Filter and manufacturing method thereof

Abstract

A filter (10) is provided with: a base material (20) that is formed from a porous polyurethane having a plurality of micropores formed therein; and an active group (28) that is immobilized on the surface (26) of the substrate (20) and that can specifically bind to an object to be separated.

Description

Technical Field

[0001] The present invention relates to a filter for capturing cells or proteins contained in a liquid and a method for manufacturing the same. Background Art

[0002] Examples of a method for separating cells from a cell suspension include a method using a filter, a centrifugal separation method, etc. For example, Japanese Patent Application Laid-Open No. 2011-24575 discloses a centrifugal separation method. Summary of the invention

[0003] Problems to be solved by the invention

[0004] A cell suspension may contain multiple types of cells of similar size. It is difficult to separate specific cells from multiple types of cells of similar size using a filter having fine pores or a centrifugal separation method.

[0005] An object of the present invention is to solve the above-mentioned problems.

[0006] Means for solving problems

[0007] The filter of the present invention is (1) a filter that captures the separation object by allowing a liquid containing cells or proteins as the separation object to pass therethrough, and comprises: a substrate formed of a porous polyurethane having a plurality of micropores; and an active group that is fixed to the surface of the substrate and is capable of specifically binding to the separation object.

[0008] According to the above configuration, specific cells can be separated from a plurality of cells of similar size contained in a cell fluid, and similarly, a specific protein can be separated from a plurality of proteins of similar size.

[0009] (2) In the filter of (1) above, the active group preferably includes an antibody or an aptamer capable of specifically binding to an antigen contained in the object to be separated.

[0010] (3) In the filter of (2) above, the active group preferably includes: a conjugate of the antibody or aptamer and biotin; and a biotin-binding protein immobilized on the substrate and capable of binding to the biotin.

[0011] (4) In the filter described in (2) or (3) above, the active group preferably comprises: a conjugate formed by the antibody or the aptamer and the first biotin; a second biotin fixed to the substrate; and a biotin-binding protein capable of binding to each of the first biotin and the second biotin.

[0012] (5) In the filter described in (4) above, the second biotin is preferably fixed to the substrate via a structural unit A derived from a compound A having an epoxy group and an ethylenically unsaturated group, and a structural unit B derived from a diamine compound B bonded to the structural unit A, wherein the structural unit A is bonded to the substrate, and the structural unit B is bonded to the second biotin.

[0013] (6) In the filter described in (5) above, the compound A is preferably at least one selected from the group consisting of glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexylmethyl acrylate, 3,4-epoxycyclohexylmethyl methacrylate, β-methylglycidyl acrylate, and β-methylglycidyl methacrylate.

[0014] (7) In the filter described in (5) or (6) above, the compound B is preferably at least one selected from the group consisting of ethylenediamine, trimethylenediamine, 1,2-diaminopropane, tetramethylenediamine, 1,3-diaminobutane, 2,3-diaminobutane, pentamethylenediamine, 2,4-diaminopentane, hexamethylenediamine (HMDA), octamethylenediamine, nonamethylenediamine, decamethylenediamine, undecamethylenediamine, dodecamethylenediamine, tridecamethylenediamine, octadecamethylenediamine, xylenediamine, and phenylenediamine.

[0015] (8) The filter according to any one of (1) to (7) above preferably comprises a container that accommodates the plurality of substrates to which the active groups are fixed, wherein the plurality of substrates are stacked in the container from upstream to downstream of the flow of the liquid.

[0016] According to the above configuration, the cell fluid can be brought into contact with each substrate.

[0017] (9) The filter of (8) above preferably comprises a plurality of the containers arranged in a row from upstream to downstream of the flow of the liquid, and the type of the active group fixed to the substrate is different for each container.

[0018] According to the above configuration, a plurality of separation targets can be separated by allowing the cell fluid to flow through the filter once.

[0019] (10) In the filter described in any one of (1) to (9) above, the plurality of fine pores formed in the substrate are preferably larger than the separation target.

[0020] According to the above configuration, clogging of the filter can be prevented. In addition, according to the above configuration, the recovery rate of target cells can be improved.

[0021] (11) The filter described in any one of (1) to (10) above preferably has another substrate formed by a porous component and different from the aforementioned substrate, wherein the plurality of micropores formed in the aforementioned other substrate are larger than the aforementioned separation object and smaller than a separation object different from the aforementioned separation object.

[0022] According to the above configuration, large-sized substances contained in the cell fluid can be separated.

[0023] In addition, the method for producing a filter of the present invention comprises: (1) irradiating at least a portion of a surface of a substrate formed of a porous polyurethane having a plurality of micropores with plasma; (2) bringing a compound A having an epoxy group and an ethylenically unsaturated group into contact with the substrate after the plasma irradiation, thereby bonding the compound A to the substrate; (3) bringing a diamine compound B into contact with the substrate after the contact with the compound A, thereby bonding the diamine compound B to the surface of the substrate via a structural unit A derived from the compound A; and (4) bringing biotin into contact with the surface of the substrate after the contact with the diamine compound B, thereby bonding the biotin to the surface of the substrate via the structural unit A derived from the compound A and the diamine compound B. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] [ Figure 1 ] Figure 1 A diagram showing the structure of a filter.

[0025] [ Figure 2 ] Figure 2 A~ Figure 2 C are each a partial enlarged view of the filter unit.

[0026] [ Figure 3 ] Figure 3 It is a figure which shows the structure of a separation device.

[0027] [ Figure 4 ] Figure 4 Flowchart of filter preparation and separation process using a separation device.

[0028] [ Figure 5 ] Figure 5 To show Figure 4 FIG. 5 is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in steps S1, S2, and S5.

[0029] [ Figure 6 ] Figure 6 To show Figure 4 FIG. 5 is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in step S1.

[0030] [ Figure 7 ] Figure 7 To show Figure 4 FIG. 5 is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in step S2.

[0031] [ Figure 8 ] Figure 8 To show Figure 4 FIG. 5 is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in step S3.

[0032] [ Fig. 9 ] Fig. 9 To show Figure 4 FIG. 5 is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in step S3.

[0033] [ Fig.10 ] Fig.10 To show Figure 4 FIG. 5 is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in step S4.

[0034] [ Fig.11 ] Fig.11 To show Figure 4 FIG. 5 is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in step S4.

[0035] [ Fig.12 ] Fig.12 To show Figure 4 FIG. 5 is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in step S5.

[0036] [ Fig.13 ] Fig.13 This is a diagram showing the opening and closing of the valve of the separation device and the flow of the liquid in the separation device in the cell recovery step.

[0037] [ Fig.14 ] Fig.14 This is a diagram for explaining the immobilization of an active group capable of specifically binding to a separation target object according to the present invention.

[0038] [ Fig.15 ] Fig.15 This is a fluorescent photograph showing the presence of biotin in Sample 1 in Example 1.

[0039] [ Fig.16 ] Fig.16 A schematic diagram showing the experimental setup used to evaluate cell capture. DETAILED DESCRIPTION

[0040] In one embodiment of the present invention, a filter is provided, which is a filter that captures the separation object by passing a liquid containing cells or proteins as the separation object, and comprises: a substrate formed of a porous polyurethane having a plurality of micropores; and an active group fixed to the surface of the substrate and capable of specifically binding to the separation object. According to the present invention, specific cells can be separated from a plurality of cells of similar size, and similarly, specific proteins can be separated from a plurality of proteins of similar size.

[0041] Hereinafter, the mode for implementing the present invention will be described in detail. It should be noted that the present invention is not limited to the following embodiments, and various changes can be made within the scope of the claims. In addition, the embodiments described in this specification can be combined arbitrarily to form other embodiments.

[0042] Throughout this specification, unless otherwise specified, expressions in the singular should be understood as also including the concept of the plural form. Therefore, unless otherwise specified, articles in the singular (for example, "a", "an", "the", etc. in English) should be understood as also including the concept of the plural form. In addition, in this specification, unless otherwise specified, the terms used should be understood as being used in the meaning commonly used in the art. Therefore, unless otherwise defined, all technical terms and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art in the field to which the invention belongs. In the event of a conflict, this specification (including definitions) takes precedence.

[0043] In this specification, "X to Y" indicating a range includes X and Y, and means "X or more and Y or less". Unless otherwise specified, operations and measurements of physical properties were performed at room temperature (20 to 25°C) and relative humidity of 40 to 60% RH.

[0044] In this specification, the term "(meth)acrylic-" includes both acrylic- and methacrylic-. Thus, for example, the term "(meth)acrylic" includes both acrylic and methacrylic. Similarly, the term "(meth)acryloyl" includes both acryl and methacryloyl. Thus, for example, the term "(meth)acryloyl" includes both acryl and methacryloyl.

[0045] In this specification, "A and / or B" means both A and B, or either A or B.

[0046] In the present specification, when a certain structural unit is defined as being “derived from” a certain monomer, it means that the structural unit is a divalent structural unit generated by cleaving one of the polymerizable unsaturated double bonds of the corresponding monomer.

[0047] [1 Configuration of filter 10]

[0048] Figure 1 1 is a diagram showing the structure of the filter 10. In the filter 10, a liquid is introduced from an inlet, one or more separation objects contained in the liquid are captured, and the liquid is discharged from an outlet. The liquid is, for example, a cell suspension (cell fluid). The separation object is a cell or a protein. The filter 10 has one or more filter units 12. Figure 1 The filter 10 shown includes two filter units 12 (a first filter unit 12a and a second filter unit 12b).

