JP2007101520A - Biological material complex, biological material complex carrier, method for purifying object material, container for affinity chromatography, chip for separation, separation apparatus and method for analyzing object material, and sensor chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological material complex capable of containing a desired specific material more, in comparison with the conventional types. <P>SOLUTION: A method for purifying an object material is provided and characterized by preparing a biological material complex obtained, by bonding a biological material structure which is composed of particulate clusters, each having the particle size of 10 μm or smaller and being made up by coupling a biological material to a compound couplable to the biological material, to a specific material other than the biological material and the compound; contacting the biological material complex with a sample liquid, containing the object material which can be specifically adsorbed to the specific material; separating the biological material complex from the sample liquid; and making the object material freed, that is coupled with the specific material. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biological material complex, and a biological material complex carrier using the same, a purification method for the target substance, a container for affinity chromatography, a separation chip, a method for analyzing the target substance, and a separation for analysis of the target substance The present invention relates to a device and a sensor chip.

  The biological material structure can be used for, for example, medical treatment / diagnosis, gene analysis, and proteomics, and is particularly suitable for use as an affinity purification and pharmaceutical action analysis tool. Several structures used for such affinity purification and drug action analysis tools have been reported so far.

  Examples of conventionally used carriers for affinity chromatography include inorganic silica materials such as porous silica gel particles, and natural polymers that include polysaccharide particles such as agarose, dextran, and cellulose. In synthetic polymers, particles made of polystyrene, polyacrylamide or the like are used.

  However, when affinity purification or the like is performed using these conventional affinity chromatography supports, it is often difficult to suppress nonspecific adsorption to the affinity chromatography support. The recovery efficiency was low.

  In recent years, as a method for solving these problems, an affinity purification method using latex fine particles has been proposed as described in Patent Document 1. Since this method uses the Brownian motion of latex particles, specific adsorption of the target purified product to the surface of the latex fine particles is efficiently performed, and further, the latex adsorbed by the target product by centrifugation is collected. There is an advantage that less is required.

On the other hand, a technique of performing separation by magnetic force without performing a centrifugal operation has been proposed, and in order to realize this, development of latex containing a magnetic material has also been performed (Patent Document 2, Non-Patent Document 1). .
Furthermore, Patent Document 3 reports biochemical fine particles in which antibodies or antigens are bound to an aggregate obtained by insolubilizing albumin with glutaraldehyde.

Japanese Patent Laid-Open No. 10-195099 JP 2003-327784 A Japanese Patent No. 2834609 Masaki Abe, Hiroshi Handa, BIO INDUSTRY, Vol.21, No.8, p.7.

However, in the technique described in Patent Document 1, the centrifugation operation and the like are complicated, and it is difficult to automate and unmanned, and in the fields of drug discovery and clinical tests that require high sample throughput in recent years. Can be irrational.
In addition, in the techniques described in Patent Document 2 and Non-Patent Document 1, it is necessary to completely encapsulate a magnetic material and suppress nonspecific adsorption of proteins, and when using iron oxide or the like as a magnetic material, It is necessary to suppress elution of iron ions. In addition, since it is a magnetic substance-encapsulated latex particle having a large specific surface area, it is difficult to maintain dispersion stability. Moreover, it is difficult to produce both latex particles and magnetic latex particles, and there remains a problem for mass production and quality maintenance for industrialization.

  Further, in the technique of Patent Document 3, there is a possibility that the protein is denatured by using a low molecular weight compound such as glutaraldehyde, there is a risk of non-specific adsorption to the denatured protein, and the structure is tightly bound. As a result, there is a possibility that an effective space cannot be formed in the interior and the reactivity is lowered, and there is a possibility of nonspecific adsorption by a hydrophobic compound such as glutaraldehyde.

  Therefore, in the fields of medicine, diagnosis, genetic analysis, and proteomics, especially in the development of affinity purification or drug action analysis tools, members that can be used as affinity chromatography carriers that have low non-specific adsorption and can be processed in high efficiency and in a short time The development of was requested.

  The present invention was devised in view of the above problems, and provides a biological material complex that can contain a desired specific substance in a larger amount than before and a biological material complex carrier having the same. The target substance purification method, affinity chromatography container, separation chip, target substance analysis method, target, which enables non-specific adsorption to be suppressed and enables separation or analysis of the target substance with high efficiency and ease An object of the present invention is to provide a separation device and a sensor chip for analyzing substances.

  As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention have found that a biological material structure is formed by binding together a particulate mass formed by binding a biological material and a compound capable of binding to the biological material. In addition, the present inventors have found that nonspecific adsorption can be suppressed by binding a desired specific substance that can interact with an analyte, and that highly efficient reactivity and highly efficient affinity separation can be achieved. .

  That is, the gist of the present invention is that a biological material structure formed by binding a biological material and a compound capable of binding to the biological material, in which particulate lumps having a particle size of 10 μm or less are bonded to each other, The biological substance complex is characterized in that the substance and a specific substance other than the compound are bound (claim 1). Thereby, it is possible to provide a biological material complex carrying a larger amount of the specific substance than before while maintaining the characteristics of the specific substance. In addition, the specific surface area can be increased and highly efficient reactivity can be realized.

  Moreover, it is preferable that a space is formed between the particulate lumps (Claim 2). This makes it possible to use this space as a reaction field that allows a specific substance to act effectively.

  Furthermore, the ratio of the weight of the biological material to the weight of the biological material complex is preferably 0.1 or more (claim 3). As a result, the biomaterial content ratio in the biomaterial complex can be increased, and the content ratio of the specific substance can be increased accordingly. It becomes possible to utilize it.

  Moreover, it is preferable that the biological material complex of the present invention has a diameter of 30 nm or more in a dry state. By setting the size of the biological material complex in the above range, more target substances can be interacted and separated.

  Furthermore, it is preferable that at least one of the compounds has two or more functional groups capable of binding to the biological substance (Claim 5). As a result, the biological material complex can be easily formed.

  The compound is preferably uncharged (Claim 6). Thereby, it can suppress that a biological material complex produces non-specific interaction with substances, such as a target substance. Here, the non-specific interaction is the target adsorption or interaction when a predetermined adsorption or interaction is caused between a specific substance and a target substance using a biological substance complex. This refers to interactions that occur outside of.

  Further, the compound is preferably miscible with water and miscible with at least one organic solvent (claim 7). Thereby, the range of the solvent which can be used at the time of manufacture of a biological material composite can be expanded, and a biological material composite can be designed variously. Also, when using some kind of solvent or dispersion medium when using the biological material complex, the number of types of solvents and dispersion media that can be used can be increased. Will be able to.

  The molecular weight of the compound is preferably 1000 or more (claim 8). As a result, it is possible to prevent internal cross-linking with respect to the biological material when the biological material complex is produced, and it is possible to efficiently produce the biological material complex.

  Furthermore, it is preferable that the diameter of the compound when mixed in the liquid is 1 nm or more (claim 9). This also makes it possible to prevent internal cross-linking with respect to the biological material at the time of producing the biological material complex, and to efficiently produce the biological material complex.

  The biological material is preferably a biomolecule (claim 10), more preferably a protein (claim 11). Thereby, the characteristic of protein can be utilized for the biological material complex of the present invention. Specifically, for example, when albumin is used as the biological material, nonspecific adsorption can be suppressed. In another aspect, when avidin is used as the biological material, a specific biotinylated material can be immobilized easily and in large quantities. In yet another aspect, if protein A is used as the biological material, when an antibody is used as the specific material, the antibody can be easily immobilized in a large amount.

  Furthermore, the specific substance is at least one selected from the group consisting of metal chelates, vitamins, sugars, glutathione, boronic acids, proteins, antigens, nucleic acids, physiologically active substances, lipids, hormones, environmental hormones, and chelating groups. (Claim 12). Among these, the specific substance is more preferably at least one selected from the group consisting of metal chelates, biotin, sugar, glutathione, boronic acid, antibodies, antigens, receptors, physiologically active substances, and chelate-forming groups. When a metal chelate, glutathione, and saccharide are used as the specific substance, in the purification of the affinity tag fusion protein, when polyhistidine (His-tag), glutathione-s-transferase (GST), or maltose binding protein is used as the affinity tag, Purification and separation can be performed with effective interaction. Furthermore, if sugar is used as the specific substance, the virus can specifically interact. In addition, by using boronic acid as a specific substance, a sugar chain and a compound containing a sugar, for example, a protein having a sugar chain can be effectively purified or interacted. Furthermore, when an enzyme is used as the specific substance, the substrate can interact and an effective immobilized enzyme can be provided. Further, by using lipid as a specific substance, screening, separation or purification of lipid binding protein can be performed. Furthermore, when a hormone or environmental hormone is used as the specific substance, it is possible to screen for a biological substance that binds to it. Furthermore, when a vitamin such as biotin is used as the specific substance, avidin, streptavidin, or an avidin derivative can be used as the active substance, thereby effectively purifying or interacting with the biotin-avidin bond. In addition, when an antibody or an antigen is used as the specific substance, it is possible to effectively purify or interact the antigen when the specific substance is an antibody, and the antibody when the specific substance is an antigen. Furthermore, if a nucleic acid (for example, a nucleic acid molecule such as DNA or RNA or a peptide nucleic acid such as PNA) is used as the specific substance, the biological substance complex of the present invention can be used for gene interaction analysis. In addition, by using a physiologically active substance such as FK506 (Chemistry & Biology Vol.9 691-698 (2002)) as a specific substance, a binding protein such as FKBP12 is analyzed, or a protein such as a receptor contributing to a disease is analyzed. If used, the biological material complex of the present invention can be used as a tool for elucidating the action mechanism of a drug candidate compound or as a screening tool. Furthermore, if a chelate-forming group is used as the specific substance, it can be used for separation of metal ions and the like.

  Furthermore, it is preferable that the specific substance and the biological substance are bonded via a hydrophilic molecule (claim 13). The hydrophilic molecule preferably contains ethylene oxide (claim 14). Thereby, when affinity separation is performed using the biological material complex of the present invention, the specimen and the specific substance can be effectively interacted with each other.

  Another gist of the present invention resides in a biological material complex-supporting body, wherein the biological material complex is fixed to a solid phase carrier (claim 15). Thereby, it is possible to expand the use form of the biological material complex to a chip (substrate), an array, a bead, a separation membrane, a microtiter plate, a microchannel, a porous membrane, and the like.

  Moreover, in the biological material complex carrier, the thickness of the biological material complex is preferably 5 nm or more (claim 16). By setting the size of the biological material complex within the above range, it is possible to separate more target substances and the like when performing separation using this biological material complex carrier.

  Still another aspect of the present invention is to separate the biological material complex from the sample liquid by bringing the biological material complex into contact with a sample liquid containing a target substance that can be specifically adsorbed to the specific substance. Then, the present invention resides in a method for purifying a target substance, wherein the target substance bound to the specific substance is released (claim 17). As a result, non-specific adsorption can be suppressed, separation can be performed easily with high efficiency, and analysis tools such as affinity purification or pharmaceutical action that suppress non-specific adsorption and diagnostic analysis tools can be realized. can do.

  Still another subject matter of the present invention is an eluent that causes a sample liquid containing a target substance that specifically interacts with the specific substance to flow through the flow path holding the biological material complex and flows out of the flow path. Among them, the present invention resides in a method for purifying a target substance, wherein the fraction containing the target substance is collected (claim 18). This also enables non-specific adsorption to be suppressed, enables high-efficiency and easy separation, and provides an analysis tool for affinity purification or pharmaceutical action that suppresses non-specific adsorption, as well as a diagnostic analysis tool. Can be realized.

  Still another subject matter of the present invention lies in an affinity chromatography container comprising a container main body capable of storing a fluid and the above-described biological material complex held in the container main body ( Claim 19). This also enables non-specific adsorption to be suppressed, enables high-efficiency and easy separation, and provides an analysis tool for affinity purification or pharmaceutical action that suppresses non-specific adsorption, as well as a diagnostic analysis tool. Can be realized.

  Still another subject matter of the present invention lies in a separation chip comprising a substrate having a flow channel formed thereon and the biological material complex held in the flow channel. 20). This also enables non-specific adsorption to be suppressed, enables high-efficiency and easy separation, and provides an analysis tool for affinity purification or pharmaceutical action that suppresses non-specific adsorption, as well as a diagnostic analysis tool. Can be realized.

  Still another subject matter of the present invention is the above-mentioned biological material complex, which is a biological material complex using a specific substance capable of specifically adsorbing a substance having a specific structure, and a sample liquid containing the target substance The biological substance complex and the sample liquid are separated, the amount of the target substance adsorbed on the specific substance is measured, and the structure of the target substance is analyzed. It exists in the analysis method of a substance (claim 21). This also enables non-specific adsorption to be suppressed, enables high-efficiency and easy separation, and provides an analysis tool for affinity purification or pharmaceutical action that suppresses non-specific adsorption, as well as a diagnostic analysis tool. Can be realized.

  Still another subject matter of the present invention is a biological material complex as described above, wherein the target is disposed in a flow path holding a biological material complex using a specific substance that can specifically interact with a substance having a specific structure. Analyzing the target substance characterized in that the sample liquid containing the substance is circulated, the amount of the target substance in the fraction of the eluate flowing out from the flow path is measured, and the structure of the target substance is analyzed It resides in a method (claim 22). This also enables non-specific adsorption to be suppressed, enables high-efficiency and easy separation, and provides an analysis tool for affinity purification or pharmaceutical action that suppresses non-specific adsorption, as well as a diagnostic analysis tool. Can be realized.

  Still another subject matter of the present invention is the above-described biological material composite using a substrate having a channel formed thereon, and a specific substance held in the channel and capable of specifically interacting with a substance having a specific structure. A separation chip having a body, a sample liquid supply unit for circulating a sample liquid containing the target substance in the flow path of the separation chip, and the target substance in the fraction of the eluate flowing out of the flow path And a measuring unit for measuring the amount of the target substance. This also enables non-specific adsorption to be suppressed, enables high-efficiency and easy separation, and provides an analysis tool for affinity purification or pharmaceutical action that suppresses non-specific adsorption, as well as a diagnostic analysis tool. Can be realized.

  Still another subject matter of the present invention is the above-mentioned biological material composite using a substrate having a channel formed thereon and a specific substance that is held in the channel and can specifically interact with a substance having a specific structure. A chip mounting part for mounting a separation chip comprising a body, a sample liquid supply part for circulating a sample liquid containing a target substance in the flow path when the separation chip is mounted on the chip mounting part, and And a measurement unit for measuring the amount of the target substance in the fraction of the eluate flowing out from the flow path. (Claim 24) This also enables non-specific adsorption to be suppressed, enables high-efficiency and easy separation, and provides an analysis tool for affinity purification or pharmaceutical action that suppresses non-specific adsorption, as well as a diagnostic analysis tool. Can be realized.

  Still another subject matter of the present invention lies in a sensor chip comprising the biological material complex described above (claim 25). By using the biological material complex as a sensor chip such as a DNA chip or protein chip, nonspecific adsorption can be suppressed and a highly reactive sensor chip can be realized.

According to the biological material complex and the biological material complex carrier of the present invention, a structure containing a large amount of a specific substance can be obtained while maintaining the reactivity of the specific substance.
In addition, according to the purification method of the target substance, the container for affinity chromatography, the separation chip, the analysis method of the target substance, and the separation apparatus for analysis of the target substance of the present invention, the nonspecific adsorption is suppressed and the high Efficient separation can be easily performed, and purification and analysis can be performed easily and with high accuracy.
Furthermore, according to the sensor chip of the present invention, non-specific adsorption can be suppressed and analysis can be performed with high sensitivity.

  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. .

[I. Biological material complex]
The biological material complex of the present invention is obtained by binding a specific substance to a biological material structure. The biological material complex can also be referred to as a biological material structure formed using a biological material and / or a binding compound to which a specific substance is bound.
The biological material structure is a structure composed of a biological material and a compound capable of binding to the biological material (hereinafter referred to as “binding compound” as appropriate). In the biological material complex, the specific substance is: The biological material structure is bound to the biological material and / or the binding compound. That is, the specific substance may be bonded to only one of the biological substance and the binding compound, or may be bonded to both. In the biological material structure, since there are usually more biological materials than the binding compound, from the viewpoint of binding a larger amount of the specific substance to the biological material complex, the specific substance is at least a biological substance. It is preferable to be bonded.

[I-1. Specific substances]
The specific substance is an element constituting the biological substance complex of the present invention. If the substance is a substance other than the biological substance and the binding compound constituting the biological substance structure to which the specific substance is bound, the specific substance depends on the purpose. As long as the effects of the present invention are not significantly impaired, any substance can be used.
In particular, when the biological substance complex of the present invention is used for purification or analysis of a target substance, the specific substance is usually a predetermined substance (hereinafter, a substance that interacts with the specific substance is referred to as “active substance” as appropriate). ) Should be used.

