JP4649845B2 - Modified substrate - Google Patents

Description

  The present invention relates to a modified base material that is used for treatment of a biological component having a hydrophilic surface. The modified substrate of the present invention can be suitably used as a biological component treatment substrate. Biocomponent separation membranes, medical devices, water treatment separation membranes, bio-experiment-related devices, bioreactors, molecular motors, DDS (drug delivery systems), protein chips, DNA chips, biosensors, or analytical instrument parts It can use suitably also. Especially, it is used suitably for the use used by contacting with protein, for example, a protein separation membrane and a protein storage container.

  Hydrophilization of the substrate surface is a technique used in various fields such as prevention of adhesion of biological components to the substrate, antifogging, drip-proofing, antifouling treatment of the substrate, and primer treatment of the substrate for water-based paints. . In particular, in instruments used when handling biological components such as proteins, reduction or disappearance due to adhesion of biological components to the instruments must be reduced. For example, when a very small amount of protein is handled in proteome analysis or the like, if there are many adhering components, the protein cannot be accurately analyzed, and the protein may be lost.

  In many biological component processing instruments, a polymer material is used as a base material. The reason for this is that the biological component treatment instrument is often disposable in order to prevent infection, contamination of the sample, and the like, and thus a base material that is inexpensive and excellent in moldability is more advantageous. However, the largest factor that a biological component adsorbs on the substrate surface is a hydrophobic interaction, and the interaction becomes stronger in proportion to the hydrophobicity of the substrate. In general, since the surface of the polymer material is hydrophobic, proteins are easily adsorbed.

It is known that the hydrophilic treatment on the surface of the base material is effective for such a problem of biological component adhesion. As the hydrophilization treatment, there is a method of directly generating a hydrophilic functional group on the substrate surface, such as reactive ion etching treatment, plasma treatment or ion cluster beam treatment. There are also a method of introducing a hydrophilic compound into a substrate by a coating treatment (Japanese Patent Laid-Open No. 2003-130882) and a method of introducing a hydrophilic compound by a graft treatment (Japanese Patent Laid-Open No. 58-40323, Japanese Patent No. 3297707).
JP 2003-130882 A JP 58-40323 A Japanese Patent No. 3297707

  However, reactive ion etching treatment, plasma treatment, and ion cluster beam treatment can be easily hydrophilized to the outer surface of the base material or one side of the plate-like base material. Since it is difficult to make the portion to be hydrophilic, it is not suitable for hydrophilizing multiple surfaces such as both sides of the plate-like substrate and the inner and outer surfaces of the hollow substrate in one treatment. In addition, the adsorption characteristics of the biological component of the base material depend on the surface state of the part in contact with the biological component. Generally, the higher the hydrophilicity of the surface, the more the movement of the hydrophilic molecules immobilized on the surface. As the property increases, the adsorption of the biological component to the substrate surface is suppressed. It is considered that hydrophilic molecules having high mobility exclude biological components such as proteins and platelets by their molecular motion. Hydrophilization by reactive ion etching treatment, plasma treatment or ion cluster beam treatment is due to the formation of hydrophilic functional groups such as hydroxyl groups on the surface of the substrate. In comparison, the mobility of hydrophilic molecules is low, so the adhesion suppressing effect of biological components is low, which is not preferable. Furthermore, since it may become high temperature during a process, since a base material may modify | denature, it is not preferable.

  In the hydrophilic polymer coating treatment, when the solvent used in the hydrophilic polymer solution comes into contact with the treated substrate, the coating does not peel off and the hydrophilicity decreases, but also comes into contact with blood. In the case of a medical device, there is a concern that the peeled substance accumulates in the patient. Further, when used in an analytical instrument such as a protein chip or a biosensor, there is a concern that the eluted hydrophilic polymer may be an inhibitory factor for analysis. In addition, hydrophilic polymer grafting improves hydrophilicity in proportion to the amount of grafting, but if the concentration of the hydrophilic polymer solution to be treated becomes high, the hydrophilic polymers crosslink three-dimensionally. However, there is a problem that the mobility of the hydrophilic polymer is lowered and the effect of suppressing the adhesion of biological components is lowered.

  The object of the present invention is to improve the drawbacks of the conventional technology, to efficiently hydrophilize the base material, and to provide a base material used for biological component treatment with less adhesion of biological components.

