JPH07308575A - Functional material fixing agent - Google Patents

Abstract

PURPOSE:To obtain a fixing agent capable of fixing various functional materials on an optional base material by covalent bond and selecting the length of arm or the balance of hydrophilicity to hydrophobicity to give high activity to the functional material by composing the agent from a polyalkylene oxide silanol derivative having a specific structure. CONSTITUTION:This fixing agent consists of a polyalkylene oxide silanol derivative expressed by the formula. In the formula, each of R<1> and R<2> is methyl group or ethyl group, (p) is integers of 1-3, (q) is integers of 0-2 (where, p+q=3), AO is a 2-4C oxyalkylene group, (n) is average addition mol number of the oxyalkylene groups and is integers of 2-1000 and each of the added oxyalkylene groups is the same or different and can be added in random state or blocked state. The functional material fixing agent is, after introduced into the base material by a silane coupling reaction, allowed to react with N,N'- carbonyldiimidazol or its derivative to be activated and brought into contact with the functional material to be fixed on the based material.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention shares various functional substances such as proteins, glycoproteins, saccharides and other physiologically active substances having functions of enzymes, antibodies, coenzymes and lectins, and pharmaceuticals, agricultural chemicals, pigments and the like. The present invention relates to a functional substance immobilizing agent that can be immobilized by binding, a method for immobilizing a functional substance, and an immobilized functional substance.

[0002]

2. Description of the Related Art Conventionally, the Schiff base method is well known as a method for immobilizing a functional substance on a substrate by a covalent bond, for example, a method for immobilizing an enzyme. In this method, porous alkyl aminated glass beads are used as a substrate, and the N-terminal, lysine residue, and histidine residue in the enzyme are used together with Schiff base by using glutaraldehyde as an immobilizing reagent. As seen in the method of forming and binding (Clin. Chim. Acta, 95, 411 (1979)) or immobilization of peroxidase, the sugar residue in the enzyme is oxidized with periodate to form a dialdehyde group. , A method of immobilizing it by forming a Schiff salt with this and an amino group on glass beads (J. Histochem. Cytochem.,
22, 1084 (1974)) and so on.

An example of immobilizing heparin on polyether urethane urea (PEUU) is known as a method for immobilizing a saccharide having a physiological activity by a covalent bond (J. Biomed. Mater. Res., 20, 1157 (1986)). In this method, PEUU containing L-lysine methyl ester in its chemical composition is synthesized beforehand, and after forming a film of this, the ester group on the surface is hydrolyzed and neutralized to induce a carboxyl group, and then the amino group in heparin is prepared. This is a method of condensing with a group and fixing with an amide bond.

However, according to the above-mentioned conventional techniques, there is a problem that the base material which can be used by each method is limited and a very complicated operation is required.
In addition, considering the arm (spacer) that connects the base material and the functional substance, the problem is that it is not possible to select the optimal arm length or hydrophilic / hydrophobic balance for exerting the characteristics of the functional substance to be immobilized. There is.

In order to solve such problems, JP-A-5-344885 discloses a physiologically active substance immobilizing material comprising a silanol derivative to which an allyl compound having a polyalkylene ether is bound. However, in the immobilization material of the above publication, the ester bond existing in the arm portion is easily hydrolyzed, and the thioether bond is also easily decomposed. Therefore, improvement in stability of the immobilization material and the immobilization product is required.

[0006]

SUMMARY OF THE INVENTION In order to solve the above problems, an object of the present invention is to allow various functional substances to be immobilized on any base material by covalent bonding, and the functionality to be immobilized. An object of the present invention is to provide a functional substance immobilizing agent capable of selecting the arm length and the balance between hydrophilicity and hydrophobicity for exerting the activity of a substance with high activity, and having excellent long-term stability. . Another object of the present invention is to propose a method for immobilizing a functional substance, which can easily and efficiently immobilize a functional substance on a substrate using the functional substance immobilizing agent. is there. Another object of the present invention is to provide an immobilized functional substance in which the functional substance is immobilized on a substrate by the above-mentioned functional substance immobilizing agent, which has high activity and is excellent in long-term stability. .

