JP5344835B2 - Immobilization method, physiologically active substance immobilization carrier and immobilization carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for immobilization which reduces nonspecific adsorption and enables the immobilization of a sufficient amount of a physiologically active substance, a physiologically active substance-immobilized carrier, and a carrier for immobilization. <P>SOLUTION: The method for immobilization for immobilizing the physiologically active substance on a solid phase carrier includes bringing the solid phase carrier into contact with an acid anhydride functional group-containing silane coupling agent such as, for example, a 3-(triethoxysilyl)propylsuccinic acid anhydride , and performing bonding treatment of the physiologically active substance to the acid anhydride functional group while holding the solid phase carrier after the contact at a temperature in the range of 0-60&deg;C. Also disclosed is a physiologically active substance-immobilized carrier obtained by the method for immobilization, Still further provided is a carrier for immobilization for preparing the physiologically active substance-immobilized carrier obtained by bringing the solid phase carrier into contact with the acid anhydride functional group-containing silane coupling agent such as, for example, the 3-(triethoxysilyl)propylsuccinic acid anhydride. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to an immobilization method, a physiologically active substance immobilization carrier, and an immobilization carrier.

  Biochemical reaction chips using solid phase carriers such as diagnostic chips and protein microarrays are very useful in research and development and clinical fields. For example, in a protein microarray or the like, a protein solution is brought into contact with a glass substrate to immobilize the protein on the substrate, and diagnosis and analysis are performed using the physiological activity of the immobilized protein. In this case, it is required that the protein can be immobilized with high density, and the substance to be reacted with the immobilized protein is not adsorbed nonspecifically on the carrier.

In order to eliminate such non-specific adsorption, for example, Non-Patent Document 1 discloses a method of directly adsorbing and fixing a protein via a polylysine coat layer or an amino group layer, or an active ester layer formed by a multistage reaction. A method of immobilizing via a covalent bond is described.
Patent Document 1 describes a method in which a substrate is directly carboxylated by using a specific silane coupling agent and a protein is covalently bonded by activating it. When a silane coupling agent as disclosed in Patent Document 1 is used, generally, after coating on a substrate, it is cured by baking at a temperature of 100 ° C. or higher to form a three-dimensional network structure layer. I am letting.
However, these methods can stably immobilize proteins at a relatively high density, but the immobilization density is not sufficient, and nonspecific adsorption of a substance to be reacted with the immobilized protein increases.

JP 2005-201901 A Proteomics, (2003) Vol.3, pp.254-264

  An object of the present invention is to provide an immobilization method, a physiologically active substance-immobilized carrier and an immobilization carrier that can immobilize a sufficient amount of a physiologically active substance with less nonspecific adsorption.

The immobilization method of the present invention is an immobilization method for immobilizing a physiologically active substance on a solid phase carrier, the solid phase carrier and a silane having an acid anhydride functional group represented by the following general formula (I) Contacting the coupling agent with at least one aromatic hydrocarbon solvent selected from the group consisting of benzene, toluene and xylene, and subjecting the solid support after the contact to a temperature range of 0 ° C. to 60 ° C. Holding the physiologically active substance with respect to the acid anhydride functional group.

(In Formula I, R ¹ represents a trivalent linear or branched aliphatic group or aromatic group, and R ² represents a C _{1 to} C ₂₀ substituted or unsubstituted alkylene group, arylene group, aryl group. Represents an alkylene group or an alkylarylene group, R ³ represents a hydrogen atom or a C _{1 to} C ₁₀ alkyl group, and X represents an alkoxy group (—OR ⁴ ), a halogen atom or an acyloxy group (—OOCR ⁵ ). R ⁴ and R ⁵ each independently represents a hydrogen atom or a C _{1 to} C ₁₀ alkyl group, n represents 1, 2, or 3, and m represents an integer of (3-n).)

In the above immobilization method, the binding treatment may be performed by bringing the physiologically active substance into direct contact with the acid anhydride functional group for binding.
In the immobilization method, the solid phase carrier to which the physiologically active substance is bound may be hydrolyzed.

Upper Symbol immobilization method, after the coupling process, may be one which does not perform washing of the solid phase carrier.

The physiologically active substance-immobilized carrier of the present invention is obtained by immobilizing a physiologically active substance by the above-described immobilization method.
The immobilization carrier of the present invention comprises at least a solid phase carrier and a silane coupling agent represented by the following general formula (I) having an acid anhydride functional group selected from the group consisting of benzene, toluene and xylene. It is obtained by contacting in one aromatic hydrocarbon solvent and is used for producing the physiologically active substance-immobilized carrier.

  According to the present invention, it is possible to provide an immobilization method, a physiologically active substance-immobilized carrier and an immobilization carrier that can immobilize a sufficient amount of a physiologically active substance with less nonspecific adsorption.

The immobilization method of the present invention is an immobilization method for immobilizing a physiologically active substance on a solid phase carrier, the solid phase carrier and a silane represented by the above general formula (I) and having an acid anhydride functional group Contacting the coupling agent with the coupling agent (contacting step), while maintaining the solid support after the contact within a temperature range of 0 ° C. to 60 ° C., the binding treatment of the physiologically active substance to the acid anhydride functional group To perform (combining process).
In the present invention, a specific silane coupling agent having an acid anhydride functional group is brought into contact with a solid phase carrier, and then a physiologically active substance is bound while being maintained in a temperature range of 0 ° C to 60 ° C. That is, the binding treatment of the physiologically active substance is performed without performing the baking treatment of 100 ° C. or higher, which is generally performed to fix the silane coupling agent to the solid phase carrier. As described above, since the baking treatment is not performed even when the silane coupling agent is brought into contact with the solid phase carrier, non-specific adsorption is remarkably suppressed and a physiologically active substance is bound to the acid anhydride to perform a sufficient amount of physiological treatment. The active substance can be fixed. Although not specifically bound by theory, in the present invention, non-specific adsorption can be suppressed by effectively reducing the contamination due to the reaction between the acid anhydride and the foreign matter during baking from not performing baking. Inferred.

