Surface treatment agent, substrate provided with coating layer comprising same on surface, and method for inhibiting protein adsorption

Abstract

A surface treatment agent according to the present invention contains a compound represented by formula (1) as an active ingredient.　According to the present invention, it is possible to provide a surface treatment agent which is easily produced and with which it is possible to create a surface that suppresses nonspecific adsorption of proteins without performing a pretreatment on a substrate. [In formula (1), n represents an integer of 11-23,000.]

Description

Surface treatment agent, substrate having a coating layer thereon on its surface, and method for inhibiting protein adsorption

[0001] This invention relates to a surface treatment agent. More specifically, it relates to a surface treatment agent used to prevent the adsorption of proteins onto a substrate. The invention further relates to a substrate having a coating layer made of a surface treatment agent on its surface, and to a method for suppressing protein adsorption using this surface treatment agent.

[0002] Biomaterials are materials that come into direct or indirect contact with living organisms. When a biomaterial is recognized as a foreign substance within the body, an immune response is triggered to remove it. Therefore, biomaterials need to be highly biocompatible, as they suppress the adsorption of biological components such as proteins that trigger immune responses. A widely used method for imparting biocompatibility to biomaterials is to apply a surface treatment agent to their surface to prevent the adsorption of biological components.

[0003] Surface treatment agents are used as one method to create surfaces that prevent the non-specific adsorption of proteins to biomaterials. To date, derivatives such as polyethylene glycol (PEG), poly(2-methoxyethyl acrylate), and 2-methacryloyloxyethyl phosphorylcholine have been used as surface treatment agents. Among these, many surface treatment agents containing PEG in their structure have been reported, as shown in Patent Documents 1 to 3. These surface treatment agents exert their effects by physically adsorbing or chemically bonding to the surface of the biomaterial substrate.

[0004] International Publication No. 2005 / 010529, Japanese Patent Publication No. 2011-50469, Japanese Patent Publication No. 2006-226982

[0005] However, surface treatment agents disclosed in Patent Documents 1 and 2 immobilize the surface treatment agent on the substrate surface via chemical bonding, requiring pretreatment such as modifying the substrate surface with functional groups that can chemically bond. As a result, the usable substrates are limited to those that can be pretreated. Furthermore, protein adsorption inhibitors such as those in Patent Document 3 require complicated purification processes and many synthesis steps during manufacturing. In view of the above problems, the object of the present invention is to provide a surface treatment agent that is easy to manufacture and can produce a surface that suppresses nonspecific adsorption of proteins without pretreatment of the substrate. Another object of the present invention is to provide a surface treatment agent that can detect specific reactions of proteins and can be applied to the field of biosensing.

[0006] Therefore, in view of the above problems, the present inventors conducted diligent studies and found that by using a compound that can be easily manufactured as shown in formula (1) as a surface treatment agent, the surface treatment agent can be immobilized on the substrate surface by physical adsorption, thereby suppressing the nonspecific adsorption of proteins to the substrate. Furthermore, they found that by using a substrate treated with the compound of formula (1) in an immunoassay, background noise can be reduced and signals originating from antigen-antibody complexes can be sufficiently detected, thus completing the present invention. That is, the present invention is as follows [1] to [3].

[0007] [1] A surface treatment agent containing the compound shown in formula (1) as an active ingredient. [In formula (1), n ​​is an integer between 11 and 23,000.] [2] A substrate having a coating layer on its surface made of the surface treatment agent described in [1] above. [3] A method for suppressing protein adsorption, comprising treating the surface of the substrate with the surface treatment agent described in [1] above to suppress the adsorption of proteins onto the surface of the substrate.

[0008] The compound of formula (1), which is the active ingredient of the surface treatment agent of the present invention, can effectively adsorb to the substrate surface, thereby highly suppressing the adsorption of proteins to the substrate surface. Since the compound of formula (1) is immobilized on the substrate surface by physical adsorption, it is possible to easily create a protein adsorption-suppressing surface on the substrate without pretreatment of the substrate surface. Furthermore, the manufacturing process of the compound of formula (1) is a one-step reaction, and it can be purified by adsorbent treatment, making it easy to manufacture.

[0009] <Surface Treatment Agent> The present invention will be described in further detail below. The surface treatment agent of the present invention can be used as a surface treatment agent to create a protein adsorption-inhibiting surface on a substrate surface in order to prevent biological responses caused by the adsorption of biological components such as proteins onto the surface of a biomaterial. For example, the surface treatment agent of the present invention can be applied as a surface treatment agent to medical materials such as artificial organs, medical stents, and catheters. By forming a coating layer of the surface treatment agent of the present invention on the substrate surface of these medical materials in advance, the adsorption of biological proteins onto the substrate surface is suppressed. From another viewpoint, it can be used in immunological assays that utilize various antigens, antibodies, receptors, enzymes, etc., and that measure using enzymatic reactions or antigen-antibody reactions. It can be applied to known immunological assays such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), fluorescence immunoassay (FIA), chemiluminescence immunoassay (CLIA), latex turbidimetry, and Western blotting. In these known immunological assays, after binding an antibody or antigen to the substrate surface, the portion of the substrate surface to which the antibody or antigen is not bound is treated with the surface treatment agent of the present invention to suppress nonspecific protein adsorption.