[0007]

The present invention provides the following functional substance-immobilizing agent, a method for immobilizing a functional substance, and an immobilized functional substance. (1) A functional substance immobilizing agent comprising a polyalkylene oxide silanol derivative represented by the following general formula [1].

[Chemical 2] [In the formula, R ¹ and R ² represent a methyl group or an ethyl group, p is an integer of 1 to 3, q is an integer of 0 to 2 (provided that p +
q = 3). AO is an oxyalkylene group having 2 to 4 carbon atoms, n is an average addition mole number of the oxyalkylene group and is 2 to
Represents a positive number of 1000. The added oxyalkylene groups may be the same or different, and may be added randomly or in blocks. (2) After introducing the functional substance-immobilizing agent described in (1) above into a substrate by a silane coupling reaction, N, N'-
A method for immobilizing a functional substance, which comprises activating carbonyldiimidazole or a derivative thereof by reaction, and then contacting the functional substance. (3) An immobilized functional substance, wherein the functional substance is immobilized on a base material by the functional substance immobilizing agent described in (1) above.

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 an average addition mole number of oxyalkylene groups of 2 to 1000, preferably 10 to 30.
It is 0, more preferably a positive number of 20 to 120. Even if the types of oxyalkylene groups added are the same,
They may be different, and may be added randomly or in blocks.

When imparting hydrophilicity, the AO is preferably ethylene oxide alone, and in this case, n is preferably 10 or more. When different types of alkylene oxides are added, it is desirable that ethylene oxide is added in an amount of 20 mol% or more, preferably 50 mol% or more. When imparting lipophilicity to the polyalkylene oxide chain, the number of added moles other than ethylene oxide is increased.

In the functional immobilizing agent of the present invention, the type of oxyalkylene group is selected so as to impart mobility or a balance between hydrophilicity and hydrophobicity so that the activity of the functional substance to be immobilized is best expressed. The mixing ratio, the average number of added moles, etc. are selected.

The functional substance-immobilizing agent of the present invention comprises a polyalkylene oxide silanol derivative represented by the above general formula [1], and a functional group having a silane coupling ability represented by -OR ¹ . Has a hydroxyl group. For this reason, a functional group having silane coupling ability is bonded to a base material as a carrier by silane coupling, while a hydroxyl group is covalently bonded to a functional substance after activation, so that the polyalkylene oxide chain serves as an arm to functionalize. The substance can be immobilized on the substrate.

The polyalkylene oxide silanol derivative represented by the general formula [1] can be produced, for example, by the following method. It can be produced by a method in which trichlorosilane or a derivative thereof is added to an olefin having a corresponding polyalkylene ether, and then an alcohol is allowed to act to carry out alkoxylation. The reaction formula when an allyl compound having trichlorosilane and polyalkylene ether is used is represented by the following formula [2].
Shown in. In the formula, R ¹ and n have the same meanings as described above.

[0014]

[Chemical 3]

The functional substance-immobilizing agent of the present invention is excellent in chemical stability because the polyalkylene oxide chain serving as an arm is composed only of carbon-carbon bonds and ether bonds and does not contain ester bonds or thioether bonds. , Excellent in long-term stability. Also the type of oxyalkylene,
The arm length and the balance between hydrophilicity and hydrophobicity for highly expressing the activity of the functional substance to be immobilized can be easily adjusted simply by selecting the mixing ratio, the average number of moles added, and the like.

The functional substance which can be immobilized by the immobilizing agent of the present invention is not particularly limited as long as it has an amino group, a hydroxyl group, a thiol group, especially a primary amino group. It may be either natural or non-natural, and various functional substances such as antibodies, enzymes, coenzymes, proteins such as lectins and heparins, physiologically active substances such as sugars and glycoproteins, other drugs, agricultural chemicals, pigments, etc. can give.

The method of immobilizing a functional substance according to the present invention is a method of immobilizing the functional substance-immobilizing agent on the substrate after introducing the functional substance-immobilizing agent into the substrate.