The solid phase carrier used in the present invention is not particularly limited as long as it is generally used, and is a flat substrate, a substrate with unevenness, a rough surface substrate, particles, fine particles, rod-shaped particles, thin films, fibers, columns, meshes, An arbitrary shape such as a probe tip can be selected. The material of the solid phase carrier is not limited as long as it is a material in which a metal (silicon) -oxygen-silicon-carbon bond is formed by a silane coupling reaction, and specifically, glass, silica, alumina, titania, zirconia. In addition, oxides such as indium tin oxide (ITO), nitrides such as silicon nitride, gallium nitride, aluminum nitride, and indium nitride can be used alone or as a composite thereof.
Furthermore, as long as the outermost surface is a material capable of forming a bond by the silane coupling reaction, the solid phase carrier itself may be a multilayer structure using silicon, various metals, or a polymer. The method for forming a silane coupling-reactive material on the outermost surface is not particularly limited according to a general surface modification method, and specifically, a vapor phase such as physical vapor deposition or chemical vapor deposition (CVD) sputtering. Thin film formation by sol-gel method, thin film formation in liquid phase by sol-gel method, modification of the vicinity of the surface by surface oxidation or the like. Of these, glass and silica carriers are preferred from the standpoint of stability of the carrier itself. Also, a carrier in which a silica thin film layer is formed on the surface of the polymer for ease of processing, and a silicon single crystal surface is oxidized from the viewpoint of smoothness. Preferred is a supported carrier.

  The silane coupling agent having an acid anhydride functional group is a silane coupling agent represented by the following general formula (I).

In general formula (I), R ¹ represents a trivalent linear or branched aliphatic group or aromatic group, and is preferably any of the following from the viewpoint of the stability of the acid anhydride functional group.

Among them, the following structure is more preferable from the balance between stability and reactivity.

R ² represents a C ₁ -C ₂₀ substituted or unsubstituted alkylene group, arylene group, arylalkylene group or alkylarylene group, and preferably C ₁ from the viewpoint of reaction efficiency and suppression of nonspecific adsorption after the reaction. a linear alkylene group -C _5, more preferably from propylene group.
R ³ represents a C ₁ -C ₁₀ alkyl group, preferably a C ₁ -C ₂ alkyl group, more preferably a methyl group, from the viewpoint of suppressing nonspecific adsorption.
X is a functional group that is hydrolyzed to hydroxy, and is an alkoxy group (—OR ⁴ ), a halogen atom, or an acyloxy group (—OOCR ⁵ ), and R ⁴ and R ⁵ are each independently a hydrogen atom or C It represents an alkyl group having ₁ -C _10. X is preferably a methoxy group, an ethoxy group, an isopropoxy group, a butoxy group, or chlorine from the viewpoint of the balance between stability and reactivity, and more preferably a methoxy group or an ethoxy group.
n represents 1, 2, or 3, m represents an integer of (3-n), and n is preferably 3 and m is 0 from the viewpoint of suppressing nonspecific adsorption after the reaction.

  Examples of such silane coupling agents include 3- (triethoxysilyl) propyl succinic anhydride, 4- (triethoxysilyl) butyl succinic anhydride, 3- (trimethoxysilyl) propyl succinic anhydride, 3- (triethoxysilyl) propyl glutaric anhydride, etc. can be mentioned, and 3- (triethoxysilyl) propyl succinic anhydride is preferred from the viewpoint of reactivity and suppression of nonspecific adsorption after the reaction. .

When the silane coupling agent is brought into contact with the solid phase carrier, the raw solution may be used as it is or in the form of an appropriate solution. From the viewpoint of thin and uniform surface modification, a 0.01% by mass to 10% by mass solution, A solution of 0.1% to 2% by weight is preferably selected.
As a kind of solvent, either a polar solvent or a nonpolar solvent can be used. Examples of the polar solvent include ethanol, methanol, water and the like. Nonpolar solvents include aliphatic hydrocarbons such as hexane, octane and cyclohexane, aromatic hydrocarbons such as benzene, xylene and toluene, ethers such as diethyl ether, and chlorines such as chloroform and methylene chloride. And other ester systems such as ethyl acetate. These solvents can be appropriately selected from polar solvents and nonpolar solvents based on the solvent resistance of the solid phase carrier, for example, hexane, ethanol and the like can be appropriately selected.
As the solvent in the present invention, a nonpolar solvent is preferable in order to effectively proceed dehydration condensation without baking, and among these, toluene and xylene are preferable.

In the contact step, a contact treatment between the solid phase carrier and the silane coupling agent is performed. As a result, a hydrogen bond is formed between the silane coupling agent having a silanol group and the hydroxy group of the solid phase carrier by hydrolysis, and it is speculated that a strong bond is formed by subsequent dehydration condensation. . The complete dehydration condensation does not need to proceed, and even when the dehydration condensation does not proceed completely, it can be used as it is depending on the purpose.
In addition, the hydrolysis treatment may be performed by hydrolyzing the silane coupling agent before contacting with the solid phase carrier in advance, and hydrolysis and binding may proceed simultaneously at any time during the contacting step. More preferred is a method in which hydrolysis and bonding proceed simultaneously as needed. This simultaneous processing is easily achieved by simply dissolving the silane coupling agent before hydrolysis in a solvent and bringing it into contact with a solid support.

