Antifouling member, member for in vitro diagnostic device, and method for producing antifouling member

Abstract

The present invention relates to an antifouling member, a member for an in vitro diagnostic device, and a method for producing the antifouling member. Provided is an antifouling member capable of achieving both low protein adsorption and durability. The antifouling member has a film formed on a substrate. The film comprises a condensate of a hydrolysate of a silane coupling agent or a condensate of a phosphonic acid derivative. The film does not contain halogen atoms. The zeta potential of the membrane in water having a pH of 7 is from-10.0 mV to 10.0 mV. The antifouling member has a B / A value of 2.0-5.0, where A represents the ratio of the amount of C atoms in XPS analysis of the substrate to the amount of atoms of the element X contained in the maximum amount in the surface of the substrate, and B represents the ratio of the amount of C atoms to the amount of atoms of the element X in XPS analysis of the film on the substrate.

Description

Technical Field

[0001] The present invention relates to an antifouling member, a member for an in vitro diagnostic device, and a method for producing the antifouling member. Background Art

[0002] Unlike disposable devices such as pacemakers, catheters, and guidewires, reusable components such as forceps, endoscopes, dental drills, and components for in vitro diagnostic equipment are reused after appropriate cleaning and sterilization. However, in order to ensure safety, the burden of cleaning is large, and therefore, in order to reduce the burden, reusable components with antifouling properties are desired.

[0003] In in vitro diagnostic device components for biochemical analysis and the like, which can be used multiple times, adsorption of trace amounts of protein leads to reduced detection sensitivity and reduced reproducibility. Conventional in vitro diagnostic device components are cleaned with alkali between samples to reduce the effects of protein adsorption. However, in recent years, the sensitivity of in vitro diagnostic devices has increased, thus raising concerns that the effects of protein adsorption may not be completely eliminated by conventional cleaning alone. Therefore, antifouling coatings for in vitro diagnostic device components are needed.

[0004] Under such circumstances, surface treatment agents that can impart hydrophilicity, biocompatibility, antithrombotic properties, and the like to the surfaces by treating the surfaces of various members with polymerization products of betaine monomers have been widely proposed.

[0005] In Japanese Patent No. 6,154,370, a method involving treating a medical device such as a catheter or a guide wire with a silane coupling agent having a vinyl group and then polymerizing a sulfobetaine monomer is proposed for the purpose of improving and maintaining sliding properties.

[0006] In addition, in Japanese Patent No. 5,349,873, a medical material in which a polymer brush using a carboxybetaine monomer is formed and a medical device using the medical material are proposed. Summary of the invention

[0007] However, according to studies conducted by the inventors of the present invention, the methods described in Japanese Patent No. 6,154,370 and Japanese Patent No. 5,349,873 each have problems in durability against an alkaline cleaning solution used for cleaning antifouling members.

[0008] Therefore, an object of the present invention is to provide an antifouling member that can achieve both low protein adsorption and durability. In addition, another object of the present invention is to provide a method for producing an antifouling member that can achieve both low protein adsorption and durability.

[0009] The above-mentioned object is achieved by the present invention described below. That is, according to the present invention, there is provided an antifouling member comprising a film formed on a substrate, wherein the film comprises a condensate of a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative, wherein the film does not contain a halogen atom, wherein the zeta potential of the film in water having a pH of 7 is -10.0 mV or more and 10.0 mV or less, and wherein the antifouling member has a B / A value of 2.0 or more and 5.0 or less, wherein A represents the ratio of the amount (atomic %) of C atoms to the amount (atomic %) of atoms of element X contained in the surface of the substrate in the largest amount in X-ray photoelectron spectroscopy analysis (ESCA measurement) of the substrate, and B represents the ratio of the amount (atomic %) of C atoms to the amount (atomic %) of atoms of element X in X-ray photoelectron spectroscopy analysis (ESCA measurement) of the film on the substrate.

[0010] Further features of the present invention will become apparent from the following description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is an exemplary schematic diagram for explaining an application example of the present invention.

[0012] Figure 2 is another exemplary schematic diagram for explaining an application example of the present invention. DETAILED DESCRIPTION

[0013] Now, the present invention is described in detail by way of exemplary embodiments.

[0014] [First embodiment]

[0015] The first embodiment relates to an antifouling member.

[0016] The antifouling member of the present invention is an antifouling member including a film formed on a substrate, wherein the film contains a condensate of a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative, wherein the film does not contain a halogen atom, wherein the zeta potential of the film in water having a pH of 7 is -10.0 mV or more and 10.0 mV or less, and wherein the B / A value of the antifouling member is 2.0 or more and 5.0 or less, wherein A represents the ratio of the amount (atomic %) of C (carbon) atoms to the amount (atomic %) of atoms of element X contained in the surface of the substrate in the largest amount in X-ray photoelectron spectroscopy analysis (ESCA measurement) of the substrate, and B represents the ratio of the amount (atomic %) of C atoms to the amount (atomic %) of atoms of element X in X-ray photoelectron spectroscopy analysis (electron spectroscopy for chemical analysis, ESCA measurement) of the film on the substrate.

[0017] The antifouling member satisfying the above-mentioned constitution can achieve both low protein adsorption and durability. The antifouling member of the present invention is an antifouling member having a film formed on a substrate. Conventional antifouling members are obtained by polymerizing betaine monomers with a silane coupling agent having a vinyl group. However, for members such as antifouling members that are expected to have durability against alkaline cleaning solutions, the effect is insufficient. The inventors have conducted extensive research and found that by controlling the two parameters within a specific range, durability against alkaline cleaning solutions can be exhibited.

[0018] In the antifouling member of the present invention, film does not contain halogen atoms.One of the parameters representing that film does not contain halogen atoms is that the ratio of the ion amount of halogen atoms to the total ion amount in the TOF-SIMS measurement of film is below 0.02.In the TOF-SIMS measurement, the information about the element or molecular species present at the depth below the sample surface several nanometers can be obtained with a significantly high detection sensitivity.Therefore, in the TOF-SIMS measurement of film, the information about the element or molecular species present on the outermost surface of film is obtained.The ratio of the ion amount of halogen atoms to the total ion amount is below 0.02 to indicate the existence state of halogen on the outermost surface of film.When the ratio of the ion amount of halogen atoms to the total ion amount is below 0.02, satisfactory durability can be shown. The ratio of the ion amount of halogen atoms to the total ion amount of 0.02 or less is only a guideline, and, in the sense that halogen atoms are not clearly detected in the film in the TOF-SIMS measurement, it is appropriate that the ratio of the ion amount of halogen atoms to the total ion amount in the TOF-SIMS measurement is at the background level (level at which halogen atoms are not detected).

[0019] The present inventors consider the reason for the foregoing to be as follows.

[0020] When the film in the above-mentioned constitution contains halogen, the halogen exists in a state of being bound to carbon. Here, the main component of the alkaline cleaning solution is water, so it is believed that the halogen and the carbon bound to the halogen are polarized, thereby being more easily solvated by water. In other words, it is believed that the film containing halogen on the outermost surface has a higher affinity for the alkaline cleaning solution than the film not containing halogen. Generally, it is known that the condensate of the hydrolyzate of the silane coupling agent or the condensate of the phosphonic acid derivative is dissolved in an alkaline solution. Therefore, it is believed that due to the high affinity for the alkaline cleaning solution, the film containing halogen undergoes progressive film degradation due to the dissolution of the condensate of the hydrolyzate of the silane coupling agent or the condensate of the phosphonic acid derivative, and the durability becomes insufficient. Therefore, in order to show sufficient durability, it is important that the ratio of the ion amount of the halogen atom to the total ion amount is 0.02 or less.

[0021] The B / A value of the antifouling member of the present invention is 2.0 or more and 5.0 or less, wherein A represents the ratio of the amount of C atoms (atomic %) in the X-ray photoelectron spectroscopy analysis (ESCA measurement) of the substrate to the amount of atoms of element X contained in the substrate surface in the maximum amount, and B represents the ratio of the amount of C atoms (atomic %) in the X-ray photoelectron spectroscopy analysis (ESCA measurement) of the film on the substrate to the amount of atoms of element X (atomic %). In the ESCA measurement, photoelectrons generated in the region from the outermost surface to about 10nm are detected. The value of B / A provides information about the density and thickness of the film. When the value is 2.0 or more and 5.0 or less, good low protein adsorption and durability are exhibited. When the value is less than 2.0, due to the insufficient density and thickness of the film, hydrophilicity becomes insufficient, and low protein adsorption cannot be fully exhibited. At the same time, when the value is greater than 5.0, the thickness of the film becomes large, and hydrophilicity is fully exhibited, and as a result, low protein adsorption is exhibited. However, the affinity for alkaline cleaning liquid becomes too high, and film degradation caused by the dissolution of the condensate of the hydrolyzate of the silane coupling agent or the condensate of the phosphonic acid derivative progresses, resulting in insufficient durability. Therefore, it is important that the value of B / A is 2.0 or more and 5.0 or less.