  For example, when a biological substance complex is used for purposes such as purification of a target substance, a substance that can interact with the active substance is used as the specific substance, and a substance corresponding to the active substance (that is, the specific substance and Substances that can interact) are used as target substances. Then, the target substance is separated and purified using the above interaction.

  In addition, for example, even when the biological substance complex is used for analyzing the target substance, a substance that can interact with the above-described active substance is used as the specific substance. If the target substance and the specific substance interact with each other and the target substance and the specific substance interact with each other, the target substance corresponds to one of the active substances. It can be analyzed that the target substance has a specific structure that causes interaction with the specific substance. Using this, it is possible to analyze the structure of the target substance and the structure of the active substance.

  Here, the “interaction” between the specific substance and the active substance is not particularly limited, but usually a covalent bond, an ionic bond, a chelate bond, a coordination bond, a hydrophobic bond, a hydrogen bond, a van der The effect | action by the force which acts between the substances which arise from at least 1 among the bonding of a Waals coupling | bonding and an electrostatic force 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. In addition, electrostatic coupling includes electric repulsion in addition to electrostatic coupling.

Furthermore, when the biological material complex is used for purification or analysis, the above interaction is arbitrary as long as purification and analysis are possible. Specific examples of the interaction include binding and dissociation between antigen and antibody, binding and dissociation between protein receptor and ligand, binding and dissociation between adhesion molecule and counterpart molecule, enzyme and substrate The binding and dissociation between the apoenzyme and the coenzyme, the binding and dissociation between the nucleic acid and the nucleic acid or protein that binds to it, the binding and dissociation between the proteins in the information transmission system, Binding and dissociation between glycoprotein and protein, binding and dissociation between sugar chain and protein, binding and dissociation between physiologically active substance (drug or drug candidate compound) and biological substance such as protein, avidin protein and Binding and dissociation with biotin, binding and dissociation of sugars such as metal chelates, glutathione and maltose with affinity tag fusion proteins, sugars and sugar chains Binding and dissociation of the pulse, and the like association and dissociation of the chelate-forming groups and the metal ions.
Depending on the method of purification or analysis, a target substance that can be adsorbed among the interactions may be used.

Specific examples of the specific substance include metal chelates, glutathione, sugars, vitamins, boronic acids, proteins, antigens, nucleic acids, physiologically active substances, lipids, hormones, environmental hormones, chelate-forming groups, and the like.
In addition, a specific substance may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and ratios, However, When isolate | separating a specific target substance, normally only 1 type is used.

Of these, metal chelates, glutathione, sugars, and the like are suitable for use in, for example, separation of affinity tag fusion proteins.
Expression and purification of affinity tag fusion proteins is one method for obtaining large quantities of recombinant proteins. A specific example will be briefly described. The protein is modified with an amino acid sequence serving as an affinity tag, and separation is performed using a specific substance that specifically interacts with the affinity tag, thereby purifying the target protein. And the like. Examples of such affinity tags include polyhistidine (His-tag), glutathione-S-transferase (GST), maltose binding protein, calmodulin and the like. Among these, preferred affinity tags are His-tag and GST, and particularly preferred is His-tag.

  As a specific example, when purifying a specimen having polyhistidine, for example, a recombinant protein having a His-tag, a metal chelate can be used as the specific substance. In this case, the affinity interaction between histidine and a metal chelate is used. This interaction is thought to occur because one of the nitrogen atoms present in the imidazole ring in polyhistidine is coordinated to an unsaturated coordination site of the metal.

  When a metal chelate is used as the specific substance when using the His-tag, the specific type of metal ion contained in the metal chelate as the specific substance is arbitrary, but in general, a transition metal ion Is preferred. Among them, the ions of iron, cobalt, nickel, copper, and zinc, which are the fourth periodic elements in the periodic table, are preferable in terms of manufacturing cost and difficult to leak in the separation and purification process. Particularly preferred is nickel.

  The chelating reagent for forming the metal chelate is not limited as long as the metal chelate can be formed. Specific examples of chelating reagents include iminodiacetic acid and its derivatives, nitrilotriacetic acid and its derivatives, and the like. Moreover, as a commercially available reagent, AB-NTA, Maleimido-C3-NTA, Maleimido-C7-NTA (made by Dojindo Laboratories) etc. are mentioned, for example. In addition, a chelating reagent may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

  Furthermore, there is a method of modifying a functional group of a biological substance without using a chelating reagent. For example, the amino group of a biological substance is modified with monochloroacetic acid, monobromoacetic acid, monochloropropionic acid, monobromopropionic acid, or a metal salt thereof, and a metal ion is bound to this to form a metal that is a specific substance It can also be a chelate.

  In addition, when GST, maltose binding protein, or the like is used as the affinity tag, for example, a sugar such as glutathione or maltose can be used as the specific substance.

Furthermore, when the antibody-antigen reaction is caused by using the biological material complex of the present invention, an antibody or an antigen can be used as the specific substance. The antibody and antigen used here are arbitrary, and examples thereof include immunoglobulins and derivatives thereof such as F (ab ′) ₂ , Fab ′, and Fab. When a biological substance complex using an antibody or antigen as a specific substance is used in a diagnostic analysis tool, a target substance (specimen) containing an active substance is, for example, blood, serum, plasma, spinal fluid, urine, feces, Examples include body fluids such as nasal discharge and saliva, cells, tissues, and extracts thereof.

  The biological material complex of the present invention can also be used for gene analysis. In this case, for example, nucleotides, oligonucleotides, nucleic acids (DNA, RNA, PNA) and the like can be used as the specific substance. The nucleic acid at this time may be double-stranded or single-stranded.

  Furthermore, when the biological substance complex of the present invention is used for clarifying the action mechanism of a medicine, a physiologically active substance, for example, a compound having physiologically active ability can be used as the specific substance. When a drug such as FK506 is used as the physiologically active substance, a binding protein such as FKBP12 can be analyzed. In addition, when a drug candidate compound is used as a physiologically active substance and a protein such as a receptor that contributes to a disease is used as an active substance, the biological substance complex of the present invention is used as an action mechanism elucidation tool or screening tool of the drug candidate compound. Can be used. Furthermore, conversely, when proteins such as membrane proteins and receptors are used as specific substances, and compounds such as drug candidate compounds are used as active substances, they can also be used as tools for elucidating the action mechanism of drug candidate compounds or as screening tools. .

Further, when the biological material complex of the present invention is used as a sensor for detecting viruses, sugars and sugar chains can be used as specific substances. Examples of such sugars include sialyl polysaccharides.
Furthermore, when the biological material complex of the present invention is used for separation and purification of a compound having a sugar, such as a sugar chain-containing protein, boronic acid can be used as the specific substance.

In addition, when a protein is used as the specific substance, examples of the protein include enzymes and the like in addition to the antibodies and receptors described above. By using an enzyme as the specific substance, a highly efficient immobilized enzyme can be provided. In this case, the enzyme can be used repeatedly.
In addition, when a nucleic acid such as protein, DNA, or RNA or a small molecule is used as the specific substance, a protein screening method and function modification method using a protein-nucleic acid linked molecule as the active substance can be provided. In this method, a known analysis technique such as yeast two-hybrid, TAP, phage display, IVV (in vitro virus), mRNA display, STABLE, and ribosome display can be used using the biological material complex of the present invention ( For example, see Yanagawa et al., “Protein Nucleic Acid Enzyme”, Vol. 48, No. 11, P 1474 (2003)).

Furthermore, by using lipid as a specific substance, screening, separation and purification of lipid binding protein and the like can be performed using the biological substance complex of the present invention.
In order to investigate the action mechanism of hormones and environmental hormones, hormones and environmental hormones can also be used as specific substances. In that case, the biomolecule complex of the present invention can be used to screen for biomolecules that bind to hormones or environmental hormones.
Furthermore, when using a vitamin as a specific substance, biotin etc. can be used, for example. Thereby, it can use for refinement | purification of substances, such as a protein which bound avidin as an active substance.
In addition, by using sugars and sugar chains as specific substances, for example, sugar-binding proteins such as lectins can be separated and purified. Examples of such sugars include mannose-containing sugars and sialyl polysaccharides.

  In addition, when the biological material complex of the present invention is used for separation or removal of metal ions, a chelate-forming group can be used as the specific substance. In order to form this chelate-forming group, for example, the above-mentioned chelating reagents can be used. Furthermore, for example, the chemical species exemplified in the method for modifying a functional group of a biological substance without using a chelating reagent can also be exemplified as the chelate-forming group. Specific examples of the chelate-forming group include iminodicarboxylic acid group, iminopropionic acid group, ethylenediaminetriacetic acid group, ethylenediaminetetraacetic acid group, hydroxyethyliminodiacetic acid group, hydroxyethyliminotriacetic acid group and the like.

In addition, when the specific substance structure is used for separation and purification using affinity separation techniques such as affinity purification and analysis, the specific substance is a target substance of the target substance to be separated and purified.
Among the above-mentioned substances, the specific substance is more preferably at least one selected from the group consisting of metal chelates, biotin, sugar, glutathione, boronic acid, antibodies, antigens, receptors, physiologically active substances, and chelate-forming groups. .

[I-2. Linker]
In the biological material complex of the present invention, the specific substance is bound to the biological substance and / or the binding compound, but the specific substance and the biological substance and / or the binding compound are not directly bound to each other. , They may be bonded via some kind of linker. Usually, the use of a linker makes it possible to enhance the interaction between the specific substance and the target substance. Therefore, the specific substance is indirectly bound to the biological substance and / or the binding compound using the linker. It is preferable.

There is no restriction | limiting in a linker, Although it is arbitrary unless the effect of this invention is impaired remarkably, It is preferable to use a molecule | numerator (hydrophilic molecule | numerator) which has hydrophilic property as a linker especially. That is, the linker is preferably hydrophilic.
For the linker to be hydrophilic, the molecule constituting the linker includes, for example, a hydrophilic atomic group such as an amino group, a carboxylic acid group, a hydroxyl group, a sulfonic acid group, a glycidyl group, a nitrile group, or ethylene oxide. It is desirable to be able to Among them, since polyethylene oxide chains are known to suppress nonspecific adsorption of proteins, it is desirable to use a linker containing an ethylene oxide chain when using proteins as biological substances or target substances.
In addition, a linker may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

[I-3. Biological material structure]
As shown in FIG. 1A and FIG. 1B, the biological material structure is formed by bonding particulate lumps formed by binding a biological material and a binding compound to each other. In addition, this particulate lump is usually formed into a particulate form as shown in FIG. 2B by binding a plurality of biological substances and binding compounds as shown in FIG. Although it does not necessarily have a round shape, in FIGS. 1A and 1B, the particulate lump is schematically shown as a circle.

  Specifically, a binding compound is bound to a biological substance by a binding functional group, and a particulate lump having a chain-like and / or network-like structure is formed by repeating the structure. Furthermore, the bonds between the particulate lumps are usually formed by the bond between the biological substance contained in the particulate lumps and the binding compound.

Therefore, the biological material structure usually has two or more partial structures represented by the following formula (A).
R ¹ -R ² formula (A)
{In the above formula (A), R ¹ represents a biological substance, and R ² represents a binding compound. However, when the biological material structure is bound to some solid phase carrier, R ² represents a binding compound that is not directly bound to the solid phase carrier. Also, each R ¹ and R ² may be the same or different. }

That is, the biological material structure is a structure in which a partial structure in which a biological material and a binding compound are bonded as in the above formula (A) is bonded in a linear and / or network form. Specifically, R ¹ in the above formula (A) is independently bonded to one or more other R ^{2 s} , and R ² is independently bonded to one or more other R ^{1 s} . . However, the biological material structure may include, for example, a partial structure in which the biological materials R ¹ or the binding compounds R ² are bonded to each other. Here, the bonds between R ¹ and R ² indicate bonds by physical interaction such as intermolecular attractive force, lyophobic interaction, and electrical interaction.

  Therefore, the biological material structure has a bridging structure in which the biological material exists between the binding compounds and the binding compound exists between the biological materials, at least in part. A biological material structure is constituted by both the biological material and the binding compound.

  In the biological material structure, whether the biological material and the binding compound have a bridging structure, for example, when the biological material is decomposed while preventing the binding compound from being decomposed, This can be confirmed by the large collapse of the structure. Furthermore, by investigating the collapsed compound, it is possible to specify a compound constituting the biological material structure other than the biological material constituent element. For example, when a biological material is directly bonded to conventional resin beads or the like and the resin beads form a structure, the resin beads remain after decomposition. The biological material decomposition method and analysis method can use the methods described later (see [I-5-4], etc.), but when used for the above purpose, not only biological materials but also bindings are exemplified. The use of compounds for which there is a possibility of decomposing compounds should be avoided because there is a possibility that the above-mentioned bridge structure cannot be confirmed accurately.

(1) Biological material The biological material is an element constituting the biological material structure, and is also an element constituting the biological material complex. As this biological substance, any substance can be used depending on the purpose as long as the effects of the present invention are not significantly impaired.

  However, from the viewpoint of configuring the biological material complex by immobilizing the specific substance on the biological material structure, when binding the specific substance to the biological material, the biological material that can bind to the specific substance is used. In addition, when a biological substance is bonded to a specific substance via a linker, a biological substance that can bind to the linker is used.

The bond between the biological substance and the specific substance or the linker may be any bond depending on the type and use of the biological substance complex.
Specific examples of the binding between the biological substance and the specific substance or the linker include a covalent bond, an ionic bond, a chelate bond, a coordination bond, a hydrophobic bond, a hydrogen bond, a van der Waals bond, and an electrostatic force bond. May be other combinations that do not fit into these classifications.

  On the other hand, when the specific substance is bound to the binding compound and is not bound to the biological substance, any biological substance can be used depending on the application. For example, when a predetermined interaction is to be caused between a specific substance and some active substance, a biological substance that does not cause a non-specific interaction with the active substance is used. It is preferable to do so.

  Specific examples of biological materials include enzymes, antibodies, lectins, receptors, protein A, protein G, protein A / G, avidin, streptavidin, neutravidin, glutathione-S-transferase, albumin, glycoprotein and the like, Peptides, amino acids, cytokines, hormones, nucleotides, oligonucleotides, nucleic acids (DNA, RNA, PNA), sugars, oligosaccharides, polysaccharides, sialic acid derivatives, sugar chains such as sialylated sugar chains, lipids, derived from biological substances other than those mentioned above And high molecular organic substances, low molecular compounds, inorganic substances, or fusions thereof, or biomolecules such as viruses or molecules constituting cells.

In addition, substances other than biomolecules such as cells can also be used as biological substances.
Furthermore, 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 Ligand, virus, cell and the like are also exemplified as biological materials.

  Further, among the examples of the biological material, 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. Further, the synthesized peptide chain may be a glycoprotein having a sugar chain bound thereto. Of these, a purified protein is preferable.

  By using the protein, the characteristics of the protein can be utilized for the biological material complex of the present invention. Specifically, for example, when albumin is used as the biological material, nonspecific adsorption can be suppressed. In another aspect, when avidin is used as the biological material, a specific biotinylated material can be immobilized easily and in large quantities. In yet another aspect, if protein A is used as the biological material, when an antibody is used as the specific material, the antibody can be easily immobilized in a large amount.

  Furthermore, among the examples of the biological material, the nucleic acid is not particularly limited, and in addition to DNA and RNA, nucleobases such as aptamers and peptide nucleic acids such as PNA can be used. The nucleic acid may be a known nucleic acid or an unknown nucleic acid. Among them, it is preferable to use a nucleic acid having a binding ability to a protein and having a known function and base sequence as a nucleic acid, or that has been cut and isolated from a genomic library or the like using a restriction enzyme or the like.

Moreover, among the examples of the above-mentioned biological substances, the sugar chain may be a sugar chain having 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.
Further, among the examples of the biological material, the low molecular compound is not particularly limited as long as the requirements for the biological material are satisfied. Those having an unknown function or those having an already known ability to bind to a protein can be used.
In addition, a biological material may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

(2) Binding Compound Any compound can be used as the binding compound as long as it is a compound that can bind to the biological substance. Therefore, as the binding compound, a compound having a functional group capable of binding to the biological substance (hereinafter appropriately referred to as “binding functional group”) can be arbitrarily used.
Here, the bond usually refers to a bond composed of one or more of a covalent bond, an ionic bond, a chelate bond, a coordination bond, a hydrophobic bond, a hydrogen bond, a van der Waals bond, and a bond by electrostatic force. Here, a covalent bond is preferable.