[0049] The two filter units 12 are arranged in a row from upstream to downstream of the flow of the liquid so that the liquid introduced into the filter 10 passes through each filter unit 12 in sequence. Figure 1 The liquid flows from bottom to top. The separation object captured by the first filter unit 12a is different from the separation object captured by the second filter unit 12b. Each filter unit 12 includes a container 18 and a plurality of substrates 20.

[0050] The container 18 accommodates a plurality of substrates 20. The container 18 has an inlet port 22 and an outlet port 24. The inlet port 22 can introduce liquid from the outside to the inside of the container 18. The inlet port 22 of the first filter unit 12a corresponds to the inlet of the filter 10. The outlet port 24 can discharge liquid from the inside of the container 18 to the outside. The outlet port 24 of the second filter unit 12b corresponds to the outlet of the filter 10. The outlet port 24 of the first filter unit 12a and the inlet port 22 of the second filter unit 12b are connected to each other through the connecting flow path 14.

[0051] It should be noted that if Figure 3 As shown, by merging the other flow path (first bypass flow path 88) with the connecting flow path 14, the inlet port 22 of the second filter unit 12b can become the inlet of the filter 10. Figure 3 As shown, by branching another flow path (second bypass flow path 90 ) from the connecting flow path 14 , the outlet port 24 of the first filter unit 12 a can serve as the outlet of the filter 10 .

[0052] The plurality of substrates 20 are filled in the container 18. The plurality of substrates 20 are stacked in the thickness direction of the substrate 20. In addition, the stacking direction of the plurality of substrates 20 (the thickness direction of the substrate 20) is the same as the direction from the inlet port 22 toward the outlet port 24. That is, the plurality of substrates 20 are arranged in a row from the upstream to the downstream of the flow of the liquid.

[0053] Figure 2 A~ Figure 2 C are each a partial enlarged view of the filter unit 12. Each figure schematically shows the surface 26 of the substrate 20 and the active group 28. The surface 26 of the substrate 20 refers to the portion of the substrate 20 that can contact the liquid passing through the filter unit 12. The substrate 20 is formed of a porous polyurethane having a plurality of micropores. The porous polyurethane can increase the surface area of ​​the substrate 20.

[0054] Active groups 28 capable of specifically binding to the separation object are immobilized on the surface 26 of the substrate 20. The active groups 28 include antibodies 30 capable of specifically binding to antigens possessed by the separation object. In addition, the active groups 28 may include aptamers instead of antibodies 30.

[0055] For example, Figure 2 In the filter unit 12 shown in A, the antibody 30 can be directly immobilized on the surface 26 of the substrate 20. Alternatively, as shown in Figure 2 The filter unit 12 shown in B can use a biotinylated antibody 36 formed by binding an antibody 30 to a biotin 34, and avidin 32 (biotin binding protein). For example, the biotinylated antibody 36 can be fixed to the surface 26 of the substrate 20 via the avidin 32. It should be noted that other biotin binding proteins, such as streptavidin, can be used instead of avidin 32. Figure 2 In the filter unit 12 shown in B, avidin 32 is directly immobilized on the surface 26 of the substrate 20. In addition, avidin 32 and biotinylated antibody 36 are bound to each other.

[0056] like Figure 2 The filter unit 12 shown in C can use a biotinylated antibody 36 formed by binding an antibody 30 to a second biotin 34b, avidin 32 (biotin binding protein), and a first biotin 34a. For example, the biotinylated antibody 36 can be fixed to the surface 26 of the substrate 20 via the avidin 32 and the first biotin 34a. Figure 2 In the filter unit 12 shown in C, the first biotin 34a is directly immobilized on the surface 26 of the substrate 20. In addition, the first biotin 34a and the avidin 32 are bound to each other. In addition, the avidin 32 and the biotinylated antibody 36 are bound to each other.

[0057] according to Figure 2 A~ Figure 2In each filter unit 12 shown in C, when the cell fluid flows from the inlet port 22 of the filter unit 12 toward the outlet port 24, the antibody 30 of the active group 28 binds to the antigen possessed by the separation object in the cell fluid. The type of antibody 30 is pre-selected according to the type of the separation object. Thus, the separation object in the cell fluid is captured by the substrate 20, and on the other hand, the cell fluid containing substances other than the separation object is discharged from the outlet port 24 to the outside of the filter unit 12.

[0058] [2. Method for manufacturing filter unit 12]

[0059] As described above, porous polyurethane is used as the substrate 20 of the filter 10. Usually, porous polyurethane is used as a leukocyte removal filter. The micropores of the porous polyurethane used as the substrate 20 are larger than the micropores of the leukocyte removal filter. The size of the micropores of the porous polyurethane is adjusted to be larger than the separation object contained in the cell fluid. The porous polyurethane is cut into a size that can be accommodated in the container 18 to form the substrate 20. The active group 28 is fixed to the surface 26 of the cut substrate 20 by the following production method.

[0060] [2.1 Production method 1]

[0061] Figure 2 The filter unit 12 shown in A can be produced, for example, in the following manner. The cut substrate 20 and acrylic acid are subjected to plasma treatment. Thus, acrylic groups are introduced into the surface 26 of the substrate 20. Next, a coupling agent (hexamethylenediamine) and an antibody 30 are reacted with the acrylic groups on the surface 26 of the substrate 20. Alternatively, the cut substrate 20 and a compound having an epoxy group (preferably a glycidyl group) such as (meth)acrylate are subjected to plasma treatment. Thus, epoxy groups (preferably a glycidyl group) are introduced into the surface 26 of the substrate 20. Next, a coupling agent (hexamethylenediamine) and an antibody 30 are reacted with the epoxy group (preferably a glycidyl group) on the surface 26 of the substrate 20. Thus, as Figure 2 As shown in A, the active group 28 (antibody 30) is fixed to the surface 26 of the substrate 20. The substrates 20 with the active groups 28 fixed thereto are stacked and filled into the container 18. Figure 2 A shows the filter unit 12.

[0062] [2.2 Production method 2]

[0063] Figure 2The filter unit 12 shown in B can be produced, for example, in the following manner. The cut substrate 20 and acrylic acid are subjected to plasma treatment. Thus, acrylic groups are introduced into the surface 26 of the substrate 20. Next, a coupling agent (hexamethylenediamine) and avidin 32 are reacted with the acrylic groups on the surface 26 of the substrate 20. Alternatively, the cut substrate 20 and a compound having an epoxy group (preferably a glycidyl group) such as (meth) acrylate are subjected to plasma treatment. Thus, epoxy groups (preferably a glycidyl group) are introduced into the surface 26 of the substrate 20. Next, a coupling agent (hexamethylenediamine) and avidin 32 are reacted with the epoxy group (preferably a glycidyl group) on the surface 26 of the substrate 20. Thus, the avidin 32 is fixed to the surface 26 of the substrate 20.

[0064] The substrate 20 with avidin 32 fixed thereto is stacked and filled inside the container 18. Next, an antibody solution is introduced into the container 18. The antibody solution contains biotinylated antibodies 36. The avidin 32 fixed to the substrate 20 and the biotinylated antibodies 36 in the antibody solution are bound to each other. Figure 2 As shown in B, the active groups 28 (avidin 32, biotinylated antibody 36) are fixed to the surface 26 of the substrate 20. Figure 2 B shows the filter unit 12.

[0065] [2.3 Production method 3]

[0066] Figure 2 The filter unit 12 shown in B can also be produced, for example, in the following manner. The cut substrate 20 is subjected to plasma treatment. As a result, the surface 26 of the substrate 20 becomes active. Next, the substrate 20 is immersed in a solution of a compound having an epoxy group (preferably a glycidyl group) such as GMA (glycidyl methacrylate) and glycidyl acrylate. Alternatively, the substrate 20 is contacted with a gas of a compound having an epoxy group (preferably a glycidyl group) such as GMA (glycidyl methacrylate) and glycidyl acrylate. As a result, compounds such as GMA are bound to the surface 26 of the substrate 20. Next, a coupling agent (hexamethylenediamine) and avidin 32 are reacted with the (meth) acrylic group or epoxy group of the surface 26 of the substrate 20. As a result, the avidin 32 is fixed to the surface 26 of the substrate 20. The series of treatments after the avidin 32 is fixed are the same as the series of treatments after the avidin 32 is fixed in the production method 2.

[0067] [2.4 Production method 4]

[0068] Figure 2The filter unit 12 shown in C can be produced, for example, in the following manner. A polymer to which the first biotin 34a is bound is applied to the surface 26 of the cut substrate 20. Thus, the first biotin 34a is fixed to the surface 26 of the substrate 20. The substrates 20 to which the first biotin 34a is fixed are stacked and filled in the interior of the container 18. Next, an avidin solution is introduced into the interior of the container 18. The avidin solution contains avidin 32. The first biotin 34a fixed to the substrate 20 and the avidin 32 in the avidin solution are bound to each other. Thus, the avidin 32 is fixed to the surface 26 of the substrate 20. A series of treatments after the avidin 32 is fixed are the same as a series of treatments after the avidin 32 is fixed in the production method 2.