[0022] In addition, the antifouling member of the present invention is a member that is reused after appropriate cleaning and sterilization, such as forceps, endoscopes, dental drills, or members for in vitro diagnostic devices. Among such antifouling members, members for in vitro diagnostic devices are preferred because they are easily affected by the adsorption of trace amounts of protein and can effectively exhibit the effects of the present invention.

[0023] In addition, in the antifouling member of the present invention, the film contains a condensate of a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative. It is preferred that the condensate of a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative forms a covalent bond with the substrate and has a vinyl group in the molecule. As long as these conditions are met, no particular restrictions are set, and a silane coupling agent known to date or a conventionally known phosphonic acid derivative can be used. In the antifouling member of the present invention, from the viewpoint of durability, the silane coupling agent is preferably selected from at least one of the following groups consisting of: 3-(methacryloyloxy)propyltrimethoxysilane; 3-(acryloyloxy)propyltrimethoxysilane; 3-(acryloyloxy)propyltriethoxysilane; 3-(acryloyloxy)propyltriethoxysilane; Vinyltrimethoxysilane; Triethoxyvinylsilane; Allyltrimethoxysilane; Allyltriethoxysilane; and methacrylic acid (triethoxysilyl)methyl ester. In addition, in the antifouling member of the present invention, from the viewpoint of durability, the phosphonic acid derivative is preferably at least one selected from the group consisting of 2-methacryloyloxyethyl acidic phosphate; 2-acryloyloxyethyl acidic phosphate; 2-(methacryloyloxy)ethyl phosphate; and acidic phosphooxy polyethylene glycol monomethacrylate. Among them, from the viewpoint of achieving both low protein adsorption and durability, 3-(methacryloyloxy)propyltrimethoxysilane and 2-methacryloyloxyethyl acidic phosphate are preferred.

[0024] In addition, in the antifouling member of the present invention, the zeta potential at pH 7 is -10.0 mV or more and 10.0 mV or less. The mechanism of protein adsorption to a substrate or the like is based on two interactions: electrostatic interaction and hydrophobic interaction. Therefore, in order to suppress protein adsorption caused by electrostatic interaction, it is important to control the zeta potential at pH 7 within the above range. As long as the zeta potential can be controlled within the above range, any material can be used, but it is preferred to control the zeta potential by any one or a combination of two or more of the following three structural features.

[0025] The first structural feature is that, in the antifouling member of the present invention, the film preferably comprises a copolymer of a monomer, a hydrophilic monomer and a crosslinking agent having a polarity opposite to that of the substrate. With this structural feature, the surface charge of the substrate is offset by a monomer having a polarity opposite to that of the substrate. In addition, due to crosslinking, the substrate shielding property shown by the film is improved, so that the film can be less affected by the surface charge of the substrate. In addition, when the hydrophilic monomer is also copolymerized, the low protein adsorption derived from the hydrophobic interaction can also be achieved. The copolymerization ratio in the copolymer of a monomer, a hydrophilic monomer and a crosslinking agent having a polarity opposite to that of the substrate is preferably adjusted so that the zeta potential when pH is 7 is more than -10.0mV and less than 10.0mV.

[0026] Here, in the antifouling member of the present invention, any monomer can be used as a monomer having a polarity opposite to that of the substrate, and amine monomers are preferred. In the antifouling member of the present invention, from the viewpoint of reactivity, the amine monomer is preferably selected from at least one of the following groups: 2-(dimethylamino)ethyl methacrylate; 2-(dimethylamino)ethyl acrylate; 2-(diethylamino)ethyl methacrylate; 2-(diisopropylamino)ethyl methacrylate; and 2-(tert-butylamino)ethyl methacrylate. Examples of amine monomers include 2-(dimethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl acrylate, 2-(diethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, and 2-(tert-butylamino)ethyl methacrylate. Among them, from the viewpoint of reactivity, 2-(dimethylamino)ethyl methacrylate and 2-(diethylamino)ethyl methacrylate are more preferred.

[0027] In the antifouling member of the present invention, the hydrophilic monomer is preferably electrically neutral. In the antifouling member of the present invention, the hydrophilic monomer is preferably selected from at least one of the following groups: 2-hydroxyethyl methacrylate; N-vinyl-2-pyrrolidone; and polyethylene glycol monomethyl ether methacrylate. Examples of hydrophilic monomers include 2-hydroxyethyl methacrylate, N-vinyl-2-pyrrolidone, polyethylene glycol monomethyl ether methacrylate, 2,3-dihydroxypropyl methacrylate, 2-hydroxyethyl acrylate, hydroxypropyl methacrylate, and 2-methacrylate 2-hydroxy-3-phenoxypropyl. Among them, from the viewpoint of low protein adsorption, 2-hydroxyethyl methacrylate and poly (ethylene glycol) monomethyl ether methacrylate are more preferred.

[0028] In the antifouling member of the present invention, the crosslinking agent is preferably at least one selected from the group consisting of ethylene glycol diacrylate; ethylene glycol dimethacrylate; polyethylene glycol diacrylate; polyethylene glycol dimethacrylate; and 1,6-hexanediol diacrylate. Thus, the gaps between the polymers can be filled, and low protein adsorption can be achieved. Examples of crosslinking agents include ethylene glycol diacrylate, ethylene glycol dimethacrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, 1,6-hexanediol diacrylate, divinylbenzene, neopentyl glycol diacrylate, 3-methyl-1,5-pentanediol diacrylate, 1,9-nonanediol diacrylate, acrylic acid adduct of neopentyl glycol hydroxypivalate, and methacrylate 2-hydroxy-3-acryloyloxypropyl. Among them, from the viewpoint of low protein adsorption, polyethylene glycol diacrylate and 1,6-hexanediol diacrylate are preferred.

[0029] In addition, the second structural feature is that in the antifouling member of the present invention, the film preferably contains a polymer of a betaine monomer. The polymer of the betaine monomer has positive and negative charges in the molecule. As a result, the polymer chains attract each other through electrostatic interaction to produce a shielding effect between the chains, and the zeta potential at pH 7 can be controlled to be greater than -10.0 mV and less than 10.0 mV. In the antifouling member of the present invention, the betaine monomer is preferably at least one selected from the group consisting of 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate; 2-[[2-(methacryloyloxy)ethyl]dimethylammonio]acetate; 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate; 3-[(3-acrylamidopropyl)dimethylammonio]propionate; 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate; 4-[[2-(methacryloyloxy)ethyl]dimethylammonio]butane-1-sulfonate; 3-[[2-(acryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate; 3-[(3-methacrylamidopropyl)dimethylammonio]propane-1-sulfonate; and 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonate. Among them, from the viewpoint of low protein adsorption, 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate, 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate, and 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate are more preferred.

[0030] In addition, the third structural feature is that in the antifouling member of the present invention, the membrane preferably contains a polymer of a betaine monomer and a polymer of a hydrophilic monomer, or a copolymer of a betaine monomer and a hydrophilic monomer. The membrane may have a structure in which a polymer of a hydrophilic monomer or a copolymer of a betaine monomer and a hydrophilic monomer is further contained on the polymer of the betaine monomer. As a result, the zeta potential at pH 7 can be controlled to be greater than -10.0 mV and less than 10.0 mV, and low protein adsorption can be achieved.

[0031] In the antifouling member of the present invention, the material of the base material preferably includes stainless steel or glass. Thus, the antifouling member of the present invention can be used as a member for an in vitro diagnostic device described later.

[0032] In the antifouling member of the present invention, the membrane preferably includes a charge adjustment site and a hydrophilic site having a polarity opposite to that of the substrate. As used herein, the charge adjustment site and the hydrophilic site may be present in different layers or in the same layer. The charge adjustment site is, for example, a site formed by a monomer having a polarity opposite to that of the substrate, or a site formed by a betaine monomer. The hydrophilic site is, for example, a site formed by a hydrophilic monomer or a site formed by a betaine monomer.

[0033] In the antifouling member of the present invention, it is preferred that the binding site to the substrate is represented by the following formula (1-1) or the following formula (1-2), the charge adjustment site is a copolymer of monomers represented by the following formula (2-1), the following formula (2-2) and the following formula (2-3) or represented by the following formula (2-4), and the hydrophilic site is represented by the following formula (3-1) or the following formula (3-2):

[0034]

[0035]

[0036] In formula (1-1) and formula (1-2), "*" indicates the binding position to the substrate, "**" indicates the binding position to the charge adjustment site, and R 1 represents any one of an alkylene group or an oxyalkylene group, and R 2 represents any one of CH=CH (vinylidene), CH=C(CH3) (isopropenylene), HC=CH=C(=O)-O (acryloyloxy), HC=C(CH3)-C(=O)-O (methacryloyloxy) or HC=C(CH3)-C(=O)-NH (methacryloylamino), and "l" satisfies 1≤l≤6; in formula (2-1), formula (2-2) and formula (2-3), R3 represents hydrogen or methyl, R4, R5 and R8 each independently represent an alkylene group having 1 to 6 carbon atoms, R6 represents hydrogen or an alkyl group having 1 to 3 carbon atoms, and R7 represents an oxyalkylene group having 1 to 3 carbon atoms; in formula (2-4), "**" represents the binding position to the binding site, R9 and R 10 Each independently represents an alkylene group or an oxyalkylene group, and R 11 Representation-COO - , and "m" satisfies 4≤m≤1,000; and in formula (3-1) and formula (3-2), "***" represents the binding position with the charge regulating site, R8 represents an alkylene group having 1 to 6 carbon atoms, "n" satisfies 4≤n≤1,000, R9 and R 10 Each independently represents an alkylene group or an oxyalkylene group, and R 11 Representation-COO - , and “m” satisfies 4≤m≤1,000.

