JP2014210397A - Production method of substrate having hydrophilic layer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for stably introducing a high-quality hydrophilic layer capable of sufficiently inhibiting nonspecific adsorption of a bio-related substance such as protein into a plastic surface of a substrate.SOLUTION: A production method of a substrate having a hydrophilic layer on a surface thereof is provided, which includes: a substrate preparation step of preparing a substrate having a plastic surface; a plasma treatment step 1 of exposing the plastic surface of the substrate to plasma containing rare gas; a plasma treatment step 2 of exposing the plastic surface of the substrate to plasma containing fluorocarbon gas; a primer layer formation step of forming a primer layer containing polysiloxane by polymerizing a silanol compound having an organic group that contains a carbon atom directly bonded to a silicon atom and has a functional group, on the surface of the substrate exposed to plasma; and a hydrophilic layer formation step of linking the primer layer with a hydrophilic polymer via a covalent bond by the reaction of the functional group on a side chain of the polysiloxane with a functional group of the hydrophilic polymer.

Description

  The present invention relates to a method for producing a substrate having a hydrophilic layer.

  In order to suppress the adsorption of biological substances such as proteins and cells, development of a technique for modifying the surface of a hydrophilic polymer such as MPC polymer or polyethylene glycol has been promoted (for example, Patent Documents 1 and 2). However, these polymers and a substrate made of glass or plastic cannot be directly bonded chemically. Therefore, for example, when plastic is used as the substrate, Patent Documents 3 and 4 describe a method using polysiloxane obtained by hydrolyzing a silane coupling agent as a linker.

  However, the above method alone makes it difficult to produce a uniform surface due to variations in surface quality due to the nature of the substrate. Therefore, as a pretreatment, a method has been established in which polysiloxane obtained by hydrolyzing a silane coupling agent is reacted with a surface to which fluorine has been added by plasma irradiation treatment using a fluorine-based gas. This is a technique for uniformizing and increasing the added amount due to the high affinity due to the difference in electronegativity between fluorine and silicon. However, this method has a problem that the plasma-treated surface deteriorates quickly and lacks storage stability. Non-patent document 1 suggests that the reason is that surface molecules are taken into the base material due to changes over time.

Therefore, it is essential to develop a technique for suppressing deterioration with time after plasma irradiation. For example, Patent Document 5 describes that when an oxygen plasma treatment is performed after a plasma treatment with argon or the like, a hydrophilic surface having durability is obtained as compared with a case where an oxygen plasma treatment is performed alone. . However, the pretreatment method using a fluorine-based gas is still unclear and requires technical development and verification. In addition, it has been pointed out that fluorine-based gases such as carbon tetrafluoride (CF ₄ ) are global warming gases, and it is essential to reduce the amount used. Therefore, establishment of a method for efficiently adding fluorine to the surface is desired.

Japanese Patent No. 3434891 Japanese Patent No. 5070565 Patent 5001015 JP2013-011479A JP 2012-102205 A

ThreeBond Technical News No.26 1989

  The present invention is a method of forming a primer layer after plasma treatment of the plastic surface of the substrate and introducing a hydrophilic layer through the primer layer, while reducing the amount of fluorine-based gas used for plasma treatment, The object is to form a high-quality hydrophilic layer with high storage stability.

  The inventor exposes the plastic surface of the substrate to a plasma containing a rare gas, and further to a plasma containing a fluorocarbon gas, and forms a primer layer containing polysiloxane on the surface. The present inventors have found that a high-quality hydrophilic layer having high storage stability can be formed while reducing the amount of fluorine-based gas used for plasma treatment by linking the polymer and the hydrophilic polymer via a covalent bond.

