JP5148818B2 - New solid support and use thereof - Google Patents

Description

  The present invention relates to a biological substance, a compound capable of binding to the biological substance and / or a support, and a solid phase carrier on which a matrix containing the support is formed, a method for producing the solid phase carrier, and use thereof About.

Solid-phase carriers on which biological substances are immobilized are, for example, in the fields of medical / diagnosis, gene analysis, proteomics, microelectronics, membrane separation, etc., particularly in the fields of biochips such as DNA chips and protein chips, and biosensor chips. Has been applied to.
There have been several reports so far on a method for producing a solid phase carrier in which such a biological substance is immobilized, that is, a method for immobilizing a biological substance on the surface of the solid phase carrier.

  For example, when immobilizing a biological substance on a solid phase carrier, first, the surface of the solid phase carrier is coated with a hydrophilic polymer compound to form a polymer film on the surface of the solid phase carrier, and the polymer membrane is configured. There is a method of binding a biological substance to be a ligand or the like to a polymer chain. Thus, from the advantage that the introduction amount (immobilization amount) of the biological substance per unit area can be improved as compared with the case where the solid phase carrier surface is not coated with the hydrophilic polymer compound, this method is It is applied to a wide range of fields.

  For example, Patent Document 1 describes a coating method using a hydrophilic polymer film into which a functional group having a charge is introduced. In the method of immobilizing a biological substance such as a ligand in the polymer membrane on the solid phase carrier as described above, it is difficult to immobilize the biological substance in a high density due to the penetration of the biological substance into the polymer membrane. there were. However, in the method described in Patent Document 1, the pH of the solution is controlled by electrostatic interaction so that the surface charge of the biological substance becomes the opposite charge of the functional group having the charge of the polymer film. The immobilization reaction of the related substance to the polymer membrane can be promoted, and the bio-related substance can be immobilized at a high density.

As a product using the method described in Patent Document 1, a glass plate coated with gold and surface-treated with a CM-dextran film is commercially available (SensorChip CM5 manufactured by BIACORE).
In addition, a slide glass, which is a solid support, coated with a polyacrylamide film is also commercially available (HydrGel Coated Slide manufactured by PerkinElmer). This product uses a swollen polyacrylamide gel that contains water, so it is suitable for dripping nanoliter-order samples of which drying is a concern, and it is activated to absorb bio-related substances on polyacrylamide. Has the advantage of eliminating the need for

  Furthermore, for example, in Patent Document 2, a polyurethane-based polymer and a biological substance are mixed in an organic solvent, and the polymer is polymerized by mixing a condensing agent, and then bonded to the surface of the substrate. Is described. This method does not use a conventional complicated method, and can easily form a membrane containing a biological substance on the surface of the solid phase carrier, so that the biological substance can be easily immobilized on the solid phase carrier. Has the advantage of being able to

  Also, for example, in Patent Document 3, a polymer chain having an active ester group is polymerized on a solid phase carrier to extend the polymer chain, thereby constructing a brush-like polymer chain on the solid phase carrier. And a method for binding a biological substance to the polymer chain. According to this method, in addition to immobilization of a biological substance such as a ligand, it becomes possible to introduce (immobilize) on a solid phase carrier without activating the polymer chain.

As described above, the conventional technique of constructing a hydrophilic polymer film on a solid support has individual advantages.
On the other hand, for example, in Non-Patent Document 1, in immobilizing a biological substance on a solid phase carrier, by using a solid phase carrier (microchannel wafer) having a porous shape in advance, by increasing the surface area, A method for binding more biologically related substances is described. According to this method, this method can be used in a wide range as described above from the advantage that the introduction amount (immobilization amount) of the biological substance per unit area can be improved as compared with the case where the solid support surface is flat. Applied to various fields.
US Pat. No. 5,242,828 US Pat. No. 6,174,683 International Publication No. 02/056021 Pamphlet Brandy J. Cheek, et al., Anal. Chem., 2001, 73, 5777-5783

  However, in the conventional techniques described in Patent Documents 1 to 3 and Non-Patent Document 1 described above, the introduction amount of the biological substance that can be immobilized on the solid phase carrier is not sufficient, immobilization unevenness can occur, and accuracy is increased. The immobilization could not be performed well and sufficient performance could not be obtained in biosensors. In addition, an expensive and special solid phase carrier is necessary for immobilization, and a special instrument and immobilization method are necessary for immobilization.

  In particular, when antigen and antibody, protein and ligand, etc. are used as a combination of a biological substance and a substance to be detected, the antigen-antibody reaction that occurs between them is a weak biological reaction. Not only is sufficient, but also the biological activity of the immobilized substance must be maintained and exposed to the surface so that it can participate in the reaction. However, with a solid phase carrier that has been used in the past, even if a large amount of biological material is immobilized and a sufficient film thickness can be secured, the reactivity of the biological material contained in the membrane is sufficient. In addition, it was not applicable to biosensors and diagnostic devices that require detection of weak reactions such as the antigen-antibody reaction.

For this reason, there has been a demand for a technique that can increase the amount of a biological substance to be introduced onto a solid phase carrier without impairing the reactivity (activity) of the biological substance and can accurately produce a solid phase carrier at low cost.
The present invention was devised in view of the above problems, and a solid phase in which a larger amount of a biological substance is more accurately immobilized on a solid phase carrier while maintaining the reactivity of the biological substance. An object of the present invention is to provide a carrier that is inexpensive and can be easily produced. Another object of the present invention is to provide a method for producing the solid phase carrier, a biosensor including the solid phase carrier, a diagnostic device, a biological substance immobilization kit, and a measurement method such as an immunoassay using the solid phase carrier. .

  As a result of intensive studies to solve the above problems, the present inventors have found a mixture in which a support, a biological substance, and a compound capable of binding to the biological substance and / or the support coexist in the presence of a solvent. If a matrix is formed by removing the solvent after supplying to the surface of the solid phase carrier, a solid substance in which a larger amount of the bio-related substance is more accurately immobilized than in the past while maintaining the reactivity of the bio-related substance. The present inventors have found that a phase carrier can be obtained and completed the present invention.

That is, according to the present invention,
(1) biological substance, immobilization compound, a solid phase carrier, wherein a matrix containing及beauty supporting bearing member is formed on the surface, biological-related substance, the detection in a sample A target substance for measuring an interaction between a target substance and a biological substance, and the immobilizing compound includes one or more binding functional groups capable of binding to the biological substance in one molecule , and one place have more of said support capable of binding binding functionality, and, a first immobilized compound Ru der polymer compounds, capable of binding bind to two or more locations of the biological-related substance in a molecule And a second immobilization compound having a functional group and a polymer compound , and the matrix has a main chain composed of the biological substance and the immobilization compound with the support as a nucleus. a structure, the first fixed to said support and biological-related substance Use compound takes a first crosslinked structure linked by the linking functional group, wherein the second immobilization compound takes a second crosslinked structure bonded by the bonding functional group with respect to biological-related substance A solid phase carrier having a chain-like structure and / or a network-like or block-like structure is provided by repeating the bridge structure comprising the first bridge structure and the second bridge structure .
According to a preferred aspect of the present invention ,
( 2 ) The solid phase carrier according to (1 ) above, wherein the matrix has voids,
( 3 ) The matrix is formed by supplying the mixture in which the support, the biological substance, and the immobilizing compound coexist in the presence of a solvent to the surface of the solid phase carrier, and then removing the solvent. The solid phase carrier according to (1) or (2) above, characterized in that
( 4 ) The solid phase carrier according to ( 3 ) above, wherein the solvent is water,
( 5 ) The solid phase carrier according to any one of (1) to ( 4 ) above, wherein the porosity of the matrix is 5% or more,
( 6 ) The solid phase carrier according to any one of (1) to ( 5 ) above, wherein the thickness of the matrix is 20 nm or more in a dry state,
( 7 ) The solid phase carrier according to any one of (1) to ( 6 ) above, wherein the immobilizing compound is miscible with water and miscible with at least one organic solvent. ,
( 8 ) The solid support according to any one of (1) to ( 7 ), wherein the support is particles having an average diameter of 10 nm to 100 μm.

According to another aspect of the present invention,
( 9) A method for producing the solid phase carrier according to any one of (1) to (8) above , wherein the support, the biological substance, and the immobilization compound coexist in the presence of a solvent. There is provided a method for producing a solid phase carrier , characterized in that a matrix is formed by removing the solvent after supplying the prepared mixture to the surface of the solid phase carrier. According to yet another aspect,
( 10 ) A biological material immobilization kit for producing the solid phase carrier according to any one of (1) to ( 8 ) above, comprising at least the support and the immobilizing compound. A kit featuring,
(11) biological substance, immobilization compounds, matrices containing及beauty supporting bearing member is at least two or more, of the biological substances, characterized by being arranged in separate regions on a solid support In the array, the biological substance is a target substance for measuring the interaction between the detection target substance and the biological substance in the specimen, and the immobilization compound is at least one place in one molecule. biological-related substance capable of binding binding functionality, and have a bondable functional group binding to one or more locations of the support, and a first immobilized compound Ru der polymer compound 1 molecule And a second immobilizing compound having a binding functional group capable of binding to the biological substance at two or more sites and being a polymer compound , the matrix having the support as a nucleus Structure having a main chain comprising a biological substance and the immobilizing compound , And the fixing said support and said first immobilized compound relative biological-related substance takes a first crosslinked structure linked by the linking functional groups, of the second with respect to biological-related substance A chemical compound is bonded by the binding functional group to form a second bridge structure, and a chain and / or a network by repeating the bridge structure composed of the first bridge structure and the second bridge structure , An array having a block-like structure ,
( 12 ) A biosensor comprising the solid phase carrier according to any one of (1) to ( 8 ) and / or the array according to (11) above,
( 13 ) A diagnostic device comprising the solid phase carrier according to any one of (1) to ( 8 ) and / or the array according to (11) above is provided. Also,
( 14 ) A method for assaying for at least one analyte in a sample, comprising the following steps (a) and (b):
(A) The solid phase carrier according to any one of (1) to ( 8 ) and / or the array according to (11) , wherein the specimen contains at least one kind of biological substance capable of reacting with the analyte. And (b) detecting the interaction between the biological substance and the analyte, or the reaction caused by the addition of a labeling substance that reacts with the analyte, A method comprising the step of detecting for an amount,
( 15 ) The method according to ( 14 ) above, wherein at least the specimen is a fluid, and the delivery of the specimen is performed by a flow,
( 16 ) The reaction in the solid phase carrier according to any one of (1) to ( 8 ) and / or the reference region provided on the array according to (11) above is detected, and the success or failure of the assay is determined. The method according to ( 14 ) or ( 15 ) above, further comprising a step of calibrating the presence or amount of the detected analyte,
( 17 ) In order to assay two or more analytes in parallel, the following step (c):
(C) The method according to any one of ( 14 ) to ( 16 ) above, which comprises the step of associating the presence or amount of at least two or more analytes with specific symptoms.

  According to the solid phase carrier and the method for producing the solid phase carrier of the present invention, a larger amount of biological material was immobilized on the solid phase carrier with higher accuracy than before while maintaining the reactivity of the biological material. A solid phase carrier that can be easily produced at low cost and has excellent storage stability is provided. As a result, it is possible to produce a highly sensitive diagnostic device or biosensor with higher accuracy than before, which can be used in the fields of medicine / diagnosis, genetic analysis, and proteomics.

Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the following embodiments and exemplifications, and can be arbitrarily modified without departing from the gist of the present invention. .
The solid phase carrier of the present invention is a solid phase carrier on which a biological substance is immobilized (hereinafter referred to as “the biological substance-fixing carrier of the present invention” as appropriate), and a matrix (hereinafter, appropriately, (Referred to as “matrix of the invention”). Here, the matrix of the present invention is a matrix comprising a biological substance, a compound capable of binding to the biological substance and / or the support (hereinafter referred to as “immobilization compound” as appropriate), and the support. That is, as schematically shown in FIG. 1, the matrix of the present invention includes a biological substance, an immobilizing compound and a support, and the skeleton of the matrix has the supporting substance as a nucleus and the biological substance and the immobilizing compound. And is a matrix having a structure in which it is bonded to a support and bonded in a chain, network, and / or block shape. In addition, FIG. 1 is a schematic diagram showing an enlarged cross section of an example of the biological substance-fixing carrier of the present invention in order to explain the structure of the matrix of the present invention. Moreover, in FIG. 1, the filled circular part represents the biological substance, the linear part represents the compound for immobilization, the white circular part represents the support, and the other blank part represents the void layer. .

Moreover, the array of the present invention is produced by arranging at least two kinds of the matrix of the present invention in separate regions on a solid phase carrier. The array comprises an array comprising at least about 10 the matrix, an array comprising at least about 100 the matrix, an array comprising at least about 10 ³ the matrix, an array comprising at least about 10 ⁴ the matrix. An array of biomaterial immobilization carriers covered by each of the matrices, wherein the area of the biomaterial immobilization carrier covered by each of the matrices is about 25 mm ² or less. Including an array wherein the area is between about 100 μm ² and about 4 mm ² , wherein the area of the biomaterial immobilization carrier covered by each of the matrices is contained within an area of about 20 cm ² or less.

  On the surface of such an array, the periphery of the separate regions to which the mixed solution is supplied may be a lyophobic (peripheral lyophobic) surface, and the region is a well-like shape that is a concave portion of a concavo-convex structure ( 2 (a)) or a pile shape (FIG. 2 (b)) which is a convex portion.

[I. Production method]
In the biological material-immobilized carrier of the present invention, in the presence of a solvent, a support, a biological material, and a mixture containing the biological material and / or a compound capable of binding to the substrate are supplied to the surface of the solid phase carrier. Then, it manufactures through the process of forming a matrix by removing this solvent.