In order to achieve the above object, the present invention has the following configuration.
(1) In a base material selected from a polymer material, a carbon material, and a composite material obtained by mixing a carbon material and a resin used for biological component treatment, the base material is contacted with a hydrophilic polymer aqueous solution having a molecular weight of 200 or more and alcohols. Modified substrate obtained by irradiating with radiation.
( 2 ) The modified substrate according to (1 ) , wherein the substrate is a polymer material.
( 3 ) The modified substrate according to (1) or ( 2 ), wherein the biological component is a protein.
( 4 ) The modified substrate according to any one of (1) to ( 3 ), which is used for protein separation.
(5) modifying a substrate according to any one of the protein adsorption amount, characterized in that at 1 [mu] g / cm ² or less from the (1) (4).
( 6 ) The ratio (D) / (C) of the protein adsorption amount (C) of the substrate before irradiation and the protein adsorption amount (D) of the substrate after irradiation is 0.8 or less. The modified substrate according to any one of (1) to ( 5 ).
( 7 ) The ratio (B) / (A) of the contact angle (A) with respect to water of the base material before irradiation and the contact angle (B) with respect to water of the base material after irradiation is 0.8 or less. The modified substrate according to any one of (1) to ( 6 ), which is characterized in that
( 8 ) A separation membrane using the modified substrate according to any one of (1) to ( 7 ).

  In the present invention, a substrate selected from polystyrene, polypropylene, polyethylene, polysulfone, polymethyl methacrylate, polycarbonate, silicone rubber, and polyurethane is first contacted with a hydrophilic polymer aqueous solution having a molecular weight of 200 or more and irradiated with radiation. Improvement in hydrophilicity of the substrate surface can be expected. Second, it can be expected to impart hydrophilicity to multiple surfaces of the substrate at once. Thirdly, it can be expected to suppress the adsorption of biological components. Further, a higher effect can be expected by using an antioxidant together.

  In the present invention, the hydrophilic polymer is immobilized on the surface of the base material by irradiating the base material used for the treatment of biological components with radiation in a state where the base material is in contact with a hydrophilic polymer aqueous solution having a molecular weight of 200 or more and an antioxidant. A modified substrate is obtained. The amount of biocomponent adhesion on the substrate depends on the surface state of the part in contact with the biocomponent, and in general, the higher the surface hydrophilicity, and the greater the mobility of hydrophilic molecules, the less adhesion .

  In the present invention, the base material refers to a material to be imparted with hydrophilicity. The substrate is preferably made of a polymer material. Examples of the polymer material include polysulfone, polystyrene, polyurethane, polycarbonate, polymethyl methacrylate, polyethylene, polypropylene, polyvinylidene fluoride, polyacrylonitrile, polyester, polyamide, polytetrafluoroethylene, and silicone rubber. Moreover, these copolymers may be sufficient. Furthermore, carbon plates such as carbon fibers, glassy carbon plates and carbon sheets, carbon materials such as carbon nanotubes and fullerenes, and composite materials obtained by mixing these with resins can also be used. A material in which a part of these materials is substituted with a functional group can also be used as a substrate. The reaction mechanism for imparting hydrophilicity in these carbon materials is not clear, and it is unclear whether they react directly or with trace amounts of impurities that are physically constrained by the carbon material. It is possible to make it hydrophilic. Examples of the shape of the substrate include, but are not limited to, fibers, films, sheets, plates, separation membranes, and particles.

  The term “immobilization” as used herein refers to a state in which a hydrophilic polymer is bonded to a substrate. The bond includes a covalent bond, an ionic bond, a hydrogen bond, a hydrophobic interaction, and the like. However, since the covalent bond is a relatively strong bond, a hydrophilic effect is sustained, which is preferable. Also, a combination of a plurality of these bonds may be used.

  Since the modified base material of the present invention has a hydrophilic polymer immobilized on the surface and an antioxidant is used, the hydrophilic polymer is prevented from being particularly unnecessarily cross-linked or collapsed. And adhesion of biological components such as platelets can be suppressed. Therefore, when used as a medical device, the present modified base material has high blood compatibility because the modified base material has less adhesion of coagulation-related proteins such as fibrinogen and platelets. That is, the modified substrate of the present invention can be suitably used as a medical substrate due to its high blood compatibility. Examples include those used in artificial blood vessels, catheters, blood bags, contact lenses, intraocular lenses, surgical aids, blood purification modules, and the like. Among them, it is suitable for applications used in contact with biological components, for example, blood purification modules such as artificial kidneys. Here, the blood purification module refers to a module having a function of circulating blood outside the body to remove waste and harmful substances in the blood, such as an artificial kidney and an exotoxin adsorption column. The artificial kidney module includes a coil type, a flat plate type, and a hollow fiber membrane type, and the hollow fiber membrane type is preferable from the viewpoint of processing efficiency.

