JP2014224710A - Immunoassay carrier having fine rugged surface - Google Patents

Abstract

PROBLEM TO BE SOLVED: To evenly provide a substance immobilizing carrier having a hydrophilic polymer layer, capable of realizing high detection sensitivity in immunoassay.SOLUTION: A substance immobilizing carrier for use in immunoassay includes: a base substance having a plastic surface; and a hydrophilic polymer layer arranged on the plastic surface of the base substance. The hydrophilic polymer layer includes a hydrophilic polymer chain having an active reactive group at least on one end. The arithmetic mean roughness of the hydrophilic polymer layer is 10 nm or more. The thickness of the hydrophilic polymer layer is 20 nm or more.

Description

  The present invention relates to a solid phase carrier for use in an immunoassay.

  Enzyme-linked immunosorbent assay (hereinafter referred to as ELISA), which is a kind of immunoassay, is used in a wide range of fields including drug discovery, diagnosis, environmental measurement, and food. A typical ELISA includes (1) antibody or antigen immobilization (solid phase), (2) binding of a target substance, (3) binding of an enzyme-labeled antibody, (4) enzymatic reaction, (5) optical detection. It consists of five steps (absorption, fluorescence, luminescence). Horseradish peroxidase or alkaline phosphatase is used as the labeling enzyme. ELISA is particularly sensitive among many immunoassays. However, in reality, the expected sensitivity is often not obtained due to nonspecific adsorption of biomolecules to the solid support. In particular, a complex such as an enzyme-labeled antibody is easily adsorbed on a solid support, which is a main cause of noise in ELISA. In order to prevent such non-specific adsorption, blocking treatment with bovine serum albumin (BSA) is attempted, but the effect is limited. Thus, attention has been focused on hydrophilic polymers such as polyethylene glycol (hereinafter PEG) that effectively prevent non-specific adsorption of biomolecules. That is, attempts have been made to prevent nonspecific adsorption of biomolecules to a solid support by immobilizing an antibody or antigen via a hydrophilic polymer chain.

  Patent Document 1 discloses a method of immobilizing a nucleic acid or protein on a glass surface via a PEG linker. Specifically, a silane compound having a PEG linker is synthesized, applied to a glass surface, and then a nucleic acid or protein is bonded to the end of the PEG linker. It is described that this method improves the S / N ratio (sensitivity) of a biochip.

  In general, plastics are preferably used as a carrier for bioassay because they are easy to mold and have the advantages of small transportation and disposal problems. Patent Document 2 describes a multiwell plate for embryoid body formation that simultaneously forms a plurality of embryoid bodies of uniform size from ES cells, and 96 holes of polystyrene resin are used for the production of the multiwell plate. A technique is disclosed in which oxygen plasma treatment is performed on a multi-well plate to impart wettability to the surface of the multi-well plate and a water-soluble polymer such as polyvinyl alcohol is coated. However, the water-soluble polymer coating obtained by this technique is insufficient to suppress nonspecific adsorption of biological substances such as proteins.

  Patent Document 3 describes a substance immobilization carrier for use in an immunoassay having a hydrophilic polymer layer containing a PEG chain having a functional group capable of forming a covalent bond with a substance to be immobilized on the surface. ing. However, the surface roughness and layer thickness of the hydrophilic polymer layer are not described.

JP 2006-143715 A JP 2010-94045 A JP2013-11479A

  In the case of producing a carrier for immunoassay by arranging a hydrophilic polymer layer on the plastic surface, the present inventors also use the same plastic material as the raw material even when the hydrophilic polymer layer is arranged by the same procedure. Even when a plastic substrate sold under the product name is used, the degree of surface treatment of the plastic substrate varies depending on the production lot of the plastic substrate, and as a result, the unevenness of the hydrophilic polymer layer varies. The present inventors have found that there is a problem that the S / N ratio in the immunoassay after the hydrophilic polymer layer is arranged varies.

  The present invention has been made in view of such circumstances, and provides a substance immobilization carrier having a hydrophilic polymer layer that stably realizes high detection sensitivity without variation in an immunoassay such as ELISA. Objective.