As the base material into which the immobilizing agent is introduced, any material can be used as long as it can chemically or physically support a silane coupling agent, and examples thereof include glass, silica gel, alumina, quartz and kaolin. , Mica,
Talc, clay, orastonite, iron powder, magnetic particles,
Inorganic polymers such as potassium titanate, asbestos, titanium oxide, zinc oxide, carbonized silica, silicon nitride, boron, various ceramics; urethane, nylon, polyvinyl chloride, polystyrene, polypropylene, polyethylene, acrylic resin, polycarbonate, epoxy resin , Synthetic polymers such as melamine resin, polyvinyl acetate and vinyl acetate-ethylene copolymer; cellulose derivatives such as regenerated cellulose and acetyl cellulose; naturally derived polymer materials such as chitosan. Also, its shape is not limited,
It may have any shape and size such as a bead shape, a tube shape, a sheet shape, and a cup shape.

In order to immobilize the functional substance on the substrate with the functional substance immobilizing agent of the present invention, the functional substance immobilizing agent is introduced into the substrate by a silane coupling reaction to form a polyalkylene oxide base material. To give N, N'-carbonyldiimidazole (CD
It can be carried out by a step of activating with an activating agent such as I) or a derivative thereof to obtain an activated substrate, and contacting the activated substrate with a functional substance to obtain an immobilized functional substance.

Examples of the CDI derivative include N, N'-
Carbonyl di (2-methyl imidazole) etc. are mentioned. CDI and its derivatives react with the terminal hydroxyl groups of the polyalkylene oxide chain to form an oxycarbonylimidazole bond or the oxycarbonylimidazole bond corresponding to the CDI derivative. Since these oxycarbonylimidazole bonds have high reactivity with amino groups, hydroxyl groups, thiol groups, especially primary amino groups, they easily react with functional substances having such groups. Form a covalent bond. As a result, the functional substance is fixed on the base material via the arm composed of the polyalkylene oxide chain.

The reaction scheme of the method for immobilizing a functional substance in the present invention is shown below. In the formula, R ³ represents a hydrogen atom or a lower alkyl group.

[Chemical 4]

In the above step, the polyalkylene oxide silanol derivative represented by the general formula [1] is introduced onto the arbitrary substrate by a silane coupling reaction,
In this step, a polyalkylene oxide-based material is obtained. The substrate used here was subjected to the same pretreatment as that used in the general silane coupling treatment, for example, NaOH aqueous solution treatment, plasma treatment, corona discharge treatment, UV-ozone treatment, precoat treatment and the like. It is preferably subjected to this step later.

The polyalkylene oxide silanol derivative may be used as it is, or may be added with water, ethanol, methanol, methyl ethyl ketone, toluene, benzene, dimethylformamide, dimethylacetamide, dimethyl sulfoxide or a mixed solvent thereof in an amount of 0.01 to
It may be used by diluting it to 80% by weight, or may be used as a primer by further converting it to a silanol group by hydrolysis and then polymerizing it more gently.

The method of coating the polyalkylene oxide silanol derivative on the substrate varies depending on the material and shape of the substrate used, and although it is not specified, the dipping method,
Various general methods such as a spray method and a brush coating method can be used. The silane coupling reaction after coating is performed under air drying and temperature conditions for a predetermined time. The reaction conditions at this time are 0 to 250 ° C. for 30 minutes to 1000 hours, preferably 20 to 140 ° C. for 1 to 24 hours.

The above step is a step of activating the hydroxyl group in the polyalkylene oxide silanol derivative residue introduced on the substrate by the step of with CDI or its derivative to obtain an activated substrate. CDI or a derivative thereof is dried acetonitrile, tetrahydrofuran, 1,4-dioxane, methyl ethyl ketone,
It is preferably used by dissolving it in acetone, toluene, benzene, dimethylformamide, dimethylacetamide, dimethylsulfoxide or a mixed solvent thereof. The concentration of CDI or its derivative at this time is not particularly limited, but is preferably 0.01 to 20% by weight.