[0010] The active ingredient of the surface treatment agent of the present invention is the compound shown in formula (1). In formula (1), n ​​is an integer between 11 and 23,000. If n is less than 11, it may become poorly soluble in aqueous solvents, and if it exceeds 23,000, the affinity with the substrate will be low, and when the substrate is washed with an aqueous solvent, the compound of formula (1) may peel off, potentially reducing the protein adsorption inhibitory ability. From the viewpoint of ease of solubility in aqueous solvents and affinity with the substrate, n is preferably between 11 and 2,300, more preferably between 11 and 1,000, even more preferably between 11 and 500, even more preferably between 11 and 200, and even more preferably between 15 and 150.

[0011] The compound of formula (1) can be synthesized, for example, by dissolving cholesterol in an organic solvent such as toluene in the presence of a basic catalyst such as sodium methoxide, and reacting it with ethylene oxide at 120°C. By changing the charging ratio of cholesterol and ethylene oxide used as raw materials, any compound in formula (1) between 11 and 23,000 integers can be synthesized.

[0012] In addition, compounds other than the compound shown in formula (1) that may be contained in the surface treatment agent include the following examples: other reagents commonly used in this field, such as nitrogen-containing compounds and their salts, including hydroxides, primary amines, secondary amines, and tertiary amines; amino acids and amino acid salts, including glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, and histidine; peptides, including glycylglycine; sugars, including glucose and sucrose; polyalcols, including mannitol and sorbitol; inorganic salts, including phosphates, borates, sulfates, and Tris salts; flavins; organic acids and organic acid salts, including acetic acid, carbonic acid, citric acid, malic acid, maleic acid, gluconic acid, and stearic acid; and cellulose derivatives, such as polyethylene glycol, polypropylene glycol, poly(2-methacryloyloxyethyl phosphorylcholine), and hydroxyethylcellulose, which are used as thickeners and stabilizers.

[0013] The amount of the compound represented by formula (1) contained in the surface treatment agent of the present invention may be 100% by mass, but if it is 50% by mass or more, it can exert the above-mentioned effects as an active ingredient.

[0014] The surface treatment agent of the present invention can preferably be dissolved in a solvent or buffer solution and used as a surface treatment agent solution. As the solvent, water such as purified water, pure water, or deionized water, or alcohols such as methanol, ethanol, or isopropanol can be used. As the buffer solution, any buffer solution used in the biomaterials field can be used, such as phosphate buffer, acetate buffer, carbonate buffer, citrate buffer, Tris buffer, HEPES buffer, or physiological saline. When treating the substrate surface with the surface treatment agent solution of the present invention to form a coating layer, as described later, it is preferable to use water, methanol, ethanol, isopropanol, or a mixture of these in any proportion from the above solvents, but a buffer solution may also be used. The compound represented by formula (1) contained in the surface treatment agent solution is preferably 0.001% by mass or more, and more preferably 0.01% by mass or more. As for the upper limit, there is no limit as long as it is dissolved in the solvent or buffer solution, but for example, it is 20% by mass or less, preferably 10% by mass or less. Within these ranges, the surface treatment agent solution shows an effective protein adsorption inhibitory effect.

[0015] <Substrates with a coating layer made of a surface treatment agent on their surface> Next, substrates having a coating layer of a surface treatment agent on their surface will be described. Examples of substrates used in the present invention include immunoassay vessels, measuring instruments, plates, magnetic microparticles, artificial organs, blood bags, dental materials, artificial joints, urine collection bags, sutures, and various catheters. The materials are not particularly limited, but examples include ferric oxide coated with hydroxyl groups, carboxyl groups, tosyl groups, amino groups, avidin, streptavidin, biotin, protein A, protein G, albumin, etc., nickel, cobalt, iron nitride, spinel-type ferrite containing manganese, copper, cobalt, nickel, zinc, etc. in its composition, polyethylene, polystyrene, polypropylene, (meth)acrylic resin, polymethyl methacrylate, glass, ceramic, polycarbonate, polyphthalamide, polyphenylsulfone, polyvinyl chloride, polyvinyl acetate, polyolefin, polyurethane, polyamide, polyimide, metal, silicone rubber, polyvinylidene fluoride, nylon, silica, etc. Examples of shapes include granular, plate-like, lump-like, and cylindrical. Among these, granular ferric oxide coated with carboxyl groups and polystyrene are preferred. Alternatively, plate-like polystyrene is preferred. As a method for forming a coating layer of the surface treatment agent of the present invention on the surface of these substrates, for example, one method is to immerse the substrate in a surface treatment agent solution prepared by dissolving the surface treatment agent of the present invention in water, ethanol, isopropanol, or a solvent or buffer solution prepared by mixing these in any proportion as described above. In this way, the surface of the substrate can be treated with the surface treatment agent to suppress the adsorption of proteins to the substrate surface. The concentration of the compound represented by formula (1) in the surface treatment agent solution for forming the coating layer is preferably 0.001 to 20.0% by mass, more preferably 0.005 to 10.00% by mass, and even more preferably 0.01 to 5.00% by mass.

[0016] <Method of Use> Next, we will explain how to use the surface treatment agent of the present invention in immunological assays. As described above, one form of the surface treatment agent of the present invention is to add the surface treatment agent solution to reagents used in various immunological assays, thereby forming a coating layer on the surface of the substrate and suppressing the adsorption of proteins to the substrate during various assays. In other words, it is a method of forming a coating layer of the surface treatment agent on the substrate as one step in various assays. The surface treatment agent of the present invention can also be added to any reagent or solution other than the sample to be measured when performing the assay.