  The inventors of the present invention have provided a hydrophilic polymer having a specific arithmetic average roughness and thickness, the hydrophilic polymer layer including a hydrophilic polymer chain having an active reactive group at the end disposed on a plastic surface, having minute irregularities. It has been found that the layer achieves an excellent S / N ratio in immunoassays and provides high sensitivity.

That is, the present invention includes the following inventions.
(1) A substance immobilization carrier for use in an immunoassay,
A substrate having a plastic surface, and a hydrophilic polymer layer disposed on the plastic surface of the substrate,
The hydrophilic polymer layer comprises a hydrophilic polymer chain having an active reactive group at least at one end;
The arithmetic average roughness of the hydrophilic polymer layer is 10 nm or more,
The thickness of the hydrophilic polymer layer is 20 nm or more,
The substance immobilizing carrier.
(2) The substance immobilization carrier according to (1), wherein the hydrophilic polymer chain is a polyalkylene glycol chain.
(3) The carrier for immobilizing a substance according to (1) or (2), wherein the active reactive group is a (1H-imidazol-1-yl) carbonyl group.
(4) The substance immobilization carrier according to any one of (1) to (3), wherein the water contact angle of the plastic surface of the substrate is 65 ° or less.
(5) A method for producing a substance immobilization carrier for use in an immunoassay,
Preparing a substrate having a plastic surface;
Disposing a hydrophilic polymer layer including a hydrophilic polymer chain having an active reactive group at least at one end on the plastic surface of the substrate;
Measuring the arithmetic mean roughness of the hydrophilic polymer layer,
A step of measuring the thickness of the hydrophilic polymer layer, and a step of selecting a carrier for immobilizing a substance having an arithmetic average roughness of the hydrophilic polymer layer of 10 nm or more and a thickness of the hydrophilic polymer layer of 20 nm or more. Including said method.

  By performing an immunoassay using the substance immobilization carrier of the present invention, high detection sensitivity can be stably realized without variation.

FIG. 1 shows an AFM image of a substance-immobilizing support produced in the example, arithmetic mean roughness (Ra), root mean square roughness (Rq), and layer thickness (hydrophilic polymer layer thickness) of the hydrophilic polymer layer. ) And the S / N ratio in the immunoassay for each substrate used for the production of the substance immobilization carrier.

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

  The substrate has a plastic surface. Plastic surface refers to a surface containing plastic, preferably a surface made of plastic. When the substrate is plate-like or substantially plate-like such as a multiwell plate, it is preferable that at least the surface on the side where the hydrophilic polymer layer is disposed is a plastic surface, and it is more preferable that the surface is made of plastic. Most preferably, the entire substrate is made of plastic.

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

  The plastic surface on which the hydrophilic polymer layer is disposed preferably has a water contact angle of 65 ° or less, and more preferably has a water contact angle of 40 to 55 °. By disposing a hydrophilic polymer layer on a plastic surface having a water contact angle in this range, an excellent S / N ratio can be achieved in an immunoassay, and a highly sensitive assay can be realized. In the present invention, the water contact angle refers to a water contact angle measured at 23 ° C.

(Hydrophilic polymer layer)
A hydrophilic polymer layer is disposed on the plastic surface of the substrate. The hydrophilic polymer layer includes a hydrophilic polymer chain having an active reactive group at at least one end. The hydrophilic polymer chain refers to a polymer containing a carbon component and having a hydrophilic functional group in the main chain or side chain of the polymer. The hydrophilic polymer chain is preferably a water-soluble polymer having a carbon-oxygen bond and having water solubility and water swellability. The hydrophilic polymer may be constantly water-soluble or water-swellable, or may be water-soluble or water-swellable by a predetermined stimulus such as light, temperature, and pH. From the viewpoint of use in an immunoassay, it is preferable to employ one having low biotoxicity.

