WO2017155019A1 - Protein adsorption inhibitor and method for inhibiting adsorption of protein - Google Patents

Abstract

The present invention addresses the problem of providing a protein adsorption inhibitor which can inhibit the non-specific adsorption of a protein, e.g., an antibody and an enzyme, onto a solid surface of a base material, e.g., an immune reaction vessel and a measurement tool, at a high level and which has improved wet-scrub resistance. The problem can be solved by a protein adsorption inhibitor which contains a water-soluble acrylic polymer (P) as an active ingredient, wherein the water-soluble acrylic polymer (P) contains a repeating constituent unit [A] represented by formula (1), has a number average molecular weight of 5,000 to 250,000 and has an LCST of 25 to 45ºC. [R¹ represents a hydrogen atom or a methyl group; R² represents a methyl group or an ethyl group; and n means the average number of moles of an oxyalkylene group added and represents 2 to 3.]

Description

Protein adsorption inhibitor and method for inhibiting protein adsorption

The present invention relates to a non-specific adsorption inhibitor for proteins and a method for inhibiting adsorption. Specifically, a protein adsorption inhibitor that can prevent the adsorption of impurities (proteins) in a sample to the solid phase surface of a substrate such as an immune reaction container or a measurement instrument, and has a high washing resistance, and a protein The present invention also relates to a method for suppressing adsorption, and further to a substrate treated with the adsorption inhibitor.

For early detection of diseases, measurement methods using immune reactions are widely used in the fields of clinical tests and diagnostic agents. In such a situation, it is demanded to increase the sensitivity of the test, and the improvement of the sensitivity of the clinical test and the diagnostic agent is a big problem. In order to increase sensitivity, the detection method is being switched from a method using an enzyme reaction such as peroxidase or alkaline phosphatase to a method using fluorescence or chemiluminescence. Theoretically, it is said that the presence of one molecule to be inspected can be confirmed by using fluorescence or chemiluminescence as a detection method, but in reality, the desired sensitivity cannot be obtained.
As one of the factors that influence the detection sensitivity when measuring using an immune reaction, the antibody to be measured, the antigen, or the label of these labels used for measurement, such as the substrate of an immune reaction container or measuring instrument Non-specific adsorption to the solid surface is mentioned. In addition, when a substance that coexists with multiple types of biomolecules such as serum, plasma, cell extract and urine is used as a specimen, an unspecified number of coexisting substances representing various proteins are used as a base material for immune reaction containers and measuring instruments. Generation of noise due to non-specific adsorption to the solid phase surface is also a factor preventing high sensitivity.

In order to prevent such non-specific adsorption, as shown in Non-Patent Document 1 and Patent Document 1, biologically-derived proteins such as bovine serum albumin, casein, and gelatin that have not been involved in an immune reaction are conventionally used as a buffer. There is a method for suppressing non-specific adsorption of proteins involved in immune reactions by adsorbing biologically-derived proteins on a fixed surface of a substrate such as an immune reaction container or measuring instrument using a treatment solution as a solution. It is used. Furthermore, as a protein adsorption inhibitor mainly composed of a chemically synthesized product, Patent Document 2 contains 2-methacryloyloxyethyl phosphorylcholine polymer, Patent Document 3 contains glycosylethyl (meth) acrylate, and Patent Document 4 contains an oxyalkylene group. Each method using an acrylic polymer is disclosed. In these methods, a chemical synthetic product as a protein adsorption inhibitor is physically adsorbed on a solid phase surface such as an immune reaction container or a measuring instrument, thereby producing an effect.

However, the adsorption suppression ability of the level of the non-specific adsorption inhibitor based on the above-described conventional technology may still be insufficient to suppress the generation of noise due to non-specific adsorption in the high sensitivity measurement. Many. This is not only because the function of the nonspecific adsorption inhibitor is still insufficient, but the nonspecific adsorption inhibitor can be easily removed in the washing process using a buffer solution or an aqueous activator solution after the nonspecific adsorption treatment. This is also because the non-specific adsorption inhibitor for fully exhibiting the non-specific adsorption inhibiting ability in the subsequent process does not remain in the reaction vessel.

Also, biocompatible materials that are used in contact with bodily fluids such as blood or living tissue and can reduce damage to living body components are known (for example, Patent Document 5). However, even when such a biocompatible material is applied to a solid phase surface such as an immune reaction container or a measuring instrument, there may be a problem that the biocompatible material is removed from the solid phase surface in the washing step after the above-described adsorption treatment. .

Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, "Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose.", Gene Anal. Tech.

Japanese Patent Application Laid-Open No. 07-270413 Japanese Patent Application Laid-Open No. 07-083923 JP-A-10-123135 JP 10-153599 A International Publication No. 2004/087228 Pamphlet

As described above, the object of the present invention is to suppress, at a high level, nonspecific adsorption of a protein such as an antibody or an enzyme to the solid phase surface of a substrate such as an immune reaction container or a measurement instrument, and further improve the washing resistance. It is in providing the protein adsorption inhibitor which improves.