(1. Solid phase carrier)
The solid phase carrier serves as a substrate for forming the matrix of the present invention on the surface, and the biomaterial-fixed carrier of the present invention is the one in which the matrix of the present invention is formed on the surface of the solid phase carrier. There is no restriction | limiting in the solid phase carrier used by this invention, The thing of arbitrary materials, a shape, and a dimension can be used if it becomes the object which forms the matrix of this invention.

  Examples of the material of the solid phase carrier include various resin materials such as polyolefin, polystyrene, polyethylene, polycarbonate, polyamide, and acrylic resin, and inorganic materials such as glass, alumina, carbon, and metal. In addition, the material of the solid phase carrier may be a single material used alone, or a combination of two or more materials in any combination and ratio. In addition, when a fluorescence method is used for measurement, a material suitable for the measurement method or apparatus to be used may be selected as appropriate, such as selecting a material with less autofluorescence.

  Examples of the shape of the solid phase carrier include flat plate shape, bead shape, fiber shape, filter shape, film shape, sheet shape, and well shape. Specific examples include a chip (substrate), a well chip (well substrate), a pile chip (pile substrate), a peripheral hydrophobic chip (peripheral hydrophobic substrate), a chromatography carrier, and a diagnosis that can arrange a large number of biological substances. Examples thereof include beads as a drug, and porous structures such as hollow fiber fibers and nitrocellulose membranes used for the purpose of increasing the surface area.

Further, the solid phase carrier may be used as it is, but a matrix may be formed on the surface after some surface treatment. For example, the matrix may be formed after the surface is coated with a coating material such as metal or metal oxide. Furthermore, a functional group may be introduced into the solid phase carrier in order to bond the solid phase carrier and the matrix. The functional group is arbitrary, but for example, a hydroxyl group, carboxyl group, thiol group, aminoaldehyde group, hydrazide group, carbonyl group, epoxy group, vinyl group, amino group, succinimide group, maleimide, etc. Examples include functional groups that bind the phase carrier and the matrix. In addition, when water is used as a solvent during the production of the solid body carrier of the biological material of the present invention, a functional group that binds the solid phase carrier to the matrix by physical adsorption by hydrophobic interaction such as an alkyl group or a phenyl group should be used. You can also.
Further, on these solid phase carrier surfaces, the surroundings of the separate known regions of the solid phase carrier surface to which the mixed solution is supplied may be made lyophobic (peripheral lyophobic) surfaces.

  As a specific example of the surface treatment, for example, when the surface treatment for coating with gold is performed on the surface of the solid phase carrier,

And the like are fixed to the gold surface. However, in the above structural formula, n ₁ and n ₂ each independently represents an integer of 2 or more.
Specific examples of the solid phase carrier that may be coated include metal-coated chips, glass slides, fiber slides, sheets, pins, microtiter plates, capillary tubes, beads, and the like.

(2. Support)
The support serves as a nucleus for forming the matrix of the present invention on the surface, and the biomaterial-fixing carrier of the present invention is formed by forming the matrix of the present invention on the surface of the solid support. There is no restriction | limiting in the support body used by this invention, The thing of arbitrary materials, a shape, and a dimension can be used if it becomes the object which forms the matrix of this invention.

  At this time, the bond between the biological substance, the support and the compound is formed in a chain, network, block or the like, and may have a plurality of these structures. For example, the compound formed by binding of the compound to the biological substance via a binding site is further bound to the support, and the compound is bound by binding to the support and the biological substance. Or those formed by binding a compound to a biological substance previously bound to a support. Accordingly, a matrix is formed by surrounding the support with the bio-related substance in a bridge structure by the compound, which further forms one or more aggregates.

  Examples of the material of the support include various resin materials such as polyolefin, polystyrene, polyethylene, polycarbonate, polyamide, and acrylic resin, and inorganic materials such as glass, alumina, carbon, and metal. In addition, the support may contain a substance known to cause fluorescence energy transfer (FRET), and may contain a conductive substance such as ferrite, graphite, or carbon nanotube, or an electromagnetic such as gold or silver. It may contain a metal known to give resonance. In addition, the material of the support may be one used alone, or two or more may be used in any combination and ratio.

Further, examples of the shape of the support include particulates and fibers. Moreover, any shape may be used as long as it provides a sufficient gap and film thickness to the formed matrix. Specific examples include latex carriers, latex colloids and gold colloids as chromatographic carriers and latex diagnostic agents.
Among these, latex particles are particularly preferably used. Latex particles can be arbitrarily selected according to the purpose of use, and the material, particle size, etc. are arbitrary. For example, polystyrene latex, styrene / divinylbenzene copolymer, acrylic acid / styrene copolymer, styrene / maleic acid copolymer, styrene / methacrylic acid copolymer, styrene / acrylic acid / alkyl acrylate, etc. Examples thereof include latex such as a copolymer and a copolymer of vinyl acetate and acrylic acid. The particle size may be any size as long as the matrix of the present invention is formed, but if the particle size is too small, sufficient voids and film thickness cannot be obtained, and if it is too large, sufficient film strength is obtained. Since this may cause problems such as being unable to be obtained, an appropriate one is selected according to the conditions used. For example, it is about 10 nm to 100 μm, preferably 100 nm to 10 μm. In addition, latex particles having different particle diameters can be mixed and used. By adjusting the conditions in this way, the porosity, film thickness, etc. of the formed matrix can be optimized.

  Further, the support may be used as it is, but may be used after some surface treatment. For example, the surface may be made hydrophilic with a hydrophilic polymer such as polyethylene glycol or a protein such as bovine serum albumin. By selecting an appropriate surface treatment, the degree of binding between the support and the biological substance and / or compound can be adjusted, and side reactions such as non-specific adsorption reaction to the support are suppressed. You can also.

  Moreover, in order to form the matrix firmly, a functional group may be introduced on the support surface. The functional group is arbitrary, for example, by a chemical bond such as hydroxyl group, carboxyl group, thiol group, aminoaldehyde group, hydrazide group, carbonyl group, epoxy group, vinyl group, amino group, succinimide group, maleimide group, etc. Examples include functional groups that form a matrix.

  Furthermore, a material in which a biological substance and / or an immobilizing compound are immobilized in advance may be used on the support. For example, when using particles in which a biological substance is immobilized on latex particles as a support, latex is used. A carboxyl group, an amino group, a hydroxyl group, a thiol group, or the like present on the particle surface may be bound to a binding functional group of the biological substance. Among these, when a carboxyl group is bonded to a binding functional group, specific examples of the binding functional group of the biological substance include an amino group and the like, and an amide bond may be formed using carbodiimide or the like. On the other hand, for example, when a lyophobic interaction or an electrostatic interaction exists between the latex particle and the biological substance, the biological substance can be fixed to the latex particle surface by these physical interactions. it can.

  The support is preferably uncharged. When the selective interaction between biological substances is detected using the biological substance-immobilized carrier of the present invention, the substance to be detected in the sample has the same charge as the support and / or the immobilizing compound. If the electrostatic repulsive force is excessively strong, the specific interaction with the biological substance may be hindered. In addition, when the detection target substance and the support and / or immobilization compound have opposite charges, the detection target substance and the support and / or immobilization compound are nonspecific such as nonspecific adsorption. This is because it is assumed that an interaction occurs.

(3. Bio-related substances)
The biological substance is a substance that is immobilized on a solid phase carrier, and any substance can be used depending on the purpose. Specific examples include enzymes, antibodies, lectins, receptors, protein A, protein G, protein A / G, avidin, streptavidin, neutravidin, glutathione-S-transferase, glycoproteins, peptides, amino acids, hormones , Nucleic acids, sugars, oligosaccharides, polysaccharides, sialic acid derivatives, sugar chains such as sialylated sugar chains, lipids, low molecular weight compounds, macromolecular organic substances other than those described above, inorganic substances, or fusions thereof, or viruses, Or biomolecules, such as a molecule | numerator which comprises a cell, are mentioned. In addition, for example, substances other than biomolecules such as cells can be used as the bio-related substance.

When the biologically relevant substance-immobilized carrier of the present invention is used for analysis, these biologically relevant substances measure the interaction (binding properties, etc.) between the detection target substance (analyte) in the specimen and the biologically relevant substance. It becomes a target substance when
The analyte is usually a substance that specifically interacts with a biological substance (hereinafter referred to as “active substance” as appropriate). Here, the “interaction” between the biological substance and the active substance is not particularly limited, but is usually a covalent bond, a hydrophobic bond, a hydrogen bond, a van der Waals bond, and a binding by electrostatic force. The effect | action by the force which acts between the substances which arise from at least one is shown. However, the term “interaction” in the present specification should be interpreted in the broadest sense, and should not be limitedly interpreted in any way. The covalent bond contains a coordinate bond. In addition, electrostatic coupling includes electric repulsion in addition to electrostatic coupling. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above action are also included in the interaction.

Specific examples of interactions include binding and dissociation between antigen and antibody, binding and dissociation between protein receptor and ligand, binding and dissociation between adhesion molecule and partner molecule, between enzyme and substrate Binding and dissociation, binding and dissociation between apoenzyme and coenzyme, binding and dissociation between nucleic acid and nucleic acid or protein binding to it, binding and dissociation between proteins in information transmission system, glycoprotein and Examples include binding and dissociation between proteins and binding and dissociation between sugar chains and proteins, but are not limited to this range. further,
For example, immunoglobulins and their derivatives F (ab ') 2, Fab', Fab, receptors and enzymes and their derivatives, nucleic acids, natural or artificial peptides, artificial polymers, carbohydrates, lipids, inorganic substances or organics Examples include ligands, viruses, cells, and drugs.

  Moreover, among the examples of the above-described biological substances, the protein may be the full length of the protein or a partial peptide including a binding active site. Further, the protein may be a protein whose amino acid sequence and its function are known or unknown. These can also be used as target substances, such as synthesized peptide chains, proteins purified from living bodies, or proteins that have been translated from a cDNA library or the like using an appropriate translation system and purified. The synthesized peptide chain may be a glycoprotein having a sugar chain bound thereto. Of these, a purified protein is preferable.

  Furthermore, there is no restriction | limiting in particular as a nucleic acid, In addition to DNA and RNA, nucleobases, such as an aptamer, and peptide nucleic acids, such as PNA, can also be used. The nucleic acid may be a known nucleic acid or an unknown nucleic acid. Preferably, a protein having a binding ability and having a known nucleic acid function and base sequence, or one that has been cleaved and isolated from a genomic library or the like using a restriction enzyme or the like can be used.

The sugar chain may be either a known sugar chain or an unknown sugar chain. Preferably, a sugar chain that has already been separated and analyzed and whose sugar sequence or function is known is used.
The low molecular compound is not particularly limited as long as it has the ability to interact. Although those with unknown functions or those with already known ability to bind to proteins can be used, drug candidate compounds and the like are preferably used.

When preparing a biological substance, the biological substance is usually prepared as a solution or dispersion in which the biological substance is dissolved or dispersed in some solvent. In this case, the solvent or dispersion medium for diluting the biological substance is prepared. Is preferably adjusted in consideration of the activity of the biological substance and the stability of the structure.
Moreover, a biological substance may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

  In addition, the bio-related substance, the compound capable of binding to the bio-related substance and / or the support, and the matrix containing the support are prepared by disposing at least two or more kinds in separate regions on the solid phase carrier. In the case of an array of biologically related substances characterized in that all of the biologically related substances that are arranged interact with functionally related active substances, It may be an array of biological materials that interact with related agents or an array of biological materials that interact with agents that are members of the same family.

  In addition, specific examples of the functionally related agents include diseases-specific agents. For example, metabolic functions, medical diseases, gastrointestinal regions, cardiovascular regions, endocrine regions, tumor regions, infectious diseases And combinations of markers for allergies and regions. Assaying in parallel the interaction with such a combination of agents related to a specific disease is very useful in "panel test" and "profile test" that comprehensively associate with the disease. is there.

  Specific examples of the above family include, for example, growth factor receptor, hormone receptor, neurotransmitter receptor, catecholamine receptor, amino acid derivative receptor, cytokine receptor, extracellular matrix receptor, antibody, lecithin, cytokine, serpin, protease, kinase, phosphatase , Ras-like GTPase, hydrolase, steroid hormone receptor, transcription factor, heat shock transcription factor, DNA binding protein, zinc finger protein, leucine zipper protein, homeodomain protein, intracellular signal transduction modulator and effector, apoptosis-related factor, DNA synthesis Factor, DNA repair factor, DNA recombination factor, cell surface antigen, hepatitis C virus (HCV) protea Ze, and group and the like consisting of HIV protease.

(4. Immobilization compound)
As the compound capable of binding to the biological substance and / or support (hereinafter referred to as “immobilizing compound” as appropriate), any compound can be used as long as it is a compound capable of binding to the biological substance and / or support. be able to. Therefore, as the immobilization compound, a compound having a functional group that can be bound to the biological substance and / or the support (hereinafter referred to as “binding functional group” as appropriate) can be arbitrarily used. Here, the binding functional group is not particularly limited as long as it is a functional group capable of binding to the biological substance or the support described above, and any functional group can be used. Usually, it is preferable to select an appropriate substance according to the type of the biological substance or the use of the biological substance fixing carrier of the present invention. In addition, a bond functional group may be used individually by 1 type, and may be used 2 or more types by arbitrary combinations and ratios.

The binding functional group is usually a reactive group that binds to a biological substance and / or support via a covalent bond and a bond that binds to the biological substance and / or support via a non-covalent bond. Broadly divided. In the case of bonding by a covalent bond, specific examples of the bonding functional group include a succinimide group, an epoxy group, an aldehyde group, and a maleimide group.
Hereinafter, the binding with the biological substance will be specifically described. Examples of the biological substance that binds to the binding functional group include proteins, nucleic acids, sugars, and the like.