Specific examples of the hydrophilic polymer chain include a polyalkylene glycol chain, a polyvinylpyrrolidone chain, a polypropylene glycol chain, a polyvinyl alcohol chain, a polyethyleneimine chain, a polyallylamine chain, a polyvinylamine chain, a polyvinyl acetate chain, a polyacrylic acid chain, a poly Examples thereof include hydrogel polymer chains such as (meth) acrylamide chains and poly-N-isopropylacrylamide chains, copolymers of these with other monomers, and graft polymers. Among these, polyalkylene glycol chains are excellent in biocompatibility and can be preferably used. The polyalkylene glycol chain has the following formula:
_{- ((CH 2) n -O} ) m -
(N represents the carbon number of the alkylene chain, m is an integer indicating the degree of polymerization)
Refers to the structure represented by n is an integer of 1-10 normally, Preferably it is an integer of 1-4. m is preferably 4 or more. The number average molecular weight of the polyalkylene glycol chain is preferably 25000 or less, more preferably 10,000 or less, and even more preferably 1000 or less. This is because when the number average molecular weight is increased, the viscosity is increased, so that handling is difficult and arrangement at a high density becomes difficult. Among the polyalkylene glycol chains, the following formula:
_{- (CH 2 -CH 2 -O)} m -
(M is as defined above, and is an integer indicating the degree of polymerization)
A polyethylene glycol chain (PEG chain) represented by

  It is preferable that at least one active reactive group capable of forming a covalent bond with the substance to be immobilized is directly or indirectly linked to at least one end of the hydrophilic polymer chain. As such an active reactive group, a (1H-imidazol-1-yl) carbonyl group, a succinimidyloxycarbonyl group, an epoxy group, an aldehyde group, which can form a covalent bond with an immobilized substance, Amino group, thiol group, carboxyl group, azide group, cyano group, active ester group (1H-benzotriazol-1-yloxycarbonyl group, pentafluorophenyloxycarbonyl group, paranitrophenyloxycarbonyl group, etc.), carbonyl halide Group (carbonyl chloride group, carbonyl fluoride group, carbonyl bromide group, carbonyl iodide group) and the like. These active reactive groups may be directly linked to the hydrophilic polymer chain or bonded to the end of the hydrophilic polymer chain as a substituent for replacing the hydrogen of the hydroxyl group at the end of the hydrophilic polymer chain. The active reactive group bonded to the linker structure may be indirectly linked. In consideration of the balance between the reactivity to the substance to be immobilized and the storage stability, a (1H-imidazol-1-yl) carbonyl group and a succinimidyloxycarbonyl group are preferable. These active reactive groups can react with a functional group such as an amino group of the substance to be immobilized to form a covalent bond. The hydrophilic polymer layer may further contain a hydrophilic polymer chain to which no active reactive group is added or other hydrophilic compounds.

  The hydrophilic polymer layer can be immobilized on the substrate by covalent bonding with a functional group chemically or physically immobilized on the substrate surface. For example, the hydrophilic polymer layer can be disposed on the plastic surface by introducing a functional group onto the plastic surface of the substrate using a primer layer and linking a hydrophilic polymer chain to the functional group.

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

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

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

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

(Arrangement of hydrophilic polymer layer)
A hydrophilic polymer chain is arranged by reacting a hydrophilic polymer on the plastic surface into which a functional group has been introduced by a primer layer or the like. At this time, a hydrophilic polymer containing a catalytic amount of concentrated sulfuric acid is brought into contact. Here, when polyethylene glycol (PEG) having a number average molecular weight exceeding 1000 is used as the hydrophilic polymer, it is previously melted by heating. If necessary, PEG may be diluted with tert-butyl alcohol or the like. The PEG solution is brought into contact with the plastic surface and heated. The heating temperature can be set in the range of 60 to 100 ° C., but preferably around 80 ° C. (75 ° C. to 85 ° C.) considering the heat resistance of the plastic. The heating time can be set in the range of 10 minutes to 24 hours, but when the heating temperature is around 80 ° C., 10 to 60 minutes is preferable. Through the above operation, the PEG chain is covalently bonded to the primer layer. At this time, the binding density of the PEG chain depends on the coating density of the primer layer.