  The modified base material of the present invention makes use of the feature of suppressing the adhesion of biological components, biological component separation membrane, separation membrane for water treatment, bio-experiment related equipment, bioreactor, molecular motor, DDS, protein chip, DNA It can also be suitably used for chips, biosensors, analytical instrument parts, and the like. Moreover, since the modified base material of the present invention has a hydrophilic polymer having a low degree of three-dimensional cross-linking on its surface, it can be expected to be applied to a material that requires slipperiness.

  In the present invention, the hydrophilic polymer refers to a polymer containing a hydrophilic functional group in the main chain or side chain of the polymer. A hydrophilic polymer having a solubility in water at 25 ° C. of preferably 0.0010% by weight or more, more preferably 0.01% by weight or more, and still more preferably 0.1% by weight or more is easy to apply to the present technology. Specific examples include polyvinylpyrrolidone, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyethyleneimine, polyallylamine, polyvinylamine, polyvinyl acetate, polyacrylic acid, polyacrylamide, and copolymers of these with other monomers, Examples thereof include graft polymers. Two or more hydrophilic polymers may be used in combination. Nonionic hydrophilic polymers such as polyalkylene glycol and polyvinyl pyrrolidone exhibit a nonspecific adsorption inhibiting effect. The nonionic hydrophilic polymer as referred to in the present invention refers to a hydrophilic polymer having a charge of less than 1 meq / g at both pH 4.5 and pH 9.5. Cationic hydrophilic polymers such as polyethyleneimine are highly effective in suppressing adsorption of acidic substances such as oxidized LDL. Anionic polymers such as dextran sulfate and polyvinyl sulfate are highly effective in suppressing adsorption of basic substances such as lysozyme. Among these, polyalkylene glycols such as polyethylene glycol and polypropylene glycol or polyvinyl pyrrolidone are preferable because of their high adsorption suppressing effect. Polyvinylpyrrolidone has a particularly high adsorption suppressing effect. Moreover, polyalkylene glycol has the merit of exhibiting a high adsorption suppressing effect without adding an antioxidant described later.

  In the present invention, the molecular weight refers to the weight average molecular weight. When the molecular weight of the hydrophilic polymer is small, the molecular mobility of the hydrophilic polymer is small, so it is 200 or more, preferably 500 or more, and more preferably 1000 or more.

  As the radiation, α rays, β rays, γ rays, X rays, ultraviolet rays, electron beams and the like are used. In addition, medical devices such as artificial kidneys need to be sterilized, and in recent years, radiation sterilization methods using γ rays and electron beams are frequently used from the viewpoint of low residual toxicity and simplicity. That is, it is preferable to use the method of the present invention for a medical substrate because sterilization and modification of the substrate can be achieved at the same time. In particular, the artificial kidney is mainly in the so-called wet type in which the separation membrane is in a state of immersing water. Therefore, the method of the present invention can be simply performed by changing this water to an aqueous solution containing a hydrophilic polymer solution. Since it can be used, it is preferable.

  The absorbed dose of radiation is preferably 5 kGy or more, more preferably 10 kGy or more, and further preferably 15 kGy or more. If the absorbed dose is small, the hydrophilic polymer graft amount is small, so that a sufficient effect cannot be obtained.

The term “antioxidant” as used in the present invention refers to a molecule that has the property of easily giving electrons to other molecules. Antioxidants have the property of suppressing a reaction when a hydrophilic polymer such as polyvinylpyrrolidone causes a radical reaction by radiation. Antioxidants include, for example, water-soluble vitamins such as vitamin C, polyphenols, alcohols such as methanol, ethanol, propanol, ethylene glycol and glycerin, sugars such as glucose, galactose, mannose and trehalose, sodium hydrosal phosphite, sodium metabisulfite, inorganic salts such as sodium hydrosulfite, uric acid, cysteine, and the like glutathione. These antioxidants may be used alone or in combination of two or more. When the method of the present invention is used for a medical device, it is necessary to consider its safety, and therefore, an antioxidant having low toxicity is preferably used. In particular, alcohols, sugars and inorganic salts are preferred. By adding an antioxidant, it is possible to fix the hydrophilic polymer with high mobility to the substrate while immobilizing the hydrophilic polymer, preventing unnecessary crosslinking and collapse of the hydrophilic polymer.