[0037] <Method for measuring membrane zeta potential>

[0038] The zeta potential of the membrane can be measured by the electrophoretic light scattering method using a zeta potential measurement system ELSZ-2000 (manufactured by Otsuka Electronics Co., Ltd.). The membrane is attached to the upper surface of the compartment of a flat sample compartment unit (manufactured by Otsuka Electronics Co., Ltd.), and the compartment is filled with a 10 mM aqueous sodium chloride solution at pH 7 in which monitoring particles (manufactured by Otsuka Electronics Co., Ltd.) are dispersed. The monitoring particles are made to undergo electrophoresis, and the electrophoretic mobility of the monitoring particles is measured. The zeta potential (mV) of the membrane at pH 7 can be calculated by analyzing the data of the obtained electrophoretic mobility using the Mori-Okamoto equation and the Smoluchowski equation.

[0039] <Method for TOF-SIMS Measurement of Membrane>

[0040] TOF-SIMS (manufactured by ULVAC-PHI, Inc., nanoTOF II) is used to detect the halogens present on the membrane surface. The analysis conditions are as described below.

[0041] Sample preparation: The substrate on which the membrane is formed is attached to an indium sheet.

[0042] Sample pretreatment: None

[0043] Primary ion: Bi 3++

[0044] Acceleration voltage: 30 kV

[0045] Charge neutralization mode: On

[0046] Measurement mode: Negative

[0047] Raster size: 300 μm

[0048] Accumulation time: 180 seconds

[0049] The ratio of the ion amount of the halogen atoms to the total ion amount of the membrane can be determined from the ion amounts obtained by performing the above measurements.

[0050] <X-ray Photoelectron Spectroscopy (ESCA Measurement) Method>

[0051] The measuring apparatus and measuring conditions are as described below.

[0052] · Measuring apparatus: X-ray photoelectron spectrometer: Quantum 2000 (manufactured by ULVAC-PHI, Inc.)

[0053] · X-ray source: Monochromatic AlKα

[0054] X-ray settings:

[0055] Photoelectron extraction angle: 45 degrees

[0056] Neutralization conditions: Use a combination of a neutralization gun and an ion gun

[0057] Analysis area: 300μm×200μm

[0058] ·Pass energy: 58.70eV

[0059] Step size: 0.125eV

[0060] Analysis software: MaltiPak (ULVAC-PHI, Inc.)

[0061] In the measurement by the XPS method, first, qualitative analysis is performed over the entire element range by survey analysis, and then quantitative analysis is performed by narrow scanning with the number of scans shown in Table 1 for each detected element.

[0062] Table 1

[0063] C1s N1s O1s Si2P Cr2p3 Fe2p3 P2p Br3D Ca2p S Na Br 20 40 10 40 40 40 40 20 20 40 20 20

[0064] In the quantitative values ​​obtained in the ESCA measurement of the substrate, the ratio of the amount of C atoms (atomic %) to the amount of atoms of element X contained in the largest amount in the surface of the substrate (atomic %) is represented by A, and in the quantitative values ​​obtained in the ESCA measurement of the film on the substrate, the ratio of the amount of C atoms (atomic %) to the amount of atoms of element X (atomic %) is represented by B. Thus, the value of B / A can be determined.

[0065] [Second embodiment]

[0066] The second embodiment relates to a method for producing an antifouling member. The respective components of the antifouling member are as described above, and thus description thereof is omitted.

[0067] In addition, the present invention relates to a method for producing a member having a film formed on the surface of a substrate. As preferred examples of the production method, three examples are given. The first example among these examples is described.

[0068] The production method of the antifouling component of the present invention (first example) is a production method of an antifouling component having a film formed on the surface of a substrate, the method comprising: a bonding step of bonding a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative to the surface of a substrate; a charge regulating site forming step of copolymerizing a monomer having a polarity opposite to that of the substrate, a hydrophilic monomer and a cross-linking agent in the presence of a polymerization initiator after the bonding step; and a hydrophilic site forming step of adding a hydrophilic monomer after the charge regulating site forming step and polymerizing the hydrophilic monomer to form a hydrophilic site.

[0069] As the silane coupling agent, the above-mentioned types of silane coupling agents can be used respectively. The hydrolysis of the silane coupling agent can be carried out by a known method. The optimum pH for hydrolysis varies depending on the type of the silane coupling agent, but it is preferred to select a pH with a slow condensation rate. For example, in the case of 3-(methacryloyloxy)propyltrimethoxysilane, it is preferably hydrolyzed in an aqueous solution with a pH of 4±0.5 because its condensation can be suppressed. In addition, the concentration of the silane coupling agent during hydrolysis is preferably 0.5wt% or more and 70.0wt% or less. The concentration of the silane coupling agent is more preferably 0.5wt% or more and 5.0wt% or less, and when the concentration falls within such a range, the condensation of the hydrolyzate of the silane coupling agent can be effectively suppressed.

[0070] The step of bonding the hydrolyzate of the silane coupling agent to the substrate is described. The substrate is preferably used after ultrasonic cleaning the substrate using one or more of an alkali, a surfactant or an organic solvent to remove organic matter attached to the surface. In addition, it is more preferred to use the substrate after UV / O3 cleaning the substrate for more than 1 minute and less than 60 minutes to remove organic matter attached to the surface. The cleaned substrate is immersed in an aqueous solution of pH 4±0.5 in which 0.5wt% or more and 5.0wt% or less of the hydrolyzate of the silane coupling agent is dissolved for more than 30 seconds and less than 10 minutes. Thereafter, the resultant is dried for more than 30 seconds and less than 1800 seconds in an atmosphere of more than 10°C and less than 40°C. After drying, the excess hydrolyzate of the silane coupling agent is removed by water washing, and then the resultant is baked in an oven at more than 50°C and less than 150°C. Thus, the hydrolyzate of the silane coupling agent is bonded to the substrate to perform the bonding step.

[0071] In addition, as the phosphonic acid derivative, the phosphonic acid derivatives of the above-mentioned types can be used respectively. It is preferred to use the phosphonic acid derivative after dissolving it in a solvent in which the phosphonic acid derivative is soluble. Preferred examples of the solvent include alcohol, tetrahydrofuran, acetone, N-methylpyrrolidone and dimethylformamide. Among them, alcohol and tetrahydrofuran are more preferred, because alcohol and tetrahydrofuran can make the bonding step to the substrate appropriately carried out.

[0072] Describe the bonding step of bonding the condensate of phosphonic acid derivatives to a substrate. Preferably, the cleaning of the substrate is carried out in the same manner as in the bonding step using a silane coupling agent. A phosphonic acid derivative solution of 0.1 mmol / l or more and 50 mmol / l or less is prepared. Any solvent in which the phosphonic acid derivative is soluble can be used, and examples thereof include alcohol, tetrahydrofuran, acetone, toluene, N-methylpyrrolidone, and dimethylformamide. Among them, alcohol and tetrahydrofuran are more preferred. The cleaned substrate is immersed in the prepared phosphonic acid derivative solution for more than 1 minute and less than 120 minutes. Afterwards, the resultant is placed in an oven at more than 15°C and less than 150°C for more than 1 minute and less than 2880 minutes. Then, ultrasonic cleaning is performed with tetrahydrofuran and methanol to remove the multilayer film. Thus, the condensate of the phosphonic acid derivative is bonded to the substrate.

[0073] Next, the charge adjustment site formation step is described. The charge adjustment site formation step is performed by copolymerizing a monomer having a polarity opposite to that of the substrate, a hydrophilic monomer, and a crosslinking agent in the presence of a polymerization initiator and the substrate that has been subjected to the bonding step. No particular limitation is set to the copolymerization method as long as the monomer can be copolymerized under the condition where the polymerization initiator and the monomer coexist, but from the viewpoint of reactivity, a method involving copolymerizing the monomer after bringing the polymerization initiator into contact with the substrate that has been subjected to the bonding step is preferred.

[0074] Any method can be used as a method for contacting a polymerization initiator with a substrate that has been subjected to a bonding step, preferably involving the following method: the substrate that has been subjected to a bonding step is placed in a reaction vessel, nitrogen purged, and then a liquid dissolved with a polymerization initiator in a reaction solvent from which oxygen is removed by nitrogen bubbling is placed in a reaction vessel, followed by stirring. Afterwards, a monomer, a hydrophilic monomer, and a crosslinking agent having a polarity opposite to that of the substrate are dissolved in the reaction solvent, and nitrogen purged. The resulting product is placed in a reaction vessel containing a substrate and a polymerization initiator, and copolymerized by heating. Such a copolymerization method is preferred. As a monomer, a hydrophilic monomer, and a crosslinking agent having a polarity opposite to that of the substrate, those described above can be used.