  When the biological substance is a protein, an amino group, a hydroxyl group, a thiol group, etc. present on the surface layer of the protein and a binding functional group of the immobilizing compound are bonded. In this case, when the amino group is bonded to the binding functional group, specific examples of the binding functional group include a succinimide group and an epoxy group. Moreover, when a hydroxyl group couple | bonds with a coupling | bonding functional group, an epoxy group etc. are mentioned as a specific example of a coupling | bonding functional group. Furthermore, when a thiol group is bonded to a binding functional group, specific examples of the binding functional group include a maleimide group.

  When the biological substance is a nucleic acid, an amino group, a hydroxyl group, a thiol group or the like introduced at the end of the nucleic acid is bound to a binding functional group of the immobilizing compound. In this case, when the amino group is bonded to the binding functional group, specific examples of the binding functional group include a succinimide group and an epoxy group. Moreover, when a hydroxyl group couple | bonds with a coupling | bonding functional group, an epoxy group etc. are mentioned as a specific example of a coupling | bonding functional group. Furthermore, when a thiol group is bonded to a binding functional group, specific examples of the binding functional group include a maleimide group.

  When the biological substance is a sugar, an amino group, a hydroxyl group, a thiol group, etc. present in the side chain of the sugar and the binding functional group of the immobilizing compound are bonded. In this case, when the amino group is bonded to the binding functional group, specific examples of the binding functional group include a succinimide group and an epoxy group. Moreover, when a hydroxyl group couple | bonds with a coupling | bonding functional group, an epoxy group etc. are mentioned as a specific example of a coupling | bonding functional group. Furthermore, when a thiol group is bonded to a binding functional group, specific examples of the binding functional group include a maleimide group.

  On the other hand, when binding by a non-covalent bond, for example, when binding by forming a complex, specific examples of the binding functional group include a boronic acid group. Further, for example, when binding is performed by interaction between biologically relevant substances, an avidin-biotin interaction or the like can be used, and a specific example of the binding functional group used at that time is a biotin group. For example, when a virus is bound as a biological substance, specific examples of the binding functional group include sugars and polysaccharides. Furthermore, for example, when the biological substance has a lyophobic region, the binding by the lyophobic interaction is also possible.

Moreover, the coupling | bonding with a support body is demonstrated concretely. In addition, when the said biological material is previously fixed to the support body, a support body couple | bonds with the compound for fixation similarly to the above via a biological material.
For example, when the support is a latex particle, an amino group, a hydroxyl group, a thiol group, etc. present on the surface of the latex particle are bonded to the binding functional group of the immobilizing compound. At this time, when the amino group is bonded to the binding functional group, specific examples of the binding functional group include a succinimide group and an epoxy group. Moreover, when a hydroxyl group couple | bonds with a coupling | bonding functional group, an epoxy group etc. are mentioned as a specific example of a coupling | bonding functional group. Furthermore, when a thiol group is bonded to a binding functional group, specific examples of the binding functional group include a maleimide group.

For example, when a lyophobic interaction or an electrostatic interaction exists between the support and the immobilizing compound, they can be bonded by these interactions.
When the immobilizing compound has a binding functional group, the immobilizing compound usually has two or more, preferably three or more, binding functional groups with a biological substance and / or support in one molecule. Is preferred. Further, the compound can be used by mixing a plurality of types, but preferably contains at least one type having a plurality of bonding sites as described above. This is to facilitate the formation of the matrix structure of the present invention. As a specific example, the use of a compound having two or more binding functional groups in one molecule makes it possible to reliably form a matrix when concentration is performed.

  Furthermore, it is usually desirable to use a compound that is miscible with water as the immobilizing compound. Although the solvent used at the time of matrix preparation is arbitrary, usually water is used as a solvent. At this time, the compound for immobilization is combined with the support and / or the biological substance in the presence of water to bind the compound to the support and / or the biological substance. This is because the biologically relevant substance and the immobilizing compound are mixed uniformly and the binding reaction is performed smoothly. In the present specification, the form of mixing may be dissolved or dispersed.

The immobilizing compound is preferably miscible with at least one organic solvent. Thereby, the range of selection of the solvent used at the time of the synthesis | combination of the compound for fixation can be expanded, and the structure of a matrix can be designed variously. For example, if the immobilizing compound is miscible with the organic solvent, the synthesis can be performed in the organic solvent for the purpose of protecting the binding functional group during the synthesis of the immobilizing compound.

Furthermore, if the immobilizing compound is miscible with both water and an organic solvent, when using any kind of solvent when using the biologically relevant substance immobilizing carrier of the present invention, the types of solvents that can be used can be increased. Therefore, the application can be expanded.
The immobilizing compound is preferably uncharged. When the selective interaction between biological substances is detected using the biological substance-immobilized carrier of the present invention, the substance to be detected in the sample has the same charge as the support and / or the immobilizing compound. If the electrostatic repulsive force is excessively strong, the specific interaction with the biological substance may be hindered. In addition, when the detection target substance and the support and / or immobilization compound have opposite charges, the detection target substance and the support and / or immobilization compound are nonspecific such as nonspecific adsorption. This is because it is assumed that an interaction occurs.

There is no limitation on the method for confirming whether or not the immobilizing compound is uncharged. For example, when preparing the immobilizing compound as a solution before preparing the mixture, the immobilizing compound is immobilized in the solution. If the compound for use is uncharged, the compound for immobilization used in the biological material-immobilized carrier of the present invention can be treated as being uncharged.
Examples of the immobilizing compound include organic compounds, inorganic compounds, organic-inorganic hybrid materials, and the like. Moreover, the compound for fixation may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

  The organic compound used as the immobilizing compound may be a low molecular compound or a high molecular compound. Specific examples of low molecular weight compounds include glutaraldehyde, diepoxybutane, diepoxyhexane, diepoxyoctane, bismaleimide hexane, bissulfosuccimidyl suberate, disuccimidyl glutarate, ethylene glycol bissuccimidyl succinate , Sulfoethylene glycol bis succimidyl succinate, succimidyl 4-N-maleimidomethylcyclohexane 1-carboxylate, succimidyl 4-N-maleimidomethylcyclohexane 1-carboxylate, sulfosulfosuccimidyl 4-p-maleimidophenyl butyrate Succimidyl 4-p-maleimidophenyl butyrate, sulfo m-maleimidobenzoyl-N-hydroxysulfosuccimide ester, and the like.

On the other hand, when a polymer compound is used as the immobilizing compound, the polymer compound may be a synthetic polymer compound or a natural polymer compound.
When a synthetic polymer compound is used as the immobilization compound, any compound can be used as long as it satisfies the above conditions. However, it is usually desirable to have a monomer that can bind to a biological substance and / or a support. In general, it is preferable to have a hydrophilic monomer so that the synthetic polymer compound is miscible with water. Furthermore, it is preferable to use a synthetic polymer compound obtained by copolymerizing a monomer capable of binding to the biological substance and / or the support and a hydrophilic monomer. That is, in the synthesis of the synthetic polymer compound, at least as a monomer species, a conjugate formed by reacting with a biological substance and / or a support can be formed, and the conjugate can be bonded between the conjugate, in a chain and / or Alternatively, it is preferable to perform copolymerization using a monomer having a binding functional group for constructing a structure bonded in a network shape, a block shape, or the like, and a monomer having a hydrophilic or amphiphilic functional group. Furthermore, a hydrophobic monomer may be copolymerized for the purpose of controlling structures such as micelles formed in the solution of the synthetic polymer compound and the spread.

  Specific examples of the monomer constituting the synthetic polymer compound include monomers used in radical polymerization such as polymerizable unsaturated aromatics such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, vinyltoluene; Polymerizable unsaturated carboxylic acids such as meth) acrylic acid, itaconic acid, maleic acid and phthalic acid; polymerizable unsaturated sulfonic acids such as styrene sulfonic acid and styrene sulfonic acid; methyl (meth) acrylate and (meth) acrylic acid Ethyl, n-butyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, glycidyl (meth) acrylate, N- (meth) acryloyloxysuccinimide, ethylene glycol -Di- (meth) acrylic acid ester, (meth) acrylic acid trib Polymerizable carboxylic acid esters such as mophenyl, glycosyloxyethyl 2- (meth) acrylate, 2-methacryloyloxyethyl phosphorylcholine; (meth) acrylonitrile, (meth) acrolein, (meth) acrylamide, N, N-dimethylacrylamide N-isopropyl (meth) acrylamide, N-vinylformamide, 3-acrylamidophenylboronic acid, N-acryloyl-N'-biotinyl-3,6-dioxaoctane-1,9-diamine, butadiene, isoprene, vinyl acetate , Vinyl pyridine, N-vinyl pyrrolidone, N- (meth) acryloyl morpholine, vinyl chloride, vinylidene chloride, vinyl bromide and other unsaturated carboxylic acid amides, polymerizable unsaturated nitriles, vinyl halides, conjugated dienes , Polyethylene Examples thereof include macromonomers such as glycol mono (meth) acrylate and polypropylene glycol mono (meth) acrylate.

  Moreover, as a monomer of the synthetic polymer compound, a monomer used in addition polymerization can also be used. Specific examples of monomers used in the addition polymerization include diphenylmethane diisocyanate, naphthalene diisocyanate, tolylene diisocyanate, tetramethylxylene diisocyanate, xylene diisocyanate, dicyclohexane diisocyanate, dicyclohexylmethane diisocyanate. And aliphatic or aromatic isocyanates such as hexamethylene diisocyanate and isophorone diisocyanate, ketenes, epoxy group-containing compounds and vinyl group-containing compounds. Moreover, as a monomer made to react with the said compound group, the functional group which has activated hydrogen, the compound which has a hydroxyl group or an amino group as a specific example, etc. are mentioned, Specifically, ethylene glycol, diethylene glycol, propylene glycol, 1 , 4-butanediol, 1,6-hexanediol, glycerin, trimethylolpropane, pentaerythritol, sorbitol, methyleneglycoside, sucrose, polyols such as bis (hydroxyethyl) benzene; ethylenediamine, hexamethylenediamine, N, N Examples include polyamines such as' -diisopropylmethylenediamine, N, N'-di-sec-butyl-p-phenylenediamine, 1,3,5-triaminobenzene; oximes and the like.

  Further, the synthetic polymer compound may coexist with a polyfunctional compound that can serve as a crosslinking agent in addition to the monomer. Examples of the polyfunctional compound include N-methylol acrylamide, N-ethanol acrylamide, N-propanol acrylamide, N-methylol maleimide, N-ethylol maleimide, N-methylol maleamic acid, N-methylol maleamic acid ester, Examples thereof include N-alkylolamides of vinyl aromatic acids (such as N-methylol-p-vinylbenzamide) and N- (isobutoxymethyl) acrylamide. Furthermore, among the monomers described above, divinylbenzene, divinylnaphthalene, divinylcyclohexane, 1,3-dipropenylbenzene, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, butylene glycol, Polyfunctional monomers such as trimethylolethane tri (meth) acrylate and pentaerythritol tetra (meth) acrylate can also be used as a crosslinking agent.

  In addition, examples of the monomer having a binding functional group capable of binding to the support and / or biological substance described above include N- (meth) acrylic acid as examples of monomers having a succinimide group, an epoxy group, an aldehyde group, a maleimide group, and the like. Examples include leuoxysuccinimide, glycidyl (meth) acrylate, acrolein, and maleimide acrylate. Examples of the monomer having a boronic acid group as a binding functional group include 3-acrylamidophenylboronic acid. Furthermore, examples of the monomer having a biotin group as a binding functional group include N-acryloyl-N′-biotinyl-3,6-dioxaoctane-1,9-diamine. Examples of the monomer having a sugar or a polysaccharide as a binding functional group include glycosyloxyethyl 2- (meth) acrylate.

  Furthermore, specific examples of the hydrophilic monomer include (meth) acrylic acid, itaconic acid, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, maleic acid, sulfonic acid, sodium sulfonate, (Meth) acrylamide, N, N-dimethyl (meth) acrylamide, N-isopropylacrylamide, N-vinylformamide, (meth) acrylonitrile, N- (meth) acryloyl morpholine, N-vinylpyrrolidone, N-vinylacetamide, N -Vinyl-N-acetamide, polyethylene glycol mono- (meth) acrylate, glycidyl (meth) acrylate, 2-methacrylooxyethyl phosphorylcholine and the like.

  The immobilizing compound is preferably an uncharged one as described above. Therefore, when the synthetic polymer compound used as the immobilizing compound is uncharged, the monomer used in the uncharged synthetic polymer compound is not particularly limited as long as it is uncharged. ) 2-hydroxyethyl acrylate, 2-hydroxypropyl (meth) acrylate, meth) acrylamide, N, N-dimethyl (meth) acrylamide, N-isopropylacrylamide, N-vinylformamide, (meth) acrylonitrile, N- ( Examples include meth) acryloyl morpholine, N-vinyl pyrrolidone, N-vinyl acetamide, N-vinyl-N-acetamide, polyethylene glycol mono- (meth) acrylate, glycidyl (meth) acrylate, and the like.