  The concentration of the antioxidant in the aqueous solution varies depending on the type of the antioxidant and the radiation dose. If the concentration of the antioxidant is too low, three-dimensional crosslinking or disintegration of the hydrophilic polymer occurs, which may reduce blood compatibility. In addition, if a large amount of an antioxidant is added, the efficiency of immobilization on the base material is lowered, so that sufficient blood compatibility may not be obtained.

  For the evaluation of the hydrophilization of the substrate, the contact angle of water with the substrate is used. In general, the smaller the water contact angle, the higher the wettability of the substrate, and the higher the hydrophilicity. In the substrate modification of the present invention, the ratio (B) / (A) of the contact angle (A) of the substrate before irradiation with water and the contact angle (B) of the substrate after irradiation with water after irradiation is 0.8. Hereinafter, it is further preferably 0.7 or less, more preferably 0.6 or less.

  The contact angle of water in the present invention is a static contact angle, and a detailed measurement method will be described later.

The biological component in the present invention refers to at least a part of the components of living organisms. Specifically, it is a body fluid such as blood, lymph, cell fluid, sweat, urine, saliva, skin, internal tissue, etc. Part, cell, protein, sugar, lipid, etc. Among these, proteins have been actively handled in the field of proteome analysis and the like in recent years. In this case, since the amount of protein is very small, if the protein is adsorbed to the surface of the substrate such as a container, it may be below the measurement lower limit of the analyzer and analysis may not be possible. The amount of adsorption is preferably 1 μg / cm ² or less, more preferably 0.5 μg / cm ² or less and 0.1 μg / cm ² or less. Furthermore, the ratio (D) / (C) of the protein adsorption amount (C) before radiation irradiation and the protein adsorption amount (D) after radiation irradiation in the substrate modification of the present invention is preferably 0.8 or less. Is preferably 0.5 or less and 0.3 or less.

  In general, the lower the protein solution concentration, the higher the protein adsorption rate to the base material. Therefore, when using a low concentration protein solution, the present technology can be particularly suitably used. The biological component treatment refers to separation, extraction, concentration, purification, storage of biological components, transportation using a pipette, tube, pump, etc., and analysis using a spectroscope or chromatography.

  There are two methods for measuring the amount of protein adsorbed on a substrate: a method of calculating the amount of adsorption from the amount of decrease when the protein is brought into contact with the substrate, and a method of quantifying the adsorbed protein. In the latter method, protein adsorbed on the substrate is directly analyzed with infrared attenuated total reflection spectroscopy (ATR-IR), X-ray photoelectron spectroscopy (XPS), or scanning electron microscope (SEM). There are methods, amino acid analysis by hydrolyzing proteins, and methods for recovering and analyzing proteins adsorbed on the substrate electrically, chemically or physically, and have the merit of directly quantifying the amount of adsorption However, there are also drawbacks such as that the type of protein cannot be specified, and that the protein is denatured and decomposed. The former is an indirect evaluation, but since protein denaturation is small, it can be easily and highly sensitively quantified by enzyme immunoassay (ELISA), fluorescence spectroscopy and the like.

  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

(Creation of hollow fiber module 1)
Iso (isotactic) -polymethyl methacrylate 5 parts by weight and syn (syndiotactic) -polymethyl methacrylate 20 parts by weight were added to 75 parts by weight of dimethyl sulfoxide and dissolved by heating to obtain a film forming stock solution. This film-forming stock solution was discharged from the tube outside the orifice-type double cylindrical die, passed 200 mm through the air, and then led into a 100% water coagulation bath to obtain a hollow fiber. At this time, dry nitrogen was discharged from the inner tube as an internal injection gas. The resulting hollow fiber had an inner diameter of 0.2 mm and a film thickness of 0.03 mm. 10000 hollow fibers obtained were inserted into a cylindrical plastic case having a dialysate inlet and a dialysate outlet, as shown in FIG. 1, and both ends were sealed with a resin to obtain an effective membrane area of 1.6 m. ^Two hollow fiber membrane modules 1 for artificial kidneys were prepared.
(Preparation of resin disc 1)
Polymethylmethacrylate resin pellets (Acrypet VH (0621073) manufactured by Mitsubishi Rayon) were vacuum-dried at 80 ° C. for 24 hours, sandwiched between glass plates (“TLC plates” manufactured by TOP), and heated in an oven at 170 ° C. for 30 minutes. . After leaving to cool at 25 ° C. for 60 minutes, it was taken out from the glass plate, and a resin disk 1 as a substrate was prepared.
(Preparation of resin disc 2)
Polystyrene resin pellets (Styrene Polymer code: 193-04305, manufactured by Wako Pure Chemical Industries, Ltd.) were vacuum-dried at 80 ° C. for 24 hours, sandwiched between glass plates (“TLC plates” manufactured by TOP), and heated in an oven at 190 ° C. for 30 minutes. . After leaving to cool at 25 ° C. for 60 minutes, it was taken out from the glass plate, and a resin disk 2 as a substrate was prepared.
Example 1
A solution in which polyvinyl pyrrolidone (BASF K90) is 0.1% by weight and ethanol (Aldrich) 0.1% by weight is dissolved in ultrapure water that has been vacuum degassed for 60 minutes or more at 25 ° C. Was prepared. The solution and the resin disc 1 were placed in a screw cap bottle (“SV50” manufactured by Nidec Denka), covered with a lid to prevent bubbles from entering, and irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption Polyvinyl pyrrolidone was removed. The water contact angle of the disk, the bovine serum albumin adsorption amount, and the fluorescent protein adsorption rate were measured. As a result, as shown in Table 1, it was found that a substrate having high surface hydrophilicity and low protein adsorption was obtained.