That is, the present invention includes the following inventions.
(1) A method for producing a substrate having a hydrophilic layer on the surface,
A substrate preparation step of preparing a substrate having a plastic surface;
A plasma treatment step 1 in which the plastic surface of the substrate is exposed to a plasma containing a rare gas;
A plasma treatment step 2 in which the plastic surface of the substrate is exposed to a plasma containing a fluorocarbon gas;
A primer layer forming step of hydrolyzing a silicon compound on the surface of the substrate exposed to plasma, polymerizing the generated silanol compound, and forming a primer layer containing polysiloxane;
A hydrophilic layer forming step in which the primer layer and the hydrophilic polymer are linked via a covalent bond by a reaction between a functional group on the side chain of the polysiloxane and a functional group of the hydrophilic polymer;
Said method.
(2) The method according to (1), wherein the rare gas is argon gas.
(3) The method according to (1) or (2), wherein the carbon fluoride gas is a carbon tetrafluoride gas.
(4) The method according to any one of (1) to (3), wherein the fluorine / carbon ratio (F / C) on the substrate surface after the plasma treatment step 2 is 0.35 or less.
(5) The method according to any one of (1) to (4), wherein the plastic is polystyrene.

  According to the present invention, it is possible to supply a base material having a high-quality and highly durable hydrophilic layer that can sufficiently suppress nonspecific adsorption of biological substances while reducing the amount of fluorine-based gas used for plasma treatment. .

FIG. 1 is a graph showing the results of surface analysis of the dishes after plasma treatment of Example 1, Comparative Example 1 and Comparative Example 2 using XPS. FIG. 2 is a photograph showing the evaluation classification when the state of cell adhesion is observed with an optical microscope.

(Base material)
In the present invention, the shape of the substrate is not particularly limited. For example, a substrate in the form of a plate, a multiwell plate, a microplate (a plate having a plurality of recesses), particles, a slide, a plate, a film, a tube, a capillary, a microchannel, or the like is used. it can. Examples of the material for the base material include plastic, glass, quartz, silicon, and metal.

  The substrate has a plastic surface. Plastic surface refers to a surface containing plastic, preferably a surface made of plastic. At least a part of the surface of the substrate is a plastic surface, and the surface to be plasma-treated is preferably a plastic surface. When the substrate is plate-shaped or substantially plate-shaped such as a multiwell plate, it is preferable that at least the surface to be plasma-treated is a plastic surface, and the surface is made of plastic. Most preferably, the whole substrate is made of plastic.

  Specific examples of plastics include vinyl polymers such as polyvinyl chloride and polyvinylidene chloride; styrene resins such as polystyrene, acrylonitrile-styrene copolymers and ABS resins; polymethyl (meth) acrylate, polyethyl (meth) acrylate Acrylic resins such as polyacrylonitrile; polyolefin resins such as polyethylene, polypropylene, polybutene, and cyclic polyolefin; polyester resins such as polyethylene terephthalate, ethylene glycol-terephthalic acid-isophthalic acid copolymer, polybutylene terephthalate; polycarbonate resins; Polyurethane resin; epoxy resin; nylon 6, nylon 66, nylon 610, nylon 612, nylon 11, nylon 12, nylon 46, etc. Bromide resins; methylpentene resins; phenolic resins; melamine resins, epoxy resins, and fluorine resins. These may be used alone or in combination of two or more. Polystyrene, polyvinyl chloride, polypropylene, and polyethylene are particularly preferable because they easily form a primer layer and a hydrophilic layer after the plasma treatment described later.

  The substrate preferably has a hydrophobic surface. Specifically, the water contact angle on the substrate surface is preferably 60 ° or more, more preferably 70 ° or more, and further preferably 80 ° or more. In the present invention, the water contact angle refers to a water contact angle measured at 23 ° C.

(Plasma treatment)
In the method of the present invention, the plastic surface of the substrate is exposed to plasma containing a rare gas (plasma treatment step 1), and further exposed to plasma containing a fluorocarbon gas (plasma treatment step 2). By performing the plasma treatment, the primer layer and the hydrophilic layer can be more stably formed without being limited to the type of plastic. In addition, the amount of fluorocarbon gas used can be reduced by exposure to plasma containing a rare gas before exposure to plasma containing the fluorocarbon gas. Furthermore, the two-stage plasma treatment process can suppress the deterioration of the plasma treatment surface, and can provide a plasma treatment surface with high storage stability, thereby forming a highly durable hydrophilic layer.

  The plasma processing step 1 is not particularly limited as long as it is exposed to plasma containing a rare gas. Examples of the rare gas used include helium (He), neon (Ne), argon (Ar), and xenon (Xe). These may be used alone or in admixture of two or more. Preferably, the plasma is exposed to a plasma containing a rare gas selected from argon gas and helium gas.