  Furthermore, water for hydrolysis may be added to the silane coupling agent solution. However, since the water adsorbed on the solid support or the water entering from the air is often sufficient, the efficiency of the subsequent dehydration condensation reaction In order to increase, it is preferable not to add water, and it is preferable to remove water dissolved in the solvent as much as possible.

Such contact is not particularly limited as long as the silane coupling agent can be brought into contact with the solid phase carrier as it is or in a solution dissolved in a solvent. Specific examples include a dipping method in which a solid support is dipped in a silane coupling agent solution and taken out from the solution after an appropriate time has elapsed. In this immersion method, since the dehydration condensation reaction can proceed during the contact step, only the bonded silane coupling agent remains even if the silane coupling agent adsorbed on the solid support after washing out from the solution is washed away. it can. As a result, thin and uniform surface modification can be performed. Moreover, since it can employ | adopt regardless of the shape of a solid-phase carrier, versatility is high.
Another contact method includes a method of coating a solution obtained by dissolving the silane coupling agent as it is or in a solvent. As a coating method, a known method can be used. Specifically, an extrusion coating method, a curtain coating method, a casting method, a screen printing method, a spin coating method, a spray coating method, a slide bead coating method, A slit-and-spin method, a slit coating method, a die coating method, a dip coating method, a knife coating method, a blade coating method, a flow coating method, a roll coating method, a wire bar coating method, a transfer printing method, and the like can be used. For these thin film formation methods, "Progress in coating technology" by Yuji Harasaki, Comprehensive Technology Center (1988), "Coating Technology" Technical Information Association (1999), "Aqueous Coating Technology" CMC (2001), "Evolution" Described in "Organic Thin Film Deposition", Sumibe Techno Research (2004), "Polymer Surface Processing" by Iwamori Satoshi, Gihodo Publishing (2005), etc. Since a coating film with a controlled film thickness can be easily produced, the method for coating on a flat solid support in the present invention is preferably a spray coating method or a spin coating method, and more preferably a spin coating method. In the case of the spin coating method, it may be performed at 300 rpm to 10,000 rpm. Among these, in order to perform surface modification thinly and uniformly, it is preferable that it is 1000 rpm or more.

  In the contact step by coating, since the progress efficiency of dehydration condensation in the coating step is low, it is preferable to perform the physiologically active substance binding treatment without immediately washing after coating. Further, when a polar solvent is used as a solvent in both the dipping method and the coating method, since the efficiency of dehydration condensation is generally low, it is preferable to perform the physiologically active substance binding treatment without washing after the contacting step. However, when a contact treatment for 10 minutes or more is performed using a nonpolar solvent, washing may be performed for the purpose of homogenizing the silane coupling agent reaction.

  The contact treatment may be continued until the acid anhydride functional group is retained with respect to the solid phase carrier. However, the contact treatment is not caused by decomposition of the acid anhydride by hydrolysis over time or multilayering of the silane coupling agent. From the viewpoint of preventing homogenization, the time is preferably less than 3 hours, more preferably from less than 2 hours from the viewpoint of processing efficiency, and even more preferably 1 hour or less.

In the binding process, when the binding treatment is performed in a high-temperature environment, the active acid anhydride functional group interacts with substances in the surrounding environment, so the target chemical structure is not maintained and non-specific Adsorption increases. For example, the organic substance contained in the air is bonded to the acid anhydride functional group, so that the organic substance is adsorbed on the solid support as dirt. Therefore, it is necessary to perform the binding treatment of the physiologically active substance to the acid anhydride functional group while maintaining the solid phase carrier after the contact treatment within the temperature range of 0 ° C to 60 ° C, and further to 0 ° C to 40 ° C. It is more preferable to hold. At this time, it is not necessary to keep this temperature range only when the physiologically active substance is bonded, but it is necessary to keep the temperature range consistently from the contact of the silane coupling agent to the bonding step. That is, the baking process generally performed in a silane coupling reaction must not be performed.
Furthermore, as described below, when a physiologically active substance is directly contacted and bonded to a succinic anhydride functional group, it is decomposed by exposing the acid anhydride functional group to high temperature, and a sufficient amount of the physiologically active substance is immobilized. Therefore, it is necessary to keep this temperature range for this purpose.

  The physiologically active substance can be immobilized by hydrolyzing the acid anhydride functional group once to form a carboxyl group, and then bringing the physiologically active substance into contact with the activated carboxyl group. However, from the viewpoint of suppressing non-specific adsorption, a physiologically active substance is directly contacted and immobilized on the acid anhydride functional group on the surface of the solid phase carrier without performing an activation treatment on the surface of the solid phase carrier. It is preferable. This method can significantly reduce the number of operations before and after activation, and the time can be greatly reduced, so that changes in chemical state (such as adsorption) due to disturbance factors can be suppressed, and nonspecific adsorption can be greatly suppressed. .

  The “activation treatment” as used in the present invention refers to a treatment in which an activator is reacted with a carboxyl group derived from an acid anhydride functional group on the surface of a solid support after the contact treatment to bring it into an active state. Examples of the activator used here include carbodiimide and derivatives thereof such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-ethyl-3- (3- Dimethylaminopropyl) carbodiimide (EDC) hydrochloride, N, N′-dicyclohexylcarbodiimide (DCC), N, N′-diisopropylcarbodiimide (DIC). Further, in order to improve reactivity and reaction stability, succinimide and the like can be used in combination, such as N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS) and 3,4-dihydro. -3-Hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt) and the like are applicable.