(Example 2)
A solution obtained by dissolving 0.1% by weight of polyvinylpyrrolidone (BASF K30) and 0.1% by weight of ethanol (made by Aldrich) in ultrapure water that has been vacuum degassed for 60 minutes or more at 25 ° C. Was prepared. The solution and the resin disc 1 were placed in a screw cap bottle (“SV50” manufactured by Nidec Denka), covered with a lid to prevent bubbles from entering, and irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption Polyvinyl pyrrolidone was removed. The water contact angle of the disk and the fluorescent protein adsorption rate were measured. As a result, as shown in Table 1, it was found that a substrate having high surface hydrophilicity and low protein adsorption was obtained.

(Example 3)
Dissolve polyethylene glycol (polyethylene glycol 20000 made by Katayama Chemical) in ultrapure water that has been vacuum degassed at 25 ° C for 60 minutes or more, and 0.1 wt% ethanol (manufactured by Aldrich) as an antioxidant. A solution was prepared. The solution and the resin disk k1 were placed in a screw cap bottle (“SV50” manufactured by Nidec Denka), and the lid was irradiated so that bubbles were not mixed, and γ rays were irradiated. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption The polyethylene glycol was removed. The water contact angle of the disk and the fluorescent protein adsorption rate were measured. As a result, as shown in Table 1, it was found that a substrate having high surface hydrophilicity and low protein adsorption was obtained.

Example 4
Dissolve 0.1 wt. Of polyethylene glycol (Macrogol 6000 manufactured by NOF Corporation) and 0.1 wt.% Of ethanol (manufactured by Aldrich) in ultra pure water that has been vacuum degassed for 60 minutes or more at 25 ° C. A solution was prepared. The solution and the resin disc 1 were placed in a screw cap bottle (“SV50” manufactured by Nidec Denka), covered with a lid to prevent bubbles from entering, and irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption The polyethylene glycol was removed. The water contact angle of the disk and the fluorescent protein adsorption rate were measured. As a result, as shown in Table 1, it was found that a substrate having high surface hydrophilicity and low protein adsorption was obtained.

(Example 5)
Dissolved in ultrapure water vacuum degassed at 25 ° C. for 60 minutes or more by dissolving 0.1 weight% of polyethyleneimine (Aldrich polyethyleneimine 25000) and 0.1% by weight of ethanol (made by Aldrich) as an antioxidant. Was prepared. The solution and the resin disc 1 were placed in a screw cap bottle (“SV50” manufactured by Nidec Denka), covered with a lid to prevent bubbles from entering, and irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption Polyvinyl pyrrolidone was removed. The water contact angle of the disk and the fluorescent protein adsorption rate were measured. As a result, as shown in Table 1, it was found that a substrate having high surface hydrophilicity and low protein adsorption was obtained.
(Comparative Example 1)
The water contact angle of the resin disk 1 and the fluorescence protein adsorption rate were measured. As a result, as shown in Table 1, it was found that a substrate having low surface hydrophilicity and high protein adsorption was obtained.
(Comparative Example 2)
Both surfaces of the resin disk 1 were subjected to hydrophilic treatment with oxygen plasma using a reactive ion etching apparatus (manufactured by Samco). The surface treatment conditions using the reactive ion etching apparatus were as follows: the reaction gas was oxygen, the supply amount was 100 cc / min, the power was 100 watts, and the treatment time was 30 seconds. The water contact angle of the disk and the fluorescent protein adsorption rate were measured. As a result, as shown in Table 1, it was found that a substrate having low surface hydrophilicity and high protein adsorption was obtained.