Other inert gases may be mixed with the rare gas. Examples of the inert gas include nitrogen (N ₂ ) and oxygen (O ₂ ).

  From the viewpoint of reducing the amount of fluorocarbon gas used, it is preferable that the plasma treatment step 1 includes at least a step of exposing to a plasma containing a rare gas but substantially not containing a fluorocarbon gas.

  The concentration of the rare gas during plasma generation is preferably 25% by volume or more. By setting the concentration of the rare gas to a certain level or more, the surface cross-linked layer is stably formed.

  The pressure at the time of plasma generation in the plasma treatment step 1 is preferably 1 Pa or less, preferably 0.2 Pa or more, more preferably 0.4 Pa or more. If the pressure is less than 0.2 Pa, the pressure control may be difficult. If the pressure is too high, the irradiation time must be shortened, and the variation after irradiation may increase.

The plasma treatment process 2 is not particularly limited as long as it is exposed to plasma containing a fluorocarbon gas. Examples of the fluorocarbon include unsaturated fluorocarbons (fluorocarbons having an unsaturated bond) such as fluoroethylene (C ₂ F ₄ ) and hexafluoropropylene (CF ₃ CFCF ₂ ), carbon tetrafluoride ( Saturated fluorocarbons such as CF ₄ ), carbon hexafluoride (C ₂ F ₆ ), octafluorocyclobutane (C ₄ F ₈ ), trifluoromethane (CHF ₃ ) and octafluoropropane (C ₃ F ₈ ) Can be mentioned. As carbon fluoride, ethylene trichloride trichloride (C ₂ ClF ₃ ), carbon trichloride monofluoride (CCl ₃ F), carbon monobromide trifluoride (CBrF ₃ ), hexafluoroacetone ((CF ₃ ) ₂ CO), hexafluoroalcohol ((CF ₃ ) ₂ CHOH) and other halogen containing elements other than fluorine may be used, but fluorocarbon consisting only of fluorine and carbon is preferred. These fluorocarbon gases may be used alone or in combination of two or more.

  Further, it is preferable to use a fluorocarbon gas having a fluorine / carbon ratio (F / C) of 2 or more. When the fluorine / carbon ratio (F / C) is 2 or more, fluorine can be stably added, and the coating quality can be further stabilized.

An inert gas may be mixed with the plasma containing the fluorocarbon gas. Examples of the inert gas include noble gases such as helium (He), neon (Ne), argon (Ar), and xenon (Xe), nitrogen (N ₂ ), oxygen (O ₂ ), and the like. These may be used alone or in admixture of two or more. The mixing ratio of the fluorocarbon gas and the inert gas is appropriately determined depending on the type of gas used. The concentration of the fluorocarbon gas during plasma generation is preferably 10% by volume or more. By setting the concentration of the fluorocarbon gas to a certain level or more, the bond density of the primer layer can be improved and the amount of hydrophilic polymer added can be increased.

  Alternatively, in the plasma treatment step 2, the treatment may be continuously performed by injecting the fluorocarbon gas little by little into the plasma containing the rare gas used in the plasma treatment step 1. Usually, when changing the gas type for plasma processing, it is necessary to exhaust and then vacuum again, but this is not necessary for continuous processing. Manufacturing time can be shortened.

  The pressure at the time of plasma generation in the plasma treatment step 2 is preferably 1 Pa or less, preferably 0.2 Pa or more, more preferably 0.4 Pa or more. If it is less than 0.2 Pa, pressure control may be difficult, and by setting it to 1 Pa or less, it is possible to suppress an excessive increase in the amount of fluorine added. If the pressure is too high, the irradiation time must be shortened to achieve the required amount of fluorine addition, and variations after irradiation may increase.

  The total flow rate during plasma generation in plasma treatment steps 1 and 2 is not particularly limited, but is usually 5 to 50 ml / min, preferably 10 to 30 ml / min. By setting the flow rate within the above range, it is possible to suppress pressure fluctuation when plasma is generated.