  The physiologically active substance is not particularly limited, and examples thereof include biologically derived substances such as proteins, peptides, nucleic acids, sugar chains, lipids, hormones, cytokines, cells, microorganisms, complexes, derivatives and artificially synthesized products thereof. Can be mentioned. Moreover, low molecular organic compounds synthesized mainly in vitro such as vitamins, drugs, and environmental hormones may also be used.

Examples of proteins include antibodies, antibody-binding proteins, enzymes, sugar chain recognition proteins, receptors, etc., which may be purified products that exist in nature, may be artificially synthesized, and may be artificially mutated. May be introduced, combined, or fragmented. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, IgD and derivatives, complexes and fragments thereof can be used. Specifically, when the solid phase carrier is used as a carrier for immunoassay, an antibody against the measurement object can be used. Moreover, protein G, protein A, protein L etc. can be used as an antibody binding protein, The antibody with respect to a measuring object can also be fix | immobilized through these.
As the enzyme, various enzymes such as oxidoreductase, hydrolase, isomerase, desorption enzyme, and synthetic enzyme can be used. Enzymes such as glucose oxidase, cholesterol oxidase, acetylcholinesterase, catecholamine esterase, noradrenaline esterase, and dopamine esterase can be used.
Examples of sugar chain recognition proteins include lectins such as ConA and WGA. Examples of the receptor include GPCR (G-proein-coupled receptor) and EGFR (Epidermal Growth Factor Receptor).
Peptides may exist in nature or may be artificially synthesized. Furthermore, a peptide introduced with an amino acid that does not exist in nature may be used.

DNA, RNA, PNA (peptide nucleic acid) of any sequence or complex of these can be used as the nucleic acid, and it can be extracted from nature, genetically engineered, or chemically synthesized Good. As the sugar chain, oligosaccharides, polysaccharides and monosaccharides having an arbitrary sequence can be used, and their length and sequence may be controlled, or those not strictly controlled may be used. In addition, amino group, acetylamide group, sulfonic acid, and carboxyl group derivatives can be used, and glycoproteins in which sugar chains and proteins are combined can also be used. As lipids, glycerides and complex lipids can be used. Hormones and cytokines may be those actually produced in nature, and synthetic products and derivatives thereof can be used. The cells and microorganisms may be natural extracts, subcultures thereof, or pseudocells using liposomes.
The low molecular weight organic compounds synthesized mainly in vitro such as vitamins, drugs and environmental hormones may be those whose effects on the living body are unknown, or those whose pharmacological action mechanisms have already been elucidated. Further, it may be an extract from the natural world or a chemical or biochemical compound.

  In the binding treatment, these physiologically active substances are used as an aqueous solution (hereinafter referred to as a physiologically active substance-containing solution) dissolved in an appropriate aqueous solvent. As the aqueous solvent that can be used here, a general solution used as a solvent for a physiologically active substance can be used as it is, for example, water, physiological saline, acetate buffer, citrate buffer, phosphate buffer. HEPES buffer solution (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), MES buffer solution (2- (N-morpholino) ethanesulfonic acid), and the like. However, acid anhydride functional groups or their hydrolyzed / activated functional groups are generally highly reactive with nucleophilic functional groups in the conjugation process, so buffers with amines such as Tris buffer (trihydroxymethylaminomethane) Is not preferred.

  When the physiologically active substance has a charge opposite to the charge on the surface of the solid phase carrier, it is concentrated on the surface of the solid phase carrier, and its binding efficiency increases. Since a part of the acid anhydride functional group has a carboxyl group when it is decomposed, the surface is considered to be negatively charged when a solution having a pH of about 3 or more is in contact. Therefore, when the pH of the solution is lower than the isoelectric point of the physiologically active substance and 3 or more, the physiologically active substance is positively charged in the solution and has a charge opposite to that of the carrier surface. Increased coupling efficiency. Therefore, it is preferable that the pH of the solution is 3 or more and less than the isoelectric point of the physiologically active substance from the viewpoint of increasing the binding amount.

After the binding treatment, it is preferable to perform a hydrolysis treatment from the viewpoint of suppressing nonspecific adsorption. By this hydrolysis treatment, since the unreacted acid anhydride functional group becomes a carboxyl group, nonspecific adsorption is effectively suppressed.
The hydrolysis treatment can be performed by a conventionally known method of decomposing an acid anhydride functional group to generate a carboxyl group, and is generally performed using an aqueous solution at 20 ° C to 100 ° C. In order to complete the hydrolysis reaction, it is preferable to use an alkaline or acidic aqueous solution, and it is more preferable to use an alkaline aqueous solution having a pH of 10 or more. Specific examples include 1 mM to 1 M NaOH aqueous solution, KOH aqueous solution, and the like. In this case, since the reactivity is high, a treatment within 10 minutes is sufficient.
However, from the viewpoint of maintaining the activity of the bound physiologically active substance, a mild aqueous solution having a pH of 5 to 9 is preferably used. Specific examples include pure water, borate buffer, citrate buffer, phosphate buffer, HEPES buffer, and acetate buffer. Furthermore, the method of hold | maintaining in water vapor | steam atmosphere from a viewpoint of operating easily is also mentioned. In the case of these mild methods, the treatment is preferably performed for 10 minutes or more. Thereby, two carboxyl groups are generated from the acid anhydride functional group. As an indicator of the progress of the hydrolysis treatment, there is a method of measuring the contact angle of the treated solid phase carrier with water or the like.

  When the physiologically active substance-immobilized carrier has insufficient ability to suppress nonspecific adsorption, such as when the pH is low, a blocking agent can be immobilized on the carrier in addition to the physiologically active substance. That is, the immobilization method of the present invention may include immobilizing the blocking agent on the solid phase carrier after contact with the silane coupling agent. A blocking agent refers to a substance that has an ability to suppress adsorption with respect to a substance that is a target for suppression of nonspecific adsorption, and an appropriate substance can be selected as a blocking agent based on the type of the target to be suppressed.