[0069] [2.5 Production method 5]

[0070] Figure 2 The filter unit 12 shown in C can also be manufactured in the following manner, for example. Figure 2 For the filter unit 12 shown in C, the active group includes: a conjugate formed by binding of an antibody or the aptamer and a second biotin; a first biotin fixed to a substrate; and a biotin-binding protein capable of binding to each of the first biotin and the second biotin.

[0071] Here, Figure 2 The first biotin 34a in C (the second biotin in the claim) is preferably fixed to the aforementioned substrate via a structural unit A derived from a compound A having an epoxy group and an ethylenically unsaturated group bonded to the aforementioned substrate, and a structural unit B derived from a diamine compound B bonded to the end of the aforementioned structural unit A.

[0072] That is, in a preferred embodiment of the present invention, a filter is provided, wherein the second biotin is fixed to the aforementioned substrate via a structural unit A derived from a compound A having an epoxy group and an ethylenically unsaturated group, and a structural unit B derived from a diamine compound B bonded to the end of the aforementioned structural unit A, the aforementioned structural unit A is bonded to the aforementioned substrate, and the aforementioned structural unit B is bonded to the aforementioned second biotin.

[0073] The filter unit 12 having such a structure is preferably manufactured by the following steps: (1) irradiating at least a portion of the surface of the substrate with plasma (irradiation step); (2) bringing a compound A having an epoxy group and an ethylenically unsaturated group into contact with the substrate after the plasma irradiation, thereby binding (fixing) the compound A to the substrate (compound A fixing step); (3) bringing a diamine compound B into contact with the substrate after the contact with the compound A, thereby binding (fixing) the diamine compound B to the surface of the substrate via a structural unit A derived from the compound A (diamine compound B fixing step); and (4) bringing biotin (the second biotin in the claim; Figure 2 The first biotin 34a) in C contacts the surface of the substrate after contacting the diamine compound B, thereby binding (immobilizing) biotin to the surface of the substrate via the structural unit A derived from the compound A and the diamine compound B (biotin immobilization step).

[0074] Therefore, the present invention also provides a method for producing a filter, which comprises: (1) irradiating at least a portion of a surface of a substrate formed of a porous polyurethane having a plurality of micropores with plasma; (2) bringing a compound A having an epoxy group and an ethylenically unsaturated group into contact with the substrate after the plasma irradiation, thereby bonding (fixing) the compound A to the substrate; (3) bringing a diamine compound B into contact with the substrate after the contact with the compound A, thereby bonding (fixing) the diamine compound B to the surface of the substrate via a structural unit A derived from the compound A; and (4) allowing biotin (the second biotin in the claim; Figure 2 The first biotin 34a) in C contacts the surface of the substrate after contacting the diamine compound B, so that the biotin is bound (fixed) to the surface of the substrate via the structural unit A derived from the compound A and the diamine compound B.

[0075] Hereinafter, regarding the above-mentioned manufacturing method, refer to Fig.14 Explain in detail.

[0076] -Irradiation process-

[0077] In this process, at least a portion of the surface of a substrate (substrate 20) formed of a porous polyurethane having a plurality of micropores is irradiated with plasma. Here, the reason for "irradiating at least a portion of the substrate surface with plasma" is that, depending on the application, it is not necessary to immobilize biotin (and thus an antibody or an aptamer) on the entire substrate surface (the entire surface), but biotin (and thus an antibody or an aptamer) is only immobilized on the surface portion (sometimes partially, sometimes entirely) where the desired cells are to be captured. Therefore, the plasma irradiation portion preferably includes at least the surface portion where the desired cells are to be captured, and more preferably substantially includes the surface portion where the desired cells are to be captured. It should be noted that the so-called "substantially including the surface portion where the desired cells are to be captured" is preferably larger than the surface where the desired cells are to be captured, and more preferably larger than the edge (outermost periphery) of the surface by more than 0.5 mm, and preferably about 10 to 20 mm.

[0078] The substrate 20 is cut into a predetermined size (substrate A-1). Here, the substrate is formed of porous polyurethane having a plurality of micropores. The size of the substrate is not particularly limited and can be appropriately selected according to the application.

[0079] Next, the cut substrate (substrate A-1) is subjected to plasma treatment (substrate A-2). As a result, the surface of substrate A-1 becomes active. Therefore, if it contacts compound A having an ethylenically unsaturated group in the next step, compound A is graft polymerized on substrate A-1. As a result, compound A can be firmly bonded (can be fixed) to substrate 20. It should be noted that the plasma irradiation process is preferably carried out by ionized gas plasma treatment, considering that even a small-caliber thin and narrow inner surface can be subjected to the desired plasma treatment.

[0080] Before irradiating the surface of substrate A-1 with plasma, the surface of the substrate can be cleaned by an appropriate method. The cleaning method is not particularly limited and can be implemented by cleaning with an appropriate solvent (e.g., ultrasonic cleaning, immersion in a cleaning solvent, rinsing with a cleaning solvent, etc.).

[0081] The pressure conditions in the plasma treatment are not particularly limited and can be any of reduced pressure and atmospheric pressure. In addition, when the substrate is tubular, the plasma irradiation nozzle can be rotated around the substrate (processed object) for one circle while irradiating the plasma gas, thereby uniformly and uniformly performing plasma treatment on the entire circumference of the substrate (processed object).

[0082] As an ionized gas that can be used in plasma treatment, it is one or more gases selected from the group consisting of helium, neon, argon, krypton, air, oxygen, carbon dioxide, carbon monoxide, water vapor, nitrogen and hydrogen. Plasma treatment is preferably carried out in an atmosphere substantially free of oxygen, more preferably in at least one inert gas atmosphere selected from the group consisting of helium, argon and nitrogen. Thus, compound A can be more efficiently combined (grafted polymerized) to the substrate in the next step. Here, the so-called "substantially free of oxygen atmosphere" refers to an oxygen content in the atmosphere of less than 3% by volume, preferably less than 0.1% by volume. The above atmosphere can be adjusted by introducing a prescribed gas into a plasma irradiation device in sufficient amount.

[0083] It should be noted that the plasma treatment conditions (irradiation time, LF output power, applied current, gas flow rate, inter-electrode gap, plasma irradiation distance) are not particularly limited and can be appropriately selected based on the binding properties of compound A to the substrate (ease of immobilization), the type and area of ​​the substrate used, etc.

[0084] As an example, the irradiation time in the plasma treatment is preferably 5 to 60 seconds, and particularly preferably 10 to 40 seconds. Under such conditions, a sufficient amount of compound A can be bound (fixed) to the surface of the substrate.

[0085] As an example, the LF output power in the plasma treatment is preferably 100 to 500W.

[0086] As an example, the gas pressure in the plasma treatment is preferably 20 to 60 Pa.

[0087] The temperature of the object to be treated (porous polyurethane substrate) during plasma treatment is not particularly limited, and may be heated or cooled other than room temperature. From an economical point of view, it is preferably carried out at a temperature that does not require a heating device or a cooling device (for example, 5 to 35° C.).

[0088] There is no particular limitation on the plasma irradiation device (system) that can be used in plasma treatment. For example, there can be exemplified a plasma irradiation device (system) having a structure that introduces gas molecules, excites them to generate plasma, and electrodes that excite the gas molecules in the plasma generating tube, and releases plasma from one end of the plasma generating tube, but there is no limitation on such a structure (system). For example, there can be cited devices that adopt a high-frequency induction method, a capacitive coupling electrode method, a corona discharge electrode-plasma jet method, a parallel plate type, a remote plasma type, an atmospheric pressure plasma type, a low-pressure plasma type, an ICP type high-density plasma type, etc. In addition, from the perspective of existing commercial products, an ionized gas plasma irradiation device (system), especially a plasma irradiation device (system) under atmospheric pressure, can be used. Specifically, a plasma irradiation device made by TRI-STAR TECHNOLOGIES: DURADYNE (trade name or trademark name), a plasma irradiation device made by DIENER ELECTRONIC: PLASMABEAM, a plasma irradiation device made by DIENER ELECTRONIC: Pico Full PC, etc., but there is no limitation on them.

[0089] -Compound A Immobilization Step-

[0090] In this step, the compound A having an epoxy group and an ethylenically unsaturated group is brought into contact with the substrate (substrate A-2) irradiated with plasma in the above-mentioned irradiation step. Thus, the compound A is graft polymerized on the substrate that has become active, and the compound A is bound (immobilized) to the substrate A-2 (substrate A-3). It should be noted that in this specification, the compound A having an epoxy group and an ethylenically unsaturated group is also referred to as "compound A".

[0091] Compound A may have an epoxy group and an ethylenically unsaturated group, preferably a glycidyl group and an ethylenically unsaturated group. As such compound A, for example, (meth)acrylates having a glycidyl group (epoxy group) such as glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexyl methyl acrylate, 3,4-epoxycyclohexyl methyl methacrylate, β-methyl glycidyl acrylate, and β-methyl glycidyl methacrylate may be cited; vinyl ethers having a glycidyl group (epoxy group) such as allyl glycidyl ether, etc., but are not limited to these. Among these, from the viewpoint of further improving the bonding property with the porous polyurethane substrate (graft polymerization property with the substrate), etc., monomer A preferably includes at least one selected from the group consisting of glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexyl methyl acrylate, 3,4-epoxycyclohexyl methyl methacrylate, β-methyl glycidyl acrylate, and β-methyl glycidyl methacrylate. Monomer A is more preferably selected from at least one of the group consisting of glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexyl methyl acrylate, 3,4-epoxycyclohexyl methyl methacrylate, β-methyl glycidyl acrylate, and β-methyl glycidyl methacrylate. Monomer A further preferably includes at least one selected from the group consisting of glycidyl acrylate and glycidyl methacrylate. Monomer A particularly preferably includes glycidyl acrylate or glycidyl methacrylate, particularly preferably includes glycidyl methacrylate, and most preferably glycidyl methacrylate. The above-mentioned compound A can be used alone or in combination of two or more. In addition, when using two or more compounds A, the structural unit A can be configured in a block shape, or can be configured randomly.