The binding functional group is not particularly limited as long as it is a functional group capable of binding to the biological material, and any functional group can be used. In general, it is preferable to select an appropriate material according to the type of the biological material and the use of the biological material complex 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 generally classified into a group that binds to a biological material as a reactive group via a covalent bond and a group that binds to a biological material via a non-covalent bond.
When binding by a covalent bond, specific examples of the binding functional group include a succinimide group, an epoxy group, an aldehyde group, a maleimide group, and a p-nitrophenyl group.

In this case, examples of the biological substance that is covalently bonded to the binding functional group include proteins, nucleic acids, sugars, and the like.
When the biological material is a protein, usually, a group such as an amino group, a hydroxyl group, or a thiol group present on the surface layer of the protein is bonded to a binding functional group of the binding compound. In this case, for example, when an amino group is bonded to a binding functional group, specific examples of the binding functional group include a succinimide group, an epoxy group, and an aldehyde group. In addition, for example, when a hydroxyl group is bonded to a binding functional group, specific examples of the binding functional group include an epoxy group. Furthermore, for example, when a thiol group is bonded to a binding functional group, a specific example of the binding functional group is a maleimide group.

  In addition, when the biological material is a nucleic acid, a group such as an amino group, a hydroxyl group, or a thiol group introduced into the end of the nucleic acid is usually bonded to a binding functional group of the binding compound. In this case, for example, when an amino group is bonded to a binding functional group, specific examples of the binding functional group include a succinimide group, an epoxy group, and an aldehyde group. For example, when a hydroxyl group is bonded to a bonding functional group, specific examples of the bonding functional group include an epoxy group. Furthermore, for example, when a thiol group is bonded to a binding functional group, a specific example of the binding functional group includes a maleimide group.

  Furthermore, when the biological material is a sugar, usually, a group such as an amino group, a hydroxyl group, or a thiol group present in the side chain of the sugar is bonded to the binding functional group of the binding compound. In this case, for example, when an amino group is bonded to a binding functional group, specific examples of the binding functional group include a succinimide group, an epoxy group, and an aldehyde group. For example, when a hydroxyl group is bonded to a bonding functional group, specific examples of the bonding functional group include an epoxy group. Furthermore, for example, when a thiol group is bonded to a binding functional group, a specific example of the binding functional group includes a maleimide group.

On the other hand, when the biological substance and the binding compound are bound by a non-covalent bond, the binding can be performed by, for example, complex formation or interaction between biological substances.
When a biological substance and a binding compound are combined to form a complex, specific examples of the binding functional group include a boronic acid group.
Further, for example, when binding is performed by an avidin-biotin interaction among the interactions between biological substances, a specific example of the binding functional group is a biotin group.

Furthermore, for example, when a virus is used as the biological material, specific examples of the binding functional group include sugars and polysaccharides.
Further, for example, when the biological material has a lyophobic region, it may be coupled by physical adsorption by the lyophobic interaction.

  In addition, when the binding compound has a binding functional group, the binding compound may contain at least one type having a binding functional group of usually 2 or more, preferably 3 or more in one molecule. preferable. This is to facilitate the formation of the structure of the present invention. For example, if two or more binding functional groups are included in one molecule, a particulate mass in which a biological substance and a binding compound are easily bonded is formed, and these particulate masses are further bonded to each other. By doing so, it becomes possible to form a biological material structure and a biological material complex which are higher-order structures using them.

However, in order for the biological material complex of the present invention to easily form the above-mentioned particulate mass, it is preferable that there is no binding between the binding compounds and the binding between the biological material and the binding compound is mainly. . This is because when the binding compounds are bonded to each other, aggregation of the binding compounds is likely to be formed, the particle size of the particulate lump tends to increase, and it is difficult to control the particle size of the particulate lump.
Further, when a polymer is used as a binding compound when there is a binding between the binding compounds, internal crosslinking of the binding compound occurs, and it is difficult to immobilize the biological substance. Here, the bond between the binding compounds refers to a bond excluding intermolecular attractive force, lyophobic interaction, and electrical interaction.

  In order to form a biological material complex that does not bond between the binding compounds in this way, select a binding functional group that does not bond the binding compounds together, and eliminate the situation where the binding compounds bind. It is desirable to do. Specific examples of such functional groups include a succinimide group, an epoxy group, an aldehyde group, a maleimide group, a boronic acid group, and a biotin group as described above. The situation where bonding functional groups are bonded to each other means that excessive heat is applied or that strong ultraviolet rays are irradiated.

  The method for examining the binding between the binding compounds contained in the biological material complex is determined by the formation of insoluble matter when the biological material in the biological material complex is decomposed by the method described below. Or it is judged by detecting the compound which suggests the coupling | bonding of the compound for coupling | bonding by analyzing a biological material composite_body | complex by a pyrolytic gas chromatography. Specifically, for example, in the step of binding a biological substance and a binding compound having a photoreactive group by ultraviolet irradiation, the binding compounds are bonded to each other by a photoreactive group (for example, an azide group) in the binding compound. In the case of binding, the binding between the compounds for binding can be inferred based on the bond involving the photoreactive group or the presence of the remaining photoreactive group (“Affinity Chromatography” published by Tokyo Chemical Doujin, author: Isao Matsumoto Take, Masatoshi Beppu, see P238 et al.).

  Furthermore, in order to make use of the characteristics of the biological material, in order to maintain the activity of the biological material, it is preferable to select the functional group of the biological material and the functional group of the binding compound so that the biological material is not deactivated. For example, when a protein is used as a biological substance and the active part of the protein has a thiol group, a group other than the thiol group (for example, an amino group) is selected as the binding functional group of the biological substance, and this amino group In order to couple | bond with, the succinimide group and an epoxy group are selected as a coupling | bonding functional group of the compound for coupling | bonding.

  When a specific substance is to be bound to the binding compound, a binding compound that can bind to the specific substance or a linker is used. In this case, specific examples of the binding between the binding compound and the specific substance or the linker include covalent bonds, ionic bonds, chelate bonds, coordination bonds, hydrophobic bonds, hydrogen bonds, van der Waals bonds, and electrostatic force bonds. Other combinations that do not fit into these classifications may be used.

Therefore, in this case, it is desirable to use a compound having a functional group capable of binding to the specific substance or the linker in addition to the binding functional group. There is no restriction | limiting in such a functional group, Arbitrary functional groups can be used, For example, the thing similar to a coupling | bonding functional group is mentioned. The specific type can be selected according to the type of the specific substance, the use of the biological material complex of the present invention, and the like.
In addition, the functional group that can be bonded to the specific substance or the linker may be used alone, or two or more may be used in any combination and ratio.

  Furthermore, it is usually desirable to use a compound that is miscible with water as the binding compound. This is because water is usually used as a medium such as a solvent or a dispersion medium during the production of the biological material complex. Specifically, when manufacturing a biological material complex, usually, a binding compound is mixed with the biological material in the presence of water, and the binding compound and the biological material are combined to produce a particulate mass. However, in such a case, the biological material and the binding compound are uniformly mixed to smoothly perform the binding reaction. In the present specification, the form of mixing may be dissolved or dispersed.

  The binding 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 coupling | bonding can be expanded, and the structure of a biological material composite_body | complex can be designed variously. For example, if the binding 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 binding compound.

  Further, it is more preferable to use a binding compound that is miscible with both water and an organic solvent. If the binding compound is miscible with both water and an organic solvent, the use of any solvent when using the biological material complex of the present invention can increase the types of solvents that can be used. Can be spread.

  The binding compound is preferably uncharged. If the binding compound has the same charge as that of the biological material (charge of the same sign), the binding between the binding compound and the biological material may be hindered by electrostatic repulsion. On the other hand, if the binding compound has a charge opposite to that of the biological substance (reverse sign charge), the biological substance and the charged part in the binding compound are bound by electrostatic attraction, resulting in binding. There is a possibility that the biological substance is prevented from effectively binding to the binding functional group of the compound for use. In addition, when the biological material complex is used for separation and purification, the binding of the binding compound and the biological material is easily broken by an additive such as pH or salt of the solution used at the time of use. Is expected and is not preferred.

  Furthermore, if the target substance has the same charge as the binding compound when it is attempted to separate the target substance using the biological substance complex, the specific substance with the specific substance contained in the biological substance complex If the target substance and the binding compound have opposite charges, non-specific adsorption occurs between the target substance and the binding compound due to electrical attraction. This is because it is estimated.

  Note that the binding compound is uncharged means that the binding compound has nonionic or zwitterionic (both positive and negative charges) at least in the structural formula, and the mutual charges are substantially The compound for binding is uncharged. However, even if the binding compound has a charge due to hydrolysis of the binding functional group or the like in the production process of the biological material complex of the present invention, such a binding compound is used as long as the effect of the present invention is not impaired. It can be used suitably.

Other examples of the binding compound include organic compounds, inorganic compounds, organic-inorganic hybrid materials, and the like.
Moreover, the compound for coupling | bonding may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and a ratio.

  Furthermore, the organic compound used as the binding compound may be a low molecular compound or a high molecular compound, but is preferably a high molecular compound.

On the other hand, when a polymer compound is used as the binding compound, the polymer compound may be a synthetic polymer compound or a natural polymer compound.
When a synthetic polymer compound is used as the binding 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 biological material. 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 material and a hydrophilic monomer. In addition, when a specific substance couple | bonds with the compound for a coupling | bonding, it is desirable to have the monomer which can couple | bond with a specific substance or a linker in addition to said monomer.

  That is, for the synthesis of a synthetic polymer compound used as a binding compound, at least as a monomer species, a monomer capable of binding to a biological substance to form a particulate lump, and the particulate lump are bonded together to form a chain. And / or a monomer having a binding functional group for constructing a network-bonded structure (that is, a structure of a biological material structure or a biological material complex), and further, hydrophilic or amphiphilic It is more preferable to use a monomer having a functional group of In addition to this, it is also preferable to contain a hydrophobic monomer for the purpose of controlling the structure such as micelles formed in the solution and the spread of the synthetic polymer compound. Further, when the specific substance and the binding compound are bonded, a monomer having a functional group capable of binding to the specific substance or the linker may be included for the purpose of binding the specific substance to the binding compound. desirable. The monomers mentioned here may be different monomers, but one monomer may have two or more of the above functions.

  Specific examples of monomers constituting a synthetic polymer compound that can be used as a binding compound include monomers such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, and vinyltoluene that can be used in radical polymerization. Unsaturated aromatics; 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 sodium styrene sulfonate; (meth) acrylic Methyl methacrylate, ethyl (meth) acrylate, (n-butyl) (meth) acrylate, 2-hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, glycidyl (meth) acrylate, N- (meta ) Acryloyloxysuccinimide, ethylene glycol di- (meth) acryl Polymerizable carboxylic acid esters such as acid esters, tribromophenyl (meth) acrylate, 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, Unsaturated carboxylic acid amides such as 9-diamine, butadiene, isoprene, vinyl acetate, vinyl pyridine, N-vinyl pyrrolidone, N- (meth) acryloyl morpholine, vinyl chloride, vinylidene chloride, vinyl bromide; polymerizable unsaturated Nitriles; halogens And vinyl monomers; conjugated dienes; and macromonomers such as polyethylene glycol mono (meth) acrylate and polypropylene glycol mono (meth) acrylate.

  Moreover, as a monomer of a synthetic polymer compound that can be used as a binding compound, for example, a monomer used in addition polymerization can also be used. Specific examples of monomers used in this addition polymerization include diphenylmethane diisocyanate, naphthalene diisocyanate, tolylene diisocyanate, tetramethylxylene diisocyanate, xylene diisocyanate, dicyclohexane diisocyanate, dicyclohexylmethane diisocyanate. Examples thereof include aliphatic or aromatic isocyanates such as nate, hexamethylene diisocyanate and isophorone diisocyanate, ketenes, epoxy group-containing compounds and vinyl group-containing compounds.

  In addition, the above compound group can be reacted with a monomer having a functional group having activated hydrogen. Specific examples thereof include a compound having a hydroxyl group or an amino group, specifically, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, glycerin, trimethylolpropane, Polyols such as pentaerythritol, sorbitol, methylene glycoside, sucrose, bis (hydroxyethyl) benzene; ethylenediamine, hexamethylenediamine, N, N′-diisopropylmethylenediamine, N, N′-di-sec-butyl-p -Polyamines such as phenylenediamine and 1,3,5-triaminobenzene; oximes and the like.

  Furthermore, in the synthetic polymer compound that can be used as the binding compound, in addition to the above-described monomers, a polyfunctional compound that can serve as a crosslinking agent may coexist. 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.
By using a polyfunctional compound that can be a crosslinking agent as a monomer, the spread and hardness of the binding compound in the medium can be controlled.

  Examples of the monomer having a binding functional group capable of binding to the above-mentioned biological substance include N- (meth) acrylic acid as an example of a monomer having a succinimide group, an epoxy group, an aldehyde group, a maleimide group, a p-nitrophenyl group, or the like. Examples include leuoxysuccinimide, glycidyl (meth) acrylate, acrolein, maleimide acrylate, p-nitrophenyloxycarbonyl polyethylene glycol methacrylate, and p-nitrophenyl methacrylate.

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.

  Examples of the monomer having a functional group capable of binding to the specific substance or linker include the same monomers as those having a binding functional group capable of binding to the biological substance.

  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.

  Further, the binding compound is preferably an uncharged one as described above. Therefore, when the synthetic polymer compound used as the binding 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- ( (Meth) acryloyl morpholine, N-vinyl pyrrolidone, N-vinyl acetamide, N-vinyl-N-acetamide, polyethylene glycol mono- (meth) acrylate, glycidyl (meth) acrylate, 2-methacrylooxyethyl phosphorylcholine It is done.

  By the way, when a synthetic polymer compound is synthesized by radical polymerization of a monomer, the polymerization is usually started by mixing a radical polymerization initiator, but the radical polymerization initiator used at that time significantly impairs the effects of the present invention. Any one can be used as long as it is not. Examples of the radical polymerization initiator that can be used include 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- Azo (azobisnitrile) type initiators such as methoxyvaleronitrile), 2,2′-azobis- (2,4-dimethylvaleronitrile), 2,2′-azobis- (2-amidinopropane) hydrochloride, Benzoyl peroxide, cumene hydroperoxide, hydrogen peroxide, acetyl peroxide, lauroyl peroxide, persulfates (eg ammonium persulfate), peracid esters ( Example, if t- butyl peroxide octoate, etc. α- cumyl peroxypivalate and t- butyl per octoate) peroxide type initiators and the like.

  Furthermore, polymerization may be initiated by mixing a redox initiator. Any redox initiator can be used as long as the effects of the present invention are not significantly impaired. Examples thereof include ascorbic acid / iron (II) sulfate / sodium peroxydisulfate, tert-butyl hydroperoxide / dioxide. Examples include sodium sulfite, 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.

When a synthetic polymer compound is used as the binding compound, the synthetic polymer compound may be a polymer synthesized by ring-opening polymerization or the like. Specific examples thereof include polyethylene glycol.
Furthermore, the synthetic polymer compound described above may use a polymer synthesized by hydrolysis or the like. Specific examples thereof include polyvinyl alcohol synthesized by hydrolyzing polyvinyl acetate and the like.
Further, the above-described synthetic polymer compound may be synthesized by modifying a functional group that binds to the aforementioned biological substance by chemical modification.

  In addition, a commercially available synthetic polymer compound can be used as the binding compound. Specific examples 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 binding compound, specific examples thereof include polysaccharides such as dextran, carboxymethyl-dextran, starch and cellulose, proteins such as albumin, collagen and gelatin, nucleic acids such as DNA and RNA. Etc. These natural compounds may be used as they are, or may be used after being chemically modified.

  When a polymer compound such as a synthetic polymer compound or a natural polymer compound is used as the binding compound, the form of the polymer compound is arbitrary. For example, it may be dissolved in an aqueous solution, or may be an aggregate such as a micelle or emulsion, or a fine particle such as a polymer latex.

  Examples of the inorganic compound used as the binding compound include metal particles such as gold colloid and inorganic fine particles such as silica. Furthermore, it is good also as a compound for coupling | bonding which has a functional group couple | bonded with a biological substance by chemically modifying these inorganic compounds.

Furthermore, examples of the organic-inorganic hybrid used as the binding compound include a colloidal silica coated with a polymer, a metal colloid coated with a polymer (for example, particles of gold, silver, platinum, etc. with a protective colloid). And a polymer obtained by adsorbing a polymer to a porous substrate such as clay. These organic-inorganic hybrids can be synthesized by a known method (see, for example, polymer nanocomposites, industrial research committee, Susumu Nakajo).
Furthermore, these organic-inorganic hybrids can be used as a binding compound by modifying the binding functional group.