As a result of intensive studies in view of the above problems, the present inventors have a specific range of lower critical shared temperature (hereinafter abbreviated as “LCST”) and a polyoxyethylene chain having a specific chain length (meth). The inventors have found that an acrylate copolymer can solve the above problems, and have completed the present invention.
That is, the present invention includes the following [1] to [5].
[1] A water-soluble acrylic polymer containing a repeating structural unit [A] represented by the following formula (1), having a number average molecular weight of 5,000 to 250,000 and an LCST of 25 to 45 ° C. A protein adsorption inhibitor containing P) as an active ingredient.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is an average addition mole number of an oxyalkylene group of 2 to 3. ]
[2] A protein adsorption inhibitor coating solution containing the protein adsorption inhibitor of [1] above and water.
[3] A base material provided with a coating layer of the protein adsorption inhibitor according to [1] on the surface.
[4] A water-soluble acrylic polymer containing a repeating structural unit [A] represented by the following formula (1), having a number average molecular weight of 5,000 to 250,000 and an LCST of 25 to 45 ° C. A method for inhibiting protein adsorption, comprising forming a coating layer on the surface of the substrate by P) and inhibiting adsorption of the protein to the substrate.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is an average addition mole number of an oxyalkylene group of 2 to 3. ]
[5] A water-soluble acrylic polymer containing a repeating structural unit [A] represented by the following formula (1), having a number average molecular weight of 5,000 to 250,000 and an LCST of 25 to 45 ° C. A water-soluble acrylic polymer (P) containing P) and having an LCST of 25 to 45 ° C. is supplied to the surface of the substrate to form a coating layer of the water-soluble acrylic polymer (P) on the surface of the substrate. The method of providing protein adsorption | suction inhibitory property to a base material by doing.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is an average addition mole number of an oxyalkylene group of 2 to 3. ]

[6] The above [4], further comprising contacting the protein adsorption inhibitor containing the water-soluble acrylic polymer (P) with the surface of the substrate at a temperature equal to or lower than the LCST of the water-soluble acrylic polymer (P). The method for inhibiting protein adsorption according to claim 1.

[7] The above [5], further comprising bringing a protein adsorption inhibitor containing the water-soluble acrylic polymer (P) into contact with the surface of the substrate at a temperature equal to or lower than the LCST of the water-soluble acrylic polymer (P). A method for imparting the protein adsorption-suppressing property described in 1.

By removing the aqueous solution or the like in which the protein adsorption inhibitor according to the present invention is dissolved in contact with the solid surface of the substrate at a predetermined temperature, proteins such as antibodies and enzymes are non-specific on the solid surface of the substrate. Adsorption is suppressed at a high level. Furthermore, even when various washings are performed after removing the aqueous solution or the like by bringing it into contact with the solid phase surface of the base material, a protein adsorption inhibitor having improved washing resistance is provided. In addition, since the protein adsorption inhibitor of the present invention is a chemically synthesized product, the protein adsorption inhibitor containing it as an active ingredient is concerned with problems such as differences between lots or biological contamination of biological protein adsorption inhibitors. It exhibits protein adsorption inhibition ability safely and stably.

FIG. 1 is a diagram showing the protein adsorption inhibiting effect of a substrate provided with the coating layer of the protein adsorption inhibitor of Example 1 on the surface.

Hereinafter, the present invention will be described in more detail.
The protein adsorption inhibitor of the present invention contains a water-soluble acrylic polymer (P) as an active ingredient.
(Water-soluble acrylic polymer (P))
The water-soluble acrylic polymer (P) used in the present invention includes a repeating structural unit [A] represented by the following formula (1), and the repeating unit [A] is represented by the following formula (2). Obtained by polymerization of the monomer.

In the formulas (1) and (2), R ¹ is a hydrogen atom or a methyl group, preferably a methyl group. R ² is a methyl group or an ethyl group, preferably a methyl group. n is the average number of added moles of the oxyalkylene group, m = 2 to 3, and preferably 2.
Specific examples of the monomer represented by the formula (2) include methyldiethylene glycol acrylate (R ¹ = H, n = 2, R ² = CH ₃ ), ethyl triethylene glycol acrylate (R ¹ = H, n = 3, R ² = C ₂ H ₅ ), methyl diethylene glycol methacrylate (R ¹ = CH ₃ , n = 2, R ² = CH ₃ ), methyl triethylene glycol methacrylate (R ¹ = CH ₃ , n = 3, R ² = CH ₃ ), methyltriethylene glycol acrylate (R ¹ = H, n = 3, R ² = CH ₃ ) and the like. Among these, methyldiethylene glycol methacrylate (R ¹ = CH ₃ , n = 2, R ² = CH ₃ ) is preferable.

The water-soluble acrylic polymer (P) used in the present invention is selected (co) polymerized by selecting one or more of the monomers represented by the formula (2), and the LCST is 25 to 45 ° C. To be. The LCST range is preferably 30 to 45 ° C, more preferably 33 to 43 ° C. The LCST design of the copolymer can be estimated from the weighted average value of the homopolymer, and the required monomer blending ratio can be estimated.
In addition, LCST (lower critical shared temperature) in the present invention refers to a temperature at which the transmittance at a wavelength of 500 nm is 50% when a 1% by mass aqueous solution is heated at a heating rate of 1 ° C./min.
Further, in the water-soluble acrylic polymer (P), as a repeating unit other than the repeating structural unit [A], other comonomers can be used in combination.