[0075] In addition, as a reaction solvent, water, alcohol, tetrahydrofuran, acetone, N-methylpyrrolidone, dimethylformamide, etc. can be used alone or in combination. Among them, water and alcohol are preferred from the viewpoint of monomer solubility. In addition, the reaction temperature is preferably determined based on the 10-hour half-life temperature of the polymerization initiator to be used, but from the viewpoint of the stability of the monomer to be used and the stability of the copolymer, it is preferably reacted at 15°C or more and 90°C or less, more preferably at 40°C or more and 80°C or less. In addition, the reaction time (heating time) is preferably 10 minutes or more and 1440 minutes or less, more preferably 30 minutes or more and 600 minutes or less.

[0076] The polymerization initiator is preferably 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate, which is common in the first to third examples of the method for producing the antifouling member of the present invention.

[0077] In addition, examples of the polymerization initiator include azobisisobutyronitrile (AIBN), 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, 2,2′-azobis[2-(2-imidazolin-2-yl)propane] disulfate dihydrate, 2,2′-azobis(2-methylpropionamidine) dihydrochloride, 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate, 2,2′-azobis[2-(2-imidazolin-2-yl)propane], 2,2′-azobis(1-imino-1-pyrrolidine-2-methylpropane) dihydrochloride, 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2′-azobis(2,4-dimethylvaleronitrile), 2,2′-azobis(2-methylpropionic acid) dimethyl ester, 2,2′-azobis(2-methylbutyronitrile), 1,1′-azobis(cyclohexane-1-carbonitrile), 2,2′-azobis[N-(2-propenyl)-2-methylpropionamide], 1,1′-azobis(1-acetoxy-1-phenylethane), 2,2′-azobisisobutyric acid dimethyl ester, and derivatives thereof. Among them, 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate is preferred from the viewpoint of reactivity.

[0078] Next, the hydrophilic site formation step is described. This step is a step performed after the above-mentioned charge adjustment site formation step, and is a step of adding a hydrophilic monomer and polymerizing the hydrophilic monomer to form a hydrophilic site. As the hydrophilic monomer, those described above can be used. The addition and polymerization method can be carried out as follows: the hydrophilic monomer is dissolved in a reaction solvent, nitrogen is purged, and then the resultant is placed in a reaction system in which the charge adjustment site formation step has been performed, and then heated. The heating temperature can be set to the same temperature range as the charge adjustment site formation step, and can be the same as or different from the charge adjustment site formation step. In addition, as a reaction solvent, the reaction solvent described in the charge adjustment site formation step can also be used, and the reaction solvent can be the same as or different from the reaction solvent in the charge adjustment site formation step. The reaction time (heating time) is preferably 10 minutes or more and 1440 minutes or less, and more preferably 30 minutes or more and 600 minutes or less.

[0079] The film may be formed on the substrate as described above, and cleaning may also be performed after the film is formed. Any method may be used as the cleaning method, and a method involving ultrasonic cleaning by using a reaction solvent is preferred.

[0080] Next, a second example of the production method is described.

[0081] The method for producing an antifouling member of the present invention (second example) is a method for producing an antifouling member having a film formed on the surface of a substrate, the method comprising: a bonding step of bonding a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative to the surface of the substrate; and a two-site forming step of polymerizing 2.0 mg / ml or more and 12.0 mg / ml or less of a betaine monomer in the presence of a polymerization initiator to form a charge regulating site and a hydrophilic site after the bonding step.

[0082] Compared with the first example, the second example is characterized in that a film is formed on a substrate by polymerizing 2.0 mg / ml or more and 12.0 mg / ml or less of a betaine monomer in the presence of a polymerization initiator after the bonding step to form a charge adjustment site and a hydrophilic site. The undescribed constituent elements are preferably carried out in the same manner as in the first example.

[0083] No particular limitation is set to the polymerization method as long as the betaine monomer can be polymerized under the condition where the polymerization initiator and the monomer coexist, and preferably involves a method of polymerizing the betaine monomer after bringing the polymerization initiator into contact with the substrate that has been subjected to the bonding step from the viewpoint of reactivity. Any method can be used as a method of bringing the polymerization initiator into contact with the substrate that has been subjected to the bonding step, but preferably involves a method of placing the substrate that has been subjected to the bonding step in a reaction vessel, performing nitrogen purging, and then placing a liquid having the polymerization initiator dissolved in a reaction solvent from which oxygen has been removed by nitrogen bubbling in the reaction vessel, followed by stirring.

[0084] Afterwards, the betaine monomer is dissolved in a reaction solvent. The resultant is placed in a reaction vessel comprising a substrate and a polymerization initiator, and polymerization is carried out by heating. This type of polymerization method is preferred. The reaction time (heating time) is preferably more than 10 minutes and less than 1440 minutes, more preferably more than 30 minutes and less than 600 minutes. As the betaine monomer, those mentioned above can be used. In addition, it is preferred to adjust the concentration of the reaction solution so that the concentration of the betaine monomer during polymerization is more than 2.0 mg / ml and less than 12.0 mg / ml. When the concentration of the monomer falls within the above range, the value of B / A can be controlled to be more than 2.0 and less than 5.0, and both low protein adsorption and durability can be achieved.

[0085] Next, a third example of the production method is described.

[0086] The method for producing an antifouling member of the present invention (third example) is a method for producing an antifouling member having a film formed on the surface of a substrate, the method comprising: a bonding step of bonding a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative to the surface of the substrate; a charge regulation site forming step of polymerizing a betaine monomer in the presence of a polymerization initiator after the bonding step; and a hydrophilic site forming step of adding a hydrophilic monomer and polymerizing the hydrophilic monomer to form a hydrophilic site after the charge regulation site forming step.

[0087] Compared with the second example, the third example is characterized in that a charge adjustment site formation step of polymerizing a betaine monomer in the presence of a polymerization initiator is performed after the bonding step, and a hydrophilic monomer is added and polymerized to form a hydrophilic site after the charge adjustment site formation step. The undescribed constituent elements are preferably carried out in the same manner as in the first example.

[0088] No particular limitation is set to the polymerization method as long as the betaine monomer can be polymerized under the condition where the polymerization initiator and the monomer coexist, and from the viewpoint of reactivity, preferably involves a method of polymerizing the betaine monomer after bringing the polymerization initiator into contact with the substrate that has been subjected to the bonding step. Any method can be used as a method of bringing the polymerization initiator into contact with the substrate that has been subjected to the bonding step, and preferably involves a method of placing the substrate that has been subjected to the bonding step in a reaction vessel, performing nitrogen purging, and then placing a liquid having the polymerization initiator dissolved in a reaction solvent from which oxygen has been removed by nitrogen bubbling in the reaction vessel, followed by stirring.

[0089] After that, the betaine monomer is dissolved in a reaction solvent. The resultant is placed in a reaction vessel containing a substrate and a polymerization initiator, and polymerization is performed by heating. Such a polymerization method is preferred. As the betaine monomer, those described above can be used. The reaction time (heating time) is preferably 10 minutes or more and 1440 minutes or less, more preferably 30 minutes or more and 600 minutes or less. Thus, the charge adjustment site formation step is performed.

[0090] After the charge adjustment site formation step, a hydrophilic monomer is added and polymerized to form a hydrophilic site. The formation of the hydrophilic site can be performed by the same method as in the first example.

[0091] In addition, in the present invention, the substrate preferably includes stainless steel or glass. Hydroxyl groups are present on the outermost surfaces of each of stainless steel and glass, so the hydrolyzate or phosphonic acid derivative of the silane coupling agent can be condensed to form a covalent bond. The above is preferred because good durability can be imparted as a result of forming a covalent bond.

[0092] In addition, in the present invention, the bonding site to be bonded to the substrate is preferably represented by formula (1-1) or formula (1-2):

[0093]

[0094] In formula (1-1) and formula (1-2), "*" indicates the binding position to the substrate, "**" indicates the binding position to the charge adjustment site, and R 1 represents any one of an alkylene group or an oxyalkylene group, and R 2 It represents any one of CH=CH (vinylidene), CH=C(CH3) (isopropenylene), HC=CH=C(=O)-O (acryloyloxy), HC=C(CH3)-C(=O)-O (methacryloyloxy) or HC=C(CH3)-C(=O)-NH (methacryloylamino), and "l" satisfies 1≤l≤6.

[0095] It is preferred that the charge adjustment site is a copolymerization product of monomers represented by formula (2-1), formula (2-2) and formula (2-3), or is represented by formula (2-4).

[0096]

[0097]

[0098] In formula (2-1), formula (2-2) and formula (2-3), R 3 represents hydrogen or methyl, R 4 , R 5 and R 8 Each independently represents an alkylene group having 1 to 6 carbon atoms, and R 6 represents hydrogen or an alkyl group having 1 to 3 carbon atoms, and R 7 represents an oxyalkylene group having 1 to 3 carbon atoms, and in the formula (2-4), "**" represents the binding position to the binding site, and R 9 and R 10 Each independently represents an alkylene group or an oxyalkylene group, and R 11 Representation-COO - , and “m” satisfies 4≤m≤1,000.