[0017] Furthermore, in addition to adding the surface treatment agent of the present invention to reagents used in various measurements, another method of using the surface treatment agent of the present invention is to first apply a coating layer of the surface treatment agent of the present invention to the surface of a substrate such as an immunoassay vessel or measuring instrument before use. That is, the compound shown in formula (1) adsorbs onto the surface of the substrate such as an immunoassay vessel or measuring instrument as a coating layer, thereby suppressing the adsorption of proteins onto the surface. In such a method of use, the concentration of the compound shown in formula (1) in each reagent and solution is preferably 0.001 to 20.0% by mass, more preferably 0.005 to 10.00% by mass, and even more preferably 0.01 to 5.00% by mass. However, when adding the surface treatment agent to a reagent or solution as a step in various measurements, it should be done before adding the sample, labeled antibody, or labeled antigen, which is the object to be measured and contains proteins. Another form of use is to first bind proteins contained in the sample, such as enzymes, labeled antibodies, or labeled antigens, to the surface of the substrate such as an immunoassay vessel or measuring instrument, and then treat the surface of the substrate with the surface treatment agent of the present invention. For example, when using magnetic microparticles having a coating layer of carboxyl groups of ferric oxide, the protein to be measured is physically adsorbed or chemically bonded to the microparticles, washed with a suitable solvent, and then the surface treatment agent solution of the present invention is brought into contact with them. In other words, after adsorbing the substance to be measured onto the surface of the microparticles, the surface treatment agent of the present invention is immobilized to suppress the adsorption of protein to the substrate surface portion where the substance has not been adsorbed. This makes it possible to obtain a substrate that has a protein adsorption suppression effect. Examples of substrates in such usage methods include substrates of the same type and shape as the "substrate having a coating layer made of a surface treatment agent on its surface" described above.

[0018] The present invention will be described in more detail by the following examples and comparative examples, but the present invention is not limited thereto. In these examples, the compound (1) synthesized as described in Synthesis Examples 1 to 3 was used as the active ingredient of each surface treatment agent.

[0019] <Synthesis of the compound shown in formula (1)> (Synthesis example 1: Chol-PEG) １０００(Synthesis) 350 g (0.905 mol) of cholesterol, 7.61 g of 28% sodium methoxide, and 1000 g of toluene were added and heated to 110°C under nitrogen, and 500 g of toluene was removed by distillation. The mixture was cooled to 80°C, and 917 g (20.8 mol) of ethylene oxide was added. The reaction was carried out at 120°C, and the reaction was stopped by cooling when the molecular weight of PEG reached 1000 by TOF-MS. The reaction solution was neutralized with phosphoric acid, and impurities were removed with an adsorbent to obtain a white powder called Chol-PEG. １０００ (The compound with n=23 in equation (1)) was obtained.

[0020] (Synthesis Example 2 Chol-PEG ２０００ (Synthesis) 350 g (0.905 mol) of cholesterol, 7.61 g of 28% sodium methoxide, and 1000 g of toluene were added and heated to 110°C under nitrogen, and 500 g of toluene was removed by distillation. The mixture was cooled to 80°C, and 2050 g (46.6 mol) of ethylene oxide was added. The reaction was carried out at 120°C, and the reaction was stopped by cooling when the molecular weight of PEG reached 2000 by TOF-MS. The reaction solution was neutralized with phosphoric acid, and impurities were removed with an adsorbent to obtain a white powder called Chol-PEG. ２０００ (The compound n=46 in equation (1)) was obtained.

[0021] (Synthesis example 3 Chol-PEG ５０００ (Synthesis) 350 g (0.905 mol) of cholesterol, 7.61 g of 28% sodium methoxide, and 1000 g of toluene were added and heated to 110°C under nitrogen, and 500 g of toluene was removed by distillation. The mixture was cooled to 80°C, and 4535 g (103 mol) of ethylene oxide was added. The reaction was carried out at 120°C, and the reaction was stopped by cooling when the molecular weight of PEG reached 5000 by TOF-MS. The reaction solution was neutralized with phosphoric acid, and impurities were removed with an adsorbent to obtain a white powder called Chol-PEG. ５０００ (The compound with n=114 in equation (1)) was obtained.