[0092] In this step, as a specific method for contacting compound A with the substrate (substrate A-2) after plasma irradiation, any of the methods of directly contacting compound A with substrate A-2 and contacting the compound A solution with substrate A-2 after preparing a compound A solution obtained by dissolving compound A in a solvent. From the viewpoint of further improving the contact efficiency (graft polymerization efficiency) between substrate A-2 and compound A, it is preferred to contact compound A directly with substrate A-2, and more preferably to contact gaseous compound A with substrate A-2. At this time, the contact between compound A and substrate A-2 can be carried out, for example, by the following method: the plasma irradiation device is kept under reduced pressure, and compound A placed under reduced pressure is introduced into the plasma irradiation device.

[0093] In addition, in the method of contacting the solution of compound A with the substrate A-2, the solvent used when dissolving compound A is not particularly limited and can be appropriately selected according to the type of compound A. From the viewpoint of high solubility, it is preferred to use water (reverse osmosis membrane water (RO water), pure water, ion exchange water, distilled water, etc.); alcohol solvents such as methanol, ethanol, isopropanol, butanol; organic solvents such as dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran (THF), dimethyl sulfoxide, N,N-dimethylformamide (DMF), acetone, dioxane, benzene, etc. These can be used alone or in combination (in the form of a mixed solvent). The concentration of compound A in the solution of compound A is not particularly limited. For example, the concentration of compound A in the solution of compound A is preferably 1 to 10% by mass, more preferably 2 to 5% by mass. When the concentration of compound A is within the above range, a sufficient amount of compound A can be bound (fixed) to the surface of the substrate.

[0094] Next, the compound A solution prepared in the above manner is brought into contact with the plasma-irradiated substrate A-2. Here, as a specific method of "contact", a conventionally known method such as a method of coating the compound A solution on the surface of the substrate or a method of immersing the substrate in the compound A solution can be appropriately adopted.

[0095] As for the contact conditions between compound A or a solution of compound A and substrate A-2, as long as compound A can be fully graft polymerized on substrate A-2, it is sufficient. For example, the contact temperature between compound A or a solution of compound A and substrate A-2 is preferably 5 to 50° C., more preferably 20 to 40° C. In addition, the contact time between compound A or a solution of compound A and substrate A-2 is preferably 30 to 90 minutes, more preferably 45 to 70 minutes, and particularly preferably 50 to 60 minutes.

[0096] In this process, if necessary, the substrate after contact with compound A can be cleaned. The method of cleaning treatment is not particularly limited, and can be carried out by cleaning with an appropriate solvent (for example, water such as ultrafiltered water) (for example, ultrasonic cleaning, immersion in a cleaning solvent, a method of pressure cleaning with a roller, a method of rinsing with a cleaning solvent, etc.). The cleaning time is not particularly limited, and is preferably 5 to 15 hours. As needed, the above-mentioned cleaning treatment can be repeated multiple times (for example, 2 to 5 times). It should be noted that when the above-mentioned cleaning treatment is repeated multiple times, each cleaning treatment may be the same or different.

[0097] -Diamine compound B fixation step-

[0098] In this step, diamine compound B is brought into contact with a substrate (substrate A-3) on which compound A is bound (immobilized) via the above-mentioned compound A immobilization step. As a result, the epoxy group (preferably glycidyl group) present in compound A is ring-opened and reacts with an amino group (-NH2) present in diamine compound B, and diamine compound B is bound (immobilized) to the substrate (substrate A-4) via compound A.

[0099] The diamine compound B may have two amino groups (-NH2). Examples of such diamine compounds B include ethylenediamine, propylenediamine, hexamethylenediamine (HMDA), 1,4-butylenediamine, cyclohexanediamine, methylcyclohexanediamine, isophoronediamine, p-phenylenediamine, m-phenylenediamine, p-phenylenediamine, m-phenylenediamine, 4,4'-diaminodiphenylmethane, 4,4'-diaminodiphenyl ether, 1,6-hexanediamine, 1,7-heptanediamine, 1,8-octanediamine, 1,9-nonanediamine, 1,10-decanediamine, 1,11-undecanediamine, 1,12-dodecanediamine, diamine, 1,13-tridecanediamine, 1,18-octadecanediamine, 1-butyl-1,2-ethylenediamine, 1,1-dimethyl-1,4-butanediamine, 1-ethyl-1,4-butanediamine, 1,2-dimethyl-1,4-butanediamine, 1,3-dimethyl-1,4-butanediamine, 1,4-dimethyl-1,4-butanediamine, 2,3-dimethyl-1,4-butanediamine, 2-methyl-1,5-pentanediamine, 3-methyl-1,5-pentanediamine, 2,5-dimethyl-1,6-hexanediamine, 2, 4-dimethyl-1,6-hexanediamine, 3,3-dimethyl-1,6-hexanediamine, 2,2-dimethyl-1,6-hexanediamine, 2,2,4-trimethyl-1,6-hexanediamine, 2,4,4-trimethyl-1,6-hexanediamine, 2,4-diethyl-1,6-hexanediamine, 2,2-dimethyl-1,7-heptanediamine, 2,3-dimethyl-1,7-heptanediamine, 2,4-dimethyl-1,7-heptanediamine, 2,5-dimethyl-1,7-heptanediamine, 2-methyl-1,8-octanediamine, 3-methyl-1,8-octanediamine, 4-methyl-1,8-octanediamine, 1,3-dimethyl-1,8-octanediamine, 1,4-dimethyl-1,8-octanediamine, 2,4-dimethyl-1,8-octanediamine, 3,4-dimethyl-1,8-octanediamine, 4,5-dimethyl-1,8-octanediamine, 2,2-dimethyl-1,8-octanediamine, 3,3-dimethyl-1,8-octanediamine, 4,4-dimethyl-1,8-octanediamine, 5-methyl-1,9-nonanediamine, etc., but are not limited to these. Among these, from the viewpoint of further improving the reactivity with compound A (and further, the bonding property of diamine compound B with the substrate), the diamine compound B preferably contains at least one selected from the group consisting of ethylenediamine, trimethylenediamine, 1,2-diaminopropane, tetramethylenediamine, 1,3-diaminobutane, 2,3-diaminobutane, pentamethylenediamine, 2,4-diaminopentane, hexamethylenediamine (HMDA), octamethylenediamine, nonamethylenediamine, decamethylenediamine, undecamethylenediamine, dodecamethylenediamine, tridecamethylenediamine, octadecamethylenediamine, xylylenediamine, and phenylenediamine.Diamine compound B is more preferably selected from at least one of the group consisting of ethylenediamine, trimethylenediamine, 1,2-diaminopropane, tetramethylenediamine, 1,3-diaminobutane, 2,3-diaminobutane, pentamethylenediamine, 2,4-diaminopentane, hexamethylenediamine (HMDA), octamethylenediamine, nonamethylenediamine, decamethylenediamine, 11methylenediamine, dodecamethylenediamine, tridecamethylenediamine, 18methylenediamine, xylene diamine, and phenylenediamine. Diamine compound B further preferably includes at least one selected from the group consisting of hexamethylenediamine (HMDA), octamethylenediamine. Diamine compound B is most preferably hexamethylenediamine (HMDA). Above-mentioned diamine compound B can be used alone or in combination of two or more.

[0100] In this step, as a specific method for contacting the diamine compound B with the substrate (substrate A-3) after contacting the compound A, any of a method of directly contacting the diamine compound B with the substrate A-3 and a method of contacting the diamine compound B solution with the substrate A-3 after preparing a diamine compound B solution obtained by dissolving the diamine compound B in a solvent. From the viewpoint of further improving the contact efficiency between the substrate A-3 and the diamine compound B (reactivity of the ring-opening epoxy group and the amino group), it is preferred to use a method of contacting the diamine compound B solution with the substrate A-3.

[0101] The solvent used in the preparation of the diamine compound B solution is not particularly limited as long as it can dissolve the diamine compound B. For example, it can be selected from water (reverse osmosis membrane water (RO water), ultrafiltration water, pure water, ion exchange water, distilled water, etc.); alcohols such as ethanol, isopropanol, n-propanol, n-butanol, isobutanol, sec-butanol, tert-butanol, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol; chloroform, tetrahydrofuran, acetone, dioxane, benzene and other organic solvents, etc. One or more, but not limited to these. In addition, the concentration of the diamine compound B contained in the diamine compound B solution is not particularly limited. As an example, the concentration of the diamine compound B in the diamine compound B solution is preferably 0.5 to 5 g / 100 mL of solvent, more preferably 1 to 3 g / 100 mL of solvent, and particularly preferably 1 to 2 g / 100 mL of solvent. If the concentration is such, the compound A and the diamine compound B are in sufficient contact with each other, so that the ring-opening epoxy group of the compound A and the amino group of the diamine compound B can react more efficiently.