Examples of the blocking agent include water-soluble proteins or water-soluble polymers. Preferred examples of water-soluble proteins include albumin, casein, and gelatin. From the viewpoint of nonspecific adsorption inhibition ability, albumin or casein is preferable. More preferably. Preferred examples of the water-soluble polymer include polyethylene glycol, phosphorylcholine group-containing polymer, polysaccharide, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylic acid, polyacrylamide, zwitterion-containing polymer, and polyvinylpyrrolidone. From the viewpoint of specific adsorption inhibiting ability, polyethylene glycol, phosphorylcholine group-containing polymer, dextran, carboxymethyldextran, and dextran sulfate are preferable.
When these water-soluble polymers are used, the water-soluble polymer may have a site that binds or adsorbs to the carrier in order to improve the immobilization ability to the carrier. The site that binds to the carrier is not particularly limited as long as it is a structure that binds to an acid anhydride, and specific examples include a functional group having an amine such as a primary amine. Examples of the site that adsorbs to the carrier include a structure that adsorbs to the carrier by electrostatic interaction. More specifically, a structure having a positive charge at the fixing pH, more specifically polyallylamine, polyethyleneimine, polyvinylamine, polylysine. And derivatives thereof. As a water-soluble polymer having such a structure, NanoBio Blocker manufactured by Naobiotech Co., Ltd. can be preferably used.

  The part for immobilizing the blocking agent in the carrier may be the same part where the physiologically active substance is immobilized in advance, that is, the gap where the physiologically active substance is immobilized but not immobilized. The portion where the physiologically active substance is not immobilized may be used, or both of them may be used. Furthermore, the timing for immobilizing the blocking agent is not particularly limited, and examples include immediately after immobilizing the physiologically active substance, after hydrolysis, and simultaneously with immobilization of the physiologically active substance. Further, the hydrolysis treatment can be performed simultaneously by performing the blocking agent immobilization treatment after immobilization of the physiologically active substance.

  The physiologically active substance-immobilized carrier obtained as described above is obtained by immobilizing a sufficient amount of physiologically active substance on the surface. The amount of the physiologically active substance immobilized on the surface of the carrier can be confirmed by a method usually used for this purpose. For example, a fluorescence measurement method using a fluorescent substance, SPR (surface plasmon resonance), LPR (Local plasmon resonance), QCM (quartz crystal microbalance) and the like.

The physiologically active substance-immobilized carrier of the present invention is obtained by the above-described immobilization method, and the physiologically active substance is immobilized on the surface at a high density.
This bioactive substance-immobilized carrier is useful for detection and analysis of molecules specific to the bioactive substance on the carrier because the bioactive substance is immobilized at high density and nonspecific adsorption is extremely low. In particular, it is suitable for various microarrays, biosensors, and the like that require high specificity for physiologically active substances, such as trace analysis and detection of fine interactions.

  The immobilization carrier of the present invention is obtained by bringing the solid phase carrier into contact with a silane coupling agent represented by the general formula (I) and having an acid anhydride functional group. It has a physical functional group. When a desired physiologically active substance is immobilized on the acid anhydride functional group on the surface of this immobilizing carrier, it is possible to have the physiologically active substance on the surface at a high density and immobilization with extremely low non-specific adsorption. A carrier can be provided. For this reason, it is useful as a material for producing an immobilization carrier for uses requiring high specificity for a physiologically active substance.

  The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.

[Example 1]
(1) Preparation of Succinic Anhydride Glass Substrate Substrate Washed glass substrate (Matsunami Glass in a dehydrated xylene solution containing 2- (triethoxysilyl) propyl succinic anhydride (manufactured by Gelest) at 2 mol / l as a silane coupling agent Industrial white plate glass) was immersed and allowed to react at room temperature for 1 hour. The reacted glass substrate was taken out from the xylene solution, immersed and washed with dehydrated xylene three times, and then dried with nitrogen gas. This glass substrate is called anhydrous succinic oxide glass.

(2) Immobilization of antibody and measurement of immobilized amount FITC-labeled antibody (manufactured by SIGMA-ALDRICH, hereinafter referred to as IgG-FITC) prepared on anhydrous succinic oxide glass to 10 mg, 0.1 mg / ml with acetic acid buffer pH 5.0 ) Was added dropwise and allowed to react for 1 hour at room temperature in a high humidity and dark environment. After removing the antibody solution by suction and immersing and washing in order of 0.1M NaOH, phosphate buffer (PBS), and pure water, the antibody-immobilized glass substrate is removed to confirm that it is stably immobilized. Stored in PBS pH 7.1 for 3 days. The fluorescence intensity of the antibody-immobilized glass substrate was measured by FLA-8000 (manufactured by Fujifilm) (excitation at 473 nm / detection at 530 nm), and the amount of immobilized antibody was estimated using a calibration curve prepared in advance.

(3) Measurement of non-specific adsorption amount of antibody Anhydrous succinic oxide glass was produced, and the glass substrate was immersed in a 10 mM borate buffer (pH 8.5) for 1 hour at room temperature and reacted (hereinafter referred to as acid anhydride). This is called a hydrolysis treatment. When the static contact angle with water was measured to confirm that the hydrolysis was sufficiently advanced, it was 36.7 ± 1.1 ° before the boric acid buffer treatment, whereas it was 7.4 after the treatment. It was ± 1.1 °, suggesting the formation of carboxyl groups by hydrolysis. After the reaction, an antibody (IgG-FITC) prepared to 100 nM (15 μg / ml) with PBS was dropped onto a substrate washed with pure water and allowed to stand at room temperature in a high humidity and dark environment for 1 hour. . The antibody solution was removed by suction, and immersed and washed in the order of PBS and pure water. The amount of antibody that was naturally dried and nonspecifically adsorbed on the substrate was measured using FLA-8000 in the same manner as described above.

