 hours with stirring. After completion of the reaction, the target compound of the following formula (8) was obtained in the same manner as in Example 1-1.

[Chemical 11]

The analysis results of ¹ H-NMR and IR are as follows. ¹ H-NMR (270 MHz, CDCl ₃ , TMS (pp
m), J (Hz)) 6.11 (b; s, 1H) 5.56 (a; s, 1H) 3.60 (d, e, g; m, about 135H) 2.65 (h; s) , 4H) 1.93 (c; s, 3H) 1.12 (f; m, 15H) i; un detect IR (liquid film method; cm ^-1 ) 2900 (carboxylic acid) 1728 (ester bond) 1655, 895 (Vinyl group)

Example 2-1 0.5 ml of the polyoxyalkylene derivative obtained in Example 1-1, 2.88 mmol of styrene and VA-as a polymerization initiator were added to 5 ml of an 80% by volume aqueous ethanol solution.
061 (manufactured by Wako Pure Chemical Industries, Ltd., trade name) 2.5 mg (10
0 μmol; 1.5 mol% based on all monomers) was charged, and polymerization was carried out in a degassed sealed tube at 60 ° C. for 48 hours while gently shaking. Next, it was dialyzed for 48 hours in a large amount of distilled water to obtain a microsphere dispersion liquid. The particle size of the resulting microspheres was measured using a COULTERN type 4 submicron particle analyzer. The average particle size and CV value are shown in Table 1.

Examples 2-2 to 2-3 Microspheres were obtained in the same manner as in Example 2-1, except that the composition of the monomers was changed as shown in Table 1. The polymerization initiator was 1.5 mol% with respect to all the monomers. Table 1 shows the average particle size and CV value of the obtained microspheres.

Examples 2-4 to 2-5 Microspheres were obtained in the same manner as in Example 2-1, except that the solvent was changed to a mixed solvent of water and methanol or propanol as shown in Table 1. The amount of solvent was 5 ml. Table 1 shows the average particle size and CV value of the obtained microspheres.
Shown in.

Example 2-6 Microspheres were obtained in the same manner as in Example 2-1, except that the polyoxyalkylene derivative obtained in Example 1-3 was used. Polymerization initiator is 1.5 mol% based on all monomers
And Table 1 shows the average particle size and CV value of the obtained microspheres.

Comparative Example 1-1 Polymerization was carried out in the same manner as in Example 2-1 except that only 4 mmol of styrene was used as the monomer. The polymerization initiator was 1.5 mol% with respect to all the monomers. As a result, a lump polymer was formed and microspheres could not be formed.

Comparative Example 1-2 As in Example 2-1, except that ethanol 5 was used as the solvent.
Polymerization was carried out using only ml. The polymerization initiator was 1.5 mol% with respect to all the monomers. As a result, a lump polymer was formed and microspheres could not be formed.

Comparative Example 1-3 As in Example 2-1, except that 5 ml of water was used as the solvent.
Polymerization was carried out using only. The polymerization initiator was 1.5 mol% with respect to all the monomers. As a result, a lump polymer was formed and microspheres could not be formed.

[0049]

[Table 1] * 1 Polyoxyalkylene derivative * 2 No microsphere formation

Example 3-1 The microspheres obtained in Example 2-1 were treated with a phosphate buffer (0.1 M, pH = 7.4) to give a solid content of 2 wt%.
Adjusted to 5 ml and 1-ethyl-
8 mg (42 μmol) of 3- (3-dimethylaminopropyl) carbodiimide hydrochloride was added, and the mixture was incubated at 4 ° C. for 30 minutes. Further, 3 ml of 20 mg / ml bovine serum albumin (hereinafter abbreviated as BSA) -0.1 M phosphate buffer (pH = 7.4) was added, and the mixture was incubated at 4 ° C. for 24 hours. After completion of the reaction, centrifuge (4000 r
pm for 10 minutes) and the supernatant was added with 0.1M phosphate buffer (p
The procedure of substituting H = 7.4) was repeated 3 times to obtain BSA-immobilized microspheres.