The molecular weight and structure of the binding compound are not particularly limited and are arbitrary. Therefore, for example, a low molecular weight compound may be used as the binding compound, but in that case, the substance is cross-linked within one biological material to be immobilized, and a biological material structure or biological material complex cannot be formed. There is a fear. From the viewpoint of preventing this, the molecular weight of the binding compound is usually 1000 or more, preferably 10,000 or more, and usually 1 million or less, preferably 500,000 or less. When a synthetic or natural polymer compound is used as the binding compound, the weight average molecular weight is preferably within the above range. This is because if it falls below this range, the biological material structure in which the particulate lumps are effectively aggregated cannot be formed, and the biological material complex may not be formed.
Various methods can be used to measure these molecular weights. For example, GPC (gel permeation chromatography), SEC (size exclusion chromatography), static light scattering measurement, viscosity measurement, etc. be able to.

  Moreover, there is no restriction | limiting in the magnitude | size of the compound for coupling | bonding, and it is arbitrary unless the effect of this invention is impaired remarkably. However, in order to effectively bind the biological substance and the binding compound, the diameter of the binding compound is usually 1 nm or more when mixed in a liquid such as a solvent or a dispersion medium (here, a medium). Preferably it is 2 nm or more, more preferably 3 nm or more. In order to satisfy this condition, when a binding compound is prepared before the production of the biological material complex of the present invention, a binding compound that falls within the above particle size range is prepared and used. It is desirable to produce a biological material complex.

  Various methods can be used to measure the size of these binding compounds, but static light scattering is possible when metal colloids, inorganic particles, fine polymer particles, etc. dispersed in a liquid are used as the binding compounds. It can be examined by a general method such as a measurement method, a dynamic light scattering measurement method, or a light diffraction method. Further, a liquid obtained by removing a liquid such as a reaction medium from a dispersion liquid in which these gold colloids, inorganic particles, polymer fine particles and the like are dispersed was observed with an electron microscope such as SEM (scanning electron microscope) or TEM (transmission electron microscope). In some cases, it may be handled as the diameter of the binding compound in the liquid.

On the other hand, when an aggregate such as a polymer or micelle dissolved in a solution is used as the binding compound, it can be obtained by a general method such as static light scattering measurement method, dynamic light scattering measurement method, or light diffraction method. Can be examined. In general, polymer particles or micelles dissolved in a solution have a difference in particle diameter depending on the measurement means and analysis method, but the particle diameter is evaluated based on the measured value obtained by any means or method. be able to.
When measuring the diameter of the binding compound in the liquid by an optical method, it is effective to ensure that the average particle size of the binding compound is within the above range. It is desirable to combine

  Furthermore, the amount of the binding functional group possessed by the binding compound is not particularly limited, and cannot be generally defined by the type of binding compound. For example, when a polymer is used as the binding compound, The mol% is usually 0.1% or more, preferably 0.5% or more, more preferably 1% or more, and usually 90% or less, preferably 80% or less, more preferably 70% or less. If the ratio is below this range, the binding compound may not be efficiently bonded to the biological substance, and if it exceeds the range, the binding compound may not be miscible with the solvent or the dispersion medium.

[I-4. Structure of biological material complex]
The biological material complex of the present invention is a biological material and / or a binding compound of a biological material structure in which a biological material and a particulate mass formed by binding a compound capable of binding to the biological material are bonded to each other. In addition, a specific substance is bound. Furthermore, in the biological material complex of the present invention, the particle size of the particulate lump is usually 10 μm or less.

  As schematically shown in FIGS. 2 (a) and 2 (b), the biological material structure has a single unit consisting of a particulate lump in which the biological material and the binding compound are bonded, as shown in FIGS. As shown schematically in (), the structures are linked to each other in a chain form and / or a network form.

  The biological material structure has a structure formed by the aggregation and / or binding of the particulate mass formed by both the biological material and the binding compound, and thus the biological material complex is the same. Have a structure in which particulate lumps are aggregated and / or combined. For this reason, it is possible to increase the ratio of the biological substance, and accordingly, the ratio of the specific substance that binds to the biological substance can be increased. Therefore, the biological material complex of the present invention can hold a larger amount of a specific substance than before. Furthermore, in the case where the specific substance can be bound to the binding compound in addition to the biological substance, a larger amount of the specific substance can be retained.

  In addition, since a large amount of a specific substance can be retained, the biological substance complex of the present invention can suppress nonspecific interactions that occur in the binding compound (and a solid phase carrier described later). Benefits can be gained. That is, if a biological material that does not cause the above-mentioned non-specific interaction is used, the binding compound that can cause the non-specific interaction can be covered with a large amount of the biological material. At this time, since a large amount of biological material is present in the biological material structure, non-specific interaction can be effectively suppressed. Therefore, affinity separation using the interaction between the specific substance and the agent can be performed while eliminating the influence of the non-specific interaction.

  On the other hand, even when the specific substance binds to the binding compound and does not bind to the biological substance, the biological substance structure has a three-dimensional structure as will be described later. It is possible to hold. In addition, the above-described advantage that non-specific interaction can be suppressed is obtained even when the binding compound and the specific substance are bound.

In the conventional technique for immobilizing a biological material on a solid phase carrier or the like, when used for affinity purification or the like, the amount of biological material immobilized on the surface of the solid phase carrier such as resin fine particles is limited to a predetermined upper limit. The value was limited (usually, protein monolayer adsorption was 0.3 to 1.0 μg / cm ² at most), and a large amount of biological material could not be retained. Therefore, even if the specific substance is bound to the biological substance, the amount of the specific substance is not sufficient.

Furthermore, the particulate lumps gather together to form a biological material structure by being brought into contact with each other by the attractive force between the particulate lumps or entangled in the carbon chain. For example, biological mass structures are formed by binding the particulate masses by intermolecular attractive force, or the biological mass structure is formed by binding the functional mass of the binding compound to the biological material. Or, the above-mentioned factors are combined to combine the particulate lumps to form a biological material structure. And a specific substance couple | bonds with this biological material structure, and the biological material composite_body | complex is comprised. Here, the bond between the particulate aggregates usually includes one or more bonds selected from covalent bonds, ionic bonds, chelate bonds, coordination bonds, hydrophobic bonds, hydrogen bonds, van der Waals bonds, and electrostatic force bonds. Among them, a covalent bond is preferable. In this case, it may be in a state where only a part of the particulate lumps are bonded, but it is preferable that as many particulate lumps as possible are bound, and that all the particulate lumps are bound. More preferred.
In general, it is presumed that the particulate mass aggregates due to a plurality of factors such as entanglement and bonding to form a biological material structure and constitutes a biological material complex.

The particle size of the particulate mass is usually 10 μm or less, preferably 5 μm or less, more preferably 1 μm or less. When the particle size of the particulate mass is large, a sufficient specific surface area cannot be obtained when used for separation and purification, and there is a possibility that a highly efficient separation and purification result cannot be obtained when used for affinity separation or the like.
Further, when the particle size of the particulate mass is measured individually, at least a part of the particulate mass in the biological material complex may have a particle size in the above range. However, it is preferable that as many particulate lumps as possible have a particle size in the above range, and it is more preferable that all the particulate lumps have a particle size in the above range.

  Here, the particle size of the particulate mass can be measured by observing with an optical microscope, an electron microscope such as SEM or TEM, or a microscope such as AFM (atomic force microscope). In addition, when observed with a microscope, the shape of the particulate mass may be observed as a shape protruding from the biological material complex into a tuft shape in addition to the particulate shape. ) Is presumed to be formed by protruding a particulate lump from the biological material complex, the diameter (usually the short diameter) of the tuft-like lump portion may be within the above range.

  As one method for examining the presence of the particulate lump, a case of analyzing Power Spectral Density (power spectral density) from the image measured by the AFM will be described in detail. The analysis using the power spectrum is used to evaluate minute irregularities on the surface of the object, and the amount of change (for example, height, depth, etc.) caused by the irregularities is decomposed into waveforms and subjected to Fourier transform. It is one of the analysis methods based on it.

  A commercially available AFM apparatus can be used for the AFM measurement, but preferably NanoScope IIIa manufactured by Digital Instruments is used. The probe preferably has a tip radius R of 5 nm or less, and specifically, SSS-NCH manufactured by Toyo Technica Co., Ltd. is preferable. Furthermore, it is desirable to measure in the tapping mode so as not to damage the sample surface. The measurement may be performed in the air or in a liquid, but in order to obtain a good image, the measurement is preferably performed in the air, and more preferably in a dry state.

  Further, the algorithm used for Power Spectral Density analysis is preferably in accordance with the recommendation of ASTM E42.14 STM / AFM subcommittee for analysis of the surface shape, and particularly preferably, Power Spectrum attached to NanoScope IIIa manufactured by Digital Instruments. The Density function is used (reference document: NanoScope command reference manual).

When analysis of a biological material structure or biological material complex is performed using these devices and algorithms, for example, measurement by AFM is performed in a field of view of 1 μm × 1 μm, and Power Spectral Density (PSD) is obtained from the obtained AFM image. When the analysis is performed, the ratio of the sum (Ic) of the power spectrum and the total power spectrum (It) excluding the value of 2D isotropic PSD of 100 (nm ⁴ ) or more and a wavelength of less than 100 nm When Ic / It is usually 30% or more, preferably 50% or more, particularly preferably 60% or more, more preferably 70% or more, the presence of a submicron-order particulate mass can be confirmed. The power spectrum represents the unevenness of the surface of the object. If the ratio Ic / It is in the above range, the unevenness caused by the structure of the particulate mass is confirmed on the surface of the biological material structure or the biological material complex. Because it can.

The particle size of the particulate mass can also be confirmed by spectroscopic techniques such as light scattering, X-rays, and neutron scattering. In this case, the average particle diameter to be measured may be in the above range. Even when the average particle size of the particulate mass is within the above range, the same advantages as when the particle size is within the above range can be obtained.
Here, as an example, a method for confirming a particulate lump by a static light scattering measurement method will be described in more detail. For example, by measuring the forward light scattered light intensity of the biological material structure or the biological material complex, the particulate mass can be evaluated by obtaining the correlation length. This method measures the scattering angle dependence of the scattering intensity of the light that has passed through the biological material complex, and based on the Debye-Bueche theory, the (light intensity) ^(-1/2) The slope plotted against the square of the wave number (= {(4πn / wavelength of light) × sin (scattering angle / 2)} ² ) is obtained, and the correlation length is obtained from (slope / intercept) ² . Here, n is the refractive index of the medium. Although a commercially available light scattering apparatus can be used for the measurement, DYNA 3000 is preferable. The biomolecule complex may be swelled in a liquid or in a dry state, but measurement in a liquid is preferable.

  Moreover, in the biological material composite of the present invention, the particulate lumps usually do not completely adhere to each other, and a space (void) is formed between the particulate lumps. When affinity separation or the like is performed using a biological material complex, a target substance that interacts with a specific substance can enter this space. Usually, a specific substance and a target substance interact with each other in this space. The action will occur. Therefore, even in the biological material complex of the present invention having a three-dimensional structure, the specific substance contained therein (inside) can interact with the target substance or the like. That is, although the biological material complex of the present invention can be provided with many specific substances three-dimensionally, the specific substances can interact without losing activity. The biological substance complex of the present invention can contain a larger amount of a specific substance than before while maintaining the reactivity of the specific substance.

Although there is no limitation on the method for examining whether the biological material complex of the present invention has a space between the particulate masses, for example, confirmation by an optical microscope, an electron microscope such as SEM or TEM, or a microscope such as AFM can do. In addition, it can also be confirmed by spectroscopic techniques such as light scattering, X-rays, and neutron scattering.
Furthermore, when the volume of the biological material complex of the present invention in a dry state is compared with the volume when the liquid is included, and the volume increases, the volume of the liquid has entered the space. It may be recognized that the biological material complex of the present invention has a space as an increase of These volume changes may be confirmed by any method. For example, when the biological material complex is formed in a film shape, the film thickness in a dry state and the film thickness when it is immersed in a liquid are calculated. Each can be measured by AFM or the like, and both can be compared and confirmed.

  Further, the size of the biological material complex of the present invention is not limited and is arbitrary, but when not bound to any solid phase carrier, the diameter of the biological material complex is usually 30 nm in a dry state. The thickness is preferably 40 nm or more, more preferably 50 nm or more. Here, the diameter of the biological material complex can be measured by an electron microscope such as SEM or TEM, or a microscope such as AFM. If the size of the biological material complex is too small, it may be difficult to recover the biological material complex by centrifugal separation or the like in purification separation when used for affinity separation or the like.

  Furthermore, the biological material complex of the present invention can be immobilized on a solid phase carrier and used as an affinity purification or pharmaceutical function analysis tool. Furthermore, surface treatment of drugs for DDS (drug delivery system), regenerative medicine It can be applied to surface treatment of carriers, surface treatment of artificial organs, surface treatment of catheters and the like. When the biological material complex of the present invention is used for these surface treatments, etc., a biological material complex carrier in which the biological material complex of the present invention is fixed to a desired solid phase carrier by an arbitrary method is formed. Will be used. In addition, although the thickness (film thickness) of the biological material complex formed by the surface treatment is arbitrary, it is usually 5 nm or more, preferably 10 nm or more, and more preferably 15 nm or more in a dry state. If the film thickness is less than this, there is a possibility that the film cannot be formed sufficiently. The above thickness can be measured by SEM, TEM, AFM, or the like.

[I-5. Composition of biological material complex]
[I-5-1. Content ratio of specific substances]
In the biological material complex of the present invention, the ratio of the specific substance contained is not limited, but it is usually desirable that a larger amount of the specific substance is contained. Specifically, the ratio of the weight of the specific substance to the weight of the biomaterial complex represented by “(weight of specific substance) / (weight of biomaterial complex)” is usually 0.001% by weight or more, preferably Is preferably 0.003% by weight or more, more preferably 0.005% by weight or more. If the ratio of the specific substance is below this range, the efficiency of the separation and purification may be reduced when the separation and purification is performed using the biological material complex.

[I-5-2. Method for measuring the content ratio of specific substances]
The content ratio of the specific substance can be measured by, for example, elemental analysis or amino acid analysis.

[I-5-3. Biomaterial content ratio]
In the biological material composite of the present invention, the ratio of the contained biological material is not limited, but it is usually desirable that a larger amount of biological material is contained. Specifically, the ratio of the weight of the biological material to the weight of the biological material structure represented by “(weight of biological material) / (weight of biological material complex)” is usually 0.1 or more, preferably 0. .3 or more, more preferably 0.5 or more, and particularly preferably 0.7 or more. When the ratio of the biological material is lower than this range, the binding compound in the configured biological material complex cannot be sufficiently covered with the biological material, which may cause nonspecific adsorption to the binding compound. In addition, when a specific substance is bound only to a biological substance, a sufficient amount of the specific substance cannot be bound. When separation and purification is performed using this biological substance complex, the efficiency of the separation and purification is increased. May decrease.

[I-5-4. Method for measuring the content ratio of biological substances]
The method for measuring the ratio of the biological substance is not particularly limited. For example, the biological substance contained in the biological substance complex of the present invention is decomposed using an enzyme, a drug, or the like, and the biological substance, the binding compound, and the specific substance are decomposed. What is necessary is just to quantify each derived substance by various methods.
Hereinafter, a method for measuring the content ratio of the biological substance by this method will be described.

(1) Degradation method of biological material When the ratio of biological material is measured by this method, an enzyme, a drug, etc. for degrading the biological material can be selected according to the type of the biological material or binding compound used. What is necessary is just to use suitably. When the specific example is given, when a biological material is a nucleic acid, nuclease, such as ribonuclease and deoxyribonuclease, etc. are mentioned, for example.

When the biological substance is a protein, examples of the enzyme and drug include proteolytic enzymes such as microbial protease, trypsin, chymotrypsin, papain, rennet, and V8 protease, cyanogen bromide, and 2-nitro-5. -Chemical substances having protein resolution such as thiocyanic benzoic acid, hydrochloric acid, sulfuric acid, sodium hydroxide and the like.
Furthermore, when the biological substance is a lipid, examples of the enzymes and chemicals include lipolytic enzymes such as lipase and phospholipase A2.

When the biological substance is sugar, examples of the enzyme and drug include glycolytic enzymes such as α-amylase, β-amylase, glucoamylase, pullulanase, and cellulase.
In addition, said enzyme, chemical | medical agent, etc. for decomposing | disassembling a biological substance may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and ratios.

(2) Method for quantifying biological substance, binding compound and substance derived from specific substance After decomposition of biological substance complex, the method for quantifying biological substance, binding compound and substance derived from specific substance is not limited and is arbitrary. Specific methods include, for example, liquid chromatography, gas chromatography, mass spectrometry (MS), infrared spectroscopy, nuclear magnetic resonance ( ¹ H-NMR, ¹³ C-NMR, ²⁹ Si-NMR), Examples include high performance liquid chromatography (HPLC), gel electrophoresis, capillary electrophoresis, absorbance measurement, fluorescence measurement, elemental analysis, and amino acid quantification. In the analysis, each measurement method may be used alone, or two or more kinds may be arbitrarily combined.