(Example 6)
The hollow fiber membrane module 1 was washed by passing 5000 ml of ultrapure water at 40 ° C. to the blood side and the dialysate side. Thereafter, 1000 ml of an aqueous solution containing 0.0025% by weight of polyvinylpyrrolidone (BASF K90) as a hydrophilic polymer and 0.1000% by weight of ethanol (Aldrich) as an antioxidant was passed through the blood side and dialysate side, respectively. The module was filled with the aqueous solution. Thereafter, the module was irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. The module was subjected to human platelet adhesion test and human interleukin 6 adsorption test. As a result, it was as shown in Table 2.
( Comparative Example 8 )
The hollow fiber membrane module 1 was washed by passing 5000 ml of ultrapure water at 40 ° C. to the blood side and the dialysate side. Thereafter, 1000 ml of an aqueous solution containing 0.0025 % by weight of polyvinylpyrrolidone (BASF K90) as a hydrophilic polymer was passed through each of the blood side and the dialysate side, and the inside of the module was filled with the aqueous solution. Thereafter, the module was irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. The module was subjected to human platelet adhesion test and human interleukin 6 adsorption test. As a result, as shown in Table 2, it was found that a substrate with a small number of platelets attached and a low amount of human interleukin adsorption was obtained.
( Comparative Example 9 )
The hollow fiber membrane module 1 was washed by passing 5000 ml of ultrapure water at 40 ° C. to the blood side and the dialysate side. Thereafter, 1000 ml of an aqueous solution containing 0.0050 % by weight of polyvinylpyrrolidone (BASF K90) as a hydrophilic polymer was passed through each of the blood side and the dialysate side, and the inside of the module was filled with the aqueous solution. Thereafter, the module was irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. The module was subjected to human platelet adhesion test and human interleukin 6 adsorption test. As a result, as shown in Table 2, it was found that a substrate with a small number of platelets attached and a low amount of human interleukin adsorption was obtained.
( Comparative Example 10 )
The hollow fiber membrane module 1 was washed by passing 5000 ml of ultrapure water at 40 ° C. to the blood side and the dialysate side. Thereafter, 1000 ml of an aqueous solution containing 0.01 % by weight of polyvinylpyrrolidone (BASF K90) as a hydrophilic polymer was passed through each of the blood side and the dialysate side, and the inside of the module was filled with the aqueous solution. Thereafter, the module was irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. The module was subjected to human platelet adhesion test and human interleukin 6 adsorption test. As a result, as shown in Table 2, it was found that a substrate with a small number of platelets attached and a low amount of human interleukin adsorption was obtained.
(Comparative Example 3)
The hollow fiber membrane module 1 was washed by passing 5000 ml of ultrapure water at 40 ° C. to the blood side and the dialysate side. Thereafter, the module was irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. The module was subjected to human platelet adhesion test and human interleukin 6 adsorption test. As a result, as shown in Table 2, it was found that a substrate having a large number of platelets adhered and a large amount of human interleukin adsorbed was obtained.