  In the plasma treatment steps 1 and 2, the time for which the substrate is irradiated with plasma is preferably 20 seconds or less, more preferably 10 seconds or less, still more preferably 5 seconds or less, and preferably 2 seconds or more. If the irradiation time is too short, it may be difficult to obtain the effect of the plasma treatment. By setting the irradiation time to 20 seconds or less, it is possible to suppress the amount of fluorine addition from increasing more than necessary.

  The power applied to the plasma generating unit is preferably 100 W or more, more preferably 200 W or more, still more preferably 300 W or more, and preferably 500 W or less. By setting the power to a certain level or more, the plasma is stabilized, so that variation can be suppressed.

  By setting it as the above plasma irradiation conditions, the unevenness | corrugation of the base-material surface becomes large, the bond density with a primer layer improves, and the addition amount of a hydrophilic polymer can be increased.

  The plasma discharge apparatus preferably used in the plasma treatment is a direct current glow discharge and low frequency discharge using an internal electrode system, a high frequency discharge using an internal electrode system, an external electrode system and a coil type system, and a micro wave system using a waveguide system. There are wave discharge and electron cyclotron resonance discharge (ECR discharge). However, the present invention is not limited to this, and other methods can be used as long as they generate plasma and thereby cause a reaction on the surface.

  Fluorine is introduced near the surface of the substrate after the plasma treatment. The fluorine is introduced at a depth that can be detected by at least a surface analyzer such as X-ray photoelectron spectroscopy (XPS). In the substrate surface exposed to plasma, the fluorine / carbon ratio (F / C) is preferably 0.35 or less, more preferably 0.3 or less, and further preferably 0.25 or less. preferable. Further, the fluorine / carbon ratio (F / C) is preferably 0.1 or more, and more preferably 0.15 or more. The fluorine / carbon ratio (F / C) on the substrate surface can be measured using X-ray photoelectron spectroscopy (XPS). Here, XPS is measured by using an X-ray spectroscopic analyzer “ESCA5600” manufactured by ULVAC-PHI, Inc. and setting the photoelectron uptake angle to 45 °. Specifically, C1s and F1s spectra are acquired by ESCA5600 (ULVAC-PHI Co., Ltd.), the area of components derived from each element is calculated by MultiPak (attached software), and the component ratio is calculated as a percentage. Then, the ratio of F1s to C1s is calculated as the fluorine / carbon ratio (F / C). By including a predetermined amount of fluorine, the amount of binding of the primer layer to be formed later on the base material is increased, and as a result, the effect of increasing the amount of hydrophilic polymer immobilized through the primer layer is obtained. .

  Although it is the knowledge which the present inventors obtained by earnestly examining, it turns out that the state of the hydrophilic layer formed depends on the state of the plastic surface. However, it is difficult to prepare a substrate having a plastic surface whose quality is always constant. On the other hand, by using a substrate obtained by plasma treatment, hydrophilic polymer can be introduced at high density through the primer layer as described later on the plasma-irradiated plastic surface. The hydrophilic layer can be formed. Thereby, the biochemical test instrument in which the nonspecific adsorption of the biological substance is suppressed can be produced. Thus, the substrate obtained by exposure to plasma can be advantageously used as a material for producing biochemical test instruments.

(Primer layer)
The primer layer can be formed of a layer containing at least polysiloxane. Here, polysiloxane is a polymer composed of repeating units of siloxane bonds (Si—O—Si) and can be obtained by condensation polymerization of silanol compounds. The condensation of the silanol compound is a reaction that occurs between the molecules of the silanol compound. When the plastic molecule on the substrate surface does not have a reactive functional group, no reaction occurs between the silanol compound and the plastic molecule on the substrate surface. That is, the silanol compound and the formed polysiloxane do not chemically react with the plastic molecules on the substrate surface, but are merely physically adsorbed. The physical adsorption power of such a silanol compound to the plastic surface is extremely weak with a monomer, but becomes stronger when condensation proceeds to some extent and becomes a polymer (polysiloxane). A primer layer containing polysiloxane is formed on the plastic surface by appropriately condensing the silanol compound.