[0102] Next, the diamine compound B solution prepared as described above is brought into contact with the substrate A-3. Here, as a specific method of "contacting", a conventionally known method such as a method of coating the diamine compound B solution on the substrate surface or a method of immersing the substrate in the diamine compound B solution can be appropriately adopted.

[0103] In the case of contacting by immersion method, the diamine compound B solution can be stirred while being carried out. Thus, the contact between the diamine compound B solution and the substrate A-3 can be further promoted. The above-mentioned stirring can be carried out by using known devices such as stirring with a stirring bar, an orbital oscillator, a rotary oscillator, a paint oscillator, etc. In addition, the stirring conditions are not particularly limited. From the viewpoint of being able to contact the treated object more efficiently, the stirring speed is, for example, 200 to 600 rpm, preferably 300 to 400 rpm. From the same viewpoint, the stirring time is, for example, 0.5 to 24 hours, preferably 1 to 12 hours. It should be noted that the temperature of the compound B solution when contacting by immersion method can be around room temperature (for example, above 20°C and less than 40°C), or it can be heated (for example, 40 to 60°C).

[0104] Moreover, when making diamine compound B bond to only a part of a base material, by immersing only a part of a base material in the diamine compound B solution, diamine compound B can be bonded to the desired surface site of the base material.

[0105] In the case where it is difficult to immerse only a part of the substrate in the diamine compound B solution, the surface part of the substrate that does not need to be bonded to the diamine compound B can be protected (coated, etc.) in advance with a detachable (loading and unloading) appropriate component, material, and then the substrate is immersed in the diamine compound B solution, and then the above-mentioned protective component (material) is removed, thereby allowing the diamine compound B to be bonded to the desired surface part of the substrate. Among them, the present invention is not subject to any limitation of these formation methods, and the diamine compound B can be appropriately bonded using a previously known method. For example, in the case where it is difficult to immerse only a part of the substrate in the diamine compound B solution, other coating methods (for example, a method of applying the diamine compound B solution to a specified surface part of the substrate using a spray device, a rod coater, a die coater, a reverse coater, a comma coater, a gravure coater, a spray coater, a scraper, etc.) can be used instead of the immersion method.

[0106] As for the contact conditions between the diamine compound B or the diamine compound B solution and the substrate A-3, it is sufficient as long as the diamine compound B can fully react with the compound A and bond to the substrate A-3. For example, the contact temperature between the diamine compound B or the diamine compound B solution and the substrate A-3 is preferably 50 to 90° C., more preferably 60 to 80° C., and particularly preferably 60 to 70° C. In addition, the contact time between the diamine compound B or the diamine compound B solution and the substrate A-3 is preferably 3 to 25 hours, more preferably 5 to 20 hours, and particularly preferably 8 to 15 hours.

[0107] In this process, if necessary, the substrate after contacting the diamine compound B can be cleaned. The method of the cleaning treatment is not particularly limited, and can be implemented by cleaning (for example, ultrasonic cleaning, immersion in a cleaning solvent, pressure cleaning with a roller, rinsing with a cleaning solvent, etc.) using an appropriate solvent (for example, water such as ultrafiltered water, aqueous hydrochloric acid solution). The cleaning time is not particularly limited, and is preferably 5 to 15 hours. If necessary, the above-mentioned cleaning treatment can be repeated multiple times (for example, 2 to 5 times). It should be noted that when the above-mentioned cleaning treatment is repeated multiple times, each cleaning treatment may be the same or different.

[0108] -Biotin Immobilization Process-

[0109] In this step, biotin is brought into contact with the surface of the substrate (substrate A-4) after contact with the diamine compound B obtained in the above-mentioned diamine compound B immobilization step. As a result, the carboxyl group (-COOH) of biotin reacts with the other amino group (-NH2) of the diamine compound B (the side that does not react with the ring-opening epoxy group of the compound A), and biotin is bound (immobilized) to the surface of the above-mentioned substrate via the structural unit A derived from the compound A and the diamine compound B.

[0110] In this step, as a specific method for bringing the biotin into contact with the substrate (substrate A-4) after contacting the diamine compound B, any of a method of bringing biotin into contact with the substrate A-4 directly or a method of preparing a biotin solution obtained by dissolving biotin in a solvent and bringing the biotin solution into contact with the substrate A-4 may be used. From the viewpoint of further improving the contact efficiency between the substrate A-4 and biotin (reactivity of the amino group and the carboxyl group), it is preferred to use a method of bringing the biotin solution into contact with the substrate A-4.

[0111] The solvent used in the preparation of the biotin solution is not particularly limited as long as it can dissolve biotin. For example, it can be selected from water (reverse osmosis membrane water (RO water), ultrafiltration water, pure water, ion exchange water, distilled water, etc.); alcohols such as ethanol, isopropanol, n-propanol, n-butanol, isobutanol, sec-butanol, tert-butanol, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol; chloroform, tetrahydrofuran, acetone, dioxane, benzene and other organic solvents, etc., one or more, but not limited to these. In addition, the concentration of biotin contained in the biotin solution is not particularly limited. As an example, the concentration of biotin in the biotin solution is preferably 0.01 to 0.30 g / 100 mL of solvent, and more preferably 0.05 to 0.15 g / 100 mL of solvent. At such a concentration, the diamine compound B is fully in contact with biotin, so the amino group of the diamine compound B and the carboxyl group of biotin can react more efficiently.

[0112] In order to improve the reaction of the diamine compound B, the biotin solution preferably further includes a reaction accelerator. As the reaction accelerator in the case of using a reaction accelerator, there is no particular limitation, for example, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide HCl (EDC), dicyclohexylcarbodiimide (DDC), N,N'-diisopropylcarbodiimide (DIC), N,N'-di-tert-butylcarbodiimide, N,N'-dicyclohexylcarbodiimide (DCC), N-tert-butyl-N-ethylcarbodiimide and other carbodiimides, etc., can be cited. In the case of using a reaction accelerator, the amount of the reaction accelerator added is as long as it can promote the reaction of the amino group of the diamine compound B with the carboxyl group of biotin, and can be appropriately selected according to the type of diamine compound B, the reaction accelerator, etc. For example, the biotin solution preferably contains a reaction accelerator in a ratio of preferably 100 to 250 parts by mass, more preferably 150 to 200 parts by mass, relative to 100 parts by mass of biotin.

[0113] Next, the biotin solution prepared as described above is brought into contact with the substrate A-4. Here, as a specific method of "contacting", a conventionally known method such as a method of coating the biotin solution on the surface of the substrate or a method of immersing the substrate in the biotin solution can be appropriately adopted.

[0114] When contact is performed by the immersion method, the biotin solution can be stirred at the same time. Thus, the contact between the biotin solution and the substrate A-4 can be further promoted. The above-mentioned stirring can be performed by using a known device such as stirring with a stirring bar, an orbital oscillator, a rotary oscillator, a paint oscillator, etc. In addition, the stirring conditions are not particularly limited. From the viewpoint of being able to contact the treated object more efficiently, the stirring speed is, for example, 200 to 600 rpm, preferably 300 to 400 rpm. From the same viewpoint, the stirring time is, for example, 0.5 to 24 hours, preferably 1 to 12 hours. It should be noted that the temperature of the biotin solution when contacted by the immersion method can be around room temperature (for example, above 20°C and less than 40°C), or it can be heated (for example, 40 to 60°C).

[0115] Furthermore, when biotin is bound to only a portion of the substrate, biotin can be bound to a desired surface site of the substrate by immersing only a portion of the substrate in a biotin solution.

[0116] In the case where it is difficult to immerse only a part of the substrate in the biotin solution, the surface part of the substrate that does not need to be bound to biotin can be protected (coated, etc.) in advance with a detachable (installable) appropriate component or material, and then the substrate is immersed in the biotin solution, and then the above-mentioned protective component (material) is removed, thereby allowing biotin to be bound to the desired surface part of the substrate. Among them, the present invention is not limited to any of these formation methods, and biotin can be appropriately bound by a previously known method. For example, in the case where it is difficult to immerse only a part of the substrate in the biotin solution, other coating methods (for example, a method of applying a biotin solution to a specified surface part of the substrate using a coating device such as a spray device, a rod coater, a die coater, a reverse coater, a comma coater, a gravure coater, a spray coater, a scraper, etc.) can be used instead of the immersion method.

[0117] The contact conditions between biotin or a biotin solution and the substrate A-4 are as long as biotin can fully react with compound A and bind to the substrate A-4. For example, the contact temperature between biotin or a biotin solution and the substrate A-4 is preferably 5 to 60° C., more preferably 15 to 35° C. In addition, the contact time between biotin or a biotin solution and the substrate A-4 is preferably 1 to 30 hours, more preferably 3 to 20 hours, and particularly preferably 5 to 15 hours.

[0118] In this process, if necessary, the substrate after contact with biotin can be cleaned. There is no particular limitation on the method of cleaning treatment, and it can be carried out by cleaning with an appropriate solvent (for example, water such as ultrafiltered water, aqueous hydrochloric acid solution) (for example, ultrasonic cleaning, immersion in a cleaning solvent, pressure cleaning with a roller, rinsing with a cleaning solvent, etc.). There is no particular limitation on the cleaning time, and it is preferably 5 to 15 hours. If necessary, the above cleaning treatment can be repeated multiple times (for example, 2 to 5 times). It should be noted that when the above cleaning treatment is repeated multiple times, each cleaning treatment may be the same or different.