  Finally, at least one active reactive group capable of forming a covalent bond with the substance to be immobilized is directly or indirectly linked to one end of the hydrophilic polymer chain. The method for introducing the active reactive group is not particularly limited. A preferred embodiment of the method for introducing the (1H-imidazol-1-yl) carbonyl group and the succinimidyloxycarbonyl group as a substituent for substituting the hydrogen of the hydroxyl group at the end of the PEG chain is as follows. 1,1′-carbonyldiimidazole (hereinafter referred to as CDI) or dicarbonate (N-succinimidyl) (hereinafter referred to as DSC) is reacted as follows on the other end of the PEG chain, one end of which is fixed to the substrate surface via the primer layer. Let

  The above reaction needs to be carried out in an organic solvent containing little water. Since plastics generally have low resistance to organic solvents, it is preferable to use acetonitrile, dimethyl sulfoxide, or a mixed solvent obtained by mixing these organic solvents in an appropriate ratio. The water content of these organic solvents is desirably 0.1% by weight or less. The final concentration of CDI or DSC can be set in the range of 0.01 to 1M. However, when the reaction is performed at room temperature or lower, it is preferably 0.1M or higher. In order to avoid damage to the plastic, it is desirable to set the reaction temperature in the range of 4 to 25 ° C. The reaction time is preferably set in the range of 10 minutes to 24 hours, and preferably 10 minutes to 60 minutes when the CDI concentration is around 0.5 M (0.4 to 0.6 M). By the above operation, a hydrophilic polymer layer containing a PEG chain to which an active reactive group is covalently bonded is disposed.

  The present inventors have an arithmetic average roughness (Ra) of 10 nm or more of a hydrophilic polymer layer including a hydrophilic polymer chain having an active reactive group at least at one end disposed on the plastic surface as described above. It has been found that when the thickness of the hydrophilic polymer layer is 20 nm or more, an excellent S / N ratio can be obtained in an immunoassay and high detection sensitivity can be realized. Therefore, by measuring the arithmetic average roughness and thickness of the hydrophilic polymer layer in the carrier for immobilizing a substance obtained by arranging the hydrophilic polymer layer on the plastic surface of the substrate, and selecting the one that satisfies the above range Thus, an excellent substance-immobilizing carrier for use in an immunoassay can be obtained without being influenced by variations inherent to the substrate to be used.

  The arithmetic average roughness of the hydrophilic polymer layer is 10 nm or more, preferably 15 nm or more, more preferably 17.5 nm or more, preferably 22 nm or less, more preferably 19.5 nm or less. The arithmetic average roughness (Ra) refers to a value measured by an atomic force microscope (AFM). More specifically, the arithmetic average roughness (Ra) defined by JIS B0601 '2001 is indicated.

  The hydrophilic polymer layer also has a root mean square roughness (Rq) of 12 nm or more, preferably 20 nm or more, more preferably 23 nm or more, preferably 35 nm or less, more preferably 25 nm or less. The root mean square roughness (Rq) is also a value measured by an atomic force microscope (AFM), and more specifically, the root mean square roughness (Rq) defined by JIS B0601 '2001.

  Non-specific adsorption of a substance by selecting a carrier for immobilizing a substance on which a hydrophilic polymer layer having an arithmetic average roughness or a root mean square roughness in such a range is disposed on a substrate. In particular, nonspecific adsorption of proteins can be suppressed, and an excellent S / N ratio, that is, excellent sensitivity can be obtained in an immunoassay.

(Immobilized substance)
The thickness of the hydrophilic polymer layer is 20 nm or more, preferably 25 nm or more, preferably 35 nm or less, more preferably 30 nm or less. The thickness (layer thickness) of the hydrophilic polymer layer refers to a value measured using a spectroscopic ellipsometer (manufactured by JOBIN YVON, UVISEL).

  In the substance immobilization carrier of the present invention, an antigen or antibody that binds to a target substance or a target substance is immobilized in accordance with the target immunoassay. In the present invention, these target substances for immobilization are called “substances to be immobilized”. In an embodiment where the “antigen or antibody that binds to a target substance” is an immobilized substance (sandwich method or direct competition method described later), the combination of the immobilized substance and the target substance is specific based on an antigen-antibody reaction. The combination is not particularly limited as long as it is a combination that can be easily combined. For example, when the target substance is an antigen (including a hapten), the immobilized substance can be an antibody (including an antibody fragment), and when the target substance is an antibody (including an antibody fragment) The immobilized substance can be an antigen (including a hapten). In an embodiment where the “target substance” is an immobilized substance (indirect competition method described later), the target substance is an antigen (including a hapten) or an antibody (including an antibody fragment).