[II. Method for producing biological material complex]
Although there is no limitation on the method for producing the biological material complex, usually, the specific substance is bound to the biological material or the binding compound before, during or after at least any step during the production of the biological material structure. Like that. Therefore, the biological material complex may be prepared, for example, by previously binding a specific substance to a biological material and / or a binding compound and then forming a biological material structure. After the biological material structure is prepared from the substance and the binding compound, the specific material may be bonded to the biological material and / or the binding compound in the biological material structure.

[II-1. Manufacturing method of biological material structure]
When producing a biological material structure, usually, a step of mixing the biological material and the binding compound (hereinafter referred to as “mixing step” as appropriate) is performed. In this mixing step, the biological material and the binding compound are mixed in a medium such as a solvent or a dispersion medium, and the biological material and the binding compound are combined by coexisting in the same system. It is formed. In order to efficiently obtain the biological material structure, a concentration step for removing the medium, a drying step, or the like may be performed after the mixing step. Furthermore, an additive may be allowed to coexist in the system as appropriate in any step of the manufacturing process of the biological material structure.

[II-1-1. Mixing process]
In the mixing step, the biological material and the binding compound are mixed. Thereby, a particulate lump is obtained. The particulate mass aggregates and binds to form a biological material structure. Normally, a process in which a biological substance and a binding compound are combined to form a particulate mass and a process in which the particulate mass is aggregated to form a biological material structure proceed as a series of processes. .

(1) Biological material The biological material is as described above.

(2) Binding compound The binding compound is as described above.

(3) Medium When mixing a biological substance and a binding compound, it is preferable to coexist a medium such as a solvent or a dispersion medium and bond the biological substance and the binding compound in the presence of the medium. As described above, the biological substance and the binding compound do not necessarily cause a chemical reaction and bind, but in this specification, a substance that forms a field when the biological substance and the binding compound are bound to each other. Is broadly called “medium”.

  As the medium, any material can be used as long as the production of the biological material structure is possible. However, it is usually desirable to use a material in which the biological material, the binding compound, and appropriate additives can be mixed. At this time, the mixing state of the biological substance, the binding compound and the additive is arbitrary, and may be in a dissolved state or a dispersed state. In order to stably bind the biological substance and the binding compound. Preferably, the biological material and the binding compound are present in a dissolved state in the medium.

  A liquid is usually used as the medium. At this time, the medium forms a field where the biological substance and the binding compound are bound to each other, which may affect the activity of the biological substance and the binding compound, the stability of the structure, etc. It is preferable to select in consideration of the above. Usually, water is used as the medium.

  Further, a liquid other than water may be used as the medium, and 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 medium other than water include THF (tetrahydrofuran), DMF (N, N-dimethylformamide), NMP (N-methylpyrrolidone), DMSO in addition to alcohol solvents such as methanol, ethanol and 1-butanol. (Dimethyl sulfoxide), dioxane, acetonitrile, pyridine, acetone, glycerin and the like.

  Further, when liquid is used as these media, salt may be added to the media. The type of the salt is arbitrary as long as the effects of the present invention are not significantly impaired, and 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 a medium, as water, an aqueous solution in which a solute other than a biological substance and a binding 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, TRIS buffer and the like.
In addition, a medium may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

(4) Additives In any step of the manufacturing process of the biological material structure, as long as the effects of the present invention are not significantly impaired with respect to the biological material, the binding compound and the medium, and the mixture obtained by mixing them. Any additive may be allowed to coexist. Examples of additives include the above-mentioned salts, moisturizers such as acids, bases, buffers, and glycerin, metal ions such as zinc as a stabilizer for biological materials, antifoaming agents, denaturing agents, and the like. .
Moreover, an additive may be used individually by 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

(5) Mixing operation When the biological material structure is manufactured, the biological material and the binding compound described above are mixed in the presence of the medium, and the mixture contains at least the biological material and the binding compound in the medium. To prepare. The resulting mixture is a mixture of a biological substance and a binding compound that can bind to the biological substance in the presence of a medium. In this mixture, the biological substance and the binding compound are bound. A particulate mass is then prepared. In the mixture, the biological material and the binding compound are preferably mixed in a solvent.

  In mixing, the biological material, the binding compound, the medium, the additive, and the like may be prepared in any state as long as the biological material structure can be produced. However, the biological material is usually prepared as a solution or dispersion in which the biological material is dissolved or dispersed in some solvent or dispersion medium. In this case, the solvent or dispersion medium for diluting the biological material is preferably adjusted in consideration of the activity of the biological material, the stability of the structure, and the like. It can be used as a medium.

  Specific operations for mixing the prepared biological material, binding compound, medium, additive and the like are also arbitrary. For example, a biological material solution (such as an aqueous solution) or dispersion may be mixed with a binding compound solution (such as an aqueous solution) or dispersion, and the biological material solution or dispersion may be mixed with a solid binding compound. It may be mixed, a solid biological substance and a binding compound solution or dispersion may be mixed, or a solid biological substance and binding compound may be mixed with a solvent. In addition, this mixture can be prepared on a solid phase carrier for the purpose of immobilizing a biological material structure on the solid phase carrier described later.

(6) Composition at the time of mixing The mixing ratio of the biological material, the binding compound, the medium, the additive and the like at the time of mixing is arbitrary as long as the biological material complex of the present invention can be obtained. However, the value of the mixing ratio represented by “(weight of biological material) / {(weight of biological material) + (weight of binding compound)}” is usually 0.1 or more, preferably 0.3 or more, More preferably 0.5 or more, and particularly preferably 0.7 or more. When the mixing ratio is lower than this, the composition of the biological material in the formed biological material structure becomes low, and the binding compound cannot be sufficiently covered with the biological material, which may cause nonspecific adsorption.

  Further, the ratio (concentration) of the biological substance and the binding compound in the medium is arbitrary as long as the biological substance complex of the present invention can be obtained, but the total concentration of the biological substance and the binding compound is usually 0.1 g. / L or more, preferably 1 g / L or more, more preferably 10 g / L or more. This is because if it falls below this range, the particulate mass and the biological material structure are not easily generated, and the biological material complex may not be obtained.

(7) Formation mechanism of biological material structure A particulate matter is formed by mixing a biological material and a binding compound, and the particulate matter aggregates constitute a biological matter structure.
The formation process of the biological material structure is not clear, but can be estimated as follows. That is, when a mixture in which a biological substance and a binding compound coexist is prepared, the biological substance and the binding compound are combined in the mixture (see FIG. 2A), and particles as shown in FIG. A clump is generated. The confirmation of the particulate mass in the process of forming such a biological material structure can be performed by dynamic light scattering measurement or the like. For example, when a protein of about 10 nm and a binding compound of about 10 nm are mixed in a solution, a submicron order particulate mass may be confirmed. As shown in FIGS. 1 (a) and 1 (b), the particulate mass is a biological material having a structure in which the particulate mass is aggregated in a chain shape and / or a mesh shape, as shown in FIGS. Presumed to form a structure.

  At this time, it is assumed that the particulate mass to be bonded may be bonded to each other by the biological material and the binding compound, and the particulate mass may be bonded to each other to form a biological material structure. Is done. In this case, since the biological material structure becomes more stable, it can be applied to a wide range of environments and uses, which is preferable. In the case where a biological material structure is prepared by the above-described method using a biological material and / or a binding compound to which a specific substance is bound in advance, the produced material is the biological material complex of the present invention. In this case, it is considered that the biological material complex is formed by the same mechanism as described above.

[II-1-2. Concentration process and drying process]
During the mixing step or after the mixing step, a concentration step or a drying step for removing the medium from the above mixture may be appropriately performed.

When the amount of the solvent in the above mixture is large, it may be difficult to generate a particulate lump or a biological material structure in the mixture, or it may not be generated. In these cases, by concentrating the mixture, a particulate mass can be efficiently formed, a biological material structure can be produced efficiently, and a biological material complex can be obtained efficiently. Become.
Furthermore, even when the above mixture contains a particulate mass or a fragment of the biological material structure, the particulate mass or the biological material structure can be further formed by concentration. Therefore, concentration may be performed for the growth of such a particulate lump or biological material structure.

  However, in order to form a uniform biological material structure and obtain a uniform biological material complex, it is necessary to uniformly mix the biological material and the binding compound in the medium in the initial stage of preparation of the mixture. preferable. Therefore, it is preferable to produce a biological material structure by once producing a particulate mass by coexisting a biological material and a binding compound in a relatively large amount of medium and concentrating them.

Further, the medium may be removed by drying after the biological material structure is formed. In general, since the mixture is concentrated in the process of drying the mixture, 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 temperature conditions for drying and concentrating the mixture are arbitrary, but from the viewpoint of avoiding denaturation of biological materials, it is usually 25 ° C. or less, preferably 10 ° C. or less.
The pressure conditions for drying and concentrating the mixture are arbitrary, but it is usually desirable to reduce the pressure below normal pressure.

  Furthermore, the concentration of the mixture increases the probability that the biological material and the binding compound are in contact with each other to facilitate the formation of a particulate mass, or increases the probability that the particulate mass is in contact with each other to form the biological material structure. The purpose is to make it easy to configure. Therefore, before concentration, during or after concentration, the mixture is precipitated by centrifugation, a poor solvent is added, or an additive such as ammonium sulfate is added to precipitate the particulate mass or biological material structure. You may make it make it.

[II-1-3. Other processes]
Moreover, in the manufacturing method of the biological material structure of this invention, you may perform processes other than the above-mentioned.
For example, after the biological material structure is manufactured, a desired functional group may be modified with respect to the biological material in the biological material structure.
Further, for example, the biological material complex of the present invention may be fixed to some solid phase carrier to produce a biological material complex carrier.

[II-2. Binding of specific substances]
By binding a specific substance to a biological substance and / or a binding compound before, during or after at least one of the above-described mixing process, concentration process, drying process and other processes. The biological material complex of the present invention is obtained.

There is no limitation on the method for binding the specific substance to the biological substance and / or the binding compound, and it is arbitrary.
Usually, it can be bound by bringing a specific substance into contact with a biological substance and / or a binding compound. When a linker is used, the linker is first brought into contact with the biological substance and / or the binding compound, and then the biological substance and / or binding compound to which the linker is bound is contacted with the specific substance. After the linker is brought into contact with the specific substance, the specific substance in contact with the linker is brought into contact with the biological substance and / or the binding compound.

  For example, by reacting a biological group and / or a functional group (including a modified group) contained in a binding compound or a biological substance structure with a functional group (including a modified group) contained in a specific substance or a linker. In the case of bonding, examples of combinations of functional groups are as follows. That is, in the case of bonding by a covalent bond, if one functional group of the bond is an amino group, examples of the other functional group include a succinimide group, an epoxy group, and an aldehyde group. Further, when one functional group of the bond is a hydroxyl group, the other functional group includes an epoxy group. Furthermore, if one functional group of the bond is a thiol group, the other functional group includes a maleimide group.

  In addition, the interaction between biological substances can be used for binding of specific substances. For example, a specific substance can be bound to a biological substance structure by an interaction between avidins and biotin. As a specific example, when the biological material contained in the biological material structure is avidin, it can be bound using biotin contained in a specific substance or a linker or modified. On the other hand, when biotin is contained or modified in a biological material structure, it can be bound using a specific substance or avidin contained or modified in a linker.

  In addition, protein A (protein G, protein A / G) and antibody (particularly Fc part) binding, antibody and antigen binding, sugar / sugar chain and virus binding, boronic acid and sugar binding, metal Examples include binding of a chelate and a tag fusion protein (for example, nickel chelate and histag fusion protein), binding of a sugar (for example, maltose) and a tag fusion protein (for example, maltose binding protein), binding of glutathione and glutathione-S-transferase, etc. It is done. Of these, a bond between avidins and biotin is preferable.

  As a specific operation when the linker or the specific substance is brought into contact with the biological substance and / or the binding compound, the biological substance or the binding compound and the specific substance or the linker used appropriately are usually present in an appropriate medium. I will let you. At this time, the temperature condition, reaction time, use device, etc. are arbitrary as long as the specific substance can be bound to the biological substance. Usually, these conditions are set according to the types of specific substances, biological substances, binding compounds, linkers, and the like. However, it is preferable to carry out the conditions under which the biological material is not deactivated as much as possible.

  For example, it is preferable that the temperature condition at the time of bonding is usually room temperature or lower. Specifically, it is usually performed at 25 ° C. or lower, preferably 10 ° C. or lower. However, when the binding of the specific substance or the linker to the biological material and / or the binding compound does not proceed within the above desirable temperature range, the binding may be performed in a temperature range where the binding is likely to proceed.

  Moreover, as a medium at the time of binding, it is preferable to mainly use water in order to suppress the deactivation of the biological material. However, when a special substance, a linker, or the like has a specific property such that it is difficult to dissolve or disperse in water, a water-soluble organic solvent may be used as a medium. In this case, the ratio of the water-soluble organic solvent is arbitrary, but is usually 80% by weight or less, preferably 70% by weight or less, more preferably 50% by weight or less.

Specific examples of the water-soluble organic solvent include carbon numbers such as methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol, tert-butyl alcohol, isobutyl alcohol, and n-amyl alcohol. 1-5 alkyl alcohols, dimethylformamide, amides of dimethylacetamides, ketones or ketoalcohols such as acetone and diacetone alcohol, ethers such as tetrahydrofuran and dioxane, polyalkylene glycols such as polyethylene glycol and polypropylene glycol , Ethylene glycol, diethylene glycol, propylene glycol, butylene glycol, triethylene glycol, 1,2,6-hexanetriol, thiodi Alkylene glycols whose alkylene group contains 2 to 6 carbon atoms such as recall, hexylene glycol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, triethylene glycol monomethyl ether, triethylene glycol Lower alkyl ethers of polyhydric alcohols such as ethylene glycol monoethyl ether, lower dialkyl ethers of polyhydric alcohols such as triethylene glycol dimethyl ether and triethylene glycol diethyl ether, glycerin, sulfolane, N-methyl-2-pyrrolidone, 1,3-dimethyl-2-imidazolidinone and the like can be mentioned. Of these, dimethylformamide is preferred. In addition, the medium at the time of coupling | bonding may be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and ratios.
Further, the medium may be removed after the production of the biological material complex.

Hereinafter, a specific method will be described with reference to an example in which a metal chelate is bound to a biological substance as a specific substance.
When binding a metal chelate as a specific substance to a biological substance, for example, first, a chelating reagent (chelate forming group) is bound to the biological substance, and then a desired metal ion is bound to the chelate forming group. Specific substances can be bound.

  Such a reaction can be appropriately modified using a known method for the purpose of suppressing non-specific adsorption or improving hydrophilicity. For example, for the purpose of suppressing nonspecific adsorption, when albumin is used as a biological material and the amino group of albumin is used as a functional group that binds to a specific substance, a succinimide group is introduced into the chelating reagent and the amino group and succinimide are introduced. By reacting with a group, a chelating reagent can be introduced into a biological substance by a covalent bond.

  In addition, for example, when hydrophobic AB-NTA (N (5-Amino-1-carboxylicoxy) -iminodiacetic acid) is used as the chelating reagent, it has hydrophilic ethylene oxide to increase the hydrophilicity. It is preferable to further intervene a linker such as EGS (Ethylene glycol-bis (succinimidyl succinate)). In addition, the use of EGS is advantageous because a succinimide group can be introduced into the chelating reagent.

  In this reaction, a biological material complex may be formed by coexisting a biological material, a binding compound, a chelating reagent, a linker, and a metal ion at a time. However, a biological material structure is first prepared, and then a reaction product obtained by reacting a chelating reagent with a linker is obtained, and the chelating reagent is introduced into the biological material structure by bringing it into contact with the biological material structure. Then, it is preferable to make a biological material complex using a metal chelate as a specific substance by contacting metal ions.

As a more specific example, the production of a biological material complex for elucidating the action mechanism of a physiologically active substance (medicine) will be described below.
When FK506, which is a physiologically active substance (medicine), is bound as a specific substance to a biological substance using an avidin-biotin interaction, avidins (eg, streptavidin, etc.) are used as the biological substance. To FK506, an ethylene oxide chain is bonded as a hydrophilic linker, and biotin is introduced into the terminal thereof to obtain biotinylated FK506 (reference document Chemistry & Biology, Vol. 9, 691-698 (2002)). At this time, the biological material (for example, streptavidin), the binding compound, and the biotinylated FK506 may coexist at the same time, but after the biological material structure is prepared, the biotinylated FK506 is contacted with the biological material structure. It is more preferable to make it.