When a synthetic polymer compound is synthesized by radical polymerization of monomers, the polymerization is usually started by mixing a radical polymerization initiator. Examples of radical polymerization initiators used here are 2, 2 '-Azobisisobutyronitrile, 2,2'-azobis- (2
-Methylpropanenitrile), 2,2'-azobis- (2,4-dimethylpentanenitrile), 2,2'-azobis- (2-methylbutanenitrile), 1,1'-azobis- (cyclohexanecarbonitrile) 2,2'-azobis- (2,4-dimethyl-4-methoxyvaleronitrile), 2,2'-azobis- (2,4-dimethylvaleronitrile), 2,2'-azobis- (2-amidino) Propane) hydrochloride and other azo (azobisnitrile) type initiators, benzoyl peroxide, cumene hydroperoxide, hydrogen peroxide, acetyl peroxide, lauroyl peroxide, persulfates (eg ammonium persulfate), peracid esters ( For example, t-butyl peroctate, α-cumyl peroxypivalate and t-butyl peroctate). Such as the flop of the initiator, and the like.

Furthermore, polymerization may be initiated by mixing a redox initiator. Examples of the redox initiator include ascorbic acid / iron (II) sulfate / sodium peroxydisulfate, tert-butyl hydroperoxide / sodium disulfite, and tert-butyl hydroperoxide / Na-hydroxymethanesulfinic acid. In addition, each component, for example, a reducing component, may be a mixture, for example, a mixture of sodium salt of hydroxymethanesulfinic acid and sodium disulfite.

The synthetic polymer compound may be a polymer synthesized by ring-opening polymerization or the like. Specific examples thereof include polyethylene glycol.
Furthermore, the polymer compound may be a polymer synthesized by hydrolysis or the like. Specific examples thereof include polyvinyl alcohol synthesized by hydrolyzing polyvinyl acetate and the like.

  Moreover, you may use a photoreactive polymer for a high molecular compound. Specific examples thereof include photoreactive polyallylamine (Bioconj. Chem., 9, 277 (1998), etc.), photoreactive polyacrylic acid (Langmuir, 14, 6610, (1998), etc.), and the like. When these are used, the matrix may be formed by irradiating light such as ultraviolet rays in the formation of the matrix with the support and the biological substance.

The above-described synthetic polymer compound may be synthesized by modifying a functional group that binds to the aforementioned biological substance and / or support by chemical modification.
In addition, a commercially available synthetic polymer compound can be used as the immobilizing compound. Specific examples thereof include SUNBRIT series DE-030AS, DE-030CS, DE-030GS, PTE-100GS, PTE-200GS, HGEO-100GS, and HGEO-200GS manufactured by NOF Corporation.

  On the other hand, when a natural polymer compound is used as the immobilizing compound, specific examples thereof include dextran, carboxymethyl-dextran, starch, cellulose, agarose, gum arabic, alginic acid and other polysaccharides, pectin, albumin, collagen, gelatin. And proteins such as DNA, RNA, and nucleic acid. These natural compounds may be used as they are, or may be used after being chemically modified.

  Of the above-mentioned compounds, compounds having the ability to crosslink biological materials are preferably used in the present invention. Specifically, for example, a synthetic polymer synthesized by a combination of a monomer having a crosslinkable functional group in the side chain and an amphiphilic monomer is preferably used. Among them, for example, a synthetic polymer synthesized by a combination of N-acryloyloxysuccinimide and N-acryloylmorpholine, N-acryloyloxysuccinimide and dimethylacrylamide, or the like is preferably used.

In the case where a polymer compound such as a synthetic polymer compound and a natural polymer compound is used as the immobilization compound, the form of the polymer compound is arbitrary. For example, it may be dissolved in an aqueous solution or an aggregate such as a micelle or emulsion.
Furthermore, examples of the organic-inorganic hybrid used as the immobilizing compound include, for example, a metal colloid coated with a polymer (for example, gold, silver, platinum, etc. coated with a protective colloid), and a clay polymer. And the like adsorbed. These organic-inorganic hybrids can be synthesized by a known method (see polymer-based nanocomposites, industrial research committee, Sumi Nakajo, etc.). Furthermore, a bio-related substance can also be used as an immobilizing compound by modifying the binding functional group of these organic-inorganic hybrids.

  In addition, the molecular weight and structure of the immobilizing compound are not particularly limited and may be arbitrary. For example, a low molecular weight compound may be used, but in that case, the compound is cross-linked within one biological substance to be immobilized. There is a risk that the matrix structure cannot be formed. From the viewpoint of preventing this, the molecular weight of the compound for immobilization is usually 1,000 or more, preferably 10,000 or more, and usually 1,000,000 or less, preferably 500,000 or less. When a synthetic or natural polymer compound is used as the immobilization compound, it is preferable that the weight average molecular weight falls within the above range. This is because if it falls below this range, the matrix may not be formed effectively.

  Furthermore, the amount of the binding functional group of the immobilizing compound is usually 0.1% or more, preferably 1% or more, more preferably 5% or more, and usually% by weight with respect to the immobilizing compound. It is 90% or less, preferably 80% or less, more preferably 70% or less. If the ratio is below this range, the immobilizing compound may not be efficiently bonded to the support or the biological substance, and if it exceeds the range, the immobilizing compound may not be miscible with the solvent.

(5. Solvent)
When the biologically relevant substance-immobilized carrier of the present invention is produced, a mixture containing at least the above-mentioned support, biologically relevant substance, and immobilizing compound is supplied to the solid phase carrier in the presence of a solvent.
In the solvent, the support, the biological substance, and the immobilizing compound can be mixed in the solvent as long as they are miscible, and may be dissolved or dispersed. In order to stably bind to the compound for use, it is preferable that the biological substance and the immobilization compound are dissolved. The support may be sufficiently mixed and dispersed.

  The solvent is a reaction medium that binds the biological substance and / or the support and the immobilizing compound, and may be any solvent as long as the support, the biological substance, and the immobilizing compound are miscible. There is no limitation, and any liquid can be used. It is preferable to select in consideration of the activity, structural stability, functionality, etc. of the support, the biological substance and the immobilizing compound, but usually water is used as the solvent. By immobilizing in the presence of water, it becomes possible to maintain the activity of the biological substance sufficiently, and the range of choices for the support, biological substance, and immobilization compound is widened and applicable range Can be expected to expand.

  Moreover, as a solvent, you may use solvents other than water, for example, an organic solvent can be used. Furthermore, among the organic solvents, an amphiphilic solvent, that is, an organic solvent miscible with water is preferable. Specific examples of the solvent other than water include THF, DMF, NMP, DMSO, dioxane, acetonitrile, pyridine, acetone, glycerin and the like in addition to alcohol solvents such as methanol, ethanol, and 1-butanol. .

Further, salts may be added to these solvents. The type of the salt is arbitrary, but specific examples include NaCl, KCl, sodium phosphate, sodium acetate, calcium chloride, sodium bicarbonate, ammonium carbonate and the like. Moreover, there is no restriction | limiting in the quantity of the salt to be used, Arbitrary quantity salt can be used according to a use.
Furthermore, when water is used as the solvent, as the water, an aqueous solution in which a medium other than a biological substance or an immobilizing compound is dissolved can be used in addition to pure water. Examples thereof include various buffer solutions, and specific examples thereof include carbonate buffer, phosphate buffer, acetate buffer, HEPES buffer and the like.
In addition, a solvent may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

(6. Mixture)
The mixture is a mixture of a support, a biological substance, and a compound capable of binding to the biological substance and / or the support in the presence of a solvent. Specifically, it is a mixture in which the support, the biological substance, and the immobilizing compound coexist in the solvent described above. In the mixture, the support, the biological substance and the immobilizing compound are preferably mixed in the solvent.

  In the mixture, the ratio of the support, the biological substance and the immobilizing compound is arbitrary, and any ratio may be used as long as the matrix of the present invention is formed. However, as described above, the biological material and the compound are preferably dissolved in a solvent, and the support is preferably mixed and dispersed. For example, when the latex particles are used as a support, the concentration of latex particles suspended in the mixture is about 0.5 to 10% [w / w].

Furthermore, there is no restriction | limiting in the method of preparing this mixture, and it is arbitrary. For example, a dispersion of a support, a solution (such as an aqueous solution) or a dispersion of a biological substance may be mixed with a solution of an immobilizing compound (such as an aqueous solution) or a dispersion. You may add another solution to the solution which mixed two types.
In addition to the support, the biological substance, the immobilizing compound, and the solvent, any additive may be coexisted in the above mixture. Examples of additives include humectants such as salts, acids, bases, buffers, glycerin, metal ions such as zinc as stabilizers for biological substances, antifoaming agents, denaturing agents, and the like.

(7. Supply)
When producing the biological material-immobilized carrier of the present invention, the above-mentioned mixture is supplied to a solid phase carrier. That is, the above-mentioned mixture is brought into contact with the solid phase carrier. Although the specific operation is arbitrary, for example, a mixture may be prepared in advance, and the mixture may be brought into contact with a solid phase carrier, or each component of the mixture may be prepared separately, and they may be placed on the solid phase carrier. A mixture may be prepared by mixing, and the mixture may be brought into contact with a solid phase carrier. Specifically, for example, a dispersion containing a support, a solution containing an organism-related substance (eg, an aqueous solution) and a solution containing a compound (eg, an aqueous solution) are supplied onto the solid phase carrier, and then both on the solid phase carrier. It can be carried out by mixing the solution. Moreover, when preparing a mixture beforehand, the conjugate and / or matrix which are mentioned later may be produced in the mixture before supply, and you may make it supply a mixture to a solid-phase carrier after that.

(8. Formation of matrix)
Next, the support, the biological substance and the compound are bound to the surface of the solid phase carrier, and the compound is bound to the biological substance and / or the support through the binding site using the support as a nucleus. In this way, a matrix formed by bonding in a chain shape, a mesh shape, a block shape or the like is formed. When the mixture is prepared, the support, the biological substance, and the immobilizing compound are combined in the mixture, and a conjugate including these three types in an arbitrary ratio is generated. This conjugate is a combination of a support, a biological substance, and an immobilization compound. Simply mix the support, the biological substance, and the immobilization compound in a solvent, and then bring the molecules into contact with each other. Can be produced. Therefore, conjugates are usually present in the mixture supplied to the solid support.

  Conjugates are formed in a chain form by combining the conjugates, biological substances, and immobilization compounds that the conjugates gather together in a process in which part or all of the solvent is removed by drying or the like. A matrix having a structure connected in a net shape, block shape or the like is formed. Therefore, by bringing the mixture supplied to the solid phase carrier into contact with the solid phase carrier, for example, the conjugates in the mixture are accumulated on the surface of the solid phase carrier, or the matrix formed by combining the conjugates in the mixture is formed. A solid phase carrier is formed by binding to a solid phase carrier or by forming a conjugate and / or a matrix by binding a support, a biological substance, and an immobilizing compound in a mixture to the surface of the solid phase carrier. A matrix can be formed on the surface.

  In a case where the amount of the solvent in the mixture is large, a conjugate or a matrix is hardly generated or not generated in the mixture. In this case, the conjugate can be efficiently formed by concentrating the mixture. Of course, even when the mixture supplied to the solid phase carrier contains a conjugate and / or a matrix as described above, concentration may be performed to further produce the conjugate and / or matrix. However, in order to form a uniform matrix, it is preferable to uniformly mix the support, the biological substance, and the immobilizing compound in a solvent in the initial stage of preparing the mixture. Therefore, it is preferable that the conjugate is formed by coexisting the support, the biological material, and the immobilizing compound in a relatively large amount of solvent, and then concentrating them.

Normally, the mixture is concentrated in the process of drying the mixture, so that concentration and drying can be performed as a series of operations. The method of drying and concentrating the mixture is arbitrary, and examples thereof include ultrafiltration and drying under reduced pressure. In addition, drying or concentration may be performed simply by evaporation under normal pressure.
The solid phase carrier of the present invention preferably has voids formed in the matrix by removing part or all of the solvent as described above. The removal of the solvent is preferably performed by drying as described above. Through this drying step, the conjugate forms a strong matrix, and voids are generated in the matrix.

For example, the temperature condition for drying and concentrating the mixture is usually 37 ° C. or lower, preferably 25 ° C. or lower, from the viewpoint of avoiding denaturation of biological substances. In addition, the humidity and pressure conditions for drying and concentrating the mixture can be appropriately set in view of the formation state of the matrix and the formation state of the voids in the matrix.
In order to sufficiently immobilize the matrix on the solid phase carrier, it is desirable that the solid phase carrier is allowed to stand for a predetermined time after the mixture is supplied. Although the standing time is arbitrary, it is usually 24 hours or less, preferably 12 hours or less. That is, after the mixture is supplied to the solid phase carrier, the mixture is allowed to stand for a certain time to sufficiently promote the formation of the conjugate and the immobilization on the surface of the carrier, and then the solvent may be removed by drying or the like. By passing through such steps, the solid phase carrier of the present invention in which the matrix of the present invention having sufficient voids and film thickness is firmly formed on the solid phase carrier can be produced.

  An example of the matrix forming process described above is schematically shown in FIGS. As shown in FIG. 3, a mixture containing a biological substance, an immobilization compound, and a support is supplied to a solid phase carrier (FIG. 3 (a)), and these three types are conjugates composed of an arbitrary ratio. (FIG. 3 (b)), and then the solvent is removed, whereby the biologically relevant substance and the immobilizing compound are bound to the support using the support as a nucleus, and the chain and / or network and A matrix having a void layer having a structure bonded in a block shape is formed (FIG. 3C). FIG. 3 is an enlarged schematic view showing an example of the biological substance-fixing carrier of the present invention in order to explain the structure of the matrix of the present invention. Further, in FIG. 3, the filled circular portion represents the biological substance, the linear portion represents the immobilizing compound, the white circular portion represents the support, the hatched portion represents the solvent, and the blank portion represents Represents a void layer.

(8. Other processes)
As described above, the above method can form a matrix on the surface of the solid phase carrier simply by contacting the solid phase carrier with a mixture in which a support, a biological substance, and an immobilization compound coexist in a solvent. Thus, the biological material-immobilized carrier of the present invention can be produced, that is, a biologically relevant substance can be immobilized on the solid phase carrier.