  The antigen as the immobilized substance and / or the target substance is not particularly limited as long as it is a substance exhibiting specific antigen-antibody reactivity with an antibody. Typical antigens include natural antigens such as proteins, peptides, saccharides, nucleic acids (DNA, RNA), lipids, coenzymes, cells, viruses, bacteria, and complexes of these, derivatives of natural antigens, and artificial synthesis. Hapten, artificial antigen and the like.

  The substance to be immobilized is not particularly limited as long as it has a functional group capable of reacting with an active reactive group of the hydrophilic polymer chain to form a covalent bond. Such functional groups typically include amino groups, but also include thiol groups, carboxyl groups, hydroxyl groups, alkoxides, secondary amines, tertiary amines, azide groups, cyano groups, and the like. Even if the substance to be immobilized does not have a functional group capable of forming a covalent bond by a reaction with a functional group linked to a hydrophilic polymer chain, such as an amino group, these substances have It can be used for immobilization by artificially introducing an amino group or the like.

(Immobilization of substances to be immobilized)
For example, the substance to be immobilized can be immobilized by a method known in the art. For example, the substance-immobilizing carrier of the present invention is brought into contact with a solution in which the substance to be immobilized is dissolved, and the solution is dried and concentrated to share the substance to be immobilized on the hydrophilic polymer chain end of the substance-immobilizing carrier. Combine. If necessary, the reactive active group at the end of the unreacted hydrophilic polymer chain is brought into contact with the amino group-containing low molecular weight compound to inactivate the reactive active group at the end of the hydrophilic polymer chain.

  Examples of the low molecular weight compound containing an amino group include 2-aminoethanol and 2- (2-aminoethoxy) ethanol, and 2- (2-aminoethoxy) ethanol is particularly preferable.

(Immunoassay)
An immunoassay using the carrier for immobilizing a substance of the present invention can be performed by a method known in the art. Specifically, the substance immobilization carrier of the present invention and the sample to be measured are immobilized on the substance to be immobilized on the carrier, in an amount of antigen or antibody (corresponding to the amount of the target substance in the sample to be measured). Here, the antigen or antibody refers to a target substance in a sample to be measured, a detectable target substance added to the antigen-antibody reaction system as necessary, a detectable antigen added to the antigen-antibody reaction system as necessary, or (Referring to a detectable antibody or the like) includes a step of contacting under conditions that bind by an antigen-antibody reaction, and a step of detecting the antigen or antibody bound to the immobilized substance in the step. The immunoassay can be in any mode such as a sandwich format, a direct competition format, an indirect competition format, and the like.

  The means for detecting the antigen or antibody in the immunoassay using the substance immobilizing carrier of the present invention is not particularly limited, and an antigen or antibody directly or indirectly labeled with any labeling substance can be used. As a labeling substance, an amplified detection signal such as an enzyme (ELISA method), a nucleic acid (immunoPCR), an electrochemiluminescent substance (electrochemiluminescent method), a fluorescent substance, a chemiluminescent substance, a radioactive substance, etc. can be generated. Possible labeling substances are mentioned. In view of safety and convenience, the immunoassay using the carrier of the present invention is preferably an enzyme-linked immunosorbent assay (ELISA) that uses an enzyme as a labeling substance and performs detection based on enzyme activity. The enzyme that can be used as the labeling substance is not particularly limited, and examples thereof include peroxidase such as horseradish peroxidase, β-galactosidase, alkaline phosphatase, and glucose oxidase. Examples of detection methods using enzyme activity include detection methods using chemiluminescent substrates that chemiluminescent by enzyme activity, chromogenic substrates that generate color by enzyme activity, chemiluminescent substrates that emit chemical fluorescence by enzyme activity, etc. A detection method using a luminescent substrate is preferred. Since detection using a chemiluminescent substrate is generally more sensitive than detection using a chromogenic substrate, the time required for the enzyme reaction can be shortened. In particular, if a highly sensitive chemiluminescent substrate used for detection of a trace amount of target substance at the femtogram level is used, a synergistic effect can be obtained in terms of sensitivity. Combining the substance-immobilizing carrier of the present invention with a chemiluminescent substrate can achieve an ELISA that is rapid, highly sensitive, and has a wide dynamic range.