Generally, it is known that polyalkylene glycol (meth) acrylate has intermediate water and hardly adsorbs proteins. At the same time, however, the presence of intermediate water also affects the adsorption to the substrate.
The above-mentioned “intermediate water” is a water molecule exhibiting a specific behavior, and is considered to exist in a material exhibiting biocompatibility according to recent research, for example, as a result of the following observation. For example, a differential scan of the endothermic amount observed when a sample containing water in PMEA (poly (2-methoxyethyl acrylate)) is cooled to −100 ° C. and then heated at a rate of 2.5 ° C./min. When measured with a calorimeter (DSC), a predetermined heat generation occurs in a specific temperature range of 0 ° C. or less (eg, around −40 ° C.), and endotherm is observed in a wide temperature range from around −10 ° C. to 0 ° C. Due to various studies, heat generation near −40 ° C. is due to the regularization of some of the water molecules contained in PMEA, and the endotherm from around −10 ° C. to 0 ° C. In this way, water molecules that exhibit behavior that does not occur in water alone are present in the water-containing PMEA, and this behavior is exhibited. Water is called “intermediate water” It is.
Such “intermediate water” is a biologically derived biocompatible material, such as biologically derived polysaccharides such as hyaluronic acid, heparin, gelatin, proteins such as albumin, nucleic acids such as DNA and RNA, and artificially synthesized materials. It is becoming clear that it is contained in substances having excellent biocompatibility, such as being contained in PEG. Thus, “intermediate water” is considered to be closely related to the development of biocompatibility in substances.
The reason why “intermediate water” is generated in the substance is not clear, but it is the result of the interaction between the polymer chain with high molecular mobility in the substance and the water molecule with a specific intermolecular force. It is thought that there is. In biocompatible materials, the presence of intermediate water between the surface of the material and the hydration shell of the biological component prevents the direct contact between the two and suppresses foreign body reaction. .

LCST is a phase transition temperature similar to a so-called cloud point, and is a temperature at which a polymer such as polyalkylene glycol (meth) acrylate causes a coil-globule transition in an aqueous solution. That is, at a temperature lower than the LCST, the hydrophilicity of the polymer increases and is soluble in water, but the polymer under the temperature exceeding the LCST increases in hydrophobicity and becomes insoluble in water.
The polyalkylene glycol (meth) acrylate according to the present invention is a polymer that has been known to exhibit predetermined biocompatibility. On the other hand, it was revealed by the present invention that the polyalkylene glycol (meth) acrylate functions as a high-performance protein adsorption inhibitor by using a composition having a specific LCST value. That is, by removing an aqueous solution or the like in which the protein adsorption inhibitor according to the present invention is dissolved at a temperature equal to or lower than the LCST of the protein adsorption inhibitor to contact with the solid phase surface of the substrate to be treated, It was revealed that non-specific adsorption of proteins such as enzymes to the solid surface of the substrate can be suppressed at a high level. Furthermore, when various washings are performed after removing the aqueous solution by bringing it into contact with the solid surface of the base material, the effects are maintained. In this case, nonspecific adsorption of protein can be suppressed. Although the mechanism of action causing such a phenomenon has not been elucidated, the specific polyalkylene glycol (meth) acrylate of the present composition is water-soluble at the LCST temperature, but particularly in the temperature range immediately below the LCST. It has a driving force for precipitation, and when the aqueous solution comes into contact with the solid surface, the polymer precipitates in a form such as adsorption to the surface, and as a result, a coating that inhibits nonspecific adsorption of proteins is formed. It is thought to form. That is, according to the present invention, it is possible to realize an excellent protein adsorption inhibitor capable of preventing nonspecific adsorption of a protein to a substrate by a film formed of a polymer containing intermediate water.

The protein adsorption inhibitor is preferably a transparent and uniform solution when used. Because it is transparent, it does not interfere with optical detection even when a protein adsorption inhibitor is contained in the liquid to be evaluated. Also, since the protein adsorption inhibitor is a homogeneous solution, This is because the coating can be easily made uniform, that is, the thickness of the coating can be made uniform. On the other hand, when a protein adsorption inhibitor is used as an opaque solution, a cloudy solution enters the evaluation system, which may harm the detection and evaluation of the in vitro diagnostic agent in the optical system, and the resulting coating Becomes non-uniform, and the ability to suppress protein adsorption may vary.
Since the protein adsorption inhibitor of the present invention contains a water-soluble acrylic polymer (P) having an LCST of 25 to 45 ° C. as an active ingredient, it can be easily transparent and uniform even when used at room temperature without heating. It is also excellent in that it can be.

The water-soluble acrylic polymer (P) used in the present invention has a number average molecular weight of 5,000 to 250,000, preferably 6,000 to 1,000,000, more preferably 7,000 to 200,000. It is. If the number average molecular weight is too low, the ability to suppress the adsorption of protein molecules will not be sufficient, and if it is too high, the water solubility will decrease, so the effects of the present invention may not be exhibited.

In the water-soluble acrylic polymer (P) used in the present invention, it is possible to eliminate a lot-to-lot variation when used in the diagnostic medicine field by aligning the molecular weight distribution. Therefore, the value of the water-soluble acrylic polymer (P) polydispersity (Mw / Mn) is preferably 1.0 to 5.0, more preferably 1.0 to 4.4. Moreover, any of random copolymer, block copolymer, and graft copolymer may be used, and the copolymerization reaction for producing the copolymer is not particularly limited, and includes free radical polymerization, ionic polymerization, It can be used by a known synthesis method such as coordinate polymerization or ring-opening polymerization. For example, it can be prepared by polymerizing a monomer by a solution polymerization method using water or an organic solvent as a solvent. More specifically, one or more monomers described in formula (1) are dissolved in purified water or an organic solvent, and a polymerization initiator is added to the solution while stirring the resulting solution. A water-soluble acrylic polymer (P) can be obtained by polymerizing a monomer in an inert gas atmosphere such as nitrogen gas or argon gas.