B of the obtained BSA-immobilized microspheres
The amount of SA immobilized was quantified using the ninhydrin method. That is, ninhydrin 400 mg, hydrin danthine 6
After dissolving 0 mg in 15 ml of methyl cellosolve, a ninhydrin solution was prepared by adding 5 ml of sodium acetate buffer (pH = 5.5). On the other hand, 1 ml of the BSA-immobilized microsphere dispersion solution obtained above was mixed with 4
After adding 1 ml of N hydrochloric acid and treating at 100 ° C. for 2 hours, it was further neutralized with a 4N sodium hydroxide solution and adjusted to exactly 5 ml with distilled water. Further centrifugation (40
(00 rpm, 10 minutes) was performed, 3 ml of the supernatant was weighed, 1 ml of ninhydrin solution was added thereto, and the mixture was incubated at 100 ° C. for 20 minutes. After cooling with water, the absorbance at 570 nm was measured with a spectrophotometer, and the BSA immobilization amount was calculated from the calibration curve previously obtained by the same operation, resulting in 25.8 μg / ml.

Example 3-2 As Example 3-1 except that BSA was replaced by 1
mg / ml horseradish peroxidase (hereinafter HRP
Abbreviated) /0.1 M phosphate buffer (pH 7.5) 3 ml
Was used to obtain HRP-immobilized microspheres. Next, the immobilization of HRP in the obtained HRP-immobilized microspheres was confirmed by a coloring method based on the enzymatic activity of HRP. That is, the obtained HRP-immobilized microsphere dispersion liquid 500
100 μl of 1,2-phenylenediamine solution (10 mmol / l), which is a substrate for HRP, was added to μl, incubated at 30 ° C. for 10 minutes, and further 10 μl of 0.1N sulfuric acid
When 1 was added, a brown color was observed, and HRP immobilization was confirmed.

Example 3-3 As Example 3-1 except that BSA was replaced by 1
0 mg / ml anti-human hemoglobin-goat IgG / 0.
Antibody-immobilized microspheres were obtained using 3 ml of 1 M phosphate buffer (pH 7.5). Next, the antibody immobilization of the obtained antibody-immobilized microspheres was assayed by the presence or absence of the agglutination reaction based on the antigen-antibody reaction. That is, 500 μl of the obtained antibody-immobilized microsphere dispersion was added to 500 μl.
When added to 1 ml of g / ml human hemoglobin-physiological saline and incubated at 37 ° C. for 10 minutes, aggregates were observed at the bottom of the reactor, and it was confirmed that the antibody was immobilized.

Comparative Example 2-1 In the same manner as in Example 3-1, except that the water-soluble dehydrating condensing agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was not used, BSA was immobilized. I went. The amount of BSA immobilized on this microsphere was quantified by the ninhydrin method in the same manner as in Example 3-1, and the amount immobilized was 0 μg / ml, confirming that BSA was not immobilized.

Comparative Example 2-2 In the same manner as in Example 3-2, except that the water-soluble dehydrating condensing agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride was not used, HRP immobilization operation was carried out. I went. This microsphere was subjected to a color development test by the enzyme activity of HRP in the same manner as in Example 3-2.
No color development was observed, confirming that HRP was not immobilized.

Comparative Example 2-3 An antibody immobilization procedure was carried out in the same manner as in Example 3-3, except that 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride which was a water-soluble dehydrating condensing agent was not used. I went. An antigen-antibody reaction was performed on this microsphere in the same manner as in Example 3-3, but no aggregation was observed, and it was confirmed that the antibody was not immobilized.

Claims (5)

[Claims] 1. A (poly) oxyalkylene derivative represented by the following general formula (1). [Chemical 1] [In formula, AO is a C2-C4 oxyalkylene group, n
Is the number of repeating units of the oxyalkylene group, and is 1 to 1000
Represents a positive number. R represents a hydrogen atom or a methyl group. ] 2. A compound represented by the general formula (1): [In formula, AO is a C2-C4 oxyalkylene group, n
Is the number of repeating units of the oxyalkylene group, and is 1 to 1000
Represents a positive number. R represents a hydrogen atom or a methyl group. ] The microsphere which consists of a copolymer of the (poly) oxyalkylene derivative represented by these, and styrene. 3. A compound represented by the general formula (1): [In formula, AO is a C2-C4 oxyalkylene group, n
Is the number of repeating units of the oxyalkylene group, and is 1 to 1000
Represents a positive number. R represents a hydrogen atom or a methyl group. ] The (poly) oxyalkylene derivative represented by these, and styrene are polymerized in the mixed solvent of alcohol and water, The manufacturing method of the microsphere characterized by the above-mentioned. 4. A functional substance-immobilized microsphere, wherein the functional substance is immobilized on the microsphere according to claim 2. 5. A method for producing a functional substance-immobilized microsphere, which comprises contacting the microsphere according to claim 2 with the functional substance in the presence of a dehydration condensation agent.