(Immunoassay kit)
The substance immobilization carrier of the present invention can constitute a kit for measuring a target substance by immunoassay. The kit can further comprise components necessary for the immunoassay. Depending on the type of immunoassay, the components required for the immunoassay include antigens or antibodies that can be detected directly or indirectly and bind to the target substance bound to the immobilized substance by the antigen-antibody reaction. Examples thereof include directly or indirectly detectable antigens or antibodies (including directly or indirectly detectable target substances) that bind to an immobilized substance by an antigen-antibody reaction. Furthermore, components necessary for the immunoassay include a labeling substance capable of binding to the indirectly detectable antigen or antibody for use in indirect detection, and a detection reagent (for example, when the labeling substance is an enzyme). Include a chemiluminescent substrate that chemiluminescents by enzyme activity, a chromogenic substrate that develops color by enzyme activity, a chemiluminescent substrate that emits chemical fluorescence by enzyme activity, and the like. The kit further includes a reagent used for immobilizing the substance to be immobilized on the substance immobilization carrier (for example, a buffer solution for dissolving the substance to be immobilized, in which sugars and nonionic surfactants are dissolved). ) May be included.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the scope of the examples.

(Example 1) Production of carrier for immobilizing a substance for immunoassay 3.6 g of GPTS (3-glycidoxypropyltrimethoxysilane; manufactured by Momentive Performance Materials) was taken, and 700 μl of diluted hydrochloric acid (pH 2. Add 4) and stir for 5 minutes. This solution is added to 156 g (200 ml) of 2-propanol (Pure Chemical), and then 1000 μl of triethylamine (Wako Pure Chemical Industries) is added. 100 μl of the above solution was added to each polystyrene 96-well plate, and the reaction was allowed to stand for 2 hours and 15 minutes. The following four types were used as 96-well plates.

Polysorb (registered trademark) (Thermo Scientific)
Nunclon (registered trademark) (Thermo Scientific)
BD351172 (registered trademark) (BD Biosciences)
BD353075 (registered trademark) (BD Biosciences)
Polysorb (registered trademark) is a polystyrene plate subjected to hydrophobic treatment called polysoap processing, Nunclon (registered trademark) is a polystyrene plate subjected to corona surface treatment, and BD1172 (registered trademark) is untreated. BD353075 (registered trademark) is a polystyrene plate subjected to plasma treatment.

  When the water contact angle before reaction of each 96-well plate was measured, it was as follows.

  After the reaction, the above solution was removed and washed with 100 μl of pure water, and the plate was heated in an oven at 80 ° C. for 15 to 60 minutes and dried. Next, 100 μl of PEG 400 containing a catalytic amount of concentrated sulfuric acid was dispensed into each well, reacted in an oven at 80 ° C. for 45 minutes, and then washed with 100 μl of pure water. Next, a CDI (Tokyo Kasei) solution with a final concentration of 0.5 M is prepared using an equal weight mixed solvent of dehydrated acetonitrile (Kanto Chemical) and dehydrated dimethyl sulfoxide (Kanto Chemical), and 10 μl is dispensed into each well. did. It was allowed to stand at room temperature for 20 minutes. Thereafter, the inside of the well was washed with pure water, heated in an oven at 80 ° C. for 15 minutes, and dried.

(Example 2) Ra measurement of hydrophilic polymer layer surface The arithmetic average roughness of the hydrophilic polymer (PEG) layer was measured using a Dimension 3000 stage system using a Digital Scope Nanoscope IIIa as an atomic force microscope. The measurement was performed under the following conditions.
・ Scanning mode: Tapping mode ・ Probe: Silicon cantilever ・ Scanning range: 10 μm × 10 μm
・ Scanning speed: 0.979Hz
-Number of pixels: 512 × 512
・ Measurement environment: Room temperature, in air

  Similarly, the root mean square roughness (Rq) was also measured by an atomic force microscope (AFM). More specifically, the measurement was performed under the conditions defined in JIS B0601 '2001.

  The results are shown in FIG.

(Example 3) Thickness measurement of hydrophilic polymer layer The thickness (layer thickness) of the hydrophilic polymer (PEG) layer was measured using UVISEL manufactured by JOBIN YVON as a spectroscopic ellipsometer, and the measurement angle was 75 °. The spectrum range was measured every 0.05 eV in the range of 1.5 to 6.5 eV.