[0099] The hydrophilic part is preferably represented by formula (3-1) or formula (3-2):

[0100]

[0101]

[0102] In formula (3-1) and formula (3-2), "***" represents the binding position with the charge adjustment site, R 8 represents an alkylene group having 1 to 6 carbon atoms, "n" satisfies 4≤n≤1000, R9 and R 10 Each independently represents an alkylene group or an oxyalkylene group, and R 11 Representation-COO - , and “m” satisfies 4≤m≤1000.

[0103] The thickness of the film in the present invention when dry is preferably 1 nm or more and 200 nm or less, more preferably 1 nm or more and less than 10 nm. The thickness of the film can be measured by any method, and can be measured by, for example, an ellipsometer. When the thickness of the film falls within the above range, both low protein adsorption and durability can be achieved.

[0104] [Application Examples]

[0105] The application examples of the present invention relate to components for in vitro diagnostic equipment. As the components for in vitro diagnostic equipment of the present invention, the above-mentioned antifouling components are preferably used. As examples of components for in vitro diagnostic equipment, a sample test analysis cell and a front end portion of a pipette are described below, but the application examples of the present invention are not limited to these two uses.

[0106] (Sample test analysis cell)

[0107] The sample test analysis cell has a rectangular tube shape. Figure 1 1 is a perspective view of a sample test analysis pool in an application example of the present invention. Figure 1 As shown, the sample test analysis cell is a glass container having a bottom surface and four side surfaces, and includes a mouth portion 11 (opening portion) on a side opposite to the bottom surface 12 .

[0108] Figure 2 1 is a top view of a sample test analysis cell in an application example of the present invention. The sample test analysis cell has a first light path surface for incident analysis light and a second light path surface opposite to the first light path surface. Figure 2 As shown, the four sides include a first light path surface 21 through which the analysis light 25 is incident, a second light path surface 22 opposite to the first light path surface 21 and from which the analysis light 26 is emitted, a non-light path surface 23 through which the analysis light does not pass, and a non-light path surface 24 opposite to the non-light path surface 23. Although the two light paths are distinguished from each other as the first light path surface 21 and the second light path surface 22, such a distinction is only made for the convenience of description, and the functions, materials, etc. of the first light path surface 21 and the second light path surface 22 are the same. Figure 2 In the embodiment, a sample testing and analyzing cell having a rectangular tube shape is shown as an example, but the sample testing and analyzing cell may also have a cylindrical shape.

[0109] In addition, the size of the sample test analysis pool is not particularly limited. An example is a rectangle with an inner dimension of 4 mm × 5 mm and an outer dimension of 6 mm × 7 mm in a plane parallel to the bottom surface and a total height of 40 mm (the distance from the mouth to the bottom surface on the outside). There is no problem with whether the inner and outer dimensions are larger or smaller than the above dimensions. However, in recent years, in order to reduce the burden on patients, it is necessary to measure with a smaller amount of samples, and the sample test analysis pool tends to be reduced in size.

[0110] The shape, size, etc. of the sample test analysis pool are examples and are not limited to those described above. In addition, even when the outer surface of the pool is chamfered or rounded (rounded), it will not cause problems. When the inner surface has a rectangular tube shape, making the inner surface round (rounded) will not cause problems.

[0111] The sample test analysis cell is made of borosilicate glass. As the material of the sample test analysis cell, a glass material having high transmittance and excellent in mechanical strength and chemical durability can be used. In the cleaning step of the sample test analysis cell in the sample test analysis device, in order to remove pollutants such as proteins, an acidic or alkaline cleaning agent is generally used. When it is assumed to be reused, the sample test analysis cell will be exposed to an acid environment or an alkaline environment for a long time. When the sample test analysis cell is made of resin, the resin is inferior to glass-based materials in terms of chemical durability. Therefore, the surface composition of the inner surface of the sample test analysis cell is easy to change, and the wettability of water thereon may change over time. As materials excellent in durability to acids and alkalis, for example, quartz glass and borosilicate glass can be given.

[0112] As described above, when the sample test analysis cell is made of glass, the antifouling member of the present invention can be applied to the sample test analysis cell. By using the absorbance measurement of the sample test analysis cell, the concentration of the detected substance (e.g., biological substances such as proteins or ions) can be detected with high accuracy.

[0113] (Tip of the pipette)

[0114] The front end of the pipette refers to the front end of the pipette head when the pipette includes a pipette tip, and refers to the front end of the syringe when the pipette does not include a pipette tip. The pipette is conductive at least at its front end. The front end of the pipette is electrically connected to a measuring portion with electrical properties. In addition, the pipette can quantitatively carry out the suction and discharge of liquid by the reciprocating motion of the plunger. Using this structure, the pipette can provide liquid to the flow path and recover liquid therefrom. From the viewpoint of preventing the mixing of impurities, etc., it is preferred that the pipette head is replaceable.

[0115] The material of the pipette is not particularly limited. Examples of the material of the pipette include resin and glass. Polypropylene is generally used as the material of the pipette. The method of imparting conductivity to the front end of the pipette is not particularly limited. For example, a pipette head made of resin may contain an additive for imparting conductivity, or may include a conductive portion made of metal. Examples of additives for imparting conductivity include: carbon-based additives formed of conductive carbon such as carbon fiber and carbon black; and metal-based additives in which fibers, powders, glass fibers, etc. are each covered with metal.

[0116] When the tip of the pipette contains metal as described above, the antifouling member of the present invention can be applied to the tip of the pipette. When the tip of the pipette contains metal, the presence or amount of the detected substance (protein, DNA, etc.) can be measured with high accuracy by using, for example, surface plasmon resonance.

[0117] (Distribution probe)

[0118] The distribution probe has a thin tube shape. The sample distribution probe performs a distribution involving aspirating a sample in a sample container and ejecting the sample into a reaction vessel, and the reagent distribution probe performs a distribution involving aspirating a reagent in a reagent container and ejecting the reagent into a reaction vessel. The material of the distribution probe is not particularly limited, but examples of the material include stainless steel, glass, and titanium. In this case, the antifouling member of the present invention can be applied to the distribution probe.

[0119] Example

[0120] The present invention is described more specifically by the examples described below. However, the present invention is by no means limited by the examples. Unless otherwise specified, all the terms "parts" and "%" in the examples and comparative examples are based on mass.

[0121] (Example 1)

[0122] A glass slide (S1111) manufactured by Matsunami Glass Ind., Ltd. was placed in a high beaker containing a 1M sodium hydroxide aqueous solution, and ultrasonically cleaned and subsequently washed with water. 3-(Methacryloyloxy)propyltrimethoxysilane was diluted to 1% by mass with dilute hydrochloric acid (pH = 4) and stirred for 1 hour for hydrolysis. The hydrolyzate was placed in a high beaker. The glass slide that had been UV / O3 cleaned for 15 minutes after ultrasonic cleaning was immersed in the hydrolyzate for 1 minute and taken out, and then dried at 30°C for 5 minutes. The glass slide was washed with pure water to remove excess hydrolysis products, and the washed glass slide was placed in an oven at 90°C and baked for 60 minutes. Thus, a condensation reaction was carried out.

[0123] The slide glass that has undergone the condensation reaction is placed in a 300mL glass container, and the glass container is covered with a lid. The interior of the glass container is purged with nitrogen, and 100mL of ultrapure water is placed in the glass container while nitrogen is flowing at 200ml / min. A needle is placed in the ultrapure water in the glass container, and nitrogen is bubbled at 150mL / min for 30 minutes. 126mg of 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate used as a polymerization initiator is dissolved in 20ml of ultrapure water purged with nitrogen, and the resultant is loaded into a 300ml glass container. Then, the resultant is stirred at room temperature for 60 minutes. 900mg of 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate used as a betaine monomer is dissolved in 30mL of ultrapure water purged with nitrogen, and the resultant is loaded into a 300mL glass container. Afterwards, a polymerization reaction is carried out at 60°C for 4 hours. Then, the slide glass subjected to the polymerization reaction was poured and washed with RO water, and ultrasonically cleaned at 28 kHz to provide a slide glass having a film.

[0124] The obtained slide glass with the film was subjected to zeta potential measurement, TOF-SIMS measurement and ESCA measurement by the above-mentioned method. In addition, low protein adsorption and durability were evaluated by the following method.

[0125] <Low protein adsorption>

[0126] Divide each hole into 400mm 2 The slide and chamber (2 wells) are attached to the slide with the membrane.

[0127] Bovine serum albumin (BSA) was dissolved in an acetate buffer (pH=4.8) having an ionic strength of 0.001 to give a concentration of 50 ppm (BSA solution). The 50 ppm BSA solution was diluted with the acetate buffer to prepare samples for a calibration curve of 40 ppm, 25 ppm, and 0 ppm.

[0128] 150 μL of 50 ppm BSA solution was added to each well. The resultant was covered with a lid and wrapped with parafilm. Thus, the BSA solution was incubated at 37° C. for 30 minutes.