[0022] (Example 1-1-1) <Preparation of Surface Treatment Agent Solution> The compound from Synthesis Example 1 was dissolved in Dulbecco's phosphorate buffered saline (Sigma Aldrich, hereafter abbreviated as D-PBS) to a concentration of 1.0% by mass to prepare a surface treatment agent solution. <Preparation of Antibody Diluent Containing Surface Treatment Agent> Goat Anti-Mouse IgG (H+L) horseradish peroxidase conjugate (Bio-Rad) was prepared by diluting it 100-fold and mixing it with the surface treatment agent solution prepared as described above to prepare an antibody diluent containing the surface treatment agent. <Evaluation of Protein Adsorption Inhibitory Effect> The protein adsorption inhibitory effect of the antibody diluent containing the above surface treatment agent was measured by the following method. Dynabeads TM MyOne TMCarboxylic Acid (manufactured by Thermo Fisher Scientific, hereafter referred to as magnetic nanoparticles) was diluted 50-fold with D-PBS. 100 μL / well of the magnetic nanoparticle suspension was dispensed into Eppendorf® Microplate 96 / V-PP (manufactured by Eppendorf). The magnetic nanoparticles were settled using a magnet, and the supernatant was completely removed. Then, 100 μL / well of D-PBS was dispensed to replace the buffer solution for the magnetic nanoparticles. The magnetic nanoparticles were settled using a magnet, and the supernatant was discarded again. Then, 100 μL / well of the antibody diluent containing the prepared surface treatment agent was dispensed. After dispersing the magnetic nanoparticles, the solution was allowed to stand at room temperature for 1 hour. After using a magnet to settle the magnetic particles and discarding the supernatant again, 100 μL / well of phosphate buffer containing 0.05% by mass Tween 20 (polyoxyethylene sorbitan monolaurate) (hereafter referred to as PBS-T) was dispensed, and the supernatant was completely removed using a magnet. The magnet was removed, 100 μL / well of PBS-T was dispensed, and the entire solution was transferred to an unused well. After completely removing the supernatant using a magnet, the solution was removed from the magnet, and 100 μL / well of a solution prepared by mixing QuantaBlue Substrate Solution and QuantaBlue Stable Peroxidase Solution (both from Thermo Fisher Scientific) in a volume ratio of 9:1 was dispensed, and the solution was allowed to stand at room temperature for 5 minutes. The reaction was stopped by dispensing 100 μL / well of QuantaBlue Stop Solution (Thermo Fisher Scientific). Using a magnet, 150 μL / well of the supernatant was collected and transferred to a 96F Nontreated Black Microwell SI (Thermo Fisher Scientific). Fluorescence measurements were then performed using a microplate reader Infinite 200 PRO M-plex (TECAN) at an excitation wavelength of 325 nm and a detection wavelength of 420 nm to detect the adsorbed antibody. A lower fluorescence intensity indicates suppressed antibody adsorption. The non-specific adsorption inhibition effect of the antibody was evaluated from the relative antibody adsorption rate calculated using the following formula from the fluorescence intensity of Example 1-1-1 and the fluorescence intensity of Comparative Example 1-1 below.Specifically, the antibody adsorption rate of the magnetic microparticles in Example 1-1-1 was evaluated as a relative adsorption rate, with the antibody adsorption rate of the magnetic microparticles in Comparative Example 1-1 set to 100%. Protein adsorption rate of Example 1-1-1 (%) = (Fluorescence intensity of Example 1-1-1 / Fluorescence intensity of Comparative Example 1-1) × 100 The results are shown in Table 1.

[0023] (Example 1-1-2) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 1 was dissolved in D-PBS at a concentration of 0.1% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0024] (Example 1-1-3) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 1 was dissolved in D-PBS at a concentration of 0.03% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0025] (Example 1-1-4) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 1 was dissolved in D-PBS at a concentration of 0.01% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0026] (Example 1-2-1) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 2 was used as a surface treatment agent instead of the compound from Synthesis Example 1. The results are shown in Table 1.

[0027] (Example 1-2-2) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 2 was used instead of the compound from Synthesis Example 1, and the compound from Synthesis Example 2 was dissolved in D-PBS to a concentration of 0.1% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0028] (Example 1-2-3) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 2 was used instead of the compound from Synthesis Example 1, and the compound from Synthesis Example 2 was dissolved in D-PBS to a concentration of 0.03% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0029] (Example 1-2-4) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 2 was used instead of the compound from Synthesis Example 1, and the compound from Synthesis Example 2 was dissolved in D-PBS to a concentration of 0.01% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0030] (Example 1-3-1) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 3 was used as a surface treatment agent instead of the compound from Synthesis Example 1. The results are shown in Table 1.

[0031] (Example 1-3-2) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 3 was used instead of the compound from Synthesis Example 1, and the compound from Synthesis Example 3 was dissolved in D-PBS at a concentration of 0.1% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0032] (Example 1-3-3) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 3 was used instead of the compound from Synthesis Example 1, and the compound from Synthesis Example 3 was dissolved in D-PBS to a concentration of 0.03% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0033] (Example 1-3-4) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that the compound from Synthesis Example 3 was used instead of the compound from Synthesis Example 1, and the compound from Synthesis Example 3 was dissolved in D-PBS to a concentration of 0.01% by mass and used as a surface treatment agent solution. The results are shown in Table 1.

[0034] (Comparative Example 1-1) The protein adsorption inhibitory effect was evaluated in the same manner as in Example 1-1-1, except that only D-PBS and antibody were used without a surface treatment agent. The results are shown in Table 1.

[0035]

[0036] Examples 1-1-1 to 1-3-4, which used the surface treatment agent of the present invention containing the compound shown in formula (1) as an active ingredient, showed a lower protein adsorption rate compared to the results of Comparative Example 1-1. This suggests that the surface treatment agent of the present invention containing the compound shown in formula (1) as an active ingredient can suppress the adsorption of proteins (antibodies) when added to a protein solution (antibody dilution).