(Example 10)
A solution obtained by dissolving 0.1% by weight of polyvinylpyrrolidone (BASF K90) and 0.1000% by weight of ethanol (manufactured by Aldrich) in ultrapure water vacuum degassed at 25 ° C. for 60 minutes or more. Was prepared. The solution and the resin disk 2 were placed in a screw mouth bottle (“SV50” manufactured by Nidec Rika Kagaku Co., Ltd.), covered with a lid so that bubbles were not mixed, and irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption Polyvinyl pyrrolidone was removed. Measurement of the water contact angle of the disc and human platelet adhesion test were performed. As a result, as shown in Table 3, it was found that a substrate having high surface hydrophilicity and low platelet adsorption was obtained.
(Comparative Example 4)
The contact angle of water on the resin disk 2 and the human platelet adhesion test were each performed. As a result, as shown in Table 3, it was found that a substrate having low surface hydrophilicity and high human platelet adsorption was obtained.
(Comparative Example 5)
A solution was prepared by dissolving 0.1% by weight of polyvinylpyrrolidone (BASF K90) in ultrapure water that had been vacuum degassed for 60 minutes or more at 25 ° C. The solution and the resin disk 2 were placed in a screw mouth bottle (“SV50” manufactured by Nidec Rika Kagaku Co., Ltd.), covered with a lid so that bubbles were not mixed, and irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption Polyvinyl pyrrolidone was removed. Measurement of the water contact angle of the disc and human platelet adhesion test were performed. As a result, as shown in Table 3, it was found that a substrate having high surface hydrophilicity but high human platelet adsorption was obtained.
(Comparative Example 6)
Ethanol (manufactured by Aldrich) was dissolved in ultrapure water that had been vacuum degassed at 25 ° C. for 60 minutes or more to prepare a solution. The solution and the resin disk 2 were placed in a screw mouth bottle (“SV50” manufactured by Nidec Rika Kagaku Co., Ltd.), covered with a lid so that bubbles were not mixed, and irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption Polyvinyl pyrrolidone was removed. Measurement of the water contact angle of the disc and human platelet adhesion test were performed. As a result, as shown in Table 3, it was found that a substrate having low surface hydrophilicity and high human platelet adsorption was obtained.
(Comparative Example 7)
A screw bottle (“SV50” manufactured by Nidec Rika) was filled with ultrapure water and resin disc 2 that had been vacuum degassed at 25 ° C. for 60 minutes or more, covered with a lid to prevent bubbles from entering, and irradiated with γ rays. The absorbed dose of γ rays was 27 kGy. After rinsing the disk with pure water, stirring in pure water at 25 ° C for 60 minutes, replacing pure water and stirring again at 25 ° C for 60 minutes, further replacing pure water and stirring at 25 ° C for 60 minutes, adsorption Polyvinyl pyrrolidone was removed. Measurement of the water contact angle of the disc and human platelet adhesion test were performed. As a result, as shown in Table 3, it was found that a substrate having low surface hydrophilicity and high human platelet adsorption was obtained.
(Test method for human platelet adhesion of hollow fiber membranes)
A double-sided tape was affixed to an 18 mmφ polystyrene circular plate, and a hollow fiber membrane was fixed thereto. The attached hollow fiber membrane was cut into a semicylindrical shape with a single blade to expose the inner surface of the hollow fiber membrane. If dirt, scratches, folds, etc. are present on the inner surface of the hollow fiber, platelets will adhere to the part and may not be evaluated correctly. The circular plate was attached to a Falcon (registered trademark) tube (18 mmφ, No. 2051) cut into a cylindrical shape so that the surface on which the hollow fiber membrane was attached was inside the cylinder, and the gap was filled with parafilm. The cylindrical tube was washed with physiological saline and then filled with physiological saline. Immediately after collecting human venous blood, heparin was added to 50 U / ml. After discarding the physiological saline in the cylindrical tube, 1.0 ml of the blood was placed in the cylindrical tube and shaken at 37 ° C. for 1 hour within 10 minutes after blood collection. Thereafter, the hollow fiber membrane was washed with 10 ml of physiological saline, blood components were fixed with 2.5% glutaraldehyde physiological saline, and washed with 20 ml of distilled water. The washed hollow fiber membrane was dried under reduced pressure at room temperature of 0.5 Torr for 10 hours. This film was attached to a sample stage of a scanning electron microscope with double-sided tape. Thereafter, a thin film of Pt—Pd was formed on the surface of the hollow fiber membrane by sputtering to prepare a sample. The inner surface of the hollow fiber membrane was observed with a field emission type scanning electron microscope (S800 manufactured by Hitachi, Ltd.) at a magnification of 1500 times. In one field of view (4.3 × 10 ³ μm ² ) The number of adherent platelets was counted. The average value of the number of adhering platelets in 10 different visual fields near the center in the longitudinal direction of the hollow fiber was defined as the number of adhering platelets (pieces / 4.3 × 10 ³ μm ² ). This is because the end portion in the longitudinal direction of the hollow fiber is likely to form a blood pool.
(Human interleukin 6 adsorption test)
Thirty hollow fiber separation membranes used for the hollow fiber membrane module 2 were bundled, and both ends were fixed to the glass tube module case with an epoxy potting agent so as not to block the hollow portion of the hollow fiber, thereby creating a mini module. . The mini module had a diameter of about 7 mm and a length of about 10 cm. The blood inlet and the dialysate outlet of the minimodule were connected by a silicone tube, and 100 ml of distilled water was allowed to flow from the blood outlet at a flow rate of 10 ml / min to wash the hollow fiber and the inside of the module. Thereafter, PBS (Dulbecco PBS (-) manufactured by Nissui Pharmaceutical Co., Ltd.) aqueous solution was filled, and the dialysate inlet and outlet were capped.