Examples of the organic solvent used in the polymerization of the monomer include alcohols such as methyl alcohol, ethyl alcohol, isopropyl alcohol, ethylene glycol, and propylene glycol; ketones such as acetone and methyl ethyl ketone; diethyl ether and tetrahydrofuran. Ethers such as benzene, toluene and xylene, and acetates such as methyl acetate and ethyl acetate, but are not limited thereto.
The concentration of the monomer in the monomer solution used in the solution polymerization method is not particularly limited, but is preferably about 10 to 80% by mass in consideration of the operability and efficiency of polymerization.
The polymerization initiator used above is not particularly limited. For example, azobisisobutyronitrile (hereinafter abbreviated as “AIBN”), azoisobutyronitrile, azoisobutyric acid methyl, azobisdimethylvaleronitrile, peroxide Light of azo polymerization initiators and peroxide polymerization initiators such as benzoyl, potassium persulfate and ammonium persulfate, benzophenone derivatives, phosphine oxide derivatives, benzoketone derivatives, phenylthioether derivatives, azide derivatives, diazo derivatives, disulfide derivatives, etc. An initiator etc. are mentioned. The amount of the polymerization initiator is not particularly limited, but usually it is preferably about 0.01 to 5 parts by mass with respect to 100 parts by mass of the monomer.
The polymer obtained by polymerization can be used after diluting and dissolving in an aqueous medium as it is, but it is preferable to carry out purification to remove impurities. Known methods can be applied for purification. Specifically, a precipitation purification method in which a 10 to 80% by mass solution of a polymer in a good solvent is mixed with a 5 to 50 times poor solvent, and the good solvent solution of the polymer is compatible with the monomer. For example, an adsorption treatment method in which a monomer is separated by contacting with a certain adsorbent, and a membrane separation purification method of a monomer utilizing size separation by membrane separation of a good solvent solution of a polymer.

The protein adsorption inhibitor of the present invention is, for example, a protein, polypeptide, steroid, lipid, hormone, etc., more specifically, an enzyme reaction using various antigens, antibodies, receptors, enzymes, or the like, or an immunoglobulin antigen-antibody reaction It can be used in an immunological measurement method for measuring using Specifically, known radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), latex turbidimetry, etc., particularly preferably enzyme immunoassay (EIA), fluorescence immunity It can be applied to measurement methods (FIA), latex turbidimetry, Western blotting, etc. In these known immunological measurement methods, antibodies or antigens are bound to the solid phase surface, and then the antibody on the solid phase surface Alternatively, protein adsorption is suppressed by treating the solid surface to which no antigen is bound with the protein adsorption inhibitor of the present invention.

The solvent for dissolving the protein adsorption inhibitor for obtaining the protein adsorption inhibitor coating solution of the present invention is not only purified water, pure water, ion-exchanged water, but also a buffer that can be used in an immunological measurement method. Any liquid can be used. For example, phosphate buffer, acetate buffer, carbonate buffer, citrate buffer, Tris buffer, HEPES buffer, physiological saline, and the like can be used.
The protein adsorption inhibitor contained in the protein adsorption inhibitor coating solution is preferably 0.01% by mass or more, and more preferably 0.1% by mass or more. The upper limit is not particularly limited as long as it dissolves in water as the main solvent, but is, for example, 20% by mass or less, preferably 10% by mass or less. This is because an effective protein adsorption suppressing effect can be obtained within these ranges.

Moreover, as a compound which can be contained in a protein adsorption inhibitor coating solution other than the protein adsorption inhibitor of the present invention, the following compounds can be exemplified.
That is, other reagents ordinarily used in this field, such as saccharides, salts, and surfactants. For example, as sugars, lactose, sucrose, trehalose and the like can be mentioned. For example, the salts include amino acids such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, histidine and the like, peptides such as glycylglycine, phosphate, borate, sulfate Examples thereof include inorganic salts such as salts, tris salts, sodium chloride and potassium chloride, flavins, organic acids such as acetic acid, citric acid, malic acid, maleic acid and gluconic acid, and salts of organic acids. Examples of the surfactant include polyoxyethylene sorbitan fatty acid ester and polyoxyethylene alkyl ether.
Furthermore, examples of compounds that can be contained in the coating solution of the protein adsorption inhibitor include water-soluble polymers such as MPC (2-methacryloyloxyethyl phosphorylcholine) polymer and polyvinyl alcohol.

Next, the base material provided with the coating layer of the protein adsorption inhibitor on the surface will be described.
The material of the base material used in the present invention is not particularly limited, but for example, polystyrene, polyvinyl chloride, polypropylene, acrylic resin, polymethyl methacrylate, polycarbonate, glass, metal, ceramic, silicon rubber, polyvinylidene fluoride, Examples thereof include nylon and nitrocellulose. Among these, polystyrene and polyvinylidene fluoride are preferable, and polystyrene is particularly preferable.
Further, the shape of the substrate is not particularly limited, but specific examples include a film (film) shape, a plate shape, a particle shape, and a test tube shape, a vial shape, a flask shape, and the like. can do.