[0037] (Example 2-1-1) <Preparation of surface treatment agent solution> The compound from Synthesis Example 1 was dissolved in D-PBS to a concentration of 1.0% by mass to prepare a surface treatment agent solution. <Evaluation of the effect of inhibiting protein (antibody) adsorption to magnetic microparticles> The effect of using the above surface treatment agent on magnetic microparticles to inhibit protein adsorption was measured by the following method. A solution of magnetic microparticles was prepared by diluting the magnetic microparticles 50 times with D-PBS. 100 μL / well of the magnetic microparticle solution prepared earlier was dispensed into Eppendorf™ Microplate 96 / V-PP (manufactured by Eppendorf). The magnetic microparticles were settled using a magnet, and after completely removing the supernatant, 100 μL / well of D-PBS was dispensed to replace the buffer solution of the magnetic microparticles with D-PBS. The magnetic microparticles were settled using a magnet, and after discarding the supernatant again, 100 μL / well of the prepared surface treatment agent solution was dispensed. After dispersing the magnetic microparticles, the mixture was shaken and stirred at room temperature for 1 hour. The magnetic microparticles were allowed to settle using a magnet, and the supernatant was discarded again. Then, 100 μL / well of Goat Anti-Mouse IgG (H+L) horseradish peroxide conjugate (Bio-Rad), diluted 2,000 times with D-PBS, was dispensed. After dispersing the magnetic microparticles, the mixture was shaken and stirred at room temperature for 1 hour. The magnetic microparticles were allowed to settle using a magnet, and the supernatant was discarded again. Then, 100 μL / well of PBS-T was dispensed, and the supernatant was completely removed using a magnet. The magnet was removed, 100 μL / well of PBS-T was dispensed, and the entire solution was transferred to an unused well. The supernatant was completely removed using a magnet, and after removing it from the magnet, 100 μL / well of a solution prepared by mixing QuantaBlue Substrate Solution and QuantaBlue Stable Peroxidase Solution (both from Thermo Fisher Scientific) in a volume ratio of 9:1 was dispensed and allowed to stand at room temperature for 5 minutes. 100 μL / well of QuantaBlue Stop Solution (from Thermo Fisher Scientific) was dispensed to stop the reaction.Using a magnet, 150 µL of the supernatant was collected per well and transferred to a 96F Nontreated Black Microwell SI (manufactured by Thermo Fisher Scientific). Then, fluorescence measurement was performed with an excitation wavelength of 325 nm and a detection wavelength of 420 nm using a microplate reader Infinite 200 PRO M-plex (manufactured by TECAN), and the adsorbed antibody was detected. A lower fluorescence intensity indicates greater inhibition of antibody adsorption. The antibody adsorption inhibition effect on the magnetic fine particles coated with the surface treatment agent of Synthesis Example 1 was evaluated based on the relative antibody adsorption rate calculated by the following formula from the fluorescence intensity of Example 2-1-1 and the fluorescence intensity of the following Comparative Example 2-1. Specifically, the antibody adsorption rate of the magnetic fine particles of Example 2-1-1 was evaluated as a relative adsorption rate when the antibody adsorption rate of the magnetic fine particles of Comparative Example 2-1 was defined as 100%. Protein adsorption rate (%) of Example 2-1-1 = (Fluorescence intensity of Example 2-1-1 / Fluorescence intensity of Comparative Example 2-1) × 100 The results are shown in Table 2.

[0038] (Example 2-1-2) The protein adsorption inhibition effect was evaluated in the same manner as in Example 2-1-1, except that the compound of Synthesis Example 1 was dissolved in D-PBS to a concentration of 0.1% by mass and used as a surface treatment agent solution. The results are shown in Table 2.

[0039] (Example 2-1-3) The protein adsorption inhibition effect was evaluated in the same manner as in Example 2-1-1, except that the compound of Synthesis Example 1 was dissolved in D-PBS to a concentration of 0.05% by mass and used as a surface treatment agent solution. The results are shown in Table 2.

[0040] (Example 2-2-1) The antibody adsorption inhibition effect on magnetic fine particles coated with the surface treatment agent of Synthesis Example 2 was evaluated in the same manner as in Example 2-1-1, except that the compound of Synthesis Example 2 was used as the surface treatment agent instead of the compound of Synthesis Example 1. The results are shown in Table 2.

[0041] (Example 2-2-2) The antibody adsorption inhibition effect on magnetic fine particles coated with the surface treatment agent of Synthesis Example 2 was evaluated in the same manner as in Example 2-1-1, except that the compound of Synthesis Example 2 was used instead of the compound of Synthesis Example 1, the compound of Synthesis Example 2 was dissolved in D-PBS to a concentration of 0.1% by mass and used as a surface treatment agent solution. The results are shown in Table 2.

[0042] (Example 2-2-3) Except that the compound of Synthesis Example 2 was used instead of the compound of Synthesis Example 1, and the compound of Synthesis Example 2 was dissolved in D-PBS to a concentration of 0.05% by mass and used as a surface treatment agent solution, the antibody adsorption suppression effect on magnetic fine particles coated with the surface treatment agent of Synthesis Example 2 was evaluated in the same manner as in Example 2-1-1. The results are shown in Table 2.

[0043] (Example 2-3-1) Except that the compound of Synthesis Example 3 was used as the surface treatment agent instead of the compound of Synthesis Example 1, the antibody adsorption suppression effect on magnetic fine particles coated with the surface treatment agent of Synthesis Example 3 was evaluated in the same manner as in Example 2-1-1. The results are shown in Table 2.