  Human interleukin 6 was added to 10 ml of human plasma to adjust the concentration to 1 ng / ml (referred to as liquid 1). The dialysate inlet and the dialysate outlet were capped, the blood side inlet and the blood side outlet were connected by a silicone tube, and the solution 1 was perfused at 37 ° C. for 4 hours at a flow rate of 1 ml / min. The amount of human interleukin 6 before and after perfusion was quantified, and the amount adsorbed on the substrate was calculated from the amount of human interleukin 6 decreased.

(Measurement of water contact angle)
The substrate was previously vacuum dried at 25 ° C. for 24 hours or more. 50 μl of water was gently dropped on the substrate surface. At this time, in the vertical cross section with respect to the base material surface passing through the top of the water droplet, the angle between the tangent at the portion of the water droplet contacting the base material and the base material surface was measured as the water contact angle. Since the contact angle changed depending on the moisture absorption state of the substrate, the measurement was performed within 1 minute after the water was dropped.

(Measurement of bovine serum albumin adsorption)
Bovine serum albumin (hereinafter referred to as BSA) manufactured by SEIKAGAKU was dissolved in an aqueous solution of PBS (Dulbecco PBS (-) manufactured by Nissui Pharmaceutical Co., Ltd.) to prepare a 0.5% BSA aqueous solution. The BSA aqueous solution per 1 cm ^{2 of the} substrate was immersed at a rate of 1 ml at 25 ° C. for 3 hours, and then the substrate was immersed at a rate of 5 ml of the PBS aqueous solution per 1 cm ^{2 of the} substrate at 25 ° C. for 1 hour. BSA adhering to the substrate was recovered using a BIO-RAD electroeluter (model 422). The recovered BSA solution was lyophilized and quantified with a Pierce Micro BCA Protein Assay Reagant to measure the amount of protein adsorbed on the substrate.

(Measurement of fluorescent protein adsorption rate)
A fluorescent protein (hereinafter abbreviated as GFP) was synthesized using a control vector of the HY kit attached to RTS-500 manufactured by Roche. The synthesized GFP was purified by Ni-NTA agarose manufactured by Qiagen. The substrate and the GFP solution were added to a glass test tube (Fisher Scientific) placed on an ice bath at a rate of 1.5 ml per 1 cm ² of the substrate. The fluorescence intensity (excitation wavelength: 395 nm, measurement wavelength: 508 nm) was measured 0 minutes and 60 minutes after the start of contact between the GFP solution and the substrate. The protein adsorption rate was determined from Equation 1.

Fluorescent protein adsorption rate = {(fluorescence intensity after 0 minutes of contact)
-(Fluorescence intensity 60 minutes after the start of contact)} / (fluorescence intensity 0 minutes after the start of contact) (Formula 1)

[1] Comparative Example 3 of the present invention, is a schematic view of the artificial kidney module employing 8-10.
[Explanation of symbols]

Explanation of symbols

1. 1. Blood inlet 2. Blood outlet 3. Blood introduction port 4. Blood outlet Hollow fiber membrane6. Blood 7. Case 8. Dialysate inlet 9. Dialysate outlet 10. Resin 11. Blood circuit

Claims (8)

In a base material selected from a polymer material, a carbon material, and a composite material obtained by mixing a carbon material and a resin, which is used for biological component treatment, the base material is brought into contact with a hydrophilic polymer aqueous solution having a molecular weight of 200 or more and alcohols. A modified substrate obtained by irradiation. The modified substrate according to claim 1 , wherein the substrate is a polymer material. The modified substrate according to claim 1 or 2 , wherein the biological component is a protein. Modified substrate according to any one of the use in the separation of proteins from claim 1, wherein 3. The modified substrate according to any one of claims 1 to 4 , wherein the protein adsorption amount is 1 µg / cm ² or less. The ratio (D) / (C) of the amount of protein adsorption (C) of the base material before irradiation and the amount of protein adsorption (D) of the base material after irradiation is 0.8 or less. The modified substrate according to any one of 1 to 5 . The ratio (B) / (A) of the contact angle (A) of the substrate before irradiation with water (A) and the contact angle (B) of the substrate after irradiation with water is 0.8 or less. The modified substrate according to any one of claims 1 to 6 . Separation film using the modified substrate according to any one of claims 1 to 7.

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