By the way, when manufacturing the biological material fixed carrier of the present invention, other steps than the above steps may be performed.
For example, a different biological substance may be bound to the biological substance in the matrix. By utilizing this, after the production of the biological substance-immobilized carrier, another biological substance modified so as to specifically bind to the biological substance in the matrix is bound later, and as a result, the above-described solid phase carrier is bonded to the above-mentioned substance. Another biological substance can be immobilized at a high density. As a specific example, avidin is used as a biological substance, and this avidin, a support and an immobilizing compound are combined to form a matrix to produce a biological substance fixing carrier. Then, another biological substance modified with biotin can be used to immobilize the other biological substance by the avidin-biotin interaction. Similarly, it is possible to immobilize a biological substance via a histidine tag or glutathione-S-transferase.

[II. Biologically related substance immobilization carrier]
The biological material-immobilized carrier of the present invention is manufactured by the method described above. In addition, the matrix possessed by the biological substance-fixing carrier of the present invention is composed of a large number of the above conjugates bonded with a support as a nucleus. Usually, the biological substance as shown in FIG. And a compound for immobilization in a chain shape and / or a network shape or a block shape.

  Furthermore, the matrix of the present invention is a structure having a main chain composed of a biological substance and an immobilizing compound with the above support as a nucleus. Here, the main chain of the matrix of the present invention constitutes the skeleton of the matrix. Specifically, the support-related substance and the immobilizing compound are surrounded around the support, and these are one or more aggregates. Specifically, the immobilizing compound is bonded to the support and / or the biological substance by a binding functional group to form a bridge structure, and the structure is repeated. A chain-like and / or network-like or block-like structure is formed.

  Therefore, the matrix of the present invention has at least a part of a bridge structure in which a biological substance or a support exists between the immobilizing compounds, and the support is formed by both the biological substance and the compound. The main chain of the matrix is made up of cores. Therefore, the matrix of the present invention is formed when the support, the biological substance and the immobilizing compound are all present in the system at the time of preparation.

  Furthermore, the biological material-immobilized carrier of the present invention preferably has voids formed in the matrix by removing part or all of the solvent as described above. As described above, the removal of the solvent is preferably performed by drying, and through this drying step, the conjugate forms a firm matrix, and voids are generated in the matrix.

The fact that the matrix of the present invention has a main chain composed of a biologically relevant substance having a support as a core and an immobilizing compound is an electron such as a scanning electron microscope (SEM) and a transmission electron microscope (TEM). It can be confirmed by a technique such as observation with a microscope. The voids and film thickness formed in the matrix can be confirmed in the same manner.
Here, as the void, it is sufficient that a space is three-dimensionally formed in the matrix of the present invention, and a space is generated between shapes such as a chain shape, a mesh shape, or a block shape that the matrix has. is there. Preferred are crack-like voids, micropore-like voids possessed by a porous material, etc., but any material can be used as long as sufficient reactivity can be secured when this is used. . In addition, the shape of these voids can be confirmed by, for example, an electron microscope.

The porosity is a value indicating the ratio of voids in the matrix of the present invention. Examples of the method for obtaining the porosity include a method of performing image analysis using an electron micrograph, a gas adsorption method, an Hg porosity method, and the like.
Although the porosity is arbitrary according to the objective, For example, it is preferable that the porosity obtained by image-analyzing the cross-sectional view of a SEM or a TEM image is 5% or more in the state which the matrix dried. As an image analysis method, a publicly known image processing method can be selected and applied as appropriate. For example, an acquired SEM photograph is captured by a scanner, noise is removed, and a background image is created. For example, there is a method of performing shading correction, binarizing an image at a threshold value set by setting a particle region, and obtaining an area ratio thereof.

  If the porosity is too low, the diffusion of the active substance into the matrix is suppressed, or the degree to which the immobilized biological related substance is exposed on the surface decreases, so that the relationship between the biological related substance and the active substance is reduced. The reaction efficiency may be lowered. There are also problems such as an increase in non-specific reactions due to a decrease in washing efficiency. On the other hand, if the porosity is too high, problems such as inability to maintain sufficient film strength may occur. Such problems can also be adjusted by optimizing the conditions for forming the matrix of the present invention as described above.

  Although the thickness of the matrix is also arbitrary, for example, the thickness measured by SEM or TEM when the matrix is dried is usually 5 nm or more, preferably 10 nm or more, more preferably 20 nm or more. If the film thickness is too small, it is difficult to make the film thickness of the matrix uniform, and it may be difficult to immobilize a large amount of biological substances with high reproducibility. In addition, the film thickness of the matrix of the present invention is the thickness from the contact surface with the solid phase carrier to the surface of the fixed carrier in the cross section of the biological substance-fixed carrier of the present invention.

  In the structure of the matrix of the present invention, the shape, void ratio, film thickness, etc. of the voids as described above are selected as appropriate compounds and supports that can bind to biological substances according to the purpose, Optimization can be achieved by appropriately selecting immobilization conditions, solvent removal conditions, and the like.

[III. effect]
The biological material-immobilized carrier of the present invention is one in which the biological material is immobilized in a larger amount with higher accuracy and at a lower cost than before.
As a technique for increasing the amount of immobilization, for example, a method of spotting a solution containing a high concentration of a biological substance directly on a solid phase carrier is frequently used. However, in this method, it is known that in the process of dropping the dropped solution (droplet), the vapor flux from the droplet is not constant depending on the location, and the flux from the outside of the droplet is large. That is, when the inside of the droplet is considered, a flux of the solution is generated from the center to the end of the droplet, and the biological substance, which is a solute, flows from the center to the end. Along with evaporation, the biological substance is deposited at the end of the droplet, and localized at the periphery of the spot, the high concentration biological substance is immobilized, resulting in the formation of a ring-shaped spot. Are known. This ring-shaped spot becomes a big problem especially in the measurement process after the reaction, and becomes one of the causes of increasing the CV value and decreasing the measurement accuracy.

  In conventional techniques such as Patent Documents 1 to 3, a biological substance is bound to a polymer film (polymer film) formed on the surface of a solid support. However, in this case, the biological substance is bonded to the tip of the polymer chain or grafted to the polymer chain in the main chain formed by the polymer chain, so that the biological substance is solid-phased. Since it was bound to the carrier, it was necessary to form a polymer chain as the main chain, and it was necessary to use a certain amount of polymer for the biological substance to be immobilized, and the immobilization amount of the biological substance was limited. was there. In addition, biological materials are biased on the surface of the membrane, and there are often no voids in which the active substance can enter the polymer membrane, resulting in a decrease in reactivity. Furthermore, in the conventional technique in which the main chain is constructed of only the hydrophilic polymer, it is speculated that the hydrophilic polymer chain hinders the access of the active substance to the biological substance due to the excluded volume effect and the movement of the polymer chain ( Editing method for real-time analysis of biological material interactions-Focusing on BIACORE, edited by Kazuhiro Nagata, Handahiro, publisher Springer Fairlark Tokyo, page 258, Development and application of medical polymer materials, CMC, No. 19).

In addition, in the prior art such as Non-Patent Document 1, for example, the solid phase carrier is porous, so the surface area is large and many biological substances can be bound. However, a direct-hole filter, special immobilization equipment, etc. Therefore, there is a problem that the solid phase carrier itself needs to have a special shape, and the solid phase carrier itself becomes an expensive and limited carrier.
Furthermore, for example, when a matrix is formed using only biological substances and compounds, more biological substances can be immobilized than when only biological substances are bound to the carrier, but usually sufficient film thickness It is difficult to form a gap and a spot, and when spotting is performed, the spot becomes a ring shape, and it is difficult to obtain sufficient accuracy. Even if a large amount of biological material is immobilized and a sufficient film thickness can be secured, if the voids are not formed sufficiently, the reactivity of the biological material contained in the film may be impaired. The active substance to be reacted may not be sufficiently diffused to reduce the reaction efficiency. In particular, for example, when an antigen and an antibody, a protein and a ligand, etc. are used as a combination of a biological substance and a substance to be detected, the antigen-antibody reaction that occurs between them is a weak biological reaction. The amount of crystallization must be large, and the biological activity of the immobilized substance must be maintained and exposed to the surface so as to participate in the reaction.

  The biological substance-immobilized carrier of the present invention can solve these problems in the prior art, and includes three substances: a biological substance, a compound capable of binding to the biological substance, and a support. Compared with the conventional method, it is very advantageous in that a matrix having a sufficient thickness can be formed, a sufficient amount of immobilization can be obtained, and voids can be provided. That is, it is possible to immobilize a large amount of the biological substance on the solid support in a three-dimensional substantially uniform state. Since the voids can be a reaction field for the immobilized biological substance to react, sufficient reactivity and sensitivity can be obtained when the biological substance-fixing carrier of the present invention is used in a biosensor, a diagnostic device, or the like. Can be secured.

  In addition, the biological substance-immobilized carrier of the present invention makes it possible to remove the matrix of any property by optimizing the conditions such as the concentration of the mixture (mixing ratio of each component), temperature, humidity, pressure, etc. at the time of matrix formation. It is also advantageous in that it can be formed with high accuracy and can be easily manufactured. In the conventional method, many steps are required to immobilize the biological material with high density. Specifically, conventionally, a polymer film is formed on a solid support in advance, and a biological material is immobilized on the polymer film to construct a matrix containing a ligand on the solid support, or a porous support. It needed a special structure like However, in these methods, when preparing a polymer film on the solid phase, it is necessary to appropriately control the molecular weight of the polymer and the density of introduction to the solid support, which is very complicated and reproducible. There is a problem that it is difficult to immobilize well, and in the case of a porous carrier, there is a problem that an influence due to diffusion of a biological substance in the porous carrier occurs during the immobilization. In particular, in the method of Patent Document 3, it is technically difficult to construct a polymer chain in a brush shape from the surface of a solid phase carrier, which is not suitable for mass production.

  On the other hand, the biological substance-immobilized carrier of the present invention can be obtained by simply contacting a solid phase carrier with a mixture in which a support, a biological substance, and an immobilizing compound coexist in a solvent. It can be formed, that is, the biological material can be immobilized, and can be manufactured very easily. Further, as in the technique described in Patent Document 2, the solvent used for the production of the biological substance-fixing carrier is limited to the organic solvent, and the biological substance that can be used is not limited thereby. It is possible to expand the selection range.

  In addition, the matrix formed on the biological material-immobilized carrier of the present invention can be used under the controlled temperature, humidity, and pressure conditions, and the amount, ratio, and / or amount of the support, biological material, and immobilized compound. By changing the shape, the formation state of the matrix, the film thickness, and the porosity can be arbitrarily controlled. In the prior art, it is usually very difficult to arbitrarily control the thickness of the matrix from the submicron level to several micron level. However, according to the present invention, the thickness of the matrix can be controlled at the precise level as described above, and the degree of freedom in designing the matrix structure can be increased.

  For example, when performing an immunoassay, the membrane used for immobilization must have a large amount of biological material immobilized and suppress nonspecific reactions, and there is an optimum thickness depending on the material. To do. In addition, for example, when performing a surface treatment of a medical instrument or a regenerative medical carrier, a micro-order film thickness is required for a film used for the surface treatment in order to realize sufficient strength and coating. Further, for example, when performing surface treatment of a drug for DDS (drug delivery system), it is required to strictly control the film thickness of the film used for the surface treatment in order to control drug release. As described above, when using some kind of membrane for immobilizing a biological material, it is known that control of the thickness is important, but it has been very difficult in the prior art. According to the present invention, the film thickness according to the intended application can be arbitrarily adjusted by adjusting the concentration, amount, reaction conditions (temperature, time, etc.) of the mixture.

  Furthermore, in the biological material-immobilized carrier of the present invention, when the biological material-immobilized carrier of the present invention is used to cause the biological material and the active substance to interact, non-biological substances are caused by matrix components other than the biological material. Specific interactions can be suppressed. This can be achieved by increasing the proportion of the bio-related substance in the matrix and using a substance that suppresses non-specific interactions with substances other than the bio-related substance.

  In particular, when an uncharged compound is used as the immobilization compound, it is possible to further suppress nonspecific interactions caused by the charge. That is, for example, in the method described in Patent Document 1, since the polymer membrane has a charge, the pH and ionic strength of the buffer solution to be used greatly affect the reaction of the biological substance. Nonspecific adsorption due to electrostatic interaction could not be avoided (see Nanotechnology Basic Series, Bio-Nanotechnology, Yasuhiro Horiike, Kazunori Kataoka (co-edition), page 186). However, if a non-charged compound is used as the immobilizing compound, such a problem can be avoided and a specific interaction can be selectively generated. This can further increase the sensitivity of measurement.

[IV. Biologically related substance immobilization kit]
In order to produce the above-described biologically relevant substance-immobilized carrier, a biologically relevant substance immobilization kit in which at least a support and an immobilizing compound are made into a kit may be used. By using the biological material immobilization kit, the matrix can be easily produced on a solid phase carrier, and the biological material immobilization carrier of the present invention can be easily produced. A large amount can be immobilized.

The support provided in the biological material immobilization kit is the same as described above. In the biological material immobilization kit, the support may be provided in any state, for example, a solution dissolved in an arbitrary solvent, a dispersion liquid dispersed in an arbitrary dispersion medium, a powdery or massive solid The existence state is arbitrary.
In addition, the immobilization compound provided in the biological material immobilization kit is the same as described above. Further, in the biological material immobilization kit, the immobilization compound may be provided in any state, for example, a solution dissolved in an arbitrary solvent, a dispersion liquid dispersed in an arbitrary dispersion medium, a powder form or a lump form. The presence state of the solid is arbitrary.