[0129] Solution A and solution B of Protein Assay BCA Kit (manufactured by FUJIFILM Wako Pure Chemical Corporation) were mixed at a ratio of 50 / 1 (BCA solution), and 400 μL of the solution was put into a 1.5 ml sample tube.

[0130] After incubation, 90 μL of sample was removed from the well and placed in a sample tube containing the BCA solution.

[0131] 90 μl of samples for the calibration curve (50 ppm, 40 ppm, 25 ppm and 0 ppm) were each placed in 1.5 ml sample tubes each containing 400 μl of a separately prepared BCA solution.

[0132] After stirring each sample tube, the liquid adhering to the cap was removed by centrifugation.

[0133] Then, the resultant was incubated at 60°C for 30 minutes to perform a color development reaction.

[0134] 150 μL of each sample that had undergone color development reaction was placed in a 96-well plate ("n" number was 3), and the absorbance at 562 nm was measured.

[0135] Calculate the average absorbance for the "n" number of 3. Create a calibration curve using the average absorbance values ​​of the samples from the calibration curve.

[0136] The concentration of the BSA solution in the membrane sample was calculated from the calibration curve, and the amount of BSA adsorbed to the membrane (μg) was calculated from the change from 50 ppm.

[0137] Divide the calculated BSA adsorption amount by the area of ​​each well (400 mm 2 ) to calculate the BSA adsorption per unit area (μg / cm 2 ). The BSA adsorption amount on the slide was 0.6 μg / cm 2 The adsorption amount was regarded as 100%, and the BSA adsorption amount per unit area was divided by 0.6 μg / cm 2 The protein adsorption rate was calculated. As the BSA adsorption amount on the slide glass, the BSA adsorption amount when the number "n" was 15 or more was calculated in advance, and the average value was used.

[0138] In the present invention, a calculated protein adsorption rate of 15% or less is considered to have sufficiently low protein adsorption (low protein adsorption). When the calculated protein adsorption rate is 15% or less, protein adsorption has no effect on the reduction of detection sensitivity and reproducibility.

[0139] Durability

[0140] An alkaline detergent (Maxpia (trademark) B14 (manufactured by Canon Medical Systems Corporation)) was diluted 76 times with ultrapure water to give 150 mL.

[0141] Maxpia B14 diluted to 76 times and the slide glass with the membrane were placed in a 300 mL flat-bottom container and stirred for 3 hours.

[0142] After 3 hours, the slides were removed and washed with water.

[0143] After that, the evaluation of "low protein adsorption" was performed to calculate the protein adsorption rate.

[0144] In the present invention, a durability (protein adsorption rate after water washing) of 25% or less is regarded as having sufficient durability. When the durability (protein adsorption rate after water washing) is 25% or less, alkali resistance can be effectively exerted.

[0145] (Example 2)

[0146] The respective processes were carried out in the same manner as in Example 1 until the condensation reaction.

[0147] The slide glass that has undergone the condensation reaction is placed in a 300mL glass container, and the glass container is covered with a lid. The inside of the glass container is purged with nitrogen, and 65mL ultrapure water and 35mL ethanol are placed in the glass container while nitrogen flows at 200ml / min. A needle is placed in the liquid in the glass container, and nitrogen is bubbled at 150mL / min for 30 minutes. 126mg of 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate used as a polymerization initiator is dissolved in 20ml ultrapure water purged with nitrogen, and the resultant is loaded into a 300ml glass container. Then, the resultant is stirred at room temperature for 60 minutes. 900 mg of 2-hydroxyethyl methacrylate used as a hydrophilic monomer, 100 mg of 2-(dimethylamino)ethyl methacrylate used as a monomer having a polarity opposite to that of the substrate, and 30 mg of polyethylene glycol diacrylate (n=about 9) used as a crosslinking agent were dissolved in a mixed solvent of ultrapure water purged with nitrogen and ethanol in a ratio of 10 mL / 15 mL, and the resultant was loaded into a 300 mL glass container. Afterwards, a polymerization reaction was carried out at 60°C for 3 hours. In addition, 1000 mg of 2-hydroxyethyl methacrylate used as an additional hydrophilic monomer was dissolved in a mixed solvent of ultrapure water purged with nitrogen and ethanol in a ratio of 7 mL / 7 mL, and the resultant was loaded into a 300 mL glass container. Then, a polymerization reaction was carried out at 60°C for 3 hours. Afterwards, the slide glass subjected to the polymerization reaction was poured and cleaned with ethanol and RO water, and ultrasonic cleaning was carried out at 28 kHz to provide a slide glass with a film.

[0148] The obtained slide glass was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement. In addition, in the same manner as in Example 1, low protein adsorption and durability were evaluated.

[0149] (Examples 3, 5, 6 and 14)

[0150] Except that the betaine monomer was changed as shown in Table 2, each process up to the evaluation was performed in the same manner as in Example 1.

[0151] Table 2

[0152]

[0153] (Examples 4, 9, 11, 12 and 15)

[0154] Except that each material was changed as shown in Table 3, each process up to the evaluation was performed in the same manner as in Example 2.

[0155] Table 3

[0156]

[0157] In Table 3, VA-57 represents 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate, V50 represents 2,2′-azobis(2-methylpropionamidine) dihydrochloride, HEMA represents 2-hydroxyethyl methacrylate, CBMA represents 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate, DMAEM represents 2-(dimethylamino)ethyl methacrylate, DEAEM represents 2-(diethylamino)ethyl methacrylate, EG(n=9) represents polyethylene glycol diacrylate (n=about 9), and HDDA represents 1,6-hexanediol diacrylate.

[0158] (Example 7)

[0159] The respective processes were carried out in the same manner as in Example 1 until the condensation reaction.

[0160] The slide glass that has undergone the condensation reaction is placed in a 300mL glass container, and the glass container is covered with a lid. The interior of the glass container is purged with nitrogen, and 50mL of ultrapure water and 50mL of ethanol are placed in the glass container while nitrogen is flowing at 200ml / min. A needle is placed in the liquid in the glass container and bubbled with nitrogen at 150mL / min for 30 minutes. 126mg of 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate used as a polymerization initiator is dissolved in 20ml of ultrapure water purged with nitrogen, and the resultant is loaded into a 300ml glass container. Then, the resultant is stirred at room temperature for 60 minutes. 360mg of 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate used as a betaine monomer is dissolved in 30mL of ultrapure water purged with nitrogen, and the resultant is loaded into a 300mL glass container. Afterwards, the polymerization reaction is carried out at 60°C for 3 hours. Separately, 720 mg of 2-hydroxyethyl methacrylate used as an additional hydrophilic monomer was dissolved in 15 mL of ultrapure water purged with nitrogen, and the resultant was charged into a 300 mL glass container. Then, polymerization was performed at 60° C. for 3 hours. Thereafter, the slide glass subjected to the polymerization reaction was poured and washed with ethanol and RO water, and ultrasonically cleaned at 28 kHz to provide a slide glass having a film.

[0161] The obtained slide glass was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement. In addition, in the same manner as in Example 1, low protein adsorption and durability were evaluated.

[0162] (Example 8)

[0163] The procedures up to the evaluation were carried out in the same manner as in Example 7 except that the betaine monomer was changed to 450 mg of 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate and the additional hydrophilic monomer was changed to 900 mg of polyethylene glycol monomethyl ether methacrylate (average Mn: 950).

[0164] (Example 10)

[0165] A slide glass (S1111) manufactured by Matsunami Glass Ind., Ltd. was placed in a high beaker containing a 1M sodium hydroxide aqueous solution and ultrasonically cleaned. 2-Methacryloyloxyethyl acid phosphate was diluted to 0.5% by mass with tetrahydrofuran and placed in a high beaker. The slide glass that was ultrasonically cleaned and UV / O3 cleaned for 15 minutes was immersed in the diluted 2-methacryloyloxyethyl acid phosphate for 60 minutes and taken out, and then dried at 30°C for 5 minutes. The slide glass was cleaned with tetrahydrofuran, and the cleaned slide glass was placed in an oven at 120°C and baked for 60 minutes. Thus, a condensation reaction was carried out.

[0166] The slide glass that has undergone the condensation reaction is placed in a 300mL glass container, and the glass container is covered with a lid. The interior of the glass container is purged with nitrogen, and 100mL of ultrapure water is placed in the glass container while nitrogen is flowing at 200ml / min. A needle is placed in the ultrapure water in the glass container, and nitrogen is bubbled at 150mL / min for 30 minutes. 126mg of 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate used as a polymerization initiator is dissolved in 20ml of ultrapure water purged with nitrogen, and the resultant is loaded into a 300ml glass container. Then, the resultant is stirred at room temperature for 60 minutes. 630mg of 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate used as a betaine monomer is dissolved in 30mL of ultrapure water purged with nitrogen, and the resultant is loaded into a 300mL glass container. Afterwards, a polymerization reaction is carried out at 60°C for 4 hours. Then, the slide glass subjected to the polymerization reaction was poured and washed with RO water, and ultrasonically cleaned at 28 kHz to provide a slide glass having a film.