[0119] The filter obtained in the above manner can be sterilized by a conventionally known method such as high pressure steam sterilization, radiation sterilization, ethylene oxide sterilization, nitrogen dioxide sterilization, etc.

[0120] [3. Configuration of Separation Device 40]

[0121] By making the cell fluid Figure 1 The desired separation object can be separated from the cell fluid by allowing the fluid to flow through the filter 10 shown. The device that performs such a process is referred to as a separation device 40.

[0122] Figure 34 is a diagram showing the structure of the separation device 40. The separation device 40 can separate the desired separation object from the cell fluid. Figure 2 C is a partial process of the method for manufacturing the filter unit 12 . The separation device 40 includes a separation circuit 42 and a controller 44 .

[0123] The separation circuit 42 includes a supply unit 46, a separation unit 48, and a discharge unit 50. The supply unit 46 includes a cell solution container 52, an avidin solution container 54, a first antibody solution container 56, a second antibody solution container 58, a stripping solution container 60, a cleaning solution container 62, a first supply flow path 64 to a sixth supply flow path 74, valves 76a to 76f, and a pump 78. Each of the cell solution container 52 to the cleaning solution container 62 is, for example, a medical bag formed by molding a soft resin material into a bag shape. Each of the cell solution container 52 to the cleaning solution container 62 may also be a tank made of a hard material, etc. Each of the cell solution container 52 to the cleaning solution container 62 is detachable relative to the separation device 40.

[0124] The cell fluid container 52 is filled with cell fluid. The cell fluid is a solution containing two separation objects. In this embodiment, the two separation objects are recorded as the first cell and the second cell. The avidin solution container 54 is filled with avidin solution. The avidin solution is a solution containing avidin 32. The first antibody solution container 56 is filled with the first antibody solution. The first antibody solution is a solution containing the first biotinylated antibody 36a ( Figure 2 C). The first antibody 30a can specifically bind to the antigen possessed by the first cell. The second antibody solution is filled in the second antibody solution container 58. The second antibody solution contains a second biotinylated antibody 36b ( Figure 2 C) solution. The second antibody 30b can specifically bind to the antigen possessed by the second cell. The stripping solution container 60 is filled with a stripping solution. The stripping solution is, for example, a deoxyribonuclease solution. The cleaning solution container 62 is filled with a cleaning solution. The cleaning solution is, for example, water, phosphate buffered saline (PBS), etc.

[0125] The cell solution container 52 is connected to the suction port 80 of the pump 78 via the first supply channel 64. A valve 76a is provided on the first supply channel 64. The avidin solution container 54 is connected to the suction port 80 of the pump 78 via the second supply channel 66. A valve 76b is provided on the second supply channel 66. The first antibody solution container 56 is connected to the suction port 80 of the pump 78 via the third supply channel 68. A valve 76c is provided on the third supply channel 68. The second antibody solution container 58 is connected to the suction port 80 of the pump 78 via the fourth supply channel 70. A valve 76d is provided on the fourth supply channel 70. The stripping solution container 60 is connected to the suction port 80 of the pump 78 via the fifth supply channel 72. A valve 76e is provided on the fifth supply channel 72. The cleaning solution container 62 is connected to the suction port 80 of the pump 78 via the sixth supply channel 74. The sixth supply flow path 74 is provided with a valve 76 f.

[0126] The separation unit 48 includes a filter 10 , an upstream flow path 84 , a downstream flow path 86 , a first bypass flow path 88 , a second bypass flow path 90 , and valves 92 a to 92 d . Figure 3 The filter 10 shown has two filter units 12. Each filter unit 12 is detachable relative to the separation device 40. That is, the filter unit 12 can be replaced according to the type of separation object. The separation part 48 is detachable relative to the separation device 40. Alternatively, the filter 10 is detachable relative to the separation device 40.

[0127] As described above, the filter 10 includes a first filter unit 12a, a second filter unit 12b, and a connecting flow path 14. The inlet port 22 of the first filter unit 12a is connected to the discharge port 82 of the pump 78 via the upstream flow path 84. A valve 92a is provided on the upstream flow path 84. The upstream flow path 84 located upstream of the valve 92a is connected to the connecting flow path 14 through the first bypass flow path 88. A valve 92b is provided on the first bypass flow path 88. The outlet port 24 of the second filter unit 12b is connected to the downstream flow path 86. A valve 92c is provided on the downstream flow path 86. The downstream flow path 86 located downstream of the valve 92c is connected to the connecting flow path 14 through the second bypass flow path 90. A valve 92d is provided on the second bypass flow path 90.

[0128] The discharge unit 50 includes a recovery container 94, a waste liquid container 96, a recovery flow path 98, a waste liquid flow path 100, and valves 102a and 102b. The recovery flow path 98 and the waste liquid flow path 100 are each connected to the downstream flow path 86 of the separation unit 48. The recovery flow path 98 is provided with a valve 102a. The waste liquid flow path 100 is provided with a valve 102b.

[0129] The recovery container 94 is connected to the outlet port 24 of the second filter unit 12b via the recovery flow path 98 and the downstream flow path 86. The recovery container 94 is connected to the outlet port 24 of the first filter unit 12a via the recovery flow path 98, the downstream flow path 86 and the second bypass flow path 90.

[0130] The waste liquid container 96 is connected to the outlet port 24 of the second filter unit 12b via the waste liquid flow path 100 and the downstream flow path 86. The waste liquid container 96 is connected to the outlet port 24 of the first filter unit 12a via the waste liquid flow path 100, the downstream flow path 86 and the second bypass flow path 90.

[0131] The controller 44 has a processing circuit and various memories (volatile memory, non-volatile memory, etc.). The processing circuit can be a processor such as a CPU. The processing circuit can be an integrated circuit such as an ASIC or FPGA. The processor can perform various processes by executing programs stored in the non-volatile memory. As volatile memory, for example, RAM, etc. can be cited. The volatile memory is used as a working memory of the processor. The volatile memory temporarily stores data required for processing or calculation, etc. As non-volatile memory, for example, ROM, flash memory, etc. can be cited. The non-volatile memory is used as a storage memory. The non-volatile memory stores programs, tables, maps, etc.

[0132] The controller 44 controls the opening and closing of each valve of the separation circuit 42. In addition, the controller 44 controls the operation of the pump 78. The controller 44 can automatically perform the operation by a program. Figure 4 A series of processing shown.

[0133] [4 Operation of Separation Device 40]

[0134] Figure 4 This is a flow chart of the filter preparation process and the separation process performed using the separation device 40 . Figure 5 to Figure 13 4 is a diagram showing the opening and closing of the valve of the separation device 40 and the flow of the liquid in the separation device 40 in each step. Figure 5 to Figure 13 In the figure, the valves painted black indicate the closed state, and the valves painted white indicate the open state. Figure 4 The steps shown are performed by the controller 44 controlling the valves and the pump 78 .

[0135] User in Figure 4 The following preparation work is performed before the series of treatments shown. The user fills the substrate 20 fixed with the first biotin 34a into each filter unit 12. The user prepares the first antibody solution containing the first biotinylated antibody 36a and fills it into the first antibody solution container 56. The user prepares the second antibody solution containing the second biotinylated antibody 36b and fills it into the second antibody solution container 58. Figure 4In the process described, the first biotinylated antibody 36a and the second biotinylated antibody 36b are substances that can bind to the unwanted separation objects (first cells and second cells) in the cell fluid. The user installs the cell fluid container 52 to the cleaning fluid container 62 in the separation device 40. When the user operates the controller 44, the separation starts. Figure 4 A series of processing shown.

[0136] In step S1, the controller 44 performs priming. The controller 44 sets the separation circuit 42 to Figure 5 That is, the controller 44 opens valve 76f, valve 92a, valve 92c, and valve 102b, and closes the other valves. In addition, the controller 44 activates the pump 78. Then, the cleaning liquid in the cleaning liquid container 62 flows along Figure 5 The liquid flows through the path indicated by the arrow and is recovered in the waste liquid container 96.

[0137] Next, the controller 44 causes the separation circuit 42 to be Figure 6 That is, the controller 44 opens valve 76f, valve 92b, valve 92d, and valve 102b, and closes the other valves. In addition, the controller 44 activates the pump 78. Then, the cleaning liquid in the cleaning liquid container 62 flows along Figure 6 The liquid flows through the path indicated by the arrow and is recovered in the waste liquid container 96. By the process of step S1, the separation unit 48 is cleaned.

[0138] In step S2, the controller 44 performs precoating (precoating) of the avidin 32 (first precoating). The controller 44 sets the separation circuit 42 to Figure 7 That is, the controller 44 opens valve 76b, valve 92a, valve 92c, and valve 102b, and closes the other valves. In addition, the controller 44 activates the pump 78. Then, the avidin liquid in the avidin liquid container 54 flows along Figure 7 The avidin 32 in the avidin solution flows along the path indicated by the arrow and is recovered in the waste liquid container 96. The controller 44 operates the pump 78 for a predetermined time or longer, and then stops the pump 78 for a predetermined time or longer. Through the process of step S2, the avidin solution is supplied to each filter unit 12, and the substrate 20 in the filter unit 12 is immersed in the avidin solution. The first biotin 34a fixed to the substrate 20 is bound to the avidin 32 in the avidin solution. Thus, the avidin 32 is fixed to the substrate 20.