Furthermore, the bio-related substance immobilization kit may be provided with other elements as necessary.
For example, a solvent may be further provided. The solvent is the same as the solvent described above as the solvent used for the production of the biological substance-fixing carrier. Further, in the biological material immobilization kit, the solvent may be provided separately from the support and the immobilization compound, and the support and immobilization medium as a solvent and a dispersion medium of the support and immobilization compound. It may be provided integrally with the compound for use.

  For example, you may further provide the reagent etc. which accelerate | stimulate manufacture of a matrix. As a specific example, when polyacrylic acid is used as the immobilizing compound, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (abbreviation: abbreviation :) is used to activate the carbonyl group of polyacrylic acid. EDC) may be provided as a reagent.

[V. Application]
The biological material-immobilized carrier of the present invention can be used in a wide range of industries. There is no restriction on the specific application, and it can be used for any application. Usually, an “interaction” between a biological substance and an agent (analyte) that specifically interacts with the biological substance. It is suitable for use using

  For example, the biological material-immobilized carrier of the present invention can be suitably used as a biosensor that detects an agent that interacts with the biological material as an analyte. The biosensor described above is, for example, a so-called DNA array or DNA chip, or a sensor chip called a protein array or protein chip on which DNA or protein is immobilized. The biological substance-fixing carrier of the invention can be applied to this sensor chip. That is, when a biological substance is immobilized on the sensor chip, a matrix can be formed on the sensor chip body by the above-described method, and the sensor chip can be used as the biological substance-fixing carrier of the present invention.

  Thus, specific examples of biosensors to which the biological substance-fixing carrier of the present invention can be applied include fluorescence method, chemiluminescence method, RI method, FRET method, SPR (surface plasmon resonance) method, QCM (quartz crystal) (Oscillator microbalance) method, piezo cantilever method, laser cantilever method, mass spectrometry, electrode method, field effect transistor (FET) method, FET and / or single electron transistor method using carbon nanotube, electrochemical Examples of the sensor include a method. Among these, detection by the chemiluminescence method is preferably used because the specimen can be analyzed with high sensitivity.

  For example, when the biological substance-immobilized carrier of the present invention is applied to the chemiluminescence method, the surface of the sensor chip body may have a functional group in order to firmly bond the matrix to the surface of the sensor chip body. preferable. In this case, the functional group is arbitrary, but examples include hydroxyl group, carboxyl group, thiol group, aminoaldehyde group, hydrazide group, carbonyl group, epoxy group, vinyl group, amino group, succinimide group and the like.

In addition, for example, when the biologically relevant substance-immobilized carrier of the present invention is applied to the fluorescence method, it is desirable to use a material that is less affected by autofluorescence for the solid phase carrier, support, and immobilization compound. When it occurs, it is preferable to employ a time-resolved fluorescence method.
In addition, for example, the biological material-immobilized carrier of the present invention can be suitably used for a diagnostic device or the like by utilizing the properties as the biosensor. In the case of diagnostic devices, for example, proteins such as antibodies, antigens and enzymes, nucleic acids such as DNA and RNA, and low molecular compounds such as drugs and antibiotics are often detected. Preferably, a chemiluminescence method, a fluorescence method, an RI method or the like is used.

  The biological substance-immobilized carrier of the present invention is suitably used for assay of an analyte in a specimen, for example, as a biosensor as described above. The method for assaying an analyte of the present invention is a method for assaying at least one analyte in a specimen, and (a) the specimen contains at least one biological substance capable of reacting with the analyte. (B) by detecting the reaction caused by adding the labeling substance that reacts with the biological substance or the analyte, or the interaction between the biological substance and the analyte or the analyte. Detecting the presence or amount of the analyte.

Here, the sample may be any sample containing or possibly containing an analyte, but is preferably a sample derived from a living body such as a human. Examples of such samples include blood, serum, plasma, urine, feces, nasal discharge, saliva and other body fluids, cells, tissues, and extracts thereof. The analyte is as described above.
Hereinafter, the assay method using the biologically relevant substance-immobilized carrier of the present invention will be described more specifically with reference to an example in which an antigen-antibody reaction is detected using a chemiluminescence method.

  A biological substance-immobilized carrier on which an antibody against an antigen as an analyte (primary antibody) is immobilized together with a support and an immobilizing compound is reacted with the antigen in a specimen for a certain period of time. Next, unreacted antigen and coexisting substances other than the antigen are removed by washing or the like, and an antibody against the antigen labeled with the enzyme (labeled secondary antibody) is allowed to react for a certain period of time. Remove next antibody. As a result, a sandwich is formed between the primary antibody, antigen, and enzyme-labeled secondary antibody (sandwich method). In addition, the method of assaying a detection target using an antigen-antibody reaction is not limited to the sandwich method, and a method widely used in the area of immunoassay such as an inhibition method can be used.

After the reaction, a chemiluminescent substrate corresponding to the enzyme is added to cause chemiluminescence based on the presence or amount of the analyte antigen, and a light signal emitted from the test region is detected by a detector.
Examples of enzymes used at this time include horseradish peroxidase (HRP) and alkaline phosphatase (ALP). As substrates, luminol is used when HRP is used, and 1,2-dioxetane is used when ALP is used. Etc. are used.

In addition, as a detector, a charge coupled device (CCD) camera, a photomultiplier tube (PMT), a photodiode (PD) or the like is used, and two or more types of biological substances are fixed in one or two dimensions. In the case of an array, the detector is preferably a CCD camera, a photodiode array, or a switching type PMT.
In addition, such an assay method can also deliver a specimen or the like to the biological substance-immobilized carrier or array of the present invention under flow conditions. At this time, it is preferable that at least the specimen is a fluid, and more preferably, other reagents are fluids. First, a cell (flow cell) including a biological substance-immobilized carrier in which an antibody (primary antibody) against an antigen as an analyte is immobilized together with a support and an immobilizing compound is prepared, and a sample or the like is flowed into the cell. And the antigen in the specimen reacts for a certain period of time. Next, the unreacted antigen and coexisting substances other than the antigen are removed by delivering the washing solution to the flow cell in the same flow. Thereafter, a solution containing an antibody against the antigen labeled with an enzyme (labeled secondary antibody) is delivered to the flow cell for a certain period of time by the same flow. Again, the washing solution is delivered to the flow cell by flow to remove excess labeled secondary antibody, and a sandwich is formed between the primary antibody, antigen, and enzyme-labeled secondary antibody (sandwich method). As for the method, enzyme, detector and the like used, the same ones as described above are used.

  In either case, a reference region is provided in advance on the biological substance-immobilized carrier of the present invention, and the reaction in this reference region is detected and compared to determine the success or failure of the assay. Calibration for the presence or amount of the analyzed analyte can be performed. For example, an antigen, which is an analyte prepared in advance at a known concentration, is immobilized on a known region (reference region) on a solid phase carrier on which a biological substance is immobilized, and the same method as described above is used. After performing the assay, the reference region is also chemiluminescent, the light signal emitted from the reference region is detected by the above detector, and this is compared with the light signal emitted from the reaction by the analyte in the sample. By doing so, the concentration of the analyte can be calculated or calibrated. In addition, since an optical signal emitted from the reference region can grasp the emission intensity expected in advance, the success or failure of the assay can be determined from the intensity of the optical signal.

  In addition, a method of assaying two or more analytes in parallel in the assay method of the present invention as described above, wherein the presence or amount of the measured analyte is associated with a specific symptom is also preferably used. It is done. Such a method is very useful, for example, as “panel inspection” or “profile inspection”. Panel inspection means performing a more detailed inspection for a certain disease or symptom by combining a plurality of inspection markers. For example, when diagnosing cancer, it is difficult to specify which cancer is just because the value of a single tumor marker is high, but by examining multiple tumor markers, You can narrow down the types and parts. In addition, “profile test” is different for each patient by measuring a plurality of test markers including conventional test markers in the determination of prognosis and treatment effect in disease treatment, and using multivariate analysis of the obtained results. A reference value is set and the fluctuation is analyzed in detail. Such an analysis result is useful as knowledge for performing appropriate treatment without overlooking even a slight change in a disease state, or avoiding side effects or the like at the time of medication.

  Specific examples of such tests include, for example, three types of markers that vary independently, that is, neurohormones, as tests performed for stratifying the risk level of patients with acute coronary syndrome when diagnosing myocardial infarction A method that can be used to determine prognosis by measuring all of B-type natriuretic peptide (BNP) as a marker, troponin T or I as an ischemic marker, and C-reactive protein (CRP) as an inflammatory marker is considered. It has been. In addition, a test method combining various test markers is being proposed. In such a test, the biological substance-immobilized carrier, the array, and the assay method of the present invention are very suitably used.

Furthermore, the biological substance-immobilized carrier of the present invention can be applied to a surface treatment of a drug for DDS (drug delivery system), a surface treatment of a regenerative medical carrier, a surface treatment of an artificial organ, a surface treatment of a catheter or the like. .
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples, and may be arbitrarily modified and implemented without departing from the gist of the present invention. it can. In the description of the examples, unless otherwise specified,% represents% by weight.

[Synthesis of polymer (immobilization compound)]
First, two types of polymers A and B were synthesized as compounds capable of binding to a biological substance for producing the biological substance-fixing carrier of the present invention.
[Production Example 1: Synthesis of Polymer A]
0.564 parts by weight of N-acryloylmorpholine (NAM, manufactured by KOHJIN) as a monomer, 0.169 parts by weight of N-acryloyloxysuccinimide (NAS, manufactured by ACROS ORGANICS), and dehydrated dioxane (Wako Pure Chemical) as a solvent 8.75 parts by weight (manufactured by Kogyo Co., Ltd.) were mixed well, poured into a 50 mL four-necked flask, and deaerated with nitrogen at room temperature for 30 minutes to prepare a monomer solution. The monomer solution was heated to 60 ° C. in an oil bath, and a solution obtained by dissolving 0.008 parts by weight of a polymerization initiator azobisisobutyronitrile (AIBN, manufactured by Kishida Chemical Co., Ltd.) in 0.5 g of dehydrated dioxane was added. The polymerization was started. The polymerization was carried out for 8 hours under a nitrogen atmosphere.

After the polymerization, the solution in which the polymer was produced was reprecipitated by dropping it into 0.5 L of diethyl ether (manufactured by Kokusan Chemical Co., Ltd.), and then pulverized by removing the solvent to obtain polymer A.
As a result of performing SEC measurement calibrated with standard polystyrene, the obtained polymer A was estimated to have a weight average molecular weight (Mw) of about 86000.
The molar ratio (NAS / NAM) between NAS and NAM contained in the obtained polymer A was estimated to be NAS / NAM = 30/70 from NMR measurement.

[Production Example 2: Synthesis of polymer B]
Instead of NAM and NAS, 0.793 parts by weight of dimethylacrylamide (DMAA, manufactured by KOHJIN) and 0.338 parts by weight of NAS were used, and the amount of dehydrated dioxane used as a solvent was 18.37 parts by weight. A polymer B was obtained in the same manner as in Production Example 1 (synthesis of polymer A) except that the amount of AIBN used as a polymerization initiator was 0.00164 parts by weight.

As a result of performing SEC measurement calibrated with standard polystyrene, the obtained polymer B was estimated to have a weight average molecular weight (Mw) of about 26000.
The molar ratio (NAS / DMAA) between NAS and DMAA contained in polymer B was estimated to be NAS / DMAA = 43/57 from NMR measurement.

[Surface treatment of sensor chip]
Next, a solid phase carrier serving as a substrate of the biological substance-fixing carrier of the present invention was prepared.
[Production Example 3: Sensor chip A]
For the sensor chip for measurement, a gold-coated sensor chip in which gold is deposited at a thickness of about 80 nm on the surface of a smooth, flat plastic substrate having a size of 2.5 cm long × 2.5 cm wide × 1.2 mm thick. Using.
The gold-coated sensor chip was immersed in 10 mM 16-mercaptohexadecanoic acid (16-MERCAPTOCHEXADECANIC ACID; manufactured by ALDEICH) ethanol solution, reacted at 60 ° C. for 2 hours, and subjected to surface treatment. After completion of the reaction, the gold-coated sensor chip was washed with ethanol and demineralized water. In this surface treatment, a carboxyl group is introduced into the gold-coated sensor chip surface via a gold-thiol bond.

  Next, 1 mL of 0.1 M N-hydroxysuccinimide (NHS, manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution and 0.4 M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC, A gold-coated sensor chip having a carboxyl group introduced therein was immersed in a solution mixed with 1 mL of an aqueous solution (produced by Dojindo Laboratories) and diluted with 18 g of demineralized water, and allowed to react for 15 minutes. The obtained gold-coated sensor chip into which the succinimide group was introduced was referred to as sensor chip A.

[Production of support]
A support for preparing the biological material-immobilized carrier of the present invention was prepared by the following method.
[Production Example 4: Latex immobilized with bovine serum albumin]
200 μL of 10% polystyrene latex aqueous solution (manufactured by Nippon Synthetic Rubber Co., Ltd., particle size 0.101 μm), 0.1% bovine serum albumin (manufactured by SIGMA), 0.1M phosphate buffer (pH 7.4: 0.15 M sodium chloride) (Hereinafter, phosphate dilution buffer) 800 μL was mixed, stirred at room temperature for 30 minutes, centrifuged, and the supernatant was removed. Then, 0.1% bovine serum albumin (manufactured by SIGMA), 0.15 M was added to the precipitate. 200 μL of 0.1 M phosphate buffer (pH 7.4) containing sodium chloride was added to obtain a 10% bovine serum albumin-immobilized latex solution (support).