  The measurement was performed twice. The results are shown in FIG.

Example 4 Immunoassay Evaluation Anti-BSA antibody was added to 10 mM acetate buffer (pH 4.8) (final concentration 5 μg / ml). A lysozyme solution was added to 10 mM phosphate buffer (pH 8.0) (final concentration 5 μg / ml). 50 μl of anti-BSA antibody solution (1-6 rows) and 50 μl of lysozyme solution (7-12 rows) were dispensed into each 96-well plate prepared in Example 1 and allowed to stand for 10 minutes. Thereafter, the buffer in the well was removed, 50 μl of a phosphate buffer solution (PBS) adjusted to a final concentration of 10 mM diglycolamine was added, the mixture was allowed to stand for 10 minutes, and then the buffer in the well was removed. Next, 50 μl of a phosphate buffer solution prepared so that the final concentration of the HRP-labeled anti-IgG antibody was 1 μg / ml was added to each well and allowed to stand for 10 minutes. Thereafter, all the buffer in the well was removed, 200 μl of 0.05% Triton (registered trademark) X-100 phosphate buffer solution was added, and the mixture was allowed to stand for 5 minutes, and then all the buffer was removed again. 50 μl of 0.1 M phosphate buffer (pH 6.0) containing SAT-3 (Dojindo Chemical) (final concentration 1 mM) and hydrogen peroxide (final concentration 0.5 mM) was added to each well and allowed to stand for 10 minutes. 100 μl of 0.5 M sulfuric acid aqueous solution was added. Thereafter, the absorbance (474 nm) was measured with a microplate reader.

  A value obtained by dividing the absorbance of the system containing the anti-BSA antibody solution by the absorbance of the system containing the lysozyme solution was calculated as the S / N ratio. The results are shown in FIG.

  As a result of disposing a hydrophilic polymer layer containing a PEG chain having (1H-imidazol-1-yl) carbonyl group by the same treatment on four kinds of polystyrene 96-well plates, the arithmetic average roughness of the hydrophilic polymer layer It was shown that the S / N ratio was different depending on the thickness. That is, by selecting a carrier having an arithmetic average roughness and thickness within a specific range, an excellent sensitivity with an S / N ratio of 20 or more and further an S / N ratio of 30 or more can be obtained in an immunoassay. It has been shown. It has also been shown that selecting a substrate having a specific range of water contact as the substrate also affects the S / N ratio after forming the hydrophilic polymer layer.

  Therefore, it has been shown that the present invention can stably provide a carrier having excellent sensitivity in an immunoassay without being affected by variations among substrates.

Claims (5)

A carrier for immobilizing a substance for use in an immunoassay,
A substrate having a plastic surface, and a hydrophilic polymer layer disposed on the plastic surface of the substrate,
The hydrophilic polymer layer comprises a hydrophilic polymer chain having an active reactive group at least at one end;
The arithmetic average roughness of the hydrophilic polymer layer is 10 nm or more,
The thickness of the hydrophilic polymer layer is 20 nm or more,
The substance immobilizing carrier.   The substance-immobilizing carrier according to claim 1, wherein the hydrophilic polymer chain is a polyalkylene glycol chain.   The carrier for immobilizing a substance according to claim 1 or 2, wherein the active reactive group is a (1H-imidazol-1-yl) carbonyl group.   The carrier for immobilizing a substance according to any one of claims 1 to 3, wherein the water contact angle of the plastic surface of the substrate is 65 ° or less. A method for producing a substance immobilization carrier for use in an immunoassay, comprising:
Preparing a substrate having a plastic surface;
Disposing a hydrophilic polymer layer including a hydrophilic polymer chain having an active reactive group at least at one end on the plastic surface of the substrate;
Measuring the arithmetic mean roughness of the hydrophilic polymer layer,
A step of measuring the thickness of the hydrophilic polymer layer, and a step of selecting a carrier for immobilizing a substance having an arithmetic average roughness of the hydrophilic polymer layer of 10 nm or more and a thickness of the hydrophilic polymer layer of 20 nm or more. Including said method.

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