[0044] (Example 2-3-2) Except that the compound of Synthesis Example 3 was used instead of the compound of Synthesis Example 1, and the compound of Synthesis Example 3 was dissolved in D-PBS to a concentration of 0.1% by mass and used as a surface treatment agent solution, the antibody adsorption suppression effect on magnetic fine particles coated with the surface treatment agent of Synthesis Example 3 was evaluated in the same manner as in Example 2-1-1. The results are shown in Table 2.

[0045] (Example 2-3-3) Except that the compound of Synthesis Example 3 was used instead of the compound of Synthesis Example 1, and the compound of Synthesis Example 3 was dissolved in D-PBS to a concentration of 0.05% by mass and used as a surface treatment agent solution, the antibody adsorption suppression effect on magnetic fine particles coated with the surface treatment agent of Synthesis Example 3 was evaluated in the same manner as in Example 2-1-1. The results are shown in Table 2.

[0046] (Comparative Example 2-1) Except that no surface treatment agent was used and only D-PBS and an antibody were used, the antibody adsorption suppression effect on magnetic fine particles was evaluated in the same manner as in Example 2-1-1. The results are shown in Table 2.

[0047]

[0048] Examples 2-1-1 to 2-3-3, which used the surface treatment agent of the present invention containing the compound shown in formula (1) as an active ingredient, showed a lower protein adsorption rate compared to the results of Comparative Example 2-1. This suggests that it is possible to suppress the adsorption of proteins (antibodies) by coating a substrate with the surface treatment agent of the present invention containing the compound shown in formula (1) as an active ingredient.

[0049] (Example 3-1-1) <Preparation of surface treatment solution> The compound from Synthesis Example 1 was dissolved in D-PBS to a concentration of 0.1% by mass to prepare a surface treatment solution. <Evaluation when used as a protein adsorption inhibitor in ELISA> The blocking effect when the above surface treatment agent was used as a blocking agent in ELISA using a microwell plate was evaluated by the following procedure. 100 μL / well of normal mouse IgG, whole molecule, purified product (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereafter referred to as antigen diluent), prepared to a concentration of 1.5 μg / mL in D-PBS, was dispensed into a polystyrene F96 CERT. MAXISORP NUNC-IMMUNO PLATE (manufactured by Thermo Fisher Scientific). After standing overnight at 4°C, the antigen diluent was aspirated, and PBS-T was dispensed at 200 μL / well and aspirated. This process was repeated three times to wash away any unimmobilized antigen. The surface treatment solution prepared above was dispensed at 200 μL / well and left to stand at room temperature for 1.5 hours. The surface treatment solution was aspirated and left to stand overnight in a desiccator at room temperature to completely dry the surface treatment solution. Goat Anti-Mouse IgG (H+L) horseradish peroxide conjugate (manufactured by Bio-Rad, hereafter referred to as antibody diluent), diluted 40,000 times with D-PBS, was dispensed at 100 μL / well and shaken at room temperature for 1 hour. After aspirating the antibody diluent, PBS-T was dispensed at a rate of 200 μL / well and aspirated three times, and the excess antibody diluent was washed away. A chromogenic solution prepared by mixing KPL TMB Peroxidase Substrate (Sera Care) and KPL Peroxidase Substrate Solution B (Sera Care) in a 1:1 ratio (by volume) was dispensed at a rate of 100 μL / well and allowed to stand at room temperature for 10 minutes. 50 μL / well of 1 mol / L sulfuric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dispensed, and after stopping the reaction, the absorbance at 450 nm was measured using a microplate reader Infinite 200 PRO M-plex (manufactured by TECAN), and the amount of antigen detected (hereafter referred to as signal intensity) was evaluated. The results are shown in Table 3.

[0050] (Example 3-1-2) The amount of non-specific adsorption of the antibody (hereinafter referred to as noise intensity) was evaluated in the same manner as in Example 3-1-1, except that D-PBS was used instead of the antigen diluent in Example 3-1-1. The results are shown in Table 3. The signal / noise ratio (hereinafter referred to as S / N ratio) was calculated by dividing the signal intensity obtained in Example 3-1-1 by the noise intensity obtained in Example 3-1-2. An evaluation system with low noise intensity and a high S / N ratio indicates that the amount of non-specific adsorption of the labeled antibody is small and that a large amount of antigen-antibody complex is detected. The surface treatment agent of the present invention was evaluated by comparing it with the noise intensity and S / N ratio obtained from Comparative Examples 3-1-1 to 3-1-2. The results are shown in Table 3. S / N ratio = (Signal intensity of Example 3-1-1 / Noise intensity of Example 3-1-2)

[0051] (Example 3-2-1) The compound from Synthesis Example 2 was used instead of the compound from Synthesis Example 1, and a surface treatment solution was prepared in the same manner as in Example 3-1-1. Furthermore, the signal intensity was evaluated in the same manner as in Example 3-1-1. The results are shown in Table 3.

[0052] (Example 3-2-2) The compound from Synthesis Example 2 was used instead of the compound from Synthesis Example 1, and a surface treatment solution was prepared in the same manner as in Example 3-1-2. Furthermore, the noise intensity was evaluated in the same manner as in Example 3-1-2. The results are shown in Table 3.

[0053] (Example 3-3-1) The compound from Synthesis Example 3 was used instead of the compound from Synthesis Example 1, and a surface treatment solution was prepared in the same manner as in Example 3-1-1. Furthermore, the signal intensity was evaluated in the same manner as in Example 3-1-1. The results are shown in Table 3.