The activation reaction is carried out by immersing or dipping the substrate obtained in the previous step in a solution of CDI or its derivative.
Alternatively, the derivative solution is applied onto the base material,
120 ° C for 1 minute to 1000 hours, preferably 0 to 40
It is desirable to carry out at 10C for 24 hours. At this time, in order to prevent hydrolysis of CDI or its derivative due to moisture, it is preferable to carry out in an atmosphere of dried air, carbon dioxide, helium, nitrogen, argon gas or the like. After completion of the reaction, the obtained activated substrate is water, ethanol, methanol, acetonitrile, tetrahydrofuran, 1,4-dioxane, methyl ethyl ketone, acetone, toluene,
It is washed with benzene, dimethylformamide, dimethylacetamide, dimethylsulfoxide, a mixed solvent thereof, or the like, and is used as it is or after being dried and then subjected to the next step.

The above-mentioned step is a step of contacting and immobilizing the functional substance with the activated base material obtained in the step to obtain the immobilized functional substance. The functional substance is covalently immobilized by bringing the activated base material into contact with the functional substance.

In the immobilization reaction, the functional substance is treated with water or various buffer aqueous solutions, physiological saline, ethanol, methanol,
Acetonitrile, tetrahydrofuran, 1,4-dioxane, methyl ethyl ketone, acetone, toluene, benzene, dimethylformamide, dimethylacetamide,
After being dissolved in dimethyl sulfoxide or a mixed solvent thereof, etc., the activated substrate is immersed in this solution or coated with this solution, and at -20 to 120 ° C for 1 minute to 10 minutes.
It is desirable to carry out the treatment for 00 hours, preferably at 0 to 40 ° C. for 10 minutes to 200 hours.

After the reaction, water or various buffer aqueous solutions,
After washing with physiological saline, ethanol, methanol, acetonitrile, tetrahydrofuran, 1,4-dioxane, methyl ethyl ketone, acetone, toluene, benzene, dimethylformamide, dimethylacetamide, dimethyl sulfoxide or a mixed solvent thereof, or the like,
As it is or by drying, the immobilized functional substance in which the target functional substance is immobilized on the substrate can be obtained.

The immobilized functional substance thus obtained is selected in terms of the length of the polyalkylene oxide chain, the balance between hydrophilicity and hydrophobicity, etc. so that the activity of the functional substance is maximized. Therefore, it has high activity. Further, since the polyalkylene oxide chain of the arm portion is excellent in chemical stability, the middle of the arm is not hydrolyzed and the long-term stability is also excellent. Such immobilized functional substances are used as antibody immobilization systems, immobilized enzymes, analytical / purifying column packing materials, antithrombotic materials, antibacterial materials, and various hybrid biomaterials.

[0031]

The functional substance-immobilizing agent of the present invention has a site that causes a silane coupling reaction and a site that is activated by N, N'-carbonyldiimidazole or a derivative thereof, and is a polyalkylene. Since the length of the oxide chain and the balance between hydrophilicity and hydrophobicity can be easily adjusted, various functional substances can be immobilized on any substrate by covalent bonding, and the function of immobilizing It is possible to exert the activity of a volatile substance with high activity, and is also excellent in long-term stability.

In the method for immobilizing a functional substance of the present invention, since the functional substance is immobilized by using the functional substance immobilizing agent, the immobilization can be performed easily and efficiently.

Since the immobilized functional substance of the present invention is immobilized on the substrate by the functional substance immobilizing agent, it has high activity and is excellent in long-term stability.

[0034]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited thereto. In each example,% means% by weight. Example 1-1 1) Introduction of functional substance immobilizing agent onto glass beads 10 g of 10 g of 300 mesh glass beads (base material)
After treatment in an aqueous solution of sodium hydroxide at 60 ° C. for 30 minutes, it was thoroughly washed with distilled water and dried. On the other hand, 50 ml of ethanol / water (8/2 (V / V)) containing 5% of a functional substance immobilizing agent composed of a polyalkylene oxide silanol derivative represented by the following formula [3] was adjusted to pH = 1 with 1N acetic acid.
It was adjusted to 4.5 and stirred at room temperature for 30 minutes. The above glass beads were dipped in this solution and filtered to obtain 10
Air dry at room temperature for 1 minute and then in an oven at 110 ° C for 1
Heat treatment was carried out for an hour to obtain polyalkylene oxide-modified glass beads.