Examples of the method for forming a protein adsorption inhibitor coating layer on these substrates include the following methods. That is, the substrate is immersed in a protein adsorption inhibitor coating solution prepared by dissolving the protein adsorption inhibitor of the present invention in water or a solvent in which water and methanol, ethanol, and isopropanol are mixed at any ratio. Then, it can be dried at room temperature or by heating. Thereby, the coating layer is formed.
In the protein adsorption inhibitor coating solution for forming a coating layer of the protein adsorption inhibitor, the concentration of the protein adsorption inhibitor is preferably 0.1 to 5.0% by mass, more preferably 1.0 to 3%. 0.0% by mass. The solvent for diluting the protein adsorption inhibitor of the present invention is preferably water or a water / alcohol mixed solvent, more preferably water.

Next, a method for using the protein adsorption inhibitor of the present invention will be described.
In the protein adsorption inhibitor of the present invention, as described above, by supplying a coating layer of the adsorption inhibitor on the substrate by supplying it to the surface of the substrate, the protein adsorption to the substrate during various measurements is performed. A method of suppressing adsorption is one form of use. That is, the water-soluble acrylic polymer (P) used in the present invention is adsorbed as the coating layer on the solid phase surface of a substrate such as an immune reaction container or a measurement instrument, so that the protein is deposited on the solid phase surface. It suppresses adsorbing.
In addition to a method in which a substrate having a protein adsorption inhibitor coating layer on its surface is prepared in advance and the substrate is used in an immune reaction container, a measuring instrument or the like, the reagent of the present invention is used in various measurements. The method of adding a protein adsorption inhibitor is mentioned. That is, it is a method of forming a coating layer of the adsorption inhibitor on the substrate as one step of various measurements. In addition, the protein adsorption inhibitor of this invention can also be added and used for all the reagents and solutions at the time of measuring.
In such a method of use, the concentration of the protein adsorption inhibitor in each reagent and solution is preferably 0.0125 to 5.0% by mass, more preferably 0.1 to 5.0% by mass. . However, when a protein adsorption inhibitor is added as one step of various measurements, the protein adsorption inhibitor is added before adding a protein-containing sample such as serum, labeled antibody, or labeled antigen as the measurement target.

Moreover, the following method can be mentioned as another usage pattern.
That is, a protein or the like that can bind to a target component contained in a sample such as an antibody or an antigen is first bound to a fixed surface of a substrate such as the above-described immune reaction container or measurement instrument, and then the protein adsorption of the present invention is performed. This is a method of treating with an inhibitor. For example, when using a polystyrene plate, the protein binding inhibitor of the present invention is physically or chemically adsorbed to a protein that binds to the target component, for example, a substance that reacts with the measurement target, on the plate. It is the method of processing with. In other words, after adsorbing a substance that can bind to the measurement object on the plate surface, the adsorption inhibitor of the present application is adsorbed to suppress adsorption of proteins to the plate surface on which the substance that binds to the measurement object is not adsorbed. It is a method to make it. As a result, a solid surface having a protein adsorption inhibiting effect can be obtained.
As a base material in such a usage method, the base material of the same kind and shape as the above-mentioned "base material provided with a coating layer of a protein adsorption inhibitor on its surface" can be exemplified.

<Synthesis Example 1>
<Synthesis of methoxydiethylene glycol monoacrylate homopolymer>
15 g of methoxydiethylene glycol monoacrylate as a monomer was weighed into a four-necked flask, 15 mg of azobisisobutyronitrile as a radical polymerization initiator was added, and dissolved in 60 g of 1,4-dioxane at room temperature. After confirming dissolution of the initiator, the temperature was raised to 75 ° C. with an oil bath, and after reaching 75 ° C., a polymerization reaction was carried out for 8 hours. After the completion of the polymerization reaction, 1,000 mL of the reaction solution was diluted and washed with about 1,000 mL of hexane, the organic phase was removed, and the product was recovered. This product was redissolved in 70 mL of tetrahydrofuran and washed again with n-hexane. As a result, the product was recovered and dried under reduced pressure all day and night to obtain the target oxyalkylene polymer. ¹ H-NMR measurement confirmed that the product was a methoxydiethylene glycol monoacrylate homopolymer. In addition, as a result of GPC molecular weight analysis, the number average molecular weight (Mn) was 13,000. Furthermore, an aqueous solution containing 1% by mass of the polymer was prepared, and the LCST of the polymer in the aqueous solution was confirmed to be 43.1 ° C. by UV measurement at a wavelength of 500 nm.
As described above, LCST (lower critical shared temperature) in the present invention refers to a temperature at which the transmittance at a wavelength of 500 nm is 50% when a 1% by mass aqueous solution is heated at a rate of temperature increase of 1 ° C./min. . The LCST measurement was performed using a UV-visible spectrophotometer V-650 manufactured by JASCO Corporation. 3 mL of a 1 wt% aqueous solution was placed in a cell with an optical path length of 1.0 cm, and the temperature was raised at a rate of temperature increase of 1 ° C / min. Measured based on the transmittance value of the aqueous solution.
In the water-soluble acrylic polymer (P) used in the present invention, one or more of the monomers represented by the formula (2) are selected (co) polymerized, and the LCST is 25 to 45 ° C. To be. The LCST range is preferably 30 to 45 ° C, more preferably 33 to 43 ° C. The LCST design of the copolymer can be estimated from the weighted average value of the homopolymer, and the required monomer blending ratio can be estimated.