[Production of sensor chip for measurement]
As described below, biosensor chips 1 to 6 were prepared as the biological substance-immobilizing carrier of the present invention. In addition, comparative biosensor chips 1 to 4 were prepared.
[Production Example 5: Biosensor chip 1]
The biosensor chip 1 is fixed by dropping a mixture of polymer A (compound capable of binding to a biological substance), 1% mouse IgG (biological substance), and latex (support) onto the sensor chip A. It was made to make.
First, 10% of the 10% bovine serum albumin-immobilized latex solution (particle size: 0.101 μm: support) is mixed with the polymer A (immobilizing compound) using 0.1 M phosphate buffer (pH 7.2). 10 μL of the aqueous polymer A solution prepared to 1% and 100 μL of 1% mouse IgG (LAMPiREBIOLOGICAL LABORATORIES; bio-related substance) aqueous solution were mixed, and 0.5 μL of the mixed solution was dropped onto the sensor chip A, 37 The mixture was fixed at 30 ° C. under saturated steam pressure for 30 minutes. Thereafter, the solvent was naturally dried at room temperature for 15 hours to form a matrix, and then 0.5% ethanolamine hydrochloride (SIGMA, pH 8.5) containing 3% bovine serum albumin (SIGMA) was formed. ) It was immersed in an aqueous solution for 30 minutes to block unreacted succinimide groups, and the substrate was washed with demineralized water and dried to obtain a biosensor chip 1 as a biological material fixing carrier of the present invention.

[Production Example 6: Biosensor chip 2]
The biosensor chip 2 was produced in the same manner as the biosensor chip 1, but the latex concentration was set to 4 levels, and 4 types of biosensor chips 2 (a) to (d) were produced.
First, 10 μL of the above-described bovine serum albumin-immobilized latex solution (particle size: 0.101 μm: support) prepared in 0%, 2%, 10%, and 40% using the phosphate dilution buffer and the polymer A (Immobilization compound) 10 μL of an aqueous solution prepared to 1% using the above phosphate buffer and 100 μL each of the above mouse IgG prepared to 1% using the above phosphate buffer, respectively, 0.5 μL of the mixed solution was dropped onto the sensor chip A and fixed at 37 ° C. under saturated water vapor pressure for 30 minutes. Thereafter, natural drying, blocking, and washing were performed in the same manner as in Production Example 5 (production of biosensor chip 1) to produce a biosensor chip. The biosensor chip as the biomaterial-fixing carrier of the present invention thus produced was designated as biosensor chip 2.

[Production Example 7: Biosensor chip 3]
The biosensor chip 3 is obtained by immobilizing polymer A, mouse antibody F (ab ′) 2 (biologically related substance), and latex on the sensor chip A. Polymer A and latex were each set to two concentrations, and four types of biosensor chips 3 (a) to (d) were produced.

  First, 10 μL of the aqueous solution prepared by fixing the bovine serum albumin-immobilized latex solution (particle size: 0.101 μm: support) to 0% and 10% using the phosphate dilution buffer and the polymer A (immobilizing compound), respectively. 10 μL of an aqueous solution prepared to 1% using the above-mentioned phosphate buffer and 3.8% respectively, and mouse antibody F (ab ′) 2 (manufactured in-house, bio-related substance) using the above-mentioned phosphate buffer Each of them was mixed with 100 μL of an aqueous solution prepared to 1%, and 0.5 μL of the mixed solution was dropped onto the sensor chip A and fixed at 37 ° C. under saturated water vapor pressure for 30 minutes. Thereafter, natural drying, blocking, and washing were performed in the same manner as in Production Example 5 (production of biosensor chip 1) to produce a biosensor chip. The biosensor chip as the biomaterial-fixing carrier of the present invention thus produced was designated as biosensor chip 3.

[Production Example 8: Biosensor chip 4]
The biosensor chip 4 was produced in the same manner as the production method of the biochip 1 described in the above Production Example 5, but a mixture of two types of particle sizes was used as the latex used as the support.
As polystyrene latex aqueous solution, 7.5% of 20% bovine serum albumin immobilized latex solution (manufactured by Nippon Synthetic Rubber, particle size 0.101 μm) and 40% bovine serum albumin immobilized latex solution (manufactured by Nippon Synthetic Rubber, particle size) 3.26 μm) A mixed latex solution with 2.5 μL was used. Preparation of 40% bovine serum albumin-immobilized latex solution (particle size 3.26 μm) was a method of preparing 20% bovine serum albumin-immobilized latex solution (manufactured by Nippon Synthetic Rubber Co., Ltd., particle size 0.101 μm) in Production Example 4. And performed in the same manner.
Otherwise, biosensor chip 4 was produced in the same manner as in Production Example 5 (production of biosensor chip 1).

[Production Example 9: Biosensor chip 5]
The biosensor chip 5 was produced in the same manner as the production of the biochip 4 described in Production Example 8 above, but polymer B was used as the compound.
A biosensor chip 5 was produced in the same manner as in Production Example 8 (production of the biosensor chip 4) except that the polymer B was used in place of the polymer A as the immobilizing compound.

[Production Example 10: Biosensor chip 6]
The biosensor chip 6 was produced using the sensor chip A mixed with the polymer A, anti-HBs antigen mouse monoclonal antibody F (ab ′) 2 (biologically related substance), and latex.

  As a biological substance, 100 μL of anti-HBs antigen mouse monoclonal antibody F (ab ′) 2 (manufactured) was adjusted to 1% aqueous solution using the phosphate buffer instead of mouse IgG aqueous solution. The concentration of the polymer A aqueous solution was 1%. Otherwise, biosensor chip 6 was produced in the same manner as in Production Example 5 (production of biosensor chip 1).

[Comparative Production Example 1: Biosensor chip 1 for comparison]
The biosensor chip 1 for comparison is one in which only 1% mouse IgG (biologically related substance) is directly immobilized on the sensor chip A without using a compound and a support.
Production Example 5 (Biosensor) except that 0.5 μL of 1% mouse IgG aqueous solution (aqueous solution of biological substances) was used instead of the mixed solution of bovine serum albumin-immobilized latex solution, polymer A aqueous solution, and mouse IgG aqueous solution. A biosensor chip was produced in the same manner as in Production of Chip 1). The biosensor chip thus produced was used as a comparative biosensor chip 1.

[Comparative Production Example 2: Biosensor chip 2 for comparison]
The comparative biosensor chip 2 was prepared by mixing 1% mouse IgG (biologically relevant substance) and polymer A and immobilizing the sensor chip A without using a support.
Instead of the mixed solution of bovine serum albumin-immobilized latex solution, polymer A aqueous solution, and mouse IgG aqueous solution, 10 μL of 1% polymer A aqueous solution and 100 μL of 1% mouse IgG aqueous solution are mixed, and 0.5 μL of the mixed solution is used. Thus, a biosensor chip was produced in the same manner as in Production Example 5 (production of biosensor chip 1). The biosensor chip thus produced was used as a comparative biosensor chip 2.

[Comparative Production Example 3: Biosensor chip 3 for comparison]
The biosensor chip 3 for comparison was prepared by mixing 1% mouse IgG (biologically relevant substance) and latex solution (support) and immobilizing the sensor chip A without using a compound.
200 μL of 10% polystyrene latex aqueous solution (manufactured by Nippon Synthetic Rubber Co., Ltd., particle size 0.101 μm), 0.1% mouse IgG (manufactured by LAMPiREBIOLOGICAL LABORATORIES; bio-related substance), and 0.1M phosphate buffer containing 0.15M sodium chloride (PH 7.4: phosphoric acid dilution buffer) 800 μL was mixed, stirred at room temperature for 30 minutes, and then centrifuged to remove the supernatant. 0.1% bovine serum albumin (manufactured by SIGMA) and 0.1 μM sodium chloride-containing 0.1M phosphate buffer (pH 7.4) (200 μL) were added to the resulting precipitate to obtain a 10% mouse IgG immobilized latex solution. .

  Instead of the mixed solution of bovine serum albumin-immobilized latex solution, polymer A aqueous solution, and mouse IgG aqueous solution, 10 μL of the above 10% mouse IgG-immobilized latex solution and 100 μL of 0.1 M phosphate buffer (pH 7.2) are mixed. A biosensor chip was produced in the same manner as in Production Example 5 (production of biosensor chip 1) except that 0.5 μL of the mixed solution was used. The biosensor chip thus produced was used as a comparative biosensor chip 3.

[Comparative Production Example 4: Biosensor chip 4 for comparison]
The biosensor chip 4 for comparison was prepared by mixing the anti-HBs antigen mouse monoclonal antibody F (ab ′) 2 (biologically relevant substance) and the polymer A and immobilizing the sensor chip A without using a support.
Instead of the mixed solution of bovine serum albumin-immobilized latex solution, polymer A aqueous solution and anti-HBs antigen mouse monoclonal antibody F (ab ′) 2 aqueous solution, 10 μL of polymer A aqueous solution prepared to 1% and 1% anti-HBs antigen mouse A biosensor chip was produced in the same manner as the production method of biochip 6 described in Production Example 10 except that 100 μL of the monoclonal antibody F (ab ′) 2 aqueous solution was mixed and 0.5 μL of the mixed solution was used. The biosensor chip thus produced was used as a comparative biosensor chip 4.

[Example 1: Measurement of mouse IgG by chemiluminescence method]
Using the biosensor chip produced in Production Example 5 (Biosensor chip 1) to 8 (Biosensor chip 4) and Comparative Production Example 1 (Comparative biosensor chip 1) to 3 (Comparative biosensor chip 3) Then, chemiluminescence measurement for detecting antigen-antibody reaction between mouse IgG and anti-mouse IgG was performed.

As a specimen (analyte, active substance), rabbit anti-mouse IgG (manufactured by Immunoprobe) labeled with biotin was used. Rabbit anti-mouse IgG was biotinylated as follows. First, EZ-Link ^™ Sulfo-NHS-LC-Biotin (manufactured by PIERCE) was added to 100 μL of a biotin aqueous solution prepared to 1 mg / mL with MilliQ water, and 1 mg / mL with 0.05 M carbonate buffer (pH 8.5). 1 mL of the prepared rabbit anti-mouse IgG was added and reacted in ice water for 2 hours. After the reaction, 100 μL of 1M glycine buffer (pH 8.0) was added to this solution, reacted for 2 hours in ice water, and then dialyzed overnight at 4 ° C. against 0.1M phosphate buffer (pH 7.4). To remove unreacted labeling reagent to obtain biotinylated rabbit anti-mouse IgG.

  Each biosensor chip is immersed in 4 mL of a solution prepared by adding the above biotinylated anti-mouse IgG to a predetermined concentration using the above-mentioned phosphate dilution buffer, and reacted for 30 minutes at room temperature with shaking. The biosensor chip was washed with 0.01 M phosphate buffer (pH 7.4: hereinafter wash buffer) containing 01% Tween 20 and MilliQ water. This chip was immersed in 4 mL of Neutra-Avidin-HRP (manufactured by PIERCE) aqueous solution adjusted to 50 ng / mL with a phosphate dilution buffer, reacted at room temperature for 30 minutes with shaking, and then washed with washing buffer and MilliQ. The biosensor chip was washed with water and dried. To this was added 1 mL of SuperSignal ELISA Femto Maximum Sensitivity Substrate (manufactured by PIERCE), and the amount of chemiluminescence was measured by imaging using a CCD camera (ORCAII-ER: manufactured by Hamamatsu Photonics) at an integration time of 60 seconds.

[Example 2: Measurement of HBs antigen by chemiluminescence method]
Using the biosensor chip produced in Production Example 10 and Comparative Production Example 4, chemiluminescence measurement was performed to detect antigen-antibody reaction between HBs antigen and anti-HBs antibody.
Rabbit anti-HBs antigen F (ab ′) 2 (immunoprobe) labeled with biotin as a labeled body for HBs antigen measurement using a recombinant HBs antigen (subtype adw: self-made) as a specimen (analyte, agent) Used). Rabbit anti-HBs antigen F (ab ′) 2 was biotinylated in the same manner except that rabbit anti-HBs antigen F (ab ′) 2 was used instead of rabbit anti-mouse IgG in Example 1.

  Each biosensor chip is immersed in 4 mL of a solution prepared by adding the above-mentioned recombinant HBs antigen to a predetermined concentration using the above-mentioned phosphate dilution buffer, and reacted for 60 minutes at room temperature with shaking. The biosensor was washed with MilliQ water. The biotinylated rabbit anti-HBs antigen F (ab ′) 2 is immersed in 4 mL of an aqueous solution adjusted to 2 μg / mL with a phosphate dilution buffer, reacted at room temperature for 30 minutes with shaking, and washed with a buffer. And the biosensor chip was washed with MilliQ water. Next, after immersing Neutra-Avidin-HRP (manufactured by PIERCE) in 4 mL of an aqueous solution adjusted to 50 ng / mL with a phosphoric acid dilution buffer and reacting at room temperature for 30 minutes with shaking The biosensor chip was washed with a washing buffer and MilliQ water and dried. To this was added 1 mL of SuperSignal ELISA Femto Maximum Sensitivity Substrate (manufactured by PIERCE), and the amount of chemiluminescence was measured by imaging using a CCD camera (ORCAII-ER: manufactured by Hamamatsu Photonics) at an integration time of 60 seconds.

[Chemiluminescence Measurement Result 1: Measurement of Mouse IgG by Chemiluminescence Method: Analysis of Biosensor Chip of the Present Invention]
Table 1 shows the chemiluminescence measurement results measured using the biosensor chip 1 and the comparative biosensor chips 1 to 3. From these results, it was confirmed that the biosensor chip 1 has high emission intensity and high reproducibility (small CV value) compared to any of the comparison biosensor chips 1, 2, and 3. .
Each emission intensity is an average value of 8 spots obtained by dropping each mixed solution in 8 different areas for each chip, and CV is CV of 8 spots.