Furthermore, the binding of the chelating reagent to the biological material and the binding of the specific substance to the biological material to which the chelating reagent is bound are usually performed in the medium, but the medium to be used is arbitrary. For example, the biological material structure Examples of the medium used at the time of the production include those similar to those exemplified as examples and aqueous organic solvents exemplified. In addition, the solvent in this case may also be used individually by 1 type, and may use 2 or more types together by arbitrary combinations and ratios.
Moreover, the reaction conditions at the time of coupling | bonding are arbitrary unless the effect of this invention is impaired remarkably.

[III. Explanation of affinity separation using biological material complex]
The biological material complex of the present invention is suitable for use in affinity separation. Therefore, the biological material complex of the present invention can be used as, for example, affinity purification, a pharmaceutical function analysis tool, or a diagnostic analysis tool.

  Specifically, for example, when a physiologically active substance is used as a specific substance for the purpose of affinity purification or a drug action mechanism analysis tool, a biological substance such as a minute amount of protein contained in serum or the like is to be purified. It becomes a substance, and it can be obtained from impurities by the interaction between the specific substance and the target substance. For example, when analyzing the action mechanism of a drug candidate compound for a specific substance contained in a biological substance complex, a plurality of drug candidate compounds can be analyzed by using a protein that contributes to a disease such as a receptor as the specific substance. The drug candidate compound can be selected or the mechanism of action can be elucidated by analyzing the drug candidate compound that is specifically bound to the target substance that is to be contacted with the biological substance complex.

Furthermore, for example, when affinity purification is performed using nickel chelate as a specific substance, a His-tag fusion protein synthesized by genetic engineering becomes a target substance, and a His-tag synthesized from impurities contained in a culture solution. The fusion protein can be purified and separated.
In addition, for example, when using an antibody or antigen as a specific substance as a diagnostic analysis tool, blood, serum, plasma, spinal fluid, urine, feces, nasal discharge, saliva and other body fluids, cells, tissues, and extracts thereof A substance that specifically binds to an antibody or antigen, which is a specific substance, contained in a specimen is a target substance, and a disease can be diagnosed by analyzing the amount or presence of the target substance.

  When affinity separation is performed as described above, separation and purification, analysis of structure and function, and the like are performed by using the above-described specific interaction between the specific substance contained in the biological substance complex and the target substance. At this time, first, a sample liquid (specimen) containing the target substance is brought into contact with the biological substance complex to separate the target substance and other substances in the sample liquid.

(1) Target substance that is subject to separation and purification The target substance that is subject to separation and purification refers to an agent that specifically interacts (affinity binds) with a specific substance. As an example of such a target substance, the same biological substance as the above-mentioned biological substance complex can be used.
As a specific example, when a substance that can be a drug candidate (drug candidate substance) is separated, a substance that can cause a desired interaction to occur in the drug candidate substance is used as a specific substance, and the drug candidate substance is contained. It is possible to separate a compound that can be a drug candidate substance as a target substance from a specimen that may possibly cause the above.

(2) Sample solution containing the target substance The target substance usually coexists with other substances in the specimen as a composition. The specimen may be a gas, but is usually prepared as a liquid. In this case, the specimen is often a solution or dispersion containing the target substance in some solvent. Hereinafter, a liquid specimen existing as a solution or a dispersion is referred to as a “sample liquid”.

The solvent and dispersion medium of the sample solution are arbitrary as long as the effects of the present invention are not significantly impaired. For example, those described above as the medium for producing the biological material complex can be used.
The concentration of the target substance in the sample solution is also arbitrary, but it is usually 1 μg / L or higher, preferably 5 μg / L or higher, more preferably 10 μg / L or higher. If the concentration is lower than this range, the purification efficiency is lowered, and the advantages of the present invention may not be fully exhibited.

  Furthermore, it is desirable that substances other than the target substance have a concentration in the sample solution of usually 50% by weight or less, preferably 40% by weight or less, and more preferably 30% by weight or less. If it exceeds this range, the viscosity of the sample solution becomes too high, and there is a possibility that the target substance will sufficiently enter the inside of the biological material complex and cannot contact the specific substance.

(3) Method for contacting biological material complex with sample liquid The method for bringing the sample liquid containing the target substance into contact with the biological material complex of the present invention is not particularly limited. Among them, there is a method in which a sample solution containing a target substance is brought into contact with the mobile phase by flowing it together with the mobile phase. At this time, the biological material complex may be in a dry state, but is preferably wetted with a liquid serving as a mobile phase before contacting the sample liquid.

  In another method, for example, a sample liquid containing a target substance is put in a container such as a microtube, and a biological material complex is added to the sample liquid, and the container is filled with the biological material complex. It is also possible to make contact by adding a sample solution containing the target substance to the liquid crystal.

  Furthermore, the efficiency of separation and purification can be further improved by immobilizing the biological material complex of the present invention on a solid phase carrier. For example, a small and highly efficient separation and purification apparatus can be configured by immobilizing the biological material complex of the present invention on the surface of a microchannel and feeding a sample solution containing the target substance into the channel.

In addition, when the sample liquid containing the target substance is brought into contact with the biological material complex, any conditions may be set as appropriate. However, it is preferably 25 ° C. or lower, more preferably 10 ° C. or lower. .
Furthermore, the sample solution can be appropriately dried and concentrated before, during and after contact. The pressure condition at that time is also arbitrary, but it is usually preferable to be equal to or lower than normal pressure.

(4) Separation of target substance It is arbitrary how the target substance is separated after contacting the biological material complex of the present invention with the sample liquid depending on the specific substance, the type of the target substance, and the like.
For example, if there is a difference in retention time between the target substance and other substances due to specific interaction between the specific substance and the target substance, use this difference in retention time. Thus, fractionation and purification can be performed to separate the target substance from other substances (affinity chromatography).

  In addition, in particular, when the target substance is adsorbed by an interaction specific to the specific substance, the target substance is adsorbed on the specific substance, and the sample liquid is separated from the biological substance complex in that state, and then By separating the target substance from the specific substance and collecting it, the target substance can be separated from other substances. The method for releasing the target substance from the specific substance is not particularly limited. For example, in addition to the added salt, pH, temperature rise, etc., a known chemical substance having a higher adsorption power than the target substance to be separated or purified, Alternatively, even if it has a weak adsorption force, the target substance can be released from the biological substance complex and collected by adding a high concentration chemical substance.

  These separated target substances can be diluted or concentrated according to the purpose. For example, when diluting, a solvent according to the purpose may be added. When concentrating, the solvent is evaporated using reduced pressure or warming or both, using an ultrafiltration filter, or using a freeze-drying method. To remove the solvent completely.

In addition, when analyzing a target substance separated by the biological material complex of the present invention, the method is not limited, but generally, for example, liquid chromatography, gas chromatography, mass spectrometry (MS), infrared, Examples include spectroscopy, nuclear magnetic resonance ( ¹ H-NMR, ¹³ C-NMR, ²⁹ Si-NMR), high performance liquid chromatography (HPLC), gel electrophoresis, capillary electrophoresis, absorbance measurement, and fluorescence measurement. In the analysis, each measurement method may be used alone, or two or more kinds may be arbitrarily combined. When measuring the amount of the target substance adsorbed to the specific substance in the second embodiment to be described later, or when measuring the amount of the target substance in the fractionation by the measuring unit 21 in the fourth embodiment, A measuring instrument using the exemplified method can be used.

(5) Advantages of affinity separation using biological material complex In affinity separation according to the prior art, the specific substance is immobilized on the surface of the carrier for affinity chromatography used for the separation, so the amount of the specific substance introduced is limited. (Normally, monolayer adsorption of protein is 0.3 to 1.0 μg / cm ² ). Therefore, conventionally, in the affinity chromatography carrier, when the specific substance interacts with the target substance, the amount of the target substance that can interact in the unit volume is small.

  Further, conventionally, the affinity chromatography carrier could not be completely covered with a specific substance, and therefore non-specific interaction with the affinity chromatography carrier was likely to occur, resulting in low separation and purification efficiency. Therefore, in order to increase the purification purity, multiple purifications are required, and separation and purification take time and effort.

  In contrast, in the biological material complex of the present invention, the reactivity of the specific substance introduced into the biological material complex can be maintained. Further, by increasing the ratio of the specific substance in the biological substance complex, it is possible to increase the amount of the target substance that specifically interacts with the specific substance in the unit volume of the biological substance complex. Furthermore, since the ratio of the binding compound can be kept low by increasing the ratio of the specific substance in the biological material complex, nonspecific adsorption based on the binding compound can be suppressed. As a result, non-specific adsorption can be suppressed as compared with the prior art, and the time required for separation and purification can be further shortened.

  Further, the biological material complex of the present invention can be formed on the surface of an arbitrary solid phase, and can be applied to microchips, microchannels, and the like that have been actively studied in recent years. Therefore, the fact that it can be applied to a wide range of uses is one of the advantages of using the biological material complex of the present invention.

  Furthermore, the biological material complex of the present invention can be suitably used as a sensor for detecting an agent that interacts with a specific substance. At this time, specifically, for example, a sensor chip used for each sensor may be provided with the biological material complex of the present invention. As the sensor, for example, when DNA, protein, or sugar is used as a specific substance, a so-called DNA array or DNA chip, or an array or sensor chip such as a protein array or protein chip, or a virus detection chip is used. Examples include biosensors and diagnostic analysis tools.

  As described above, specific examples of biosensors to which the biological material complex of the present invention can be applied include fluorescence method, ELISA method, chemiluminescence method, RI method, SPR (surface plasmon resonance) method, QCM (quartz crystal oscillation). Child 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 nanotubes, electrochemical method Sensor. Among them, detection by the QCM method and the SPR method is preferably used because the sample can be easily analyzed without labeling.

(6) Embodiment Hereinafter, an embodiment in which a target substance is separated from a sample solution using a biological material complex will be described. However, the present invention is not limited to the following embodiments.

(6-1) First Embodiment In this embodiment, a target substance is separated and purified from a sample solution in which the target substance (separation target substance) and other substances coexist in the liquid.
Moreover, FIG. 3 is sectional drawing which shows typically the container for affinity chromatography used for this embodiment.

  In this embodiment, as shown in FIG. 3, an affinity chromatography container 3 (hereinafter referred to as “affinity container” as appropriate) 3 holding a biological material complex 2 in a container body 1 is used. Here, there is no restriction | limiting in the shape of the container main body 1, If it can accommodate fluids, such as a sample solution, it is arbitrary.

Further, in the affinity container 3 of the present embodiment, the biological material complex 2 is fixed to the inner surface of the container body 1, thereby preventing the biological material complex 2 from coming out of the container body 1. It is held in the main body 1. However, the biological material complex 2 may be simply stored in the container body 1 without being fixed to the container body 1.
Furthermore, the specific substance of the biological material complex 2 used in the present embodiment and the target substance to be purified specifically interact, so that the target substance can be specifically adsorbed to the specific substance. It shall be.

When the target substance is separated from the sample liquid using the affinity container 3, first, the sample liquid is injected into the affinity container 3. Thereby, the biological material complex 2 comes into contact with the sample liquid, and the target substance is adsorbed to the specific substance.
Next, the sample solution is discharged out of the affinity container 3 in order to separate the sample solution and the biological material complex 2. Thereby, components other than the target substance contained in the sample solution are discharged. On the other hand, the target substance is held in the affinity container 3 by adsorbing to the specific substance of the biological substance complex 2.
Then, the target substance is released from the biological substance complex 2 by, for example, injecting a recovery solution adjusted to a predetermined pH that can weaken the interaction between the target substance and the specific substance into the affinity container 3, and the released target The material is recovered with the recovery solution described above.

  As described above, the target substance in the sample solution can be separated and purified. In this case, a specific substance that specifically interacts with the target substance is used, and the biological substance complex of the present invention contains a very large amount of the specific substance that retains its activity. Furthermore, non-specific adsorption can be suppressed and high-efficiency separation and purification can be easily performed.

In addition, when it is unclear which agent causes a given interaction with the specific substance using this, it is necessary to screen what substance interacts with the biological material. Also, the technique of the present embodiment can be applied. That is, for example, in the above embodiment, if a sample liquid containing a substance that causes some interaction is used, the substance (target substance) obtained by separating and purifying from other substances in the sample liquid is Since it is an agent that causes the above interaction, the above screening can be appropriately performed.
If the biological material complex 2 is not fixed to the container body 1 in the configuration of the present embodiment, the biological material complex 2 is collected using a centrifuge before discharging the sample liquid. It is preferable because efficiency and recovery efficiency are improved.

(6-2) Second Embodiment In this embodiment, a target substance is analyzed by separating and purifying the target substance in a sample solution containing the target substance (analysis target substance) in the liquid. .
In addition, although this embodiment is demonstrated using FIG. 3 similarly to 1st Embodiment, in this embodiment, the site | part similar to 1st Embodiment is shown using the same code | symbol.

The affinity container 3 used in this embodiment specifically interacts with a substance (active substance) having a specific structure (molecular structure) as a specific substance that the biological substance complex 2 has, thereby The one that is specifically adsorbed is used.
Other configurations are the same as those in the first embodiment.

  When analyzing the target substance in the sample liquid using the affinity container 3, as in the first embodiment, the sample liquid is injected into the affinity container 3 to bring the biological material complex into contact with the sample liquid. Subsequently, the sample liquid is discharged out of the affinity container 3 in order to separate the biological material complex 2 and the sample liquid. Thereby, when the target substance has a specific structure capable of specifically interacting with the specific substance, the target substance is adsorbed by the specific substance of the biological material complex 2, and thereby the affinity container. 3 is held. Conversely, if the target substance does not have the specific structure, the target substance is discharged out of the affinity container 3 together with the sample solution.

Therefore, whether or not the target substance has the above-mentioned specific structure by examining whether or not the target substance is adsorbed on the specific substance, that is, whether or not the target substance remains in the affinity container 3. It turns out whether or not.
In order to check whether or not the target substance is adsorbed on the specific substance, specifically, the amount of the target substance adsorbed on the specific substance may be measured. For example, in the same manner as in the first embodiment, the target substance is introduced into the biological material complex 2 by injecting into the affinity container 3 a recovery solution adjusted to a predetermined pH that can weaken the interaction between the target substance and the specific substance. The recovery solution may be recovered, and the amount of the target substance contained in the recovered recovery solution may be measured by the above-described measurement method or the like.

By doing so, it is possible to analyze whether or not the target substance in the sample solution has a specific structure corresponding to at least the specific substance.
Furthermore, the technique of the present embodiment can also be applied to the case where the molecular structure that the active substance should have in order to interact with the specific substance is examined using this. That is, for example, when a group of substances (target substances) is separated from other substances by interacting with a specific substance, if the group of substances has a common molecular structure, It can be assumed that the molecular structure is the molecular structure that the active substance should have in order to interact with the specific substance.

Moreover, since the biological material complex of the present invention can easily perform highly efficient separation and purification by suppressing non-specific adsorption as in the first embodiment, analysis of the target substance is possible. Can be performed accurately and with high sensitivity.
The present embodiment can be modified in the same manner as the first embodiment.

(6-3) Third Embodiment In this embodiment, the target substance is separated and purified from a sample solution in which the target substance (purification target substance) and other substances coexist in the liquid.
FIG. 4 is a diagram schematically showing an outline of the affinity chromatography apparatus used in the present embodiment.

  In this embodiment, an affinity chromatography apparatus (hereinafter referred to as “affinity chromatography apparatus” as appropriate) 10 as shown in FIG. 4 is used. The affinity chromatography apparatus 10 includes a tank 11, a pump 12, an autoinjector 13, an affinity separation chip 14, a flow path switching valve 15, recovery bottles 16 and 17, and a control unit 18.

The tank 11 stores a carrier liquid serving as a mobile phase.
The pump 12 is for flowing the carrier liquid stored in the tank 11 at a predetermined flow rate in accordance with the control of the control unit 18.
Further, the autoinjector 13 injects the sample liquid into the carrier liquid flow under the control of the control unit 18.
Accordingly, the carrier liquid stored in the tank 11 is supplied to the affinity separation chip 14 through the autoinjector 13 by the pump 12 at a predetermined speed, and the sample liquid is injected into the carrier liquid by the autoinjector 13. It has come to be. Accordingly, the tank 11, the pump 12, and the autoinjector 13 constitute a sample liquid supply unit 19 that distributes the sample liquid to the flow path 14B of the affinity separation chip 14.