[Chemical 5]

2) CDI activation of polyalkylene oxide-oxidized glass beads In a well-dried, three-necked flask filled with argon gas, 50 ml of dry tetrahydrofuran, 10.0 g of the above polyalkylene oxide-oxidized glass beads and N, N '.
-Carbonyldiimidazole (CDI) 100 mg was added, and after stirring at room temperature for 4 hours, distilled water 10 ml was added,
Excess CDI was hydrolyzed. Further, decantation was repeated 3 times with distilled water, and activated beads (activated substrate) were obtained by filtration. The obtained activated beads were freeze-dried and used in the following reaction.

3) Immobilization of bovine serum albumin (BSA) 500 mg of the above-mentioned activated beads was added to a BSA solution having a concentration of 500 μg / ml (0.1 M phosphate buffer pH = 7.4) 1
Soak in 0 ml and incubate at 4 ° C for 3 days B
SA was immobilized on activated beads to obtain immobilized BSA.
After filtration, in order to hydrolyze unreacted activated sites on the beads of the immobilized BSA, 0.1 M phosphate buffer of pH = 8.5 was added, and the mixture was further incubated at room temperature for 12 hours. After the reaction was completed, the immobilized BSA was thoroughly washed with a 0.1% aqueous solution of dodecylbenzenesulfonic acid and purified water, and then the immobilization was confirmed by a general xanthoprotein reaction as a protein detection method. That is, 1 ml of concentrated nitric acid was added to 10 mg of immobilized BSA beads, and
After boiling for 1 minute, the mixture was cooled to room temperature and 0.1% aqueous sodium hydroxide solution was added. At this time, since the surface of the beads and the solution were colored orange yellow, it was confirmed that BSA was immobilized.

Example 1-2 Activation was carried out in the same manner as in steps 1) and 2) of Example 1-1, except that the compound represented by the following formula [4] was used as the functional substance immobilizing agent. Obtained beads.

[Chemical 6]

The activated beads (500 mg) were added to 80
Alkaline phosphatase (ALP) solution derived from bovine small intestine at a concentration of μg / ml (0.1 M phosphate buffer pH = 7.
4) Immersed in 5 ml and incubated at 4 ° C. for 3 days to obtain immobilized ALP. Furthermore, to block unreacted activated sites, BSA at a concentration of 150 μg / ml was used.
1 ml of the solution (0.1 M phosphate buffer pH = 7.4) was added, and the mixture was further incubated at room temperature for 1 day. After completion of the reaction, confirmation of immobilization of ALP was carried out by a colorimetric detection method of ALP using 4-nitrophenyl phosphate. That is, immobilized ALP in a quartz glass cell with an optical path length of 1 cm.
About 10 mg of wet beads of 0.1M glycine-
Add 0.1 ml of sodium hydroxide aqueous solution, and add 5 at 37 ℃.
Incubate for 2 minutes, add 0.5 ml of 4-nitrophenyl phosphate solution with a concentration of 2 g / l, and incubate at 37 ° C.
And incubated for 30 minutes. Further, 0.1 ml of 2N sodium hydroxide aqueous solution was added, and the absorbance at 405 nm was measured. As a result, since the absorbance of 0.83 was obtained, ALP kept its enzyme activity high,
It was confirmed that the immobilized ALP was obtained after being immobilized on the substrate.

Example 1-3 As a functional substance immobilizing agent, ethanol / water (8/2 (V /
V)) was used, but 1) and 2) of Example 1-1.
Activated beads were obtained in the same manner as the step.

[Chemical 7]

50 mg of the obtained activated beads was added to 80 μm.
g / ml concentration of anti-human Hb rabbit IgG solution (0.1M
It was immersed in 5 ml of a phosphate buffer (pH = 7.4) and incubated at 4 ° C. for 3 days to obtain an immobilized antibody. 150 μg to block unreacted activated sites
/ Ml concentration of BSA solution (0.1M phosphate buffer pH =
7.4) 1 ml was added and further incubated at room temperature for 1 day. After the reaction, in order to confirm the immobilization of the antibody, the beads of the immobilized antibody were thoroughly washed with purified water and then used for the following evaluation. Confirmation of immobilization of anti-human Hb-rabbit IgG was performed by confirming that the immobilized anti-human Hb-rabbit IgG and human Hb were immobilized.
And a three-way sandwich complex with anti-human Hb-rabbit IgG labeled with ALP were formed, and the colorimetric detection of ALP with 4-nitrophenyl phosphate was performed.