<Synthesis Example 2>
<Synthesis of ethoxytriethylene glycol monoacrylate homopolymer>
The same procedure as in Synthesis Example 1 was performed except that ethoxytriethylene glycol monoacrylate was used as a raw material. It was confirmed by ¹ H-NMR measurement that it was an ethoxytriethylene glycol monoacrylate homopolymer. The number average molecular weight (Mn) was 14,000 and the LCST was 33.8 ° C.

<Synthesis Example 3>
<Synthesis of methoxytriethylene glycol monomethacrylate homopolymer>
The same procedure as in Synthesis Example 1 was performed except that methoxytriethylene glycol monomethacrylate was used as a raw material. It was confirmed by ¹ H-NMR measurement that it was a methoxytriethylene glycol monomethacrylate homopolymer. The number average molecular weight (Mn) was 105,000 and the LCST was 42.5 ° C.

<Synthesis Example 4>
<Synthesis of Methoxydiethylene Glycol Monomethacrylate-Methoxytriethylene Glycol Monoacrylate Copolymer (50/50)>
The procedure was the same as in Synthesis Example 1 except that 6.9 g of methoxydiethylene glycol monomethacrylate and 8.1 g of methoxytriethylene glycol monoacrylate were used as raw materials, for a total of 15 g. ¹ H-NMR measurement confirmed that the product was a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monoacrylate = 47/53; molar ratio). did. The number average molecular weight (Mn) was 48,000, and the LCST was 37.2 ° C.

<Synthesis Example 5>
<Synthesis of Methoxydiethylene Glycol Monomethacrylate-Methoxytriethylene Glycol Monomethacrylate Copolymer (60/40)>
The procedure was the same as in Synthesis Example 1 except that 8.2 g of methoxydiethylene glycol monomethacrylate and 6.8 g of methoxytriethylene glycol monomethacrylate were used as raw materials, for a total of 15 g. ¹ H-NMR measurement confirmed that the product was a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monomethacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monomethacrylate = 58/42; molar ratio). did. The number average molecular weight (Mn) was 188,000 and the LCST was 35.1 ° C.

<Synthesis Example 6>
<Synthesis of Methoxydiethylene Glycol Monomethacrylate-Methoxytriethylene Glycol Monoacrylate Copolymer (40/60)>
The procedure was the same as in Synthesis Example 1 except that 5.5 g of methoxydiethylene glycol monomethacrylate and 9.5 g of methoxytriethylene glycol monoacrylate were used as raw materials, for a total of 15 g. ^{According to 1} H-NMR measurement, the product is a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monoacrylate copolymer = 35/65; molar ratio). It was confirmed. The number average molecular weight (Mn) was 7,000 and the LCST was 37.2 ° C.

<Synthesis Example 7>
<Synthesis of methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (50/50, low molecular weight)>
The same procedure as in Synthesis Example 1 was performed except that 3.5 g of methoxydiethylene glycol monomethacrylate and 4.0 g of methoxytriethylene glycol monoacrylate were used as raw materials, for a total of 7.5 g and 30 mg of initiator. ^{According to 1} H-NMR measurement, the product is a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monoacrylate copolymer = 47/53; molar ratio). It was confirmed. The number average molecular weight (Mn) was 17,000, and the LCST was 37.2 ° C.

<Comparative Synthesis Example 1>
<Synthesis of methoxytriethylene glycol monoacrylate homopolymer>
The same procedure as in Synthesis Example 1 was performed except that methoxytriethylene glycol monomethacrylate was used as a raw material. ¹ H-NMR measurement confirmed that the product was a methoxytriethylene glycol monoacrylate copolymer. The number average molecular weight (Mn) was 12,000 and the LCST was 63.8 ° C.

<Comparative Synthesis Example 2>
<Synthesis of 2-methoxyethyl vinyl ether homopolymer>
15 g of 2-methoxyethyl vinyl ether monomer as a raw material is diluted with 15 g of dry ethyl acetate, weighed into a four-necked flask, added with 15 mL of a 1-butoxyethyl acetate 40 mM toluene solution as a polymerization initiator, and argon in 180 mL of dry toluene After cooling to 0 ° C. under a stream of air, 3.75 mL of a 1M toluene solution of Et _1.5 AlCl _1.5 was added as a Lewis acid while stirring. After stirring this at 0 ° C. for 30 minutes, 22.5 mL of ethanol was added to the reaction solution to stop the reaction. After completion of the polymerization reaction, the reaction solution was diluted with toluene and washed, and the solvent was distilled off from the organic phase to obtain a product. Subsequently, it was washed three times with warm water at 80 ° C. The product was collected and dried under reduced pressure all day and night to obtain the desired oxyalkylene polymer. The structure of the obtained polymer was confirmed to be 2-methoxyethyl vinyl ether homopolymer by ¹ H-NMR measurement. The number average molecular weight (Mn) was 26,000, and the LCST was 69.0 ° C.

<Comparative Synthesis Example 3>
<Synthesis of Methoxydiethylene Glycol Monomethacrylate-Methoxytriethylene Glycol Monoacrylate Copolymer (20/80, Low Molecular Weight)>
The same procedure as in Synthesis Example 1 was performed except that 1.3 g of methoxydiethylene glycol monomethacrylate and 6.2 g of methoxytriethylene glycol monoacrylate were used as raw materials to make a total of 7.5 g and the initiator was 30 mg. ^{According to 1} H-NMR measurement, the product is a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monoacrylate copolymer = 15/85; molar ratio). It was confirmed. The number average molecular weight (Mn) was 4,000 and the LCST was 52.0 ° C.