  The primer layer containing polysiloxane can be formed by, for example, a method described in JP2013-11479A. Specifically, it can be formed by a step of polymerizing a silanol compound on the plastic surface after the plasma treatment. The step preferably includes a step of hydrolyzing the silicon compound to produce a silanol compound, and a step of bringing a solution in which the produced silanol compound and a base are dissolved in an alcohol into contact with the surface of the substrate. As the silicon compound, a commercially available compound as a silane coupling agent can be suitably used, and 3-glycidoxypropyltrimethoxysilane or 3-glycidoxypropyltriethoxysilane is particularly preferable. Hydrolysis conditions are not particularly limited, but for example, the following method is possible. First, dilute hydrochloric acid is added to the silicon compound. The pH of the diluted hydrochloric acid is desirably adjusted to 2.0 to 3.0. The molar ratio of water molecules to silicon compound is 2-4.

  A silanol compound is then applied to the substrate surface to form a polysiloxane by condensation polymerization. Silanol compounds are dissolved in alcohol together with a base. It is desirable to adjust the final concentration of the silanol compound in the range of 0.1 to 10% (v / v). As the base, triethylamine, N, N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine and the like can be used, but the base is not limited thereto. It is desirable to adjust the final concentration of the base in the range of 0.1 to 10% (v / v). As the alcohol, ethanol, 2-propanol, tert-butyl alcohol, and the like can be used, but the alcohol is not limited thereto. This silanol compound solution is brought into contact with the plastic surface of the substrate and left for 10 minutes to 24 hours. The reaction temperature can be set in the range of 4 to 80 ° C, but room temperature (20 to 25 ° C) is particularly preferable. By the above operation, a primer layer containing polysiloxane is formed on the plastic surface by physical adsorption.

  The formed polysiloxane can be converted to a functional group of a hydrophilic polymer on the side chain, for example, a functional group capable of reacting with a hydroxyl group of polyalkylene glycol to form a covalent bond, or can be converted to such a functional group. It is preferable to have a functional group. Examples of such functional groups include (1H-imidazol-1-yl) carbonyl group, succinimidyloxycarbonyl group, glycidyl group, epoxy group, aldehyde group, amino group, thiol group, carboxyl group, and azido group. , Cyano group, active ester group (1H-benzotriazol-1-yloxycarbonyl group, pentafluorophenyloxycarbonyl group, paranitrophenyloxycarbonyl group, etc.), halogenated carbonyl group, isocyanate group, maleimide group, etc. Of these, a glycidyl group or an epoxy group is preferred.

(Hydrophilic layer)
The hydrophilic layer disposed on the primer layer includes a hydrophilic polymer. The hydrophilic polymer refers to a polymer containing a carbon component and having a hydrophilic functional group in the main chain or side chain of the polymer. The hydrophilic polymer is preferably a water-soluble polymer containing a carbon-oxygen bond and having water solubility and water swellability. The hydrophilic polymer may be constantly water-soluble or water-swellable, or may be water-soluble or water-swellable by a predetermined stimulus such as light, temperature, and pH.

  Specific examples of the hydrophilic polymer include polyalkylene glycol, polyvinyl pyrrolidone, polypropylene glycol, polyvinyl alcohol, polyethylene imine, polyallylamine, polyvinyl amine, polyvinyl acetate, polyacrylic acid, poly (meth) acrylamide, poly-N-isopropyl. Examples thereof include hydrogel polymers such as acrylamide, copolymers of these with other monomers, and graft polymers. Among them, polyalkylene glycols having various molecular weights are commercially available and are excellent in biocompatibility, so that they can be suitably used.

  Specific examples of the polyalkylene glycol include polyethylene glycol (PEG), polypropylene glycol, and a copolymer of ethylene glycol and propylene glycol. In the present invention, polyethylene glycol is particularly preferably used as the hydrophilic polymer. The molecular weight of polyethylene glycol is not particularly limited, and can be appropriately set according to the use of the substrate. The number average molecular weight of polyethylene glycol is preferably 176 or more, more preferably 300 or more, and still more preferably 350 or more. The upper limit of the number average molecular weight of the polyethylene glycol is not particularly limited, but the number average molecular weight of the polyethylene glycol is difficult because the viscosity increases as the number average molecular weight increases, and it is difficult to arrange the polyethylene glycol at a high density. Is preferably 25000 or less, more preferably 10,000 or less, and even more preferably 1000 or less.