[0054] (Example 3-3-2) The compound from Synthesis Example 3 was used instead of the compound from Synthesis Example 1, and a surface treatment solution was prepared in the same manner as in Example 3-1-2. Furthermore, the noise intensity was evaluated in the same manner as in Example 3-1-2. The results are shown in Table 3.

[0055] (Comparative Example 3-1-1) The signal intensity was evaluated in the same manner as in Example 3-1-1, except that only D-PBS was added without using a surface treatment agent. The results are shown in Table 3.

[0056] (Comparative Example 3-1-2) Noise intensity was evaluated in the same manner as in Example 3-1-2, except that only D-PBS was added without using a surface treatment agent. The results are shown in Table 3.

[0057]

[0058] The signal-to-noise ratios (S / N ratios) obtained from Examples 3-1-1, 3-1-2 to 3-3-1, and 3-3-2, using the surface treatment agent of the present invention with the compound shown in formula (1) as an active ingredient, were larger than those obtained from Comparative Examples 3-1-1 and 3-1-2. Furthermore, the noise intensity obtained from Examples 3-1-2 to 3-3-2 was smaller than that obtained from Comparative Example 3-1-2. This suggests that the surface treatment agent of the present invention, with the compound shown in formula (1) as an active ingredient, can be used as a protein adsorption inhibitor in the ELISA method to suppress non-specific adsorption of proteins (antibodies) and selectively detect antigen-antibody complexes.

[0059] (Example 4-1-1) <Preparation of surface treatment solution> The compound from Synthesis Example 1 was dissolved in Tris-buffered saline (manufactured by Nippon Gene Co., Ltd., hereafter abbreviated as TBS) at a concentration of 0.1% by mass to prepare a surface treatment solution. <Preparation of captured antibody-immobilized magnetic microparticles> 400 μL of Magnosphere MX100 (manufactured by JSR Co., Ltd., hereafter referred to as Magbeads) was dispensed into Eppendorf Safe-Lock Tubes (manufactured by Eppendorf Co., Ltd., hereafter referred to as tubes), and the Magbeads were settled using a magnet, and the supernatant was removed. 800 μL of D-PBS was added, and the Magbeads were dispersed. Then, the Magbeads were settled again using a magnet, and the supernatant was removed. MES (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in ultrapure water, and the pH was adjusted to 5.5 using sodium hydroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to create a buffer (hereinafter referred to as MES buffer). Using this buffer, N-ethyl-N'-3-dimethylaminopropylcarbodiimide was dissolved to a concentration of 10 mg / mL. 200 μL of this conjugation reagent solution was dispensed into a tube and shaken for 30 minutes at room temperature. Magbeads were allowed to settle using a magnet, and the supernatant was removed. 800 μL of Anti-Human IgG Fab Fragment antibody [4A11] (manufactured by Abcam, hereinafter referred to as capture antibody), diluted 40-fold with MES buffer, was added, and the mixture was shaken for 3 hours at room temperature. Magbeads were precipitated using a magnet, and after removing the supernatant, 800 μL of MES buffer was added. Magbeads were precipitated using a magnet, and after removing the supernatant, the excess capture antibody was washed away. 400 μL of MES buffer was added to prepare a suspension of magnetic microparticles immobilized with capture antibody.

[0060] <Evaluation when used as a protein adsorption inhibitor in the CLEIA method> The amount of antigen-antibody complex detected when the above surface treatment agent was used as a protein adsorption inhibitor in the CLEIA method, which is also used as an actual diagnostic agent, was evaluated by the following procedure. A suspension of magnetic microparticles immobilized with captured antibodies was dispensed into a tube, the magnetic microparticles immobilized with captured antibodies were allowed to settle using a magnet, the supernatant was discarded, TBS was added to disperse the magnetic microparticles immobilized with captured antibodies, and the buffer solution of the suspension of magnetic microparticles immobilized with captured antibodies was replaced. The magnetic microparticles immobilized with captured antibodies were allowed to settle using a magnet, the supernatant was discarded again, TBS was added to dilute the suspension of magnetic microparticles immobilized with captured antibodies 100 times to the original volume, and the suspension was dispensed at a rate of 100 μL / well into Eppendorf™ Microplate 96 / V-PP (manufactured by Eppendorf). Using a magnet, the captured antibody-immobilized magnetic microparticles were allowed to settle, and the supernatant was discarded. Then, 200 μL / well of the surface treatment agent solution was dispensed and shaken for 1 hour at room temperature. Using a magnet, the captured antibody-immobilized magnetic microparticles were allowed to settle, and the supernatant was discarded. Then, 200 μL / well of TBS was dispensed to disperse the captured antibody-immobilized magnetic microparticles, and the captured antibody-immobilized magnetic microparticles were allowed to settle again using a magnet. This process was repeated twice, and the excess surface treatment agent was washed away. Normal human IgG, whole molecule, purified product (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted with TBS to a concentration of 1 μg / mL. 100 μL / well of the antigen dilution was dispensed and the captured antibody-immobilized magnetic microparticles were dispersed. The mixture was shaken for 1 hour at room temperature. The captured antibody-immobilized magnetic microparticles were precipitated using a magnet, and the supernatant was discarded. Then, 200 μL / well of TBS was dispensed and the captured antibody-immobilized magnetic microparticles were dispersed. The captured antibody-immobilized magnetic microparticles were precipitated using a magnet again, and the supernatant was discarded. This procedure was repeated twice, and the excess antigen diluent was washed. 100 μL / well of anti-human IgG (FC-specific)-alkaline phosphatase goat host antibody (Sigma-Aldrich), diluted 10,000 times with TBS, was dispensed and the captured antibody-immobilized magnetic microparticles were dispersed. The mixture was shaken and stirred at room temperature for 1 hour.Capture antibody-immobilized magnetic microparticles are sedimented using a magnet, after discarding the supernatant, 200 μL / well of TBS containing 0.05% by mass of Tween 20 is aliquoted to disperse the capture antibody-immobilized magnetic microparticles, the capture antibody-immobilized magnetic microparticles are sedimented using a magnet, the operation of discarding the supernatant is performed twice again, and excess antigen diluent is washed away. CDP-Star. ＴＭ Substrate (0.4 mM Ready-to-Use) with Sapphire-II ＴＭ 100 μL / well of Enhancer (manufactured by Thermo Fisher Scientific) is aliquoted to disperse the capture antibody-immobilized magnetic microparticles, followed by shaking and stirring at room temperature for 10 minutes. The capture antibody-immobilized magnetic microparticles are sedimented using a magnet, and the entire amount of the supernatant is transferred to a 96 Well Black / Clear Bottom Plate, TC Surface (manufactured by Thermo Fisher Scientific), luminescence measurement is performed with a microplate reader Infinite 200 PRO M-plex (manufactured by TECAN), and the detected amount of antigen (hereinafter, signal intensity) is evaluated. The results are shown in Table 4.