<Comparative Synthesis Example 4>
<Synthesis of methoxytetraethylene glycol monoacrylate homopolymer>
The same procedure as in Synthesis Example 1 was performed except that methoxytetraethylene glycol monoacrylate was used as a raw material. ¹ H-NMR measurement confirmed that the product was a methoxytetraethylene glycol monoacrylate homopolymer. The number average molecular weight (Mn) was 87,000, and the LCST was 75 ° C. or higher.

<Comparative Synthesis Example 5>
<Synthesis of methoxytetraethylene glycol monomethacrylate homopolymer>
The same procedure as in Synthesis Example 1 was performed except that methoxytetraethylene glycol monomethacrylate was used as a raw material. ¹ H-NMR measurement confirmed that the product was a methoxytetraethylene glycol monomethacrylate homopolymer. The number average molecular weight (Mn) was 122,000 and the LCST was 75 ° C. or higher.
<Comparative Synthesis Example 6>
<Synthesis of methoxydiethylene glycol monomethacrylate homopolymer>
The same procedure as in Synthesis Example 1 was performed except that methoxydiethylene glycol monomethacrylate was used as a raw material. It was confirmed by ¹ H-NMR measurement that the product was a methoxydiethylene glycol monomethacrylate homopolymer. The number average molecular weight (Mn) was 25,000 and the LCST was 23.5 ° C.

The meanings of the abbreviations in the table are as follows.
* Mn: Number average molecular weight * * LCST: Lower Critical Solution Temperature

<Example 1>
<Preparation of protein adsorption inhibiting solution>
The polymer of Synthesis Example 1 was mixed in Dulbecco's phosphate buffer solution (manufactured by Sigma-Aldrich, hereinafter abbreviated as D-PBS) so as to be 0.1% by mass, and stirred and dissolved in a vortex mixer for 1 hour to adsorb protein. An inhibitor coating solution was obtained. The coating solution was transparent at room temperature, and it was considered that the whole amount of the charged polymer was dissolved.

<Evaluation of protein adsorption inhibition effect>
About the protein adsorption inhibitor coating liquid of the said Example 1, the protein adsorption | suction inhibitory effect was measured with the following methods.
A protein adsorption inhibitor coating solution was dispensed at 200 μL / well on MaxiSoap plate (a polystyrene plate manufactured by Thermofisher Scientific) and allowed to stand at room temperature for 2 hours. Thereafter, the solution was completely removed with an aspirator, one of which was left as it is (the result is shown in the column labeled “not washed” in Table 3), and the other was immediately dispensed with 200 μL / well of D-PBS. The operation of removing the liquid with an aspirator was repeated 5 times each (in Table 3, the result is shown in the column labeled “After washing”). Subsequently, POD-IgG (peroxidase-labeled immunoglobulin G, manufactured by Biorad) diluted 20,000 times with D-PBS was dispensed at 100 μL / well and allowed to stand at room temperature for 2 hours. Thereafter, D-PBS containing Tween 20 at a concentration of 0.05% was dispensed at 200 μL / well, and the solution was completely removed with an aspirator. This operation was repeated 4 times to wash the plate surface. After washing, 100 μL / well of a color developing solution prepared using TMB Microwell Peroxidase Substrate (manufactured by KPL) was added and reacted at room temperature for 7 minutes. The coloring reaction was stopped by dispensing a 1 mol / L sulfuric acid solution into 50 μL / well, the absorbance at 450 nm was measured, and the adsorbed protein was detected. A smaller absorbance indicates that protein adsorption is suppressed. For absorbance measurement, Spectra Max M3 (Molecular Device) was used. The evaluation results are shown in Table 3.

The effect of the protein adsorption inhibitor according to the present invention will be described based on the schematic diagram of FIG. For example, when the wall surface 12 of the immune reaction container 10 is used as a base material, a sample to be measured such as the antigen 14 is adsorbed to the antibody 16 on the left side of FIG. On the other hand, when the protein adsorption inhibitor 18 exerts an adsorption inhibiting effect, it is considered that adsorption of a specimen such as the antigen 14 is prevented like the antibody 16 on the right side of FIG. As will be described in detail later, according to the protein adsorption inhibitor of the present invention, as shown on the right side of FIG. 1, adsorption of the specimen to the antibody 16 is effectively suppressed.

<Evaluation of washing resistance>
The washing resistance of the protein adsorption inhibitor coating solution was evaluated based on the “difference in protein adsorption rate” between “when not washed” and “after washing”. It can be said that the adsorption inhibitor having a small value of “difference in protein adsorption rate” (Δ%) was well retained on the surface of the base material even after being washed, and can be said to exhibit good washing resistance.

<Example 2 to Example 7>
Synthesis Example 2-7 A protein adsorption inhibitor coating solution was prepared in the same manner as in Example 1 except that the polymer was used. Each coating solution was transparent at room temperature, and it was considered that the total amount of each charged polymer was dissolved. Furthermore, the protein adsorption inhibitory effect and washing resistance were evaluated in the same manner as in Example 1. The results are shown in Table 3.