[0167] The obtained slide glass with the film was subjected to zeta potential measurement, TOF-SIMS measurement and ESCA measurement by the above method. In addition, low protein adsorption and durability were evaluated by the above method.

[0168] (Example 13)

[0169] The respective processes were carried out in the same manner as in Example 1 until the condensation reaction.

[0170] The glass slide that has undergone the condensation reaction is placed in a 300mL glass container, and the glass container is covered with a lid. The interior of the glass container is purged with nitrogen, and 50mL of ultrapure water and 20mL of ethanol are placed in the glass container while nitrogen is flowing at 200ml / min. A needle is placed in the liquid in the glass container, and nitrogen is bubbled at 150mL / min for 30 minutes. 50mg of 2,2′-azobis(isobutyronitrile) used as a polymerization initiator is dissolved in 50ml of ethanol purged with nitrogen, and the resultant is loaded into a 300ml glass container. Then, the resultant is stirred at room temperature for 60 minutes. 900mg of 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate used as a betaine monomer is dissolved in 30mL of ultrapure water purged with nitrogen, and the resultant is loaded into a 300mL glass container. Afterwards, a polymerization reaction is carried out at 60°C for 4 hours. Thereafter, the slide glass subjected to the polymerization reaction was poured and washed with ethanol and RO water, and ultrasonically cleaned at 28 kHz, thereby providing a slide glass having a film.

[0171] The obtained slide glass was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement. In addition, in the same manner as in Example 1, low protein adsorption and durability were evaluated.

[0172] (Example 16)

[0173] Each process from preparation to evaluation was performed in the same manner as in Example 1 except that the slide glass (S1111) manufactured by Matsunami Glass Ind., Ltd. was changed to a SUS304 plate (width×length×thickness=26 mm×60 mm×1 mm).

[0174] (Example 17)

[0175] Each process from preparation to evaluation was performed in the same manner as in Example 2 except that the slide glass (S1111) manufactured by Matsunami Glass Ind., Ltd. was changed to a SUS304 plate (width×length×thickness=26 mm×60 mm×1 mm).

[0176] (Comparative Example 1)

[0177] A slide glass (S1111) manufactured by Matsunami Glass Ind., Ltd. was placed in a beaker containing a 1M sodium hydroxide aqueous solution and subjected to ultrasonic cleaning, followed by water washing, and then subjected to UV / O3 cleaning for 15 minutes.

[0178] Lipidure-S (manufactured by NOF CORPORATION) was diluted with toluene to 1 mass % and placed in a high beaker. A slide glass after UV / O3 cleaning was immersed in the diluted Lipidure-S for 1 minute and taken out, followed by drying at 30°C for 24 hours to form a film.

[0179] The slide glass having the film formed thereon was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement in the same manner as in Example 1. Furthermore, low protein adsorption and durability were evaluated.

[0180] (Comparative Example 2)

[0181] The procedures up to the evaluation were performed in the same manner as in Example 2 except that the hydrophilic monomer was changed to 800 mg of 2-hydroxyethyl methacrylate and the monomer having a polarity opposite to that of the substrate was changed to 200 mg of 2-(dimethylamino)ethyl methacrylate.

[0182] (Comparative Example 3)

[0183] The respective processes were carried out in the same manner as in Example 1 until the condensation reaction.

[0184] The slide glass that has undergone the condensation reaction is placed in a 300mL glass container, and the glass container is covered with a lid. The interior of the glass container is purged with nitrogen, and 65mL of ultrapure water and 35mL of ethanol are placed in the glass container while nitrogen flows at 200ml / min. A needle is placed in the liquid in the glass container and bubbled with nitrogen at 150mL / min for 30 minutes. 126mg of 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate used as a polymerization initiator is dissolved in 20ml of ultrapure water purged with nitrogen, and the resultant is loaded into a 300ml glass container. Then, the resultant is stirred at room temperature for 60 minutes. 900mg of 2-hydroxyethyl methacrylate used as a hydrophilic monomer is dissolved in a mixed solvent of ultrapure water purged with nitrogen and ethanol in a ratio of 10mL / 15mL, and the resultant is loaded into a 300mL glass container. Afterwards, the polymerization reaction is carried out at 60°C for 6 hours. Thereafter, the slide glass subjected to the polymerization reaction was poured and washed with ethanol and RO water, and ultrasonically cleaned at 28 kHz, thereby providing a slide glass having a film.

[0185] The obtained slide glass was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement. In addition, in the same manner as in Example 1, low protein adsorption and durability were evaluated.

[0186] (Comparative Example 4)

[0187] A slide glass (S1111) manufactured by Matsunami Glass Ind., Ltd. was placed in a beaker containing a 1M sodium hydroxide aqueous solution and subjected to ultrasonic cleaning, followed by water washing, and then subjected to UV / O3 cleaning for 15 minutes.

[0188] LAMBIC-1000W (manufactured by OSAKA ORGANIC CHEMICAL INDUSTRY LTD.) was diluted 5 times with ultrapure water and put into a high beaker. A slide glass after UV / O3 cleaning was immersed in the diluted LAMBIC-1000W for 1 minute and taken out, followed by drying at 80°C for 15 minutes to form a film.

[0189] The slide glass having the film formed thereon was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement in the same manner as in Example 1. Furthermore, low protein adsorption and durability were evaluated.

[0190] (Comparative Example 5)

[0191] A slide glass (S1111) manufactured by Matsunami Glass Ind., Ltd. was placed in a beaker containing a 1M sodium hydroxide aqueous solution and subjected to ultrasonic cleaning, followed by water washing, and then subjected to UV / O3 cleaning for 15 minutes.

[0192] The cleaned slide glass is placed in a vacuum dryer containing 10 drops of (3-aminopropyl) triethoxysilane. The system (in the vacuum dryer) is decompressed to less than 1mbar, and placed under reduced pressure for 30 minutes after being disconnected from the pump. Afterwards, the slide glass is baked at 110 ℃ for 30 minutes under normal pressure. The baked slide glass is placed in a reaction vessel, and nitrogen is purged. 50mL dehydrated tetrahydrofuran, 1.3mL 2-bromoisobutyryl bromide and 1.5ml triethylamine are added to the reaction vessel, and stirred for 1 hour. The slide glass is taken out from the reaction vessel, and cleaned with tetrahydrofuran, methanol and ultrapure water, and then dried.

[0193] A dried glass slide and 143 mg copper (I) bromide were placed in a reaction vessel and purged with nitrogen. 156 mg 2,2′-bipyridine and 874 mg 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate were placed in a pear-shaped flask and purged with nitrogen. Then, 5 mL of nitrogen-purged methanol and 5 mL of nitrogen-purged ultrapure water were added to the pear-shaped flask to dissolve it. The solution in the pear-shaped flask was placed in a reaction vessel containing a glass slide and copper (I) bromide by using a syringe and stirred for 1 hour. Afterwards, the resulting product was cleaned with ethanol, phosphate buffered saline (PBS) and ultrapure water to provide a glass slide with a polymer brush formed thereon.

[0194] The resulting slide glass having the polymer brush formed thereon was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement. In addition, low protein adsorption and durability were evaluated in the same manner as in Example 1.

[0195] (Comparative Example 6)

[0196] The procedures up to the evaluation were performed in the same manner as in Example 1 except that the amount of 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate was changed to 270 mg.

[0197] (Comparative Example 7)

[0198] The respective processes were carried out in the same manner as in Example 1 until the condensation reaction.

[0199] The slide glass that has undergone the condensation reaction is placed in a 300mL glass container, and the glass container is covered with a lid. The inside of the glass container is purged with nitrogen, and 65mL ultrapure water and 35mL ethanol are placed in the glass container while nitrogen flows at 200ml / min. A needle is placed in the liquid in the glass container, and nitrogen is bubbled at 150mL / min for 30 minutes. 126mg of 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate used as a polymerization initiator is dissolved in 20ml ultrapure water purged with nitrogen, and the resultant is loaded into a 300ml glass container. Then, the resultant is stirred at room temperature for 60 minutes. 900 mg of 2-hydroxyethyl methacrylate used as a hydrophilic monomer, 100 mg of 2-(dimethylamino)ethyl methacrylate used as a monomer having a polarity opposite to that of the substrate, and 30 mg of polyethylene glycol diacrylate (n=about 9) used as a crosslinking agent were dissolved in a mixed solvent of ultrapure water purged with nitrogen and ethanol in a ratio of 10 mL / 15 mL, and the resultant was charged into a 300 mL glass container. Thereafter, a polymerization reaction was performed at 60° C. for 3 hours. The slide glass subjected to the polymerization reaction was poured and washed with ethanol and RO water, and ultrasonically cleaned at 28 kHz to provide a slide glass having a film.

[0200] The obtained slide glass was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement. In addition, in the same manner as in Example 1, low protein adsorption and durability were evaluated.