[Example 2]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and the amount of antibody immobilized thereon and the amount of non-specific adsorption were measured. However, after the reaction with the silane coupling agent, only drying with nitrogen gas was performed without washing with xylene.

[Example 3]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and the amount of antibody immobilized thereon and the amount of non-specific adsorption were measured. However, a silane coupling agent prepared as an ethanol solution and a cleaning solution as ethanol was used.

[Example 4]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 2, and the amount of antibody immobilized thereon and the amount of nonspecific adsorption were measured. However, a silane coupling agent prepared as an ethanol solution was used.

[Example 5]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and the glass substrate was immersed in 10 mM borate buffer (pH 8.5) for 1 hour at room temperature for hydrolysis.
DCC / NHS mixed DMF solution (0.1 M each) is brought into contact with this glass substrate for 1 hour at room temperature to activate (hereinafter referred to as activation treatment), and then immersed and washed twice with ethanol and then nitrogen gas. Dried. The amount of antibody immobilized on the activated glass substrate was measured in the same manner as in Example 1.
Further, the activated succinic oxide glass subjected to the activation treatment in the same manner was immersed in 10 mM HCl at room temperature for 1 hour to completely hydrolyze the activated ester (this operation is hereinafter referred to as active ester hydrolysis treatment). . Using this substrate, the nonspecific adsorption amount of the antibody was measured in the same manner as in Example 1.

[Example 6]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 5, subjected to hydrolysis treatment and activation treatment, and the amount of antibody immobilized thereon and the amount of nonspecific adsorption after the hydrolysis treatment were measured. However, a silane coupling agent prepared as an ethanol solution and a cleaning solution as ethanol was used.

[Example 7]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 5, subjected to hydrolysis treatment and activation treatment, and the amount of antibody immobilized thereon and the amount of nonspecific adsorption after the hydrolysis treatment were measured. However, instead of the DCC / NHS mixed solution, an EDC (PIERCE 0.4M) and Dhbt (Tokyo Chemical Industry 2.8 mM) mixed aqueous solution was used.

[Example 8]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and the amount of antibody immobilized thereon was measured. However, a silane coupling agent was used as a hexane solution and a cleaning liquid was used as hexane.

[Comparative Example 1]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and then baked in an oven at 120 ° C. for 1 hour. After returning to room temperature, the amount of antibody immobilized on the substrate and the amount of non-specific adsorption were measured in the same manner as in Example 1.

[Comparative Example 2]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 2 and then baked in an oven at 120 ° C. for 1 hour. After returning to room temperature, the amount of antibody immobilized on the substrate and the amount of non-specific adsorption were measured in the same manner as in Example 1.

[Comparative Example 3]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 3 and then baked in an oven at 120 ° C. for 1 hour. After returning to room temperature, the amount of antibody immobilized on the substrate and the amount of non-specific adsorption were measured in the same manner as in Example 1.

[Comparative Example 4]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 4 and then baked in an oven at 120 ° C. for 1 hour. After returning to room temperature, the amount of antibody immobilized on the substrate and the amount of non-specific adsorption were measured in the same manner as in Example 1.

[Comparative Example 5]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 5, subjected to hydrolysis treatment and activation treatment, and the amount of antibody immobilized and the amount of nonspecific adsorption thereto were measured. However, after the silane coupling agent reaction, baking treatment was performed in an oven at 120 ° C. for 1 hour before the hydrolysis treatment.

[Comparative Example 6]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 6, hydrolyzed and activated, and the amount of antibody immobilized and nonspecifically adsorbed thereto were measured. However, after the silane coupling agent reaction, baking treatment was performed in an oven at 120 ° C. for 1 hour before the hydrolysis treatment.

[Comparative Example 7]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and then baked in an oven at 70 ° C. for 1 hour. The amount of antibody immobilized on the substrate returned to room temperature and the amount of non-specific adsorption were measured.

[Comparative Example 8]
FITC-labeled antibody prepared to 0.1 mg / ml with PBS was dropped onto an aldehyded glass substrate (Genetics), and reacted in a high humidity environment for 1 hour. The antibody solution was removed by suction, immersed and washed in the order of 0.1 M NaOH, phosphate buffer (PBS), and pure water, and then stored in PBS for 3 days. The amount of antibody immobilized and the amount of non-specific adsorption were estimated using FLA-8000.
Another aldehyded glass substrate was immersed in BSA (bovine serum albumin: manufactured by SIGMA-ALDRICH) 2% PBS solution and reacted for 1 hour for blocking treatment. After soaking and washing with PBS, it was naturally dried. Using this substrate, the nonspecific adsorption amount of the antibody to the substrate was measured in the same manner as in Example 1.

[Test Example 1]
About said Example 1-8 and Comparative Examples 1-8, the antibody immobilization amount, the nonspecific adsorption amount, and the antibody immobilization amount / nonspecific adsorption amount were compared. The results are shown in Table 1. In addition, Example 3, Example 4, Example 6, and Example 8 are reference examples of the present invention.
As shown in Table 1, in the examples of the present invention, a carrier having a high immobilization amount and a high immobilization amount / nonspecific adsorption amount (over 100) can be obtained.
Further, it was found that a higher immobilization amount and an immobilization amount / non-specific adsorption amount ratio can be realized by directly binding an antibody without performing an activation treatment (Examples 1 to 4).
Moreover, it turned out that it is more effective to use a nonpolar solvent as an immobilization solvent (Example 1, 2, 8).
Regarding the washing after the silane coupling reaction, it was found that the antibody immobilization amount was higher without washing (Examples 2 and 4).