  PEG is bonded to the primer layer via a covalent bond formed by reaction of a hydroxyl group at one end of PEG with a functional group on the side chain of the polysiloxane or a functional group derived from the functional group. The hydroxyl group at the other end of PEG may be in a state of being not derivatized and blocked with a hydroxyl group, and a functional group capable of forming a covalent bond with another substance is directly or indirectly It may be in a state of being introduced (through the linker).

  The functional group introduced at the other end of PEG is typically (1H-imidazol-1-yl) carbonyl group, succinimidyloxycarbonyl group, epoxy group, aldehyde group, amino group, thiol group. , Carboxyl group, azide group, cyano group, active ester group (1H-benzotriazol-1-yloxycarbonyl group, pentafluorophenyloxycarbonyl group, paranitrophenyloxycarbonyl group, etc.), halogenated carbonyl group (carbonyl chloride group) Carbonyl fluoride group, carbonyl bromide group, carbonyl iodide group) and the like. These functional groups may be directly linked to PEG as a substituent that replaces the hydrogen of the hydroxyl group at the terminal of PEG, or as a functional group bonded to a linker structure bonded to the terminal of PEG, as PEG. It may be indirectly connected to. Examples of the linker structure include a divalent group having 0 to 3 carbon atoms and 0 to 3 identical or different heteroatoms selected from nitrogen, oxygen and sulfur. The hydrophilic layer may further contain other hydrophilic compounds.

(Formation method of hydrophilic layer)
The base material of the present invention can be produced by a method including at least a step of forming a primer layer and a step of linking a hydrophilic polymer to the polysiloxane of the primer layer.

  The hydrophilic layer reacts the functional group on the side chain of the polysiloxane of the primer layer (the functional group corresponding to the functional group of the silanol compound or the functional group derived from the functional group) with the functional group of the hydrophilic polymer. Can be formed. The reaction conditions are appropriately selected based on the functional group on the side chain of the polysiloxane and the kind of the hydrophilic polymer.

  For example, when the hydroxyl group of polyalkylene glycol is reacted with a functional group on the side chain of polysiloxane, an oxidation catalyst, preferably a polyalkylene glycol containing a catalytic amount of concentrated sulfuric acid, is contacted with the primer layer. Here, the polyalkylene glycol having a number average molecular weight exceeding 1000 is previously melted by heating. If necessary, polyalkylene glycol may be diluted with tert-butyl alcohol or the like. The polyalkylene glycol solution is brought into contact with the plastic surface and heated. The heating temperature can be set in the range of 60 to 100 ° C., but preferably around 80 ° C. (75 ° C. to 85 ° C.) considering the heat resistance of the plastic. The heating time can be set in the range of 10 minutes to 24 hours, but preferably 10 minutes to 60 minutes when the heating temperature is around 80 ° C.

  The density and hydrophilicity of the hydrophilic polymer in the hydrophilic layer can be easily evaluated using the contact angle of water on the surface of the hydrophilic layer as an index. For example, if the water contact angle on the surface of the hydrophilic layer is typically 48 ° or less, preferably 40 ° or less, more preferably 30 ° or less, the hydrophilic polymer material is present in sufficient density, It is thought to have.

(Use of base material)
The substrate having a hydrophilic layer on the surface obtained by the present invention is suitably used for a biochemical test instrument because non-specific adsorption of biomolecules or bio-related substances is suppressed. Biochemical test instruments include proteins, peptides, saccharides, nucleic acids (DNA, RNA), lipids, coenzymes, cells, viruses, bacteria, and other test instruments that handle biomolecules or related substances, and cell culture Equipment. Specific examples include instruments for detecting the interaction of biomolecules or bio-related substances, such as solid phase carriers for immunoassays, carriers for microarrays, carriers for DNA chips, and the like. It can also be used for multiwell plates, microplates, pipettes, syringes, cell culture bags, and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to the range of an Example.

<Test Example 1>
Plasma treatment was performed on a cell culture dish (BD Palcon) having a diameter of 3.5 cm. The details are described below.