[0139] Next, the controller 44 causes the separation circuit 42 to be Figure 5 Thus, the portion through which the avidin solution flows is cleaned.

[0140] In step S3, the controller 44 performs pre-coating of the first biotinylated antibody 36a (second pre-coating). The controller 44 sets the separation circuit 42 to Figure 8That is, the controller 44 opens valve 76c, valve 92a, valve 92d, and valve 102b, and closes the other valves. In addition, the controller 44 activates the pump 78. Then, the first antibody liquid in the first antibody liquid container 56 flows along Figure 8 The first antibody solution is supplied to the first filter unit 12a through the process of step S3, and the substrate 20 in the first filter unit 12a is immersed in the first antibody solution. The avidin 32 fixed to the substrate 20 is bound to the first biotinylated antibody 36a in the first antibody solution. As a result, in the first filter unit 12a, as shown in FIG. Figure 2 As shown in FIG. 3C , the active groups 28 (the first biotin 34 a , the avidin 32 , and the first biotinylated antibody 36 a ) are immobilized on the surface 26 of the substrate 20 .

[0141] Next, the controller 44 causes the separation circuit 42 to be Fig. 9 That is, the controller 44 opens valve 76f, valve 92a, valve 92d, and valve 102b, and closes the other valves. In addition, the controller 44 activates the pump 78. Then, the cleaning liquid in the cleaning liquid container 62 flows along Fig. 9 The antibody flows through the path indicated by the arrow and is recovered in the waste liquid container 96. By this treatment, the portion through which the first antibody solution flows is cleaned.

[0142] In step S4, the controller 44 performs pre-coating of the second biotinylated antibody 36b (third pre-coating). The controller 44 sets the separation circuit 42 to Fig.10 That is, the controller 44 opens valve 76d, valve 92b, valve 92c, and valve 102b, and closes the other valves. In addition, the controller 44 activates the pump 78. Then, the second antibody liquid in the second antibody liquid container 58 flows along Fig.10 The second antibody solution is supplied to the second filter unit 12b through the process of step S4, and the substrate 20 in the second filter unit 12b is immersed in the second antibody solution. The avidin 32 fixed to the substrate 20 is bound to the second biotinylated antibody 36b in the second antibody solution. As a result, in the second filter unit 12b, as shown in FIG. Figure 2 As shown in FIG. 3C , the active groups 28 (the first biotin 34 a , the avidin 32 , and the second biotinylated antibody 36 b ) are immobilized on the surface 26 of the substrate 20 .

[0143] Next, the controller 44 causes the separation circuit 42 to be Fig.11That is, the controller 44 opens valve 76f, valve 92b, valve 92c, and valve 102b, and closes the other valves. In addition, the controller 44 activates the pump 78. Then, the cleaning liquid in the cleaning liquid container 62 flows along Fig.11 The second antibody solution flows through the path shown by the arrow and is recovered in the waste liquid container 96. By this treatment, the portion through which the second antibody solution flows is cleaned.

[0144] In step S5, the controller 44 separates the desired cells from the cell fluid. The controller 44 controls the separation circuit 42 to Fig.12 That is, the controller 44 opens valve 76a, valve 92a, valve 92c, and valve 102a, and closes the other valves. In addition, the controller 44 activates the pump 78. Then, the cell fluid in the cell fluid container 52 flows along Fig.12 The cell fluid flows along the path indicated by the arrow and is recovered in the recovery container 94. The controller 44 operates the pump 78 until the cell fluid in the cell fluid container 52 is exhausted, and then stops the pump 78. Through the process of step S5, the cell fluid is supplied to each filter unit 12, and the substrate 20 in each filter unit 12 is immersed in the cell fluid. The first biotinylated antibody 36a fixed on the substrate 20 of the first filter unit 12a binds to the antigen of the first cell in the cell fluid. The second biotinylated antibody 36b fixed on the substrate 20 of the second filter unit 12b binds to the antigen of the second cell in the cell fluid. As a result, the cell fluid from which the first cell and the second cell are removed is recovered in the recovery container 94.

[0145] Next, the controller 44 causes the separation circuit 42 to be Fig.13 The state shown in FIG. 4 is achieved. Thus, the cells remaining in the separation circuit 42 can be recovered without waste. This completes a series of treatments.

[0146] It should be noted that when using Figure 4 In the process described, the unnecessary separation objects (first cell, second cell) are removed from the cell fluid, and the cell fluid containing the desired cells is recovered. That is, the separation device 40 performs the so-called negative selection. Alternatively, the desired separation object can be extracted from the cell fluid. That is, the separation device 40 can also perform the so-called positive selection. In this case, after the desired separation object is captured by the filter 10, the stripping liquid in the stripping liquid container 60 is passed through the filter 10, and the stripping liquid and the separation object are recovered by the recovery container 94.

[0147] [5 Others]

[0148] The filter 10 may include other units for physically separating the separation object. The other units are formed of porous components and include other substrates different from the filter unit 12. The multiple micropores formed in the other substrates are larger than the separation object captured by the filter unit 12 and smaller than the separation object to be captured by the other units.

[0149] Example

[0150] The present invention is specifically described below using examples, but the present invention is not limited to these examples. It should be noted that the parts and % in each example are based on mass. The room temperature storage conditions are 23°C / 55%RH unless otherwise specified.

[0151] Example 1

[0152] 1. Graft polymerization of GMA onto the surface of polyurethane filter material

[0153] 1) Preparation of substrate (substrate A-1)

[0154] A polyurethane filter material (manufactured by Terumo Corporation, product name: Imugard, thickness: 2 mm) was cut into a size of 15 cm×5 cm to prepare a PU film.

[0155] 2) Plasma treatment (substrate A-2) and immobilization of GMA (substrate A-3)

[0156] The PU film obtained in 1) was placed in a graft polymerization reaction chamber (plasma irradiation device, Pico Full PC, manufactured by DIENER ELECTRONIC, low pressure type), the chamber was evacuated, and 0.2 torr of Ar gas (100%) was introduced into the chamber. Plasma irradiation (argon ionized gas plasma irradiation) was performed for 40 seconds under the conditions of LF output power: 400 W and gas pressure: 45 Pa (substrate A-2).

[0157] Next, the valve between the chamber and the vacuum pump was closed, and the valve of the reservoir of glycidyl methacrylate (GMA) which had been evacuated in advance was opened to introduce GMA into the chamber.

[0158] After leaving it in this state for 1 hour, the pressure in the chamber was returned to normal pressure, and the PU film was taken out of the chamber. Thus, a PU film (GMA-PU film) having GMA graft-polymerized on the surface was obtained (substrate A-3).

[0159] 2. Immobilization of biotin on GMA-PU membrane (substrate A-4)

[0160] The GMA-PU film obtained in 1. above was immersed in an HMDA aqueous solution of the following composition and incubated at 70° C. overnight to react the epoxy group of GMA with one amine group of hexamethylenediamine (HMDA) (HMDA-GMA-PU film) (substrate A-4).

[0161] (HMDA aqueous solution composition)

[0162] Ultrafiltered water: 186mL

[0163] Ethanol: 14 mL

[0164] Hexamethylenediamine (HMDA): 4g

[0165] After the incubation, the HMDA-GMA-PU membrane was immersed in a 0.01N-HCl aqueous solution for 8 hours (immersion of the HMDA-GMA-PU membrane).

[0166] While the HMDA-GMA-PU impregnation membrane was impregnated in 2 L of ultrafiltrated water, the HMDA-GMA-PU impregnation membrane was pressure-washed with a roller (HMDA-GMA-PU membrane washing).

[0167] The cleaned HMDA-GMA-PU membrane after pressure cleaning was immersed in a biotin aqueous solution of the following composition at room temperature (25° C.) overnight to react the other amine group of hexamethylenediamine (HMDA) with the carboxyl group of biotin (biotin-HMDA-GMA-PU membrane).

[0168] (Biotin aqueous solution composition)

[0169] Ultrafiltered water: 200mL

[0170] Biotin: 0.16g

[0171] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl (EDC): 0.26 g

[0172] After the impregnation was completed, the biotin-HMDA-GMA-PU membrane was washed under pressure using a roller while being immersed in 2 L of ultrafiltrated water (washing of the biotin-HMDA-GMA-PU membrane).

[0173] The washed biotin-HMDA-GMA-PU membrane was left to stand overnight at room temperature (25° C.) to be fully dried, thereby obtaining a polyurethane filter material (Sample 1) to which biotin was bonded via GMA and HMDA.

[0174] 3. Fluorescence Observation

[0175] The sample 1 obtained in 2. above was punched into a 1 mm diameter

[0176] Separately, Tween 20 (0.05 (w / v)%) was added to 10 mL of PBS to prepare T-PBS.

[0177] Streptavidin-FITC (manufactured by Funakoshi Co., Ltd., trade name: Streptavidin Conjugates, hereinafter referred to as "StA-F") was added to 2 mL of PBS at a concentration of 5 μg / mL to prepare StA-F-PBS.

[0178] The sample 1 having a diameter of 1 mm was placed in a commercially available filter housing (manufactured by EMD Millipore Corp., trade name: SWINNEX), and was fully pre-filled with T-PBS to remove air bubbles in the housing.

[0179] 2 mL of StA-F-PBS was injected into the pre-filled shell, and the mixture was incubated at 37°C for 30 minutes.