  Further, the affinity separation chip 14 has a channel 14B formed on a substrate 14A, and the channel 14B is filled with a biological material complex (not shown). Here, the specific substance of the biological material complex used in the present embodiment and the target substance to be purified change specifically, thereby changing the retention time of the target substance flowing through the flow path 14B. It is assumed that Note that a filter (not shown) for preventing the biological material complex from flowing out is formed at the downstream end of the flow channel 14B, so that the biological material complex is reliably held in the flow channel 14B. It has become so.

  Accordingly, the supplied carrier liquid (including the liquid into which the sample liquid is injected) flows through the flow path 14B filled with the biological material complex. Further, the carrier liquid eluted from the flow path 14B (hereinafter, the liquid flowing out from the flow path 14B is referred to as “eluent” as appropriate) is sent to the downstream flow path switching valve 15.

  By the way, in this embodiment, the affinity separation chip 14 is assumed to be detachable from the affinity chromatography apparatus 10. Specifically, the affinity chromatography apparatus 10 includes a chip mounting portion 14C for mounting the affinity separation chip 14, and when used, the affinity separation chip 14 is mounted on the chip mounting portion 14C for separation. ing.

The flow path switching valve 15 switches the flow path according to the control of the control unit 18 and switches the recovery bottle 16 or the recovery bottle 17 to recover the eluate sent from the affinity separation chip 14. is there. Therefore, the eluate from the flow path 14B of the affinity separation chip 14 is fractionated by the flow path switching valve 15 and recovered in one of the recovery bottles 16 and 17.
In the present embodiment, it is assumed that the carrier liquid containing the target substance is collected in the collection bottle 16 and the carrier liquid not containing is collected in the collection bottle 17.

The control unit 18 controls the pump 12, the auto injector 13, and the flow path switching valve 15. In the present embodiment, the control unit 18 is configured by causing a computer to read a control program.
Further, the control unit 18 records information on the retention time due to the interaction between the specific substance and the target substance included in the biological substance complex used in the present embodiment. Based on this information, the auto injector 13 is recorded. The switching timing of the flow path switching valve 15 is controlled in accordance with the amount of the supplied carrier liquid after the sample liquid is injected from, and the elapsed time.
In FIG. 4, the control by the control unit 18 is indicated by a one-dot chain line arrow.

When the target substance is separated from the sample solution using the affinity chromatography apparatus 10, first, the affinity separation chip 14 is mounted on the chip mounting section 14 </ b> C, and the control section 18 operates the pump 12. However, the flow path switching valve 15 is initially switched to collect the eluate in the collection bottle 17. In this case, the carrier liquid in the tank 11 flows out downstream, passes through the auto injector 13, the flow path 14 </ b> B of the affinity separation chip 14, and the flow path switching valve 15, and is recovered in the recovery bottle 17.
Thereafter, the control unit 18 controls the autoinjector 13 to cause the autoinjector 13 to inject the sample liquid into the carrier liquid. Further, the control unit 18 starts counting time simultaneously with the injection.

  The injected sample liquid flows into the flow path 14B and flows through the flow path 14B. At this time, the sample liquid comes into contact with the biological material complex in the flow path 14B, the specific substance and the target substance interact, and the flow rate of the target substance decreases. Therefore, components other than the target substance in the sample liquid flow through the flow path 14B together with the carrier liquid, but the target substance flows out of the flow path 14B with a delay corresponding to the change in the retention time due to the interaction. For this reason, the fraction corresponding to the retention time of the target substance after the time when components other than the target substance pass through the flow path switching valve 15 in the eluate flowing out of the flow path 14B includes the carrier liquid and , And other substances that are not targeted for separation are included.

  Therefore, the control unit 18 sets the flow path switching valve 15 after the amount of change in the retention time due to the above interaction from the time when the components other than the target substance pass through the flow path switching valve 15 depending on the counting time. By switching, the fraction containing the target substance is collected in the collection bottle 16.

  Thereafter, when performing separation and purification continuously, the control unit 18 switches the flow path switching valve 15 again so that the fraction of the eluate not containing the target substance is collected in the collection bottle 17, and the same as described above. Perform the operation. When the separation / purification is stopped, the control unit 18 stops the pump 12 and stops the supply of the carrier liquid.

  As described above, the target substance in the sample solution can be separated and purified. In this case, a specific substance that specifically interacts with the target substance is used, and the biological substance complex of the present invention contains a very large amount of the specific substance that retains its activity. Furthermore, it is possible to easily perform highly efficient separation and purification by suppressing non-specific interaction.

Further, by using this, as in the first embodiment, when an active substance that causes a predetermined interaction with the specific substance is unknown, what kind of substance is It can also be screened for interaction.
In the configuration of the present embodiment, the biological material complex may be fixed to the affinity separation chip 14 as in the first embodiment.

  In the affinity chromatography apparatus 10, the affinity separation chip 14 is configured to be detachable from the affinity chromatography apparatus 10. However, the affinity separation chip 14 is appropriately integrated with the affinity chromatography apparatus 10. You may do it.

(6-4) Fourth Embodiment In this embodiment, a target substance is analyzed by separating and purifying the target substance in a sample solution containing the target substance (analysis target substance) in the liquid. .
FIG. 5 is a diagram schematically showing the outline of the affinity chromatography apparatus used in the present embodiment. However, in FIG. 5, the same parts as in FIG. 4 are denoted by the same reference numerals as in FIG.

  In this embodiment, an affinity chromatography apparatus 20 as shown in FIG. 5 is used. The affinity chromatography apparatus 20 includes a tank 11, a pump 12, an autoinjector 13, an affinity separation chip 14, a control unit 18, and a measurement unit 21.

The sample liquid supply unit 19, that is, the tank 11, the pump 12, and the auto injector 13 are the same as those in the third embodiment.
Further, the affinity separation chip 14 circulates in the flow path 14B by specifically interacting with a substance (active substance) having a specific structure (molecular structure) as a specific substance possessed by the biological material complex. The third embodiment is the same as the third embodiment except that a material that changes the retention time of the target substance is used.
Further, the control unit 18 is the same as the third embodiment except that the control valve 18 is not controlled because the affinity chromatography apparatus 20 of the present embodiment does not have the switching valve.

  Further, the affinity chromatography device 20 includes a measurement unit 21 that measures the amount of the detection target substance in the fraction of the eluate from the flow path 14B downstream of the affinity separation chip 14. Specifically, the measurement unit 21 measures the amount of the target substance in the eluate over time, so that the eluate flowing into the measurement unit 21 at each time is included in each fraction as a fraction. The amount of the target substance is measured. In addition, as a specific example of the measurement part 21, the thing similar to what was mentioned above is mentioned.

When the target substance in the sample solution is analyzed using the affinity chromatography apparatus 20, the affinity separation chip 14 is mounted on the chip mounting section 14C, and the control section 18 is connected to the pump 12 as in the third embodiment. Is operated to cause the carrier liquid in the tank 11 to flow. At this time, the measurement unit 21 also starts measuring the target substance in the eluate.
Thereafter, the controller 18 causes the autoinjector 13 to inject the sample liquid into the carrier liquid, as in the third embodiment. The injected sample liquid flows into the flow path 14B together with the carrier liquid and flows through the flow path 14B. At this time, the sample solution comes into contact with the biological material complex in the flow path 14B.
Then, the eluate flowing out from the flow path 14B flows into the measurement unit 21. And in the measurement part 21, the quantity of the target substance contained in the fraction of each time of an eluate is measured.

  Here, when the biological material complex and the sample liquid come into contact with each other, if the target substance has the specific structure described above, the retention time of the target substance changes in the flow path 14B. The time when the target substance flows out from the path 14B is delayed. On the other hand, if the target substance does not have the specific structure, the retention time of the target substance does not change.

Therefore, depending on which fraction of the eluate fraction the target substance was detected, whether or not the target substance has a specific structure and how much is there. Can be analyzed.
That is, if the target substance is measured in the fraction of the time to be observed when the retention time does not change, it can be determined that the target substance does not have the specific structure.

  In addition, if the target substance is measured in a fraction after the time that should be observed when the retention time does not change, it can be determined that the target substance has the specific structure described above. it can. Further, by measuring how much the change in the retention time was (ie, how long it was measured later), the magnitude of the interaction is sensed, and the specific structure of the target substance is detected. Numbers can also be analyzed. In addition, if the target substance contains two or more types of substances, the amount of the target substance contained in each fraction can be measured to determine which target substance has how much specific structure. It is also possible to do.

As described above, according to the affinity chromatography apparatus 20 of the present embodiment, it is possible to analyze whether or not the target substance in the sample solution has a specific structure corresponding to the specific substance. . Moreover, since the biological material complex of the present invention can easily perform highly efficient separation and purification by suppressing non-specific interactions as in the third embodiment, Analysis can be performed accurately and with high sensitivity.
Furthermore, using this, as in the second embodiment, it is also possible to investigate the molecular structure that the active substance should have in order to interact with the specific substance.

It is also possible to output the measurement result of the measurement unit 21 to the analysis unit that performs the analysis, and cause the analysis unit to analyze the target substance. For example, the analysis unit reads the measurement result output from the measurement unit 21, and the analysis unit has measured the target substance in the fraction of time to be observed when the retention time does not change as described above. It can be configured to determine whether or not the target substance has a specific structure depending on whether or not. However, in this case, it is preferable that the analysis unit is provided with a storage unit that stores the type of biological material complex, a specific structure corresponding to the type, and retention time information used for determination. Note that the analysis unit can be configured as hardware, for example, by causing a computer to read a program that causes the computer to function as the analysis unit.
Note that this embodiment can be modified in the same manner as the third embodiment.

(7) Biological substance complex carrier As in the affinity container and affinity separation chip used in the first to fourth embodiments, the biological material of the present invention can be used as a tool for affinity purification or for analysis of drug action mechanism. When a substance complex is used, it is desirable to use it as a biological substance complex carrier by fixing it to a solid support depending on conditions.

  The biological material complex carrier refers to a substance in which the biological material complex of the present invention is fixed to some solid phase carrier. In this case, the solid phase carrier is a substrate for immobilizing the biological material complex of the present invention on the surface. 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 biological material composite_body | complex 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.

Examples of the shape of the solid phase carrier include a flat plate shape, a particle shape, a fiber shape, a film shape, and a sheet shape. Specific examples include chips (substrates) on which a large number of biological material complexes can be arranged, flat members such as glass slides, fiber slides, microtiter plates, beads as chromatographic carriers and latex diagnostic agents, separation Examples thereof include hollow fiber fibers and porous membranes used as membranes.
In particular, when forming a chip having a flow channel as in the third and fourth embodiments, the shape and dimensions of the flow channel can be arbitrarily formed according to the application. However, when the biological material complex is immobilized on a substrate or the like, it is not necessary. However, when the biological material complex is simply filled and held in the flow path 14B, the flow path 14B can be used. In order to prevent the biological material complex from flowing out, it is preferable to provide an outflow prevention means such as a filter.

Furthermore, the solid phase carrier may be used as it is, or after performing some surface treatment, a biological material complex may be formed on the surface.
For example, the biological material composite may be formed after the surface is coated with a coating material such as metal or metal oxide. Specific examples of the solid phase carrier that may be subjected to such coating treatment include metal-coated chips, glass slides, fiber slides, sheets, pins, microtiter plates, capillary tubes, beads, and the like.

  Further, for example, a functional group may be introduced into the solid phase carrier as a surface treatment in order to bind the solid phase carrier and the biological material complex. The functional group is optional. For example, a hydroxyl group, carboxyl group, thiol group, aminoaldehyde group, hydrazide group, carbonyl group, epoxy group, vinyl group, amino group, succinimide group, etc. And a functional group that binds the biological material complex.

  In addition, when water is used as a medium (solvent) at the time of manufacturing the biological material complex of the present invention, the solid phase carrier and the biological material complex are bound by physical adsorption by lyophobic interaction such as alkyl group and phenyl group. Functional groups can also be used. In addition, the functional group to introduce may be 1 type and may use 2 or more types together by arbitrary combinations and a ratio.

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

  Furthermore, a specific operation for binding the biological material complex of the present invention to the solid phase carrier described above is also arbitrary. For example, a biological material complex may be prepared in advance and bound to a solid phase carrier, or each component of the biological material complex such as a specific substance, biological material, and binding compound may be separately prepared and fixed. They may be combined with a solid phase carrier at the same time while producing a biological material complex by mixing them on the phase carrier. Specifically, for example, a solid phase carrier comprising a solution containing a specific substance (eg, an aqueous solution), a solution containing a linker, a solution containing a biological substance (eg, an aqueous solution), and a solution containing a binding compound (eg, an aqueous solution). After each supply, the two solutions can be mixed on a solid support. When preparing a mixture in advance, the conjugate, particulate mass or biological material complex described above is prepared in the mixture before supply, and then the mixture is supplied to the solid phase carrier. good.

The method and conditions for binding the biological material complex to the solid phase carrier are arbitrary, but from the viewpoint of avoiding denaturation of the biological material, the temperature condition is usually 25 ° C. or lower, preferably 10 ° C. or lower.
In addition, when the mixture after being fixed to the solid phase carrier is dried and concentrated, the pressure condition at that time is also arbitrary, but usually it is preferably not more than normal pressure.

  Furthermore, in order to fix the biological material complex to 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.

  EXAMPLES Hereinafter, the present invention will be specifically described 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. Can do.

[Example 1: Biological material complex using albumin]
(1) Synthesis of Compound for Binding (Polymer A) 1.13 parts by weight of monomer N-acryloyl morpholine (NAM, manufactured by KOHJIN) and N-acryloyloxysuccinimide (NAS, manufactured by ACROS ORGANICS) 0.33 Part by weight and 18.03 parts by weight of dehydrated dioxane (manufactured by Wako Pure Chemical Industries, Ltd.) as a solvent are mixed well, poured into a 50 mL four-necked flask, and deaerated with nitrogen at room temperature for 30 minutes. A monomer solution was prepared.

The monomer solution was heated to 60 ° C. in an oil bath, and a solution prepared by dissolving 0.0016 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 polymer-generated solution was reprecipitated by dropping it into 0.5 L of diethyl ether (made by Kokusan Chemical Co., Ltd.), and then pulverized by removing the solvent to obtain a binding compound polymer A. It was.

As a result of performing SEC (Size Exclusion Chromatography) measurement calibrated with standard polystyrene, the obtained polymer A was estimated to have a weight average molecular weight (Mw) of about 150,000.
The molar ratio (NAS / NAM) between NAS and NAM contained in the obtained polymer A was estimated to be NAS / NAM = 30/70 from ¹ H-NMR measurement.
Further, the polymer A was adjusted to a concentration of 0.2% by weight, 0.4% by weight, and 0.6% by weight with distilled water, and measured with a photon correlation meter ALV5000 (manufactured by ALV) at a measurement angle of 30 °, 40 °. When measured by dynamic light scattering at 50 ° and 60 °, the average hydrodynamic radius of polymer A was estimated to be 6.8 nm.

(2) Formation of biological material complex on QCM sensor chip (2-1) Surface treatment of QCM sensor chip Sensor chip for QCM (Quartz Crystal Micro Balance) (AFFINIX Q: manufactured by Inicium Co.) coated with gold (Initium) was immersed in 10 mM 16-mercaptohexadecanoic acid (16-MERCAPTOCHEXADENOIC ACID: ALDRICH) ethanol solution and reacted at room temperature for 12 hours for surface treatment. After completion of the reaction, the sensor chip was washed with ethanol. This surface treatment is a treatment for introducing a carboxyl group into the surface of the sensor chip via a gold-sulfur 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, The substrate (sensor chip) having the 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 reacted for 15 minutes. This is because a succinimide group capable of bonding the substrate and the biological material structure is introduced to the substrate surface.

(2-2) Formation of biological material structure Next, a 10 mg / mL albumin (ALBUMIN, PIG: manufactured by SIGMA) aqueous solution (phosphate buffer 10 mM, pH 9.0) and polymer A were mixed at a weight mixing ratio of 10: 1 ( 3 μL of the mixture mixed with the biological material (binding compound) was spotted on the surface of the sensor chip. After drying this at room temperature, it was immersed in 8 mL of 1M ethanolamine aqueous solution (pH 8.5) for 15 minutes for the purpose of blocking unreacted active ester groups. As a result, a biological material structure using albumin and polymer A as the biological material and the binding compound was formed on the surface of the sensor chip.
Thereafter, the sensor chip was thoroughly washed with demineralized water and dried at room temperature to obtain a chip for biological material structure QCM carrying the biological material structure on the surface.