[Example 9]
An anhydrous succinic oxide glass substrate was produced in the same manner as in Example 1. However, it was not immersed in a xylene solution, but after dropping the xylene solution, it was applied at 1000 rpm for 35 seconds using a spin coater and then dried at room temperature (25 ° C.). Further, in the same manner as in Example 1, the amount of immobilized antibody and the amount of nonspecific adsorption were measured.

[Example 10]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 9, and the amount of antibody immobilized thereon was measured. However, a silane coupling agent prepared as an ethanol solution was used.

[Test Example 2]
For each of the substrates of Example 2, Example 4, Example 9, and Example 10, the amount of antibody immobilized was compared. The results are shown in Table 2. Examples 4 and 10 are reference examples of the present invention.
As shown in Table 2, in the examples of the present invention, the immobilization amount is not affected greatly regardless of whether the contact method between the coupling agent and the carrier is an immersion method or spin coating. It was found that a high immobilization amount of substrate can be provided.

[Example 11]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and the amount of antibody immobilized thereon was measured. However, a pH 6.5 acetate buffer was used as a buffer for immobilizing IgG-FITC. The isoelectric point of the antibody used in this example was about 6.5.

[Test Example 3]
For Example 1 and Example 11, the antibody immobilization amount, the nonspecific adsorption amount, and the antibody immobilization amount / nonspecific adsorption amount ratio were compared. The results are shown in Table 3.
As shown in Table 3, in the examples of the present invention, the non-specific adsorption amount is low regardless of the pH of the immobilization buffer, and the ratio to the immobilization amount is good. It was shown that a better balance between the immobilization amount and the immobilization amount / non-specific adsorption amount ratio can be achieved by setting it below the isoelectric point of the active substance.

[Example 12]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and the amount of antibody immobilized thereon was measured. However, the immersion time in PBS was 14 days instead of 3 days.

[Test Example 4]
About Example 1 and Example 12, the amount of antibody immobilization was compared. The results are shown in Table 4.
As shown in Table 4, in the examples of the present invention, even when the immersion of the antibody-conjugated glass substrate in PBS was extended to 14 days, a high fixing amount was maintained, and the fixing ability was stably high. It was shown that.
Therefore, it was shown that the modified surface is stable and has a good immobilization ability in the method of the present invention without performing a dehydration treatment at a high temperature immediately after the silane coupling agent reaction, that is, a so-called baking treatment.

[Example 13]
In the same manner as in Example 1, the nonspecific adsorption amount of the antibody against anhydrous succinylated glass was measured. However, a citrate buffer of 20 mM and pH 6.3 was used as a reaction solution for hydrolysis treatment of acid anhydride.

[Example 14]
In the same manner as in Example 1, the nonspecific adsorption amount of the antibody against anhydrous succinylated glass was measured. However, a 0.1M NaOH solution was used as a reaction solution for the acid anhydride hydrolysis treatment, and the reaction time was 30 seconds.

[Example 15]
In the same manner as in Example 1, the nonspecific adsorption amount of the antibody against anhydrous succinylated glass was measured. However, the non-specific adsorption amount of the antibody was measured after standing for 1 hour at room temperature without hydrolyzing the acid anhydride using a borate buffer.

[Test Example 5]
About Example 1 and Examples 13-15, the nonspecific adsorption amount was compared. The results are shown in Table 5.
As shown in Table 5, in the examples of the present invention, a carrier having a small amount of non-specific adsorption can be obtained regardless of the type and presence of hydrolysis treatment. A carrier with less target adsorption can be obtained.

[Example 16]
In the same manner as in Example 1, the nonspecific adsorption amount of the antibody against anhydrous succinylated glass was measured. However, the reaction time of the silane coupling agent was 18 hours instead of 1 hour. At this time, the static contact angles of water after hydrolyzing the acid anhydride with borate buffer or 0.1 M NaOH were 45.1 ° and 22.9 °, respectively, after the reaction. . This suggested that the surface modification was slightly inferior to that of Example 1.

[Test Example 6]
About Example 1 and Example 16, the nonspecific adsorption amount of the antibody was compared. The results are shown in Table 6.
As shown in Table 6, in the examples of the present invention, it is possible to obtain a carrier with less nonspecific adsorption regardless of the immobilization time, and in particular, contact between the silane coupling agent and the solid phase carrier. It was found that the non-specific adsorption amount can be more effectively reduced by setting it to less than 3 hours.

[Example 17]
Anhydrous succinic oxide glass was prepared in the same manner as in Example 1, and the antibody was immobilized. However, an anti-CRP monoclonal antibody (manufactured by Fitzgerald) was used as the antibody. CRP antigen (Oriental Yeast) labeled with Cy5 (manufactured by GE Healthcare Bioscience) was prepared with PBS to 1 nM and 100 nM, respectively, and applied to the entire substrate on which the antibody was immobilized. The antigen-antibody reaction was performed by allowing to stand for 1 hour in a room temperature, high humidity and dark environment. Immersion washing was performed in the order of PBS and pure water. The amount of antigen binding to the antibody-immobilized part and the amount of non-specific adsorption to the non-immobilized antibody part were measured using FLA-8000 (excitation 635 nm / detection 675 nm) and the ratio (specific binding amount / non-specific) (Adsorption amount: S / N ratio) was determined. The results are shown in Table 7 and FIG.