  4A and 4B show images of a CCD camera at the time of chemiluminescence measurement at a biotinylated anti-mouse IgG concentration of 1 ng / mL. 4A shows the biosensor chip 1 at the time of chemiluminescence measurement, and FIG. 4B shows the comparison biosensor chip 2 at the time of chemiluminescence measurement. In FIGS. 4A and 4B, the white part is the part emitting chemiluminescence. As can be seen from this figure, in the chemiluminescence measurement, the comparative biosensor chip 2 shows ring-like light emission, and the light emission is uneven, whereas the biosensor chip 1 does not show ring-like light emission, It can be seen that there is little unevenness in light emission. This also shows that the biochip produced by this method has high accuracy.

[Chemiluminescence Measurement Result 2: Measurement of Mouse IgG by Chemiluminescence Method: Analysis of Support Concentration Dependence in Biosensor Chip of the Present Invention]
Using the biosensor chips 2 (a) to 2 (d) produced in Production Example 6 and having different support concentrations, the influence of changes in the support concentration was examined. The results of chemiluminescence measurement are shown in Table 2.
From these results, it was confirmed that as the support concentration increases, the emission intensity increases and the reproducibility (CV) increases. However, if the concentration is too high, a blank value (biotinylated anti-mouse IgG concentration is 0 ng). (luminescence intensity at / mL) also increased, and it was found that there was an optimal support concentration.
Each emission intensity is an average value of 8 spots obtained by dropping each mixed solution in 8 different areas for each chip, and CV is CV of 8 spots.

[Chemiluminescence Measurement Result 3: Measurement of Mouse F (ab ′) 2 by Chemiluminescence Method: Analysis of Compound Concentration Dependence in Biosensor Chip of the Present Invention]
Using the biosensor chips 3 (a) to 3 (d) produced in Production Example 7, the effects of the presence or absence of the support and the concentration change of the immobilizing compound were examined. The chemiluminescence measurement results are shown in Table 3.
In the absence of the support, it was confirmed that the emission intensity increased as the concentration of the immobilizing compound increased, but the reproducibility (CV) was low. In the presence of the support, it was confirmed that the emission intensity slightly increased and the reproducibility (CV) also improved as the concentration of the immobilizing compound increased. From these results, it was found that there was an optimal support and immobilized compound concentration.
Each emission intensity is an average value of 8 spots obtained by dropping each mixed solution in 8 different areas for each chip, and CV is CV of 8 spots.

[Chemiluminescence measurement result 4: Measurement of mouse IgG by chemiluminescence method: analysis of influence of particle size of support]
Table 4 shows the chemiluminescence measurement results of biosensor chip 4 using latex particles having different particle diameters produced in Production Example 8. From this result, it was confirmed that the biosensor chip 4 had higher emission intensity and higher reproducibility (CV) than the comparative biosensor chip 1.
Each emission intensity is an average value of 8 spots obtained by dropping each mixed solution in 8 different areas for each chip, and CV is CV of 8 spots.

[Chemiluminescence Measurement Result 5: Measurement of Mouse IgG by Chemiluminescence Method: Analysis of Biochips Made of Different Polymers]
Effect of immobilizing compound on blank value (luminescence intensity at biotinylated anti-mouse IgG concentration of 0 ng / mL) using biosensor chip 4 produced in Production Example 8 and biosensor chip 5 produced in Production Example 9 I investigated. The biosensor chip 4 is manufactured using the polymer A, and the biosensor chip 5 is manufactured using the polymer B. The chemiluminescence measurement results are shown in Table 5.

It was found that the polymer B had a lower blank value than the polymer A, and as a result, the S / N ratio (= luminescence intensity / blank value at a biotinylated anti-mouse IgG concentration of 1 ng / mL) was increased. From these results, it was found that the blank value can be suppressed or the S / N ratio can be increased by appropriately selecting and using a compound according to the purpose.
In addition, each light emission intensity is an average value of a pot in which each mixed solution is dropped in 8 different areas for each chip, and CV is CV of 8 spots.

[Chemiluminescence measurement result 6: Measurement of HBs antigen by chemiluminescence method]
Using the biosensor chip 6 produced in Production Example 10 and the comparative biosensor chip 4 produced in Comparative Production Example 4, chemiluminescence measurement of antigen-antibody reaction (interaction) against HBs antigen was performed. The chemiluminescence measurement results are shown in Table 6.
According to this, it was confirmed that the biosensor chip 6 had higher emission intensity and higher reproducibility (CV) than the comparative biosensor chip 4. Thus, it was confirmed that the present method is effective even when the biological material and the measurement system are different.
In addition, each light emission intensity is an average value of a pot in which each mixed solution is dropped in 8 different areas for each chip, and CV is CV of 8 spots.

[Example 3: Observation by SEM]
The matrix structure of the biosensor chip 1 produced in Production Example 5, the biosensor chip 3 (b) produced in Production Example 7 and the biosensor chip 4 produced in Production Example 8 was subjected to SEM after evaluation in the above examples. And observed. Moreover, the film thickness and porosity of the biosensor chip 1 were calculated using the obtained electron micrograph.

A photograph showing the surface of the biosensor chip 1 observed by SEM (magnification × 4000 and × 60000) is shown in FIG. 5, and a photograph showing a cross section (magnification × 50000 and × 5000) is shown in FIGS. Moreover, the cross-sectional photograph (magnification x5000) of the biosensor chip 4 is shown in FIG. 8, and the cross-sectional picture (magnification x50000) of the biosensor chip 3 (b) is shown in FIG.
In these photographs, the portion that appears white is the matrix skeleton.

(1) Calculation of film thickness The film thickness in the dry state of the biosensor chip 1 was calculated from the cross-sectional view of FIG. As a result, the thickness of the biosensor chip 1 was about 3 μm. Further, a cross-sectional view of the biosensor chip 4 in which the film thickness was changed by mixing and using two types of latex particles was calculated from FIG. 8, and the film thickness of the biosensor chip 4 was about 9 μm. .
This indicates that biosensor chips having different film thicknesses can be produced by using particles having different particle diameters as a support, and it was shown that biosensor chips having an arbitrary film thickness can be produced.

(2) Calculation of porosity The cross-sectional view of the biosensor chip 1 in FIG. 6 and the cross-sectional view of the biosensor chip 3 (b) in FIG. 9 (in the case of 10% support and 3.8% immobilizing compound) The image analysis using the image programmer (MEDIA-CYBERNETICS company: Image-Pro-Plus ver. 4.0) was performed. Based on the image density information, the porosity of each biosensor chip was measured.

(2-1) Image input The SEM photograph was input at 240 DPI from a scanner. The image size was 1019 × 764 Pixel and the calibration value was 2.17 nm / Pixel.
(2-2) Image processing The median filter 3 × 3 was used 5 times to remove noise. A background image was created (background image creation conditions: “bright” “object width · 20”), and shading correction (correction by division) was performed.
(2-3) Area ratio measurement Set the particle region, binarize the image with a fixed threshold (concentration 30%), and then change the shape of the hole by morphological processing (connection 5 × 5, once, hole filling) Correction and area ratio were measured.

From this analysis result, the porosity of the biosensor chip 1 was 23.2%. Moreover, the porosity of the biosensor chip 3 (b) (in the case of 10% support and 3.8% immobilization compound) in which the concentration of the immobilization compound is 3.8 times is about 16.3%. I understood that.
From the results of the above (1) and (2), by changing the concentrations of the support, the immobilization compound and the biological substance as appropriate, the film thickness and porosity can be changed as appropriate, and biosensors having different properties It was shown that the chip can be made arbitrarily.

  The biological substance-immobilized carrier of the present invention and the method for producing the biological substance-immobilized carrier of the present invention can be used in any industrial field, and among them, for example, are suitably used for biosensors, diagnostic devices, and the like.

It is a schematic diagram which expands and shows sectional drawing of an example of the structure of the matrix of this invention. (A) is a schematic diagram of a well-like carrier having a solid phase carrier surface with a concave portion, and (b) is a schematic diagram of a pile-like carrier with a convex portion. It is a schematic diagram which shows the formation process of the matrix structure of this invention. The image by the CCD camera at the time of the chemiluminescence measurement obtained in the chemiluminescence measurement result 1 is shown. (A) is a copy of the biosensor chip 1, and (b) is a copy of the comparative biosensor chip 2. The photograph which observed the surface of the matrix of the biosensor chip 1 of the present invention produced in Production Example 5 with an SEM is shown. (A) is a photograph at a magnification of 4000 times, and (b) is a photograph at a magnification of 60000 times. The photograph which observed the cross section of the matrix of the biosensor chip 1 of this invention produced by the manufacture example 5 of this invention by SEM is shown. The magnification is 50000 times. The photograph which observed the cross section of the matrix of the biosensor chip 1 of this invention produced by the manufacture example 5 of this invention by SEM is shown. The magnification is 5000 times. The photograph which observed the cross section of the matrix of the biosensor chip 4 of this invention produced by the manufacture example 8 of this invention by SEM is shown. The magnification is 5000 times. The photograph which observed the cross section of the matrix of the biosensor chip 3 (b) of this invention produced by the manufacture example 7 of this invention by SEM is shown. The magnification is 50000 times.

Claims (17)

Biological substance, immobilization compound, a solid phase carrier, wherein a matrix containing及beauty supporting bearing member is formed on the surface,
The biological substance is a target substance for measuring the interaction between the detection target substance in the specimen and the biological substance,
The immobilizing compound is
1 molecule bondable functional group binding to one or more locations of the biological-related substance in, and have a bondable functional group binding to one or more locations of the support, and a first Ru der polymer compound An immobilizing compound;
A second immobilizing compound having a binding functional group capable of binding to two or more biorelated substances in one molecule and being a polymer compound ;
The matrix is
A structure having a main chain composed of the biologically relevant substance and the immobilizing compound with the support as a nucleus;
The said first immobilized compound relative to the support and biological-related substance takes a first crosslinked structure linked by the linking functional group,
The second immobilizing compound is bound to the biological substance by the binding functional group to form a second bridge structure,
A solid phase carrier characterized by having a chain-like and / or network-like, block-like structure by repeating the bridge structure comprising the first bridge structure and the second bridge structure . The solid phase carrier according to claim 1, wherein the matrix has voids. The matrix is formed by supplying a mixture containing the support, the biological substance, and the immobilizing compound in the presence of a solvent to the surface of the solid phase carrier, and then removing the solvent. The solid-phase carrier according to claim 1 or 2 , wherein The solid phase carrier according to claim 3 , wherein the solvent is water. Solid support according to any one of claims 1 to 4, wherein the porosity of said matrix is 5% or more. Wherein the thickness of the matrix is 20nm or more in a dry state, the solid phase support of any one of claims 1-5. The immobilization compound, with miscible in water, and wherein the miscible with at least one organic solvent, a solid carrier according to any one of claims 1-6. Said support, a solid phase carrier according to any one of claims 1 to 7, characterized in that a particle having an average diameter of 10 nm to 100 [mu] m. A method for producing the solid phase carrier according to any one of claims 1 to 8 , wherein a mixture comprising the support, the biological substance, and the immobilizing compound coexisting in the presence of a solvent is prepared. A method for producing a solid phase carrier, wherein the matrix is formed by removing the solvent after being supplied to the surface of the solid phase carrier. A biological substance-immobilized kit for preparing a solid support according to any one of claims 1-8, characterized in that it comprises at least the support and the immobilized compound kit. Biological substance, immobilization compounds, matrices containing及beauty supporting bearing member is at least 2 or more, a an array of biological substances, characterized by being arranged in separate regions on a solid support And
The biological substance is a target substance for measuring the interaction between the detection target substance in the specimen and the biological substance,
The immobilizing compound is
1 molecule bondable functional group binding to one or more locations of the biological-related substance in, and have a bondable functional group binding to one or more locations of the support, and a first Ru der polymer compound An immobilizing compound;
A second immobilizing compound having a binding functional group capable of binding to two or more biorelated substances in one molecule and being a polymer compound ;
The matrix is
A structure having a main chain composed of the biologically relevant substance and the immobilizing compound with the support as a nucleus;
The said first immobilized compound relative to the support and biological-related substance takes a first crosslinked structure linked by the linking functional group,
The second immobilizing compound is bound to the biological substance by the binding functional group to form a second bridge structure,
Chain by repeated crosslinked structure consisting of said Ichinohashi bridged structure and said Ninohashi bridged structure and / or mesh, and having a block-like structure, the array. Characterized in that it comprises an array according to a solid support and / or claim 11 according to any one of claims 1-8, biosensor. Characterized in that it comprises an array according to a solid support and / or claim 11 according to any one of claims 1-8, diagnostic device. A method for assaying for at least one analyte in a sample comprising the following steps (a) and (b):
(A) a specimen, deliver the array according to a solid support and / or claim 11 according to any one of claims 1-8 comprising at least one of bio-related substance capable of reacting with the analyte And (b) detecting the interaction between the biological substance and the analyte, or the reaction caused by adding a labeling substance that reacts with the analyte, thereby determining the presence or amount of the analyte. A method comprising the step of detecting. 15. The method of claim 14 , wherein at least the analyte is a fluid and the analyte is delivered by flow. Detecting the reaction in the reference region provided on the array according to a solid support and / or claim 11 according to any one of claims 1-8, judging success or failure of the assay, or the detected analyzed 16. A method according to claim 14 or 15 , further comprising the step of calibrating the presence or amount of an object. In order to assay two or more analytes in parallel, the following further step (c):
(C) the presence or amount of at least two or more analytes, the method according to any one of claims 14 to 16, characterized in that it comprises a step of associating a particular condition.