[0061] (Example 4-1-2) The non-specific adsorption amount of antibody (hereinafter, noise intensity) was evaluated in the same manner as in Example 4-1-1, except that TBS was used instead of the antigen diluent of Example 4-1-1. The results are shown in Table 4. The S / N ratio was obtained by dividing the signal intensity obtained in Example 4-1-1 by the noise intensity obtained in Example 4-1-2. An evaluation system with low noise intensity and a high S / N ratio indicates a small amount of non-specific antibody adsorption and detection of a large amount of antigen-antibody complexes. The surface treatment agent of the present invention was evaluated by comparing with the noise intensity and S / N ratio obtained from Comparative Examples 4-1-1 to 4-1-2. The results are shown in Table 4. S / N ratio = (signal intensity of Example 4-1-1 / noise intensity of Example 4-1-2)

[0062] (Example 4-2-1) A surface treatment agent solution was prepared in the same manner as in Example 4-1-1, except that the compound of Synthesis Example 2 was used instead of the compound of Synthesis Example 1. Further, the signal intensity was evaluated in the same manner as in Example 4-1-1. The results are shown in Table 4.

[0063] (Example 4-2-2) The compound from Synthesis Example 2 was used instead of the compound from Synthesis Example 1, and a surface treatment solution was prepared in the same manner as in Example 4-1-2. Furthermore, the noise intensity was evaluated in the same manner as in Example 4-1-2. The results are shown in Table 4.

[0064] (Example 4-3-1) The compound from Synthesis Example 3 was used instead of the compound from Synthesis Example 1, and a surface treatment solution was prepared in the same manner as in Example 4-1-1. Furthermore, the signal intensity was evaluated in the same manner as in Example 4-1-1. The results are shown in Table 4.

[0065] (Example 4-3-2) The compound from Synthesis Example 3 was used instead of the compound from Synthesis Example 1, and a surface treatment solution was prepared in the same manner as in Example 4-1-2. Furthermore, the noise intensity was evaluated in the same manner as in Example 4-1-2. The results are shown in Table 4.

[0066] (Comparative Example 4-1-1) The signal intensity was evaluated in the same manner as in Example 4-1-1, except that only D-PBS was added without using a surface treatment agent. The results are shown in Table 4.

[0067] (Comparative Example 4-1-2) Noise intensity was evaluated in the same manner as in Example 4-1-2, except that only D-PBS was added without using a surface treatment agent. The results are shown in Table 4.

[0068] The signal-to-noise ratios (S / N ratios) obtained from Examples 4-1-1, 4-1-2 to 4-3-1, and 4-3-2, using the surface treatment agent of the present invention with the compound shown in formula (1) as an active ingredient, were larger than those obtained from Comparative Examples 4-1-1 and 4-1-2. Furthermore, the noise intensity obtained from Examples 4-1-2 to 4-3-2 was smaller than that obtained from Comparative Example 4-1-2. This suggests that the surface treatment agent of the present invention, with the compound shown in formula (1) as an active ingredient, can be used as a protein adsorption inhibitor in the CLEIA method to suppress non-specific adsorption of proteins (antigens and antibodies) without inhibiting specific adsorption of proteins (antigens and antibodies), and to selectively detect antigen-antibody complexes.

Claims

1. A surface treatment agent containing the compound shown in formula (1) as an active ingredient. [In equation (1), n ​​is an integer between 11 and 23,000.] 2. A substrate having a coating layer on its surface made of the surface treatment agent described in claim 1.

3. A method for suppressing protein adsorption, comprising treating the surface of a substrate with the surface treatment agent described in claim 1 to suppress the adsorption of proteins onto the substrate surface.

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