Using EXAM (Shinko Seiki Co., Ltd.), the dish was first irradiated with argon plasma under conditions of RF power 300 W, pressure 1 Pa, and 10 seconds. Subsequently, after degassing argon, CF ₄ plasma irradiation was performed under the same conditions. This is Example 1.

Examples comparing the contrast material subjected only CF ₄ plasma irradiation 1 was things were CF ₄ plasma irradiation after performing an oxygen plasma before instead of the argon plasma irradiation as Comparative Example 2.

  In order to see the difference in surface fluorine addition amount between Example 1, Comparative Example 1 and Comparative Example 2, surface analysis using XPS was performed. The details are described below.

  The bottom surface of the dish was cut with an ultrasonic cutter, and the spectrum measurement of each element of F1s, C1s, and O1s was repeated 10 times with K-Alpha (Thermo Fisher Scientific). Since fluorine is easily damaged by X-rays, measurement was performed at the beginning of various samples.

  The results are shown in FIG. The yellow-green line is Example 1, the red line is Comparative Example 1, and the blue line is Comparative Example 2. From this, it was found that the amount of addition of fluorine in Example 1 was increased as compared with Comparative Examples 1 and 2.

<Test Example 2>
Using the dishes of Example 1, Comparative Example 1 and Comparative Example 2, surface coating with polyethylene glycol 400 was performed according to the method described in Patent Document 4. Specifically, PEG400 containing a catalytic amount of concentrated sulfuric acid was added and heated at 80 ° C. for 45 minutes. Thereafter, the dish was washed with pure water and dried by nitrogen blowing. By this operation, a hydrophilic layer containing PEG was formed on the primer layer.

  Separately, a sample was prepared by applying a surface coating to a dish that had not been pretreated. This was designated as Comparative Example 3.

  The coating dish was sterilized with 70% ethanol and then washed twice with phosphate buffer before use.

1 × 10 ⁶ CCL cells were seeded per dish, and cultured for 24 hours in a 37 ° C. incubator using DMEM medium (Sigma) containing 10% volume ratio of fetal calf serum. Thereafter, the state of cell adhesion was observed with an optical microscope (Olympus). The observation results were classified according to the state of cell adhesion as shown in the photograph of FIG. The results are summarized in Table 1 below.

  From the above, it was found that cell adhesion suppression is improved by pretreatment with fluorocarbon plasma.

<Test Example 3>
A sample was prepared in which the dishes of Example 1 and Comparative Examples 1 to 3 that were subjected to surface coating with polyethylene glycol in Test Example 2 were placed in a polyethylene bag and heated at 40 ° C. for 2 weeks.

  Then, after sterilizing and washing according to the method described in Test Example 2, cells were similarly seeded and the state of adhesion was performed following the method of Test Example 2. The results are summarized in Table 2 below.

  From the above, it has been found that the durability of the hydrophilic layer is improved by the plasma treatment containing the rare gas argon gas and the plasma treatment containing the fluorocarbon gas.

Claims (5)

A method for producing a substrate having a hydrophilic layer on the surface,
A substrate preparation step of preparing a substrate having a plastic surface;
A plasma treatment step 1 in which the plastic surface of the substrate is exposed to a plasma containing a rare gas;
A plasma treatment step 2 in which the plastic surface of the substrate is exposed to a plasma containing a fluorocarbon gas;
A primer layer forming step of hydrolyzing a silicon compound on the surface of the substrate exposed to plasma, polymerizing the generated silanol compound, and forming a primer layer containing polysiloxane;
A hydrophilic layer forming step in which the primer layer and the hydrophilic polymer are linked via a covalent bond by a reaction between a functional group on the side chain of the polysiloxane and a functional group of the hydrophilic polymer;
Said method.   The method according to claim 1, wherein the rare gas is argon gas.   The method according to claim 1 or 2, wherein the fluorocarbon gas is a carbon tetrafluoride gas.   The method according to any one of claims 1 to 3, wherein a fluorine / carbon ratio (F / C) of the substrate surface after the plasma treatment step 2 is 0.35 or less.   The method according to claim 1, wherein the plastic is polystyrene.

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