[0180] StA-F not immobilized on the sample 1 was washed away with 30 mL of T-PBS, and then the sample 1 was taken out from the shell (measurement sample 1).

[0181] The measurement sample 1 obtained above was subjected to fluorescence observation under the following conditions using a confocal laser scanning microscope (manufactured by Olympus Corporation, trade name: FV3000).

[0182] (Observation conditions)

[0183] Magnification: 1.25 times

[0184] Excitation wavelength: 488nm

[0185] Voltage: 700V

[0186] The results are shown in Fig.15 .like Fig.15 As shown, sample 1 uniformly emits fluorescence. From this result, it can be seen that in sample 1, biotin is uniformly immobilized on the polyurethane filter material.

[0187] Example 2

[0188] 1. Install filter 200

[0189] Five biotin-HMDA-GMA-PU membranes (Sample 2) were prepared in the same manner as in Example 1 except that the polyurethane filter material (manufactured by Terumo Corporation, product name: Imugard, thickness: 2 mm) was cut into a size with a diameter (Φ) of 25 mm.

[0190] For sample 2, use Fig.16 The filter 200 shown was evaluated for cell capture as follows.

[0191] Fig.16 Schematic diagram showing the experimental setup used to evaluate cell capture. Fig.16 In the embodiment, each sample 2220 is stacked with a silicone spacer 231 (ring shape with inner diameter: 24 mm, outer diameter: 29 mm, thickness: 1.0 mm), and a polypropylene (PP) mesh (diameter (Φ): 21 mm, thickness: 0.5 mm, mesh size: 1.7 mm×1.7 mm) 230 is placed in the center of the uppermost sample 2220a and the lowermost sample 2220b to prepare a filter unit 212. The filter unit 212 is set in a container 218 in which tubes 223 and 225 are connected at the lower and upper parts via tube connectors 222 and 224, respectively, to form a filter 200.

[0192] 2. Preparation of Filters for Cell Capture

[0193] From the lower tube 223 of the above-mentioned filter 200, use a syringe pump to inject PBS buffer (phosphate buffer, composition: 136.89mM sodium chloride, 2.68mM potassium chloride, 0.147mM potassium dihydrogen phosphate, 9.58mM disodium hydrogen phosphate (anhydrous)) at a flow rate of 1mL / min, and pre-fill the filter 200 with about 10mL of PBS buffer.

[0194] A 0.05% (w / v) streptavidin (hereinafter also referred to as "StA") / PBS solution (StA / PBS solution) was prepared. Using a syringe pump, 5 mL of the StA solution was injected into the filter 200 from the lower tube 223 of the filter 200 at a flow rate of 1 mL / min. After the StA / PBS solution was injected, the upper tube 225 and the lower tube 223 of the filter 200 were clamped. In this state, incubation was performed at 37°C for 15 minutes.

[0195] A porcine CD8 antibody (manufactured by Thermo Fisher, product name: Invitrogen, CD8α monoclonal antibody (76-2-11), biotin) conjugated with biotin was added to PBS at a concentration of 0.005% (w / v) to prepare a porcine CD8 antibody / PBS solution. Using a syringe pump, 5 mL of the porcine CD8 antibody / PBS solution was injected into the filter 200 from the lower tube 223 of the filter 200 at a flow rate of 1 mL / min. After the porcine CD8 antibody / PBS solution was injected, the upper tube 225 and the lower tube 223 of the filter 200 were clamped. In this state, incubate at 37°C for 15 minutes.

[0196] 3. Cell Capture Evaluation

[0197] 1000 mL of pig blood was collected into 140 mL of CPD solution (composition: 26.30 g of sodium citrate hydrate, 3.27 g of citric acid hydrate, 23.20 g of glucose, 2.51 g of sodium dihydrogen phosphate / 1,000 mL of water for injection) to prepare pig whole blood (CPD blood collection).

[0198] After incubation for a predetermined time in 2., 5 mL of the above-mentioned pig whole blood (CPD blood collection) was injected into the filter 200 from the lower tube 223 of the filter 200 at a flow rate of 1 mL / min using a syringe pump. After the pig whole blood (CPD blood collection) was injected, the upper tube 225 and the lower tube 223 of the filter 200 were clamped. In this state, incubation was performed at 37°C for 15 minutes.

[0199] After incubation, a PBS buffer solution was injected into the filter 200 from the lower tube 223 of the filter 200 at a flow rate of 1 mL / min using a syringe pump, and 5 mL of pig whole blood in the filter 200 was recovered.

[0200] Blood cell count and flow cytometry were performed on the pig whole blood collected before and after the filter was injected, and the CD8 positive rate in the total white blood cells of the pig whole blood before and after the filter was injected was calculated. Based on these values, the CD8 positive cell capture rate by the filter was calculated according to the following formula.

[0201] [Mathematical formula 1]

[0202]

[0203] Comparative Example 1

[0204] A polyurethane filter material (manufactured by Terumo Corporation, product name: Imugard, thickness: 2 mm) was cut into a size of 25 mm in diameter (Φ), and five comparative PU films (Comparative Example 1) were prepared.

[0205] In Example 2, except that a comparative PU film was used instead of Sample 2, the same operation as in Example 2 was carried out to calculate the CD8-positive cell capture rate.

[0206] As a result, Sample 2 (Example 2) captured significantly more cells than the comparative PU film (Comparative Example 1).

[0207] It should be noted that the present invention is not limited to the above-mentioned disclosure, and various configurations can be adopted without departing from the gist of the present invention.

[0208] This application is based on Japanese patent application No. 2022-123240 filed on August 2, 2022, the disclosure of which is incorporated by reference in its entirety.

[0209] Description of Reference Numerals

[0210] 10...Filter 18...Container

[0211] 20 ... substrate 26 ... surface

[0212] 28…Active group 30…Antibody

[0213] 30a... first antibody 30b... second antibody

[0214] 32…Avidin (biotin binding protein) 34…Biotin

[0215] 34a...1st biotin 34b...2nd biotin

[0216] 36...biotinylated antibody 36a...first biotinylated antibody

[0217] 36b…Second biotinylated antibody

Claims

1. A filter for capturing a separation target by passing a liquid containing cells or proteins as the separation target, the filter comprising: A substrate formed of porous polyurethane having a plurality of fine pores; The active group is fixed to the surface of the substrate and can specifically bind to the separation object.

2. The filter according to claim 1, wherein The active group includes an antibody or an aptamer that can specifically bind to an antigen possessed by the object to be separated.

3. The filter according to claim 2, wherein: The active group includes: a conjugate formed by the antibody or the aptamer and biotin; and a biotin-binding protein fixed to the substrate and capable of binding to the biotin.

4. The filter according to claim 2, wherein: The active group includes: a conjugate formed by the antibody or the aptamer and a first biotin; a second biotin fixed to the substrate; and a biotin-binding protein capable of binding to each of the first biotin and the second biotin.

5. The filter according to claim 4, wherein The second biotin is fixed to the substrate via a structural unit A derived from a compound A having an epoxy group and an ethylenically unsaturated group and a structural unit B derived from a diamine compound B bonded to a terminal of the structural unit A. The structural unit A is bound to the substrate, and the structural unit B is bound to the second biotin.

6. The filter according to claim 5, wherein The compound A is at least one selected from the group consisting of glycidyl acrylate, glycidyl methacrylate (GMA), 3,4-epoxycyclohexylmethyl acrylate, 3,4-epoxycyclohexylmethyl methacrylate, β-methylglycidyl acrylate, and β-methylglycidyl methacrylate.

7. The filter according to claim 5, wherein The compound B is at least one selected from the group consisting of ethylenediamine, trimethylenediamine, 1,2-diaminopropane, tetramethylenediamine, 1,3-diaminobutane, 2,3-diaminobutane, pentamethylenediamine, 2,4-diaminopentane, hexamethylenediamine (HMDA), octamethylenediamine, nonamethylenediamine, decamethylenediamine, undecamethylenediamine, dodecamethylenediamine, tridecamethylenediamine, octadecamethylenediamine, xylylenediamine, and phenylenediamine.

8. The filter according to claim 1, comprising a container, wherein the container houses a plurality of the substrates to which the active groups are fixed. In the container, the plurality of substrates are stacked from upstream to downstream of the flow of the liquid.

9. The filter according to claim 8, comprising a plurality of the containers arranged in a row from upstream to downstream of the flow of the liquid, The type of the active group fixed to the substrate is different for each container.

10. The filter according to claim 1, wherein The plurality of fine pores formed in the substrate are larger than the separation target object.

11. The filter according to claim 1, comprising another substrate formed of a porous member and different from the substrate, The plurality of fine pores formed in the other substrate are larger than the separation target object and smaller than a separation target object different from the separation target object.

12. A method for manufacturing a filter, comprising: (1) irradiating at least a portion of a surface of a substrate formed of a porous polyurethane having a plurality of micropores with plasma; (2) contacting the plasma-irradiated substrate with a compound A having an epoxy group and an ethylenically unsaturated group, thereby bonding the compound A to the substrate; (3) contacting the diamine compound B with the substrate after contacting the compound A, thereby bonding the diamine compound B to the surface of the substrate via the structural unit A derived from the compound A; and (4) biotin is brought into contact with the surface of the substrate after contact with the diamine compound B, thereby binding biotin to the surface of the substrate via the structural unit A derived from the compound A and the diamine compound B.

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