That is, in a quartz glass cell having an optical path length of 1 cm, about 10 mg of wet beads of the immobilized antibody were combined with 4 mg of human Hb and ALP-labeled anti-human Hb-rabbit I.
0.1 M phosphate buffer (pH 7.
2) Immersed in 1 ml and incubated at room temperature for 30 minutes. After thoroughly washing with purified water, 0.5 ml of 0.1 M glycine-sodium hydroxide buffer (pH 10.3) was added, the mixture was incubated at 37 ° C for 5 minutes, and further 2 g / l.
0.5 ml of concentrated 4-nitrophenyl phosphate solution
Was added and the absorbance at 405 nm was measured. as a result,
Since the absorbance of 0.6 was obtained, it was confirmed that the anti-human Hb rabbit IgG was immobilized while keeping its antibody activity high, and the immobilized antibody was obtained.

Example 1-4 A bead of immobilized BSA in which BSA was immobilized in the same manner as in Example 1-1, except that the compound represented by the following formula [6] was used as the functional substance immobilizing agent. Got Further, when xanthoprotein method was applied, immobilization of BSA was confirmed.

[Chemical 8]

Example 1-5 Beads of immobilized BSA in which BSA was immobilized in the same manner as in Example 1-1, except that the compound represented by the following formula [7] was used as the functional substance immobilizing agent. Got Further, when xanthoprotein method was applied, immobilization of BSA was confirmed.

[Chemical 9]

Comparative Example 1 500 mg of the polyalkylene oxide beads obtained in 1) of Example 1-1 were added to BSA at a concentration of 500 μg / ml.
It was immersed in 10 ml of the solution (0.1 M phosphate buffer pH = 7.4) and incubated at 4 ° C. for 3 days. 0.1%
After washing well with the aqueous solution of dodecylbenzene sulfonic acid and purified water, the xanthoprotein reaction was carried out, but no coloring was observed on the bead surface or in the solution. That is, BSA was not immobilized on the beads that had not been treated with CDI.

Example 2 A compound represented by the following formula [8] was used and treated in the same manner as in Example 1-1 to obtain polyalkylene oxide-immobilized glass beads. To 1 g of this glass bead, 0.
After adding 5 ml of 5N hydrochloric acid aqueous solution and leaving it at room temperature for 24 hours, it was filtered, and the filtrate was concentrated under reduced pressure. What added 500 μl of chloroform was analyzed by capillary gas chromatography, but there was no detected substance. This result shows that the compound of the following formula [8] is not easily hydrolyzed and has excellent stability.

[Chemical 10]

Comparative Example 2

The compound represented by [9] was used for treatment in the same manner as in Example 1-1 to obtain polyalkylene oxide-immobilized glass beads. The glass beads were subjected to an acid treatment in the same manner as in Example 2 and then analyzed by capillary gas chromatography. As a result, ethylene glycol was detected. This is the formula

It is produced by hydrolyzing the ester bond in [9].

[Chemical 11]

Claims (3)

[Claims] 1. A functional substance immobilizing agent comprising a polyalkylene oxide silanol derivative represented by the following general formula [1]. [Chemical 1] Wherein, R ¹ and R ² represents a methyl group or an ethyl group, p is an integer of 1 to 3, q is an integer of 0 to 2 (provided that p + q =
3). AO is an oxyalkylene group having 2 to 4 carbon atoms, and n is an average addition mole number of the oxyalkylene group of 2 to 100.
Represents a positive number of zero. The added oxyalkylene groups may be the same or different, and may be added randomly or in blocks. ] 2. The functional substance immobilizing agent according to claim 1,
After introducing into the substrate by silane coupling reaction, N,
A method for immobilizing a functional substance, which comprises activating by reacting N'-carbonyldiimidazole or a derivative thereof, and then contacting the functional substance. 3. An immobilized functional substance, wherein the functional substance is immobilized on a substrate by the functional substance immobilizing agent according to claim 1.