<Example 8>
The polymer of Synthesis Example 4 was mixed with Dulbecco's phosphate buffer solution (manufactured by Sigma-Aldrich, hereinafter abbreviated as D-PBS) so as to be 0.04% by mass, and stirred and dissolved in a vortex mixer for 1 hour to adsorb protein. An inhibitor coating solution was obtained. The coating solution was transparent at room temperature, and it was considered that the whole amount of the charged polymer was dissolved. Otherwise, the evaluation was performed in the same manner as in Example 1.
<Comparative Examples 1 to 5>
A solution was prepared in the same manner as in Example 1 except that the compounds of Comparative Synthesis Examples 1 to 5 were used instead of the polymer of Synthesis Example 1. Each solution was transparent at room temperature, and it was considered that the total amount of each charged polymer was dissolved. Furthermore, the protein adsorption inhibitory effect and washing resistance were evaluated in the same manner as in Example 1. The results are shown in Table 4.
<Comparative Example 6>
The compound of Comparative Synthesis Example 6 was used in place of the polymer of Synthesis Example 1, but turbidity occurred, so the protein adsorption inhibitory effect and washing resistance test were stopped.
A protein adsorption inhibiting solution was prepared in the same manner as in Example 1, except that protein (BSA) derived from albumin bovine serum was used instead of the polymer of Synthesis Example 1. Furthermore, the protein adsorption inhibitory effect and washing resistance were evaluated in the same manner as in Example 1. The results are shown in Table 4.

The substrates treated with the protein adsorption inhibitor of the present invention (Examples 1 to 8) were compared with the substrates treated with other oxyalkylene group-containing acrylic polymers (Comparative Examples 1 to 5). And has the ability to suppress nonspecific protein adsorption. Such a result is that when the acrylic polymer (P) according to the present invention is brought into contact with the aqueous solution, a coating layer of the acrylic polymer (P) is formed on the surface, and protein adsorption is suppressed by the coating layer. It is considered a thing.
That is, the protein adsorption inhibitor of each example has an overall "protein adsorption rate" value of "when not washed" and "after washing" as compared with the protein adsorption inhibitor of the comparative example. Since it was low, it was confirmed that the protein adsorption inhibitor of the present invention is excellent in nonspecific protein adsorption inhibiting ability.
Furthermore, it was confirmed that the protein adsorption inhibitor of the present invention has excellent washing resistance. That is, in each Example of the present invention, the difference in protein adsorption rate from the unwashed case is also obtained after further washing after contacting the aqueous solution of the protein adsorption inhibitor with the substrate. The corresponding value of washing resistance (Δ%) is small, which indicates that the adsorption inhibitor was well retained on the surface of the substrate even after washing. On the other hand, in each comparative example, since the value of the washing resistance (Δ%) was large, it was confirmed that the adsorption inhibitor was easily detached from the surface of the substrate by washing. In particular, the substrate provided with the coating layers shown in Examples 4, 5, 7, and 8 on the surface had a protein adsorption inhibiting ability and washing resistance more predominately.
As a reference comparative example, BSA whose results are shown in Table 4 is known to be excellent in protein adsorption inhibitor and washing resistance. Even when compared with such a reference comparative example, it can be seen that the protein adsorption inhibitors of the respective examples are substantially equivalent in adsorption inhibition effect and have higher durability cleaning properties. In particular, the protein adsorption inhibitors of Examples 4, 5, 7, and 8 have better protein adsorption inhibition ability after washing than BSA as a reference example. As described above, it was confirmed that the stability of the analysis accuracy can be improved by using the protein adsorption inhibitor of the present invention.

10: Immune reaction container 12: Wall surface of the immune reaction container 14: Antigen (specimen)
16: Antibody 18: Protein adsorption inhibitor

Claims (7)

A water-soluble acrylic polymer (P) containing a repeating structural unit [A] represented by the following formula (1), having a number average molecular weight of 5,000 to 250,000 and an LCST of 25 to 45 ° C. A protein adsorption inhibitor contained as an active ingredient.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is an average addition mole number of an oxyalkylene group of 2 to 3. ] A protein adsorption inhibitor coating solution comprising the protein adsorption inhibitor of claim 1 and water. A base material provided with a coating layer of the protein adsorption inhibitor according to claim 1 on a surface thereof. A water-soluble acrylic polymer (P) containing a repeating structural unit [A] represented by the following formula (1), having a number average molecular weight of 5,000 to 250,000 and an LCST of 25 to 45 ° C. A method for inhibiting protein adsorption, wherein a coating layer is formed on the substrate surface to inhibit adsorption of protein to the substrate.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is an average addition mole number of an oxyalkylene group of 2 to 3. ] A water-soluble acrylic polymer (P) containing a repeating structural unit [A] represented by the following formula (1), having a number average molecular weight of 5,000 to 250,000 and an LCST of 25 to 45 ° C. A method for imparting protein adsorption inhibition to a substrate by supplying the substrate surface and forming a coating layer of a water-soluble acrylic polymer (P) on the substrate surface.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is an average addition mole number of an oxyalkylene group of 2 to 3. ] The protein adsorption inhibitor containing the said water-soluble acrylic polymer (P) is further contacted with the surface of the said base material at the temperature below LCST of the said water-soluble acrylic polymer (P). Of inhibiting protein adsorption. The protein adsorption inhibitor containing the water-soluble acrylic polymer (P) is further brought into contact with the surface of the substrate at a temperature equal to or lower than the LCST of the water-soluble acrylic polymer (P). A method for imparting protein adsorption inhibitory property.

Images