[0201] (Comparative Example 8)

[0202] The surface of a SUS304 plate (width×length×thickness=26mm×60mm×1mm) was cleaned with acetone and dried. The SUS304 plate was immersed in a 2 mass % aqueous solution of 3-(acryloyloxy)propyltrimethoxysilane (with 2 mass % acetic acid added) for 10 minutes and removed, followed by drying at 40°C for 24 hours. Afterwards, the SUS304 plate was washed with water and then with acetone. The treated SUS304 plate was immersed in a 1.5 mass % ethanol solution of azobisisobutyronitrile for 5 minutes and removed, followed by drying.

[0203] The SUS304 plate was placed in a glass container containing a 1.25 M aqueous solution of 3-sulfopropyl methacrylate potassium salt, and the glass container was covered with a lid.

[0204] After purging by introducing nitrogen, the glass container was immersed in a water bath at 60°C for 5 hours to perform heating and polymerization. Thereafter, the surface was washed with water to wash off unreacted monomers and the like to provide a SUS304 plate with a film. The obtained SUS304 plate with a film was subjected to zeta potential measurement, TOF-SIMS measurement, and ESCA measurement, and low protein adsorption and durability were evaluated in the same manner as in Example 1.

[0205] (Comparative Example 9)

[0206] The procedures up to the evaluation were performed in the same manner as in Example 1 except that the amount of 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate was changed to 180 mg.

[0207] Table 4 shows the physical properties and evaluation results of the films obtained in Examples 1 to 17 and Comparative Examples 1 to 9.

[0208] Table 4

[0209]

[0210]

[0211] The antifouling member of the present invention can achieve both low protein adsorption and durability. Therefore, the antifouling member of the present invention can perform measurements with high accuracy and can be used repeatedly when used as a member for an in vitro diagnostic device.

[0212] While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments.The scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. An antifouling member comprising a film formed on a substrate, wherein the film comprises a condensate of a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative, wherein the film contains no halogen atoms, wherein the zeta potential of the membrane in water having a pH of 7 is greater than -10.0 mV and less than 10.0 mV, and in, The B / A value of the antifouling member is 2.0 or more and 5.0 or less, wherein A represents the ratio of the amount of C atoms in atomic % in X-ray photoelectron spectroscopy analysis of the substrate, i.e., ESCA measurement, to the amount of atoms of the element X in atomic % contained in the surface of the substrate in the largest amount, and B represents the ratio of the amount of C atoms in atomic % in X-ray photoelectron spectroscopy analysis of the film on the substrate, i.e., ESCA measurement, to the amount of atoms of the element X in atomic %.

2. The antifouling member according to claim 1, wherein The membrane comprises a copolymer of a monomer having a polarity opposite to that of the substrate, a hydrophilic monomer, and a cross-linking agent.

3. The antifouling member according to claim 1, wherein The film comprises a polymer of betaine monomers.

4. The antifouling member according to claim 1, wherein The membrane comprises a polymer of betaine monomers and a polymer of hydrophilic monomers.

5. The antifouling member according to claim 2 or 4, wherein The hydrophilic monomer is electrically neutral. 6 . The antifouling member according to claim 2 , wherein the hydrophilic monomer is at least one selected from the group consisting of: 2-hydroxyethyl methacrylate; N-vinyl-2-pyrrolidone; and polyethylene glycol monomethyl ether methacrylate.

7. The antifouling member according to claim 3 or 4, wherein the betaine monomer is at least one selected from the group consisting of 2-(methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate; 2-[[2-(methacryloyloxy)ethyl]dimethylammonio]acetate; 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propionate; 3-[(3-acrylamidopropyl)dimethylammonio]propionate; 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate; 4-[[2-(methacryloyloxy)ethyl]dimethylammonio]butane-1-sulfonate; 3-[[2-(acryloyloxy)ethyl]dimethylammonio]propane-1-sulfonate; 3-[(3-methacrylamidopropyl)dimethylammonio]propane-1-sulfonate; and 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonate.

8. The antifouling member according to any one of claims 1 to 4, wherein the silane coupling agent is at least one selected from the group consisting of: 3-(methacryloyloxy)propyltrimethoxysilane; 3-(acryloyloxy)propyltrimethoxysilane; 3-(methacryloyloxy)propyltriethoxysilane; 3-(acryloyloxy)propyltriethoxysilane; vinyltrimethoxysilane; triethoxyvinylsilane; allyltrimethoxysilane; allyltriethoxysilane; and (triethoxysilyl)methyl methacrylate, and The phosphonic acid derivative is at least one selected from the group consisting of: 2-methacryloyloxyethyl acidic phosphate; 2-acryloyloxyethyl acidic phosphate; 2-(methacryloyloxy)ethyl phosphate; and acidic phosphooxy polyethylene glycol monomethacrylate.

9. The antifouling member according to claim 2, wherein The monomer having a polarity opposite to that of the substrate is an amine monomer.

10. The antifouling member according to claim 9, wherein the amine monomer is at least one selected from the group consisting of: 2-(dimethylamino)ethyl methacrylate; 2-(dimethylamino)ethyl acrylate; 2-(diethylamino)ethyl methacrylate; 2-(diisopropylamino)ethyl methacrylate; and 2-(tert-butylamino)ethyl methacrylate.

11. The antifouling member according to claim 2, wherein the crosslinking agent is at least one selected from the group consisting of ethylene glycol diacrylate; ethylene glycol dimethacrylate; polyethylene glycol diacrylate; polyethylene glycol dimethacrylate; and 1,6-hexanediol diacrylate.

12. The antifouling member according to any one of claims 1 to 4, wherein The material of the substrate includes stainless steel or glass.

13. The antifouling member according to any one of claims 1 to 4, wherein The membrane includes a charge regulation site having a polarity opposite to that of the substrate and a hydrophilic site.

14. The antifouling member according to claim 13, The binding site with the substrate is represented by the following formula (1-1) or the following formula (1-2): wherein the charge adjustment site is a copolymerization product of monomers represented by the following formula (2-1), the following formula (2-2) and the following formula (2-3), or is represented by the following formula (2-4), and The hydrophilic part is represented by the following formula (3-1) or the following formula (3-2): In formula (1-1) and formula (1-2), "*" indicates the binding position with the substrate, "**" indicates the binding position with the charge adjustment site, and R 1 represents any one of an alkylene group or an oxyalkylene group, and R 2 represents any one of the following: CH=CH, i.e., vinylidene, CH=C(CH3), i.e., isopropenylene, HC=CH=C(=O)-O, i.e., acryloyloxy, HC=C(CH3)-C(=O)-O, i.e., methacryloyloxy, or HC=C(CH3)-C(=O)-NH, i.e., methacryloylamino, and "l" satisfies 1≤l≤6; In formula (2-1), formula (2-2) and formula (2-3), R 3 represents hydrogen or methyl, R 4 , R 5 and R 8 Each independently represents an alkylene group having 1 to 6 carbon atoms, and R 6 represents hydrogen or an alkyl group having 1 to 3 carbon atoms, and R 7 represents an oxyalkylene group having 1 to 3 carbon atoms; In formula (2-4), "**" indicates the binding position with the binding site, R 9 and R 10 Each independently represents an alkylene group or an oxyalkylene group, and R 11 Representation-COO - , and "m" satisfies 4≤m≤1,000; and In formula (3-1) and formula (3-2), "***" represents the binding position with the charge adjustment site, R 8 represents an alkylene group having 1 to 6 carbon atoms, "n" satisfies 4≤n≤1000, R 9 and R 10 Each independently represents an alkylene group or an oxyalkylene group, and R 11 Representation-COO - , and "m" satisfies 4≤m≤1000.

15. An in vitro diagnostic device using the antifouling member according to any one of claims 1 to 4 and 9 to 11.

16. A method for producing an antifouling member having a film formed on a surface of a substrate, the method comprising: a step of bonding a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative to the surface of the substrate; a charge regulating site forming step of copolymerizing a monomer having a polarity opposite to that of the substrate, a hydrophilic monomer, and a cross-linking agent in the presence of a polymerization initiator after the combining step; and The charge regulation site forming step is followed by a hydrophilic site forming step of adding a hydrophilic monomer and polymerizing the hydrophilic monomer to form a hydrophilic site.

17. A method for producing an antifouling member having a film formed on a surface of a substrate, the method comprising: a step of bonding a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative to the surface of the substrate; and After the combining step, a two-site forming step is performed in which 2.0 mg / ml or more and 12.0 mg / ml or less of a betaine monomer is polymerized in the presence of a polymerization initiator to form a charge regulating site and a hydrophilic site.

18. A method for producing an antifouling member having a film formed on a surface of a substrate, the method comprising: a step of bonding a hydrolyzate of a silane coupling agent or a condensate of a phosphonic acid derivative to the surface of the substrate; a charge regulating site forming step of polymerizing the betaine monomer in the presence of a polymerization initiator after the combining step; and The charge regulation site forming step is followed by a hydrophilic site forming step of adding a hydrophilic monomer and polymerizing the hydrophilic monomer to form a hydrophilic site. 19 . The method for producing an antifouling member according to any one of claims 16 to 18 , wherein the polymerization initiator is 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate.

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