[Comparative Example 9]
In the same manner as in Comparative Example 8, the anti-CRP antibody was immobilized on the aldehyded glass, and the entire substrate including the antibody-immobilized portion was subjected to blocking treatment in the same manner as in Example 8. Furthermore, the amount of Cy5-conjugated CRP bound and the amount of nonspecific adsorption were measured in the same manner as in Example 21 to determine the S / N ratio. The results are shown in Table 7 and FIG.

[Example 18]
An anti-CRP antibody was immobilized in the same manner as in Example 17. After washing, a CRP antibody (manufactured by Fitzgerald, hereinafter referred to as a secondary antibody), which is different from the antibody immobilized on the substrate and capable of forming a sandwich, is labeled with FITC at 100 nM in PBS containing 1% BSA ( 15 μg / ml) and mixed with various concentrations of CRP antigen (unlabeled) solution (PBS solution containing 1% BSA). The mixed solution that had been allowed to stand for 1 hour was applied to the entire surface of the antibody-immobilized substrate. The antigen-antibody reaction was performed by allowing to stand for 1 hour in a room temperature, high humidity and dark environment. Immersion washing was performed in the order of PBS and pure water. The amount of secondary antibody bound to the antibody immobilization part was measured using FLA-8000 (excitation 473 nm / detection 530 nm), and the ratio of the amount in various antigen concentration conditions to the amount in the condition not containing antigen (specific binding amount / Nonspecific adsorption amount, S / N ratio) was determined. The results are shown in Table 7.

  As shown in Table 7, the physiologically active substance-immobilized carrier of the present invention exhibits a high S / N ratio regardless of the antigen concentration, and is an aldehyded glass most utilized in antigen-antibody reaction on glass. The S / N ratio was higher by one digit or more. When a secondary antibody was used, the S / N ratio was 9.5, which was a very good value even at an antigen concentration of 10 pM.

  Thus, according to the present invention, a physiologically active substance-immobilized carrier in which a non-specific adsorption amount is suppressed and a physiologically active substance that maintains physiological activity is sufficiently immobilized, and a carrier for immobilization useful therefor are provided. Can be obtained.

It is a fluorescence photograph image of the biologically active substance immobilization carrier (antigen amount 1 nm (A) and antigen amount 100 nm (B)) according to Example 17 of the present invention. It is a fluorescence photographic image of the bioactive substance immobilization support (antigen amount 1 nm (A) and antigen amount 100 nm (B)) concerning the comparative example 9 of this invention.

Claims (9)

An immobilization method for immobilizing a physiologically active substance on a solid phase carrier,
At least one aromatic hydrocarbon system selected from the group consisting of benzene, toluene and xylene, wherein the solid phase carrier and the silane coupling agent represented by the following general formula (I) and having an acid anhydride functional group Contacting in a solvent ,
Performing the binding treatment of the physiologically active substance to the acid anhydride functional group while maintaining the solid phase carrier after the contact within a temperature range of 0 ° C. to 60 ° C .;
Immobilization method.


(In formula (I), R ¹ represents a trivalent linear or branched aliphatic group or aromatic group, and R ² represents a C _{1 to} C ₂₀ substituted or unsubstituted alkylene group or arylene group. Represents an arylalkylene group or an alkylarylene group, R ³ represents a hydrogen atom or a C _{1 to} C ₁₀ alkyl group, and X represents an alkoxy group (—OR ⁴ ), a halogen atom or an acyloxy group (—OOCR ⁵ ). R ⁴ and R ⁵ each independently represents a hydrogen atom or a C _{1 to} C ₁₀ alkyl group, n represents 1, 2, or 3, and m represents an integer of (3-n). )   The immobilization method according to claim 1, wherein the binding treatment is performed by directly bringing the physiologically active substance into contact with the acid anhydride functional group.   The immobilization method according to claim 1 or 2, wherein the solid phase carrier to which the physiologically active substance is bound is subjected to a hydrolysis treatment.   The immobilization method according to any one of claims 1 to 3, wherein the solid phase carrier is not washed after contacting the silane coupling agent. The immobilization method according to any one of claims 1 to 4 , wherein the contact between the silane coupling agent and the solid phase carrier is set to less than 3 hours. The immobilization method according to any one of claims 1 to 5 , wherein the binding treatment uses a physiologically active substance solution having a pH of 3 or more and lower than the isoelectric point of the physiologically active substance. The immobilization method according to any one of claims 1 to 6, wherein the binding treatment is performed while maintaining the immobilization carrier after the contact in a temperature range of 0 ° C to 40 ° C.   A bioactive substance-immobilized carrier on which a bioactive substance is immobilized by the method according to any one of claims 1 to 7. At least one aromatic hydrocarbon solvent selected from the group consisting of benzene, toluene and xylene , wherein the solid phase carrier and the silane coupling agent represented by the following general formula (I) and having an acid anhydride functional group A carrier for immobilization which is obtained by contacting in the medium and used for producing the carrier for immobilizing a physiologically active substance according to claim 8.


(In formula (I), R ¹ represents a trivalent linear or branched aliphatic group or aromatic group, and R ² represents a C _{1 to} C ₂₀ substituted or unsubstituted alkylene group or arylene group. Represents an arylalkylene group or an alkylarylene group, R ³ represents a hydrogen atom or a C _{1 to} C ₁₀ alkyl group, and X represents an alkoxy group (—OR ⁴ ), a halogen atom or an acyloxy group (—OOCR ⁵ ). Wherein R ⁴ and R ⁵ each independently represents a hydrogen atom or a C _{1 to} C ₁₀ alkyl group, n represents 1, 2, or 3, and m represents an integer of (3-n). .)