(2-3) Chelate surface treatment on biological material structure 300 μL of DMSO (Dimethyl Sulfoxide: manufactured by Kanto Chemical Co.) solution of 100 mM EGS (Ethylene glycol-bis (succinimidylsuccinate): manufactured by PIERCE) and 100 mM AB-NTA ( A DMSO solution (450 μL) of N- (5-Amino-1-carboxypentyl) -iminodiacetic acid (manufactured by Dojindo Laboratories) was mixed and allowed to stand at room temperature for 24 hours. Hereinafter, the obtained solution is referred to as an EGS / AB-NTA solution as appropriate. EGS has a succinimide group at both ends, and by the above mixing operation, one end was bonded to the amino group of AB-NTA to form a compound in which EGS and AB-NTA were bonded.

  As a result of NMR measurement of a part of the EGS / AB-NTA solution, unreacted EGS in the EGS / AB-NTA solution and a compound in which one end of the succinimide group of EGS reacted with AB-NTA (EGS / AB- NTA) and the compound (AB-NTA / EGS / AB-NTA) in which both ends of the succinimide group of EGS are reacted "Unreacted EGS: EGS / AB-NTA: AB-NTA / EGS / AB-NTA" = 10:40:50 (molar ratio). As a result, the obtained EGS / AB-NTA solution was found to be a solution containing only 17 mM of the EGS / AB-NTA compound.

Further, the biological material structure QCM chip was immersed in 8 mL of HEPES buffer (10 mM, pH 7.4), and 50 μL of EGS / AB-NTA solution was added and immersed for 30 minutes.
Furthermore, after removing the chip for the biological material structure QCM from the HEPES buffer, the EGS / AB-NTA solution is diluted 10 times with the HEPES buffer in order to reliably introduce EGS / AB-NTA onto the surface of the biological material albumin. The obtained product was spotted on the biological material structure QCM chip at 3 μL.

After that, the biological material structure QCM chip was washed with demineralized water, and EGS unreacted succinimide was blocked with 8 mL of 1M ethanolamine aqueous solution (pH 8.5) for 15 minutes. Thereafter, it was washed again with demineralized water.
These operations are aimed at bonding EGS / AB-NTA to the biological material structure QCM chip. That is, the succinimide group at one end of EGS / AB-NTA is bonded to the amino group on the surface of albumin in the biological material structure. Here, EGS acts as a linker.

(2-4) Introduction of nickel ions Furthermore, a biological material structure QCM chip was placed in 8 mL of an aqueous solution of 2 mM nickel (II) sulfate hexahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) for 1 hour. It was immersed and Ni ²⁺ was introduced into the biological material structure. At this time, nickel chelate, which is a specific substance, is formed from AB-NTA and Ni ²⁺ previously introduced into the biological material structure. Furthermore, the biological material structure QCM chip was immersed in 8 mL of demineralized water for 30 minutes to wash unbound Ni ²⁺ . A chip as a biological material composite carrier obtained by this operation is referred to as a QCM chip 3D.

(3) QCM measurement Polyhistidine was used as an active substance (target substance). This utilizes the property that histidine specifically adsorbs to Ni ²⁺ of the QCM chip 3D into which Ni ²⁺ has been introduced.
The QCM used for the measurement is AFFINIX Q: (manufactured by Initiative).
Specifically, first, the QCM chip 3D was immersed in 8 mL of HEPES buffer (10 mM pH 7.4), measurement by QCM was started, and when the frequency was sufficiently stable, 5 mg / ml of poly 80 μL of a histidine (POLY-L-HISTIDINE: manufactured by SIGMA) aqueous solution (pH 1.7 glycine-HCl buffer) was injected.
The measurement results are shown in FIGS. 6 (a) and 6 (b). FIG. 6B is an enlarged view of the main part of FIG.

[Comparative Example 1]
A succinimide group was introduced on the surface of the QCM sensor chip formed of gold in the same manner as in “(2-1) QCM sensor chip surface treatment” in Example 1.
Next, the sensor chip for QCM was immersed in 8 mL of HEPES buffer (10 mM, pH 7.4), and 50 μL of a 17 mM AB-NTA DMSO solution was added and immersed for 30 minutes.

The QCM sensor chip was taken out of the HEPES buffer, and in order to reliably bind AB-NTA to the gold surface, this DM-solution of AB-NTA was diluted 10-fold with HEPES buffer, spotted at 3 μL, and allowed to stand for 15 minutes. This operation is for bonding the amine group of AB-NTA with the succinimide group on the gold surface.
Thereafter, Ni ²⁺ was introduced onto the surface of the QCM sensor chip in the same manner as in “(2-4) Introduction of nickel ions” in Example 1. The chip obtained by this operation is referred to as a QCM chip 2D.

  Using this QCM chip 2D, in the same manner as “(3) QCM measurement” in Example 1, QCM measurement is performed when polyhistidine (target substance) and nickel chelate (specific substance) are allowed to interact. It was. The results are shown in FIGS. 6 (a) and 6 (b).

[Comparative Example 2]
A succinimide group was introduced on the surface of the QCM sensor chip formed of gold in the same manner as in “(2-1) QCM sensor chip surface treatment” in Example 1.
Next, 3 μL of 10 mg / ml albumin aqueous solution (phosphate buffer 10 mM, pH 9.0) was spotted on the surface of the QCM sensor chip.
Thereafter, in the same manner as in “(2-3) Chelate surface treatment to biological material structure” and “(2-4) Introduction of nickel ions” in Example 1, Ni ²⁺ was applied to the surface of the QCM sensor chip. Introduced. The chip obtained by this operation is defined as QCM chip albumin 2D.

  Using this chip albumin 2D for QCM, QCM measurement was performed in the case where polyhistidine (target substance) and nickel chelate (specific substance) were allowed to interact in the same manner as “(3) QCM measurement” in Example 1. I did it. The results are shown in FIGS. 6 (a) and 6 (b).

[Consideration about Example 1 and Comparative Examples 1 and 2]
As can be seen from FIGS. 6A and 6B, in the result of Example 1 using the QCM chip 3D which is an example of the present invention, a very large frequency change is observed as compared with Comparative Examples 1 and 2. Has occurred. This is presumably due to the following reasons.
First, the QCM chip 2D used in Comparative Example 1 is obtained by fixing nickel chelate, which is a specific substance, on a chip from which gold is exposed. Therefore, it is considered that non-specific interaction occurs between the gold on the chip surface and polyhistidine which is the target substance, thereby reducing the frequency change.

The QCM chip albumin 2D used in Comparative Example 2 is obtained by two-dimensionally covering the chip surface with albumin and then fixing nickel chelate, which is a specific substance, on the chip. This is considered to be because a large amount of Ni ^{2+ as} a specific substance can be bound by binding albumin to the surface of the QCM chip as a single layer. Moreover, it is presumed that polyhistidine is more easily bound by the introduction of EGS. However, in Comparative Example 2, the frequency change is not yet sufficient.

  On the other hand, in Example 1, since the biological material complex having a three-dimensional structure is configured, the chip surface can be covered with a larger amount of albumin than Comparative Example 2, so that the biological material complex According to this, nickel chelate, which is a specific substance, is bound to three-dimensionally arranged albumin amino groups (usually about 60 sites per albumin molecule), so that nickel chelate can be immobilized in a larger amount than before. it can. Furthermore, since the biological material complex has a porous structure, polyhistidine can sufficiently penetrate deep into the biological material complex, thereby dramatically increasing the size of the interaction to be detected. As a result, it is considered that the frequency change is large as shown in FIG.

[Example 2: Biomaterial complex using biotin]
As a sensor chip for SPR, it has a diffraction grating with a groove pitch of about 870 nm and a groove depth of about 40 nm on the surface of a flat polycarbonate substrate having a size of 2.5 cm long × 2.5 cm wide × 1.2 mm thick. Further, a substrate was prepared by depositing gold with a thickness of about 80 nm on the surface of the substrate.

The SPR sensor chip was subjected to a surface treatment in the same manner as “(2-1) QCM sensor chip surface treatment” in Example 1 to introduce a succinimide group on the surface of the SPR sensor chip.
Next, a biological material structure was produced on the SPR sensor chip in the same manner as “(2-2. Formation of biological material structure)” in Example 1 except that the dripping amount at the time of spotting was changed to 1 μL. did.

In order to bind biotin as a specific substance to the surface of the biological material structure, 2 μL of 100 mg / mL EZ-Link NHS-PEO ₄ -Biotin (manufactured by PIRCE) was spotted on the sensor chip for SPR, and under saturated vapor pressure It was allowed to stand for 30 minutes and then dried at room temperature. This operation is to support biotin on the biological material structure by reacting and binding the succinimide group of EZ-Link NHS-PEO ₄ -Biotin to the amino group of albumin in the biological material structure. It is. Let the spot obtained by this operation be SPR3D.

Using a grating type SPR measurement device FLEX CHIPS ^™ Kinetic Analysis System (manufactured by HTS Biosystems) as a measurement device, SPR measurement was performed while flowing a liquid in contact with SPR3D as follows. HEPES buffer (10 mM, pH 7.4) was fed for 2 minutes from the start of measurement, and then 10 μg / ml of streptavidin (ImmunoPure Streptavidin: PIERCE) aqueous solution (HEPES buffer, 10 mM, pH 7.4) as an active substance. Was fed. Finally, HEPES buffer (10 mM, pH 7.4) was fed for 15 minutes. All liquid feeding speeds were 500 μL / min.
The result of SPR measurement is shown in FIG.

[Comparative Example 3]
1 μL of 10 mg / mL albumin aqueous solution (phosphate buffer, pH 9) is spotted on the same SPR sensor chip as in Example 2, and then biotin is bound to albumin in the same manner as in Example 2. It was. Let the spot obtained by this operation be SPR2D.
This spot SPR2D was subjected to SPR measurement in the same manner as in Example 2. The result of SPR measurement is shown in FIG.

[Discussion about Example 2 and Comparative Example 3]
As can be seen from FIG. 7, the resonance angle shift amount in the spot SPR3D fabricated in Example 2 is larger than the resonance angle shift amount in the spot SPR2D fabricated in Comparative Example 3, and the spot SPR3D is more than the spot SPR2D. It can be seen that it has high reactivity. This is presumably due to the following reasons.

  That is, in the spot SPR3D prepared in Example 2, since a biological material complex is formed, biotin is present in a large amount three-dimensionally, whereas in the spot SPR2D prepared in Comparative Example 3, biotin is 2 Since it exists only in a dimensional manner, its abundance is small compared to the spot SPR3D. Since the shift amount in this SPR measurement is caused by the interaction between biotin present in each of the spots SPR3D and SPR2D and the active substance streptavidin, as described above, the spot SPR3D is more sensitive than the spot SPR2D. Since a large amount of biotin is present, the interaction at the spot SPR3D is larger than the interaction at the spot SPR2D, and the resonance angle shift amount at the spot SPR3D is larger than the resonance angle shift amount at the spot SPR2D. It is thought to be a thing. Furthermore, it is thought that the biological material complex has a porous structure in which particulate lumps are gathered, which is also a cause of high reactivity.

[Reference Example 1]
A spot SPRref3D was formed in the same manner as in Example 2 except that an EZ-Link NHS-PEO ₄ -Biotin (Pirce) aqueous solution was not spotted on the same SPR sensor chip as in Example 2.
The spot SPRref3D was subjected to SPR measurement in the same manner as in Example 2. The result of SPR measurement is shown in FIG.

[Reference Example 2]
A spot SPRref2D was formed in the same manner as in Comparative Example 3 except that an EZ-Link NHS-PEO ₄ -Biotin (Pirce) aqueous solution was not spotted on the same SPR sensor chip as in Example 2.
SPR measurement was performed on the spot SPRref2D in the same manner as in Example 2. The result of SPR measurement is shown in FIG.
FIG. 8 also shows the measurement results when SPR measurement is similarly performed on the site of the sensor chip for SPR that has not been subjected to albumin treatment (in FIG. 8, “gold surface (ethanolamine treatment)” is displayed. To do). When the albumin treatment is not performed, the succinimide on the gold surface is treated with ethanolamine.

[Consideration on Reference Examples 1 and 2]
As can be seen from FIG. 8, the spot SPRref3D has the smallest shift amount, the spot SPRref2D has the next smallest shift amount, and the shift amount of the part where no processing is performed is the largest. This is presumed to be due to the difference in the amount of albumin that suppresses non-specific interaction at each measurement site (spot).

That is, non-specific interaction occurs because gold is exposed without the presence of albumin at the site where no treatment is performed, and the resonance angle is greatly shifted by the non-specific interaction. .
In the spot SPRref2D, albumin is two-dimensionally present on the surface of the sensor chip for SPR, and the chip surface is covered with albumin. Therefore, non-specific interaction is suppressed, and the resonance angle shifts. The amount is also getting smaller.

  On the other hand, in the spot SPRref3D, a large amount of albumin is three-dimensionally present on the surface of the SPR sensor chip due to the biological material structure, and thus the chip surface is covered with a large amount of albumin. Therefore, it is considered that the non-specific interaction is more reliably suppressed as compared with the spot SPRref2D, and the shift amount of the resonance angle is further reduced.

[Example 3: Structure of biological material structure]
A biological material structure was formed in the same manner as in Example 2 on the same chip as the sensor chip for SPR used in Example 2 except that no grating was applied.
The surface of this chip was observed by AFM. Nanoscope IIIa Multimode (manufactured by Digital Instruments) was used as the AFM apparatus. The tapping mode was used as the measurement mode, and SSS-NCH (manufactured by Toyo Technica, tip radius R <5 nm) was used as the probe. Data sampling was performed in the atmosphere with 512 in the x direction and 512 in the Y direction. For confirmation of the particulate mass, measurement of an AFM image of 500 nm × 500 nm was used. For the power spectrum analysis, measurement of an AFM image of 1000 nm × 1000 nm was used, and for the analysis software, the Power Spectral Density function attached to Nanoscope IIIa was used.

A drawing-substituting photograph showing the observation result is shown in FIG. 9, and the power spectrum analysis result is shown in FIG. As can be seen from FIG. 9, a submicron-order particulate lump was confirmed on the chip surface. Further, from the analysis result of the power spectrum, the value of 2D isotropic PSD at a wavelength of 100 nm is 11,300 nm ⁴ , the sum (It) of all power spectra is 39.9 nm ² , and the power spectrum excluding the wavelength less than 100 nm The sum (Ic) is 35.2 nm ² . It can be seen that the ratio (Ic / It) occupied by the sum (Ic) of the power spectrum excluding the wavelength less than 100 nm in the total power spectrum (It) is 88.2%.

[Example 4: Measurement of correlation length]
In a 15 ml centrifuge tube, 5% by weight of albumin (ALBUMIN, manufactured by PIG SIGMA) (solvent: pH 9, phosphate buffer) and polymer A (synthesized in the same manner as in Example 1. Molecular weight: Mw = 59500 5% by weight (solvent: demineralized water) was mixed so that the volume ratio was 20: 1, 10: 1, 1: 1 (albumin: polymer A). The phosphate buffer concentration of albumin was adjusted so that the phosphate buffer concentration after mixing was 10 mM, and the total volume was adjusted to 550 μl.

Thereafter, drying under reduced pressure was performed to cause PSA and polymer A to react to produce a biological material structure. To this biological material structure, 7 ml of ethanolamine aqueous solution (1M pH 8.5) was added to block unreacted succinimide groups contained in the polymer.
It was washed once with 7 mL of 1 M aqueous KCl solution (10 mM phosphate buffer, pH 7.4), and further washed twice with 7 mL of demineralized water.

  In order to confirm the internal structure of the washed biological material structure, static light scattering measurement was performed. The light scattering device was measured in demineralized water using Dyna 3000 (Otsuka Electronics Co., Ltd.). The measurement was performed by Vv scattering measurement. From the angle dependency of the forward scattered light intensity obtained, the scattering angle dependency is subjected to a Debye-Bouchet plot, and the correlation length is estimated from the good linearity, and 118.5 nm (20: 1), 70 nm (10: 1). ), 118.7 nm (1: 1). As a result, the structure of the particulate mass could be estimated to be on the order of submicrons.

[Example 5: Biological substance complex using sugar as a specific substance]
[Synthesis of sugar ligands used as specific substances]
The sugar ligand (1) was synthesized by the procedure shown in the reaction formula of FIG. Ts represents a tosyl group, THF represents tetrahydrofuran, DMF represents N, N-dimethylformamide, Ph represents a phenyl group, Me represents a methyl group, Ac represents an acetyl group, and PS represents polystyrene. R. t. Represents room temperature.

First, commercially available compound (2) and TsCl were reacted in a mixed solvent of THF and water at 0 ° C. for 4 hours in the presence of NaOH, and after treatment and purification, compound (3) was obtained in a yield of 65%. It was. This compound (3) was reacted with NaN ₃ in DMF at 50 ° C. for 2.5 hours, and after workup and purification, compound (4) was obtained in 79% yield. This compound (4) was reacted with PPh ₃ in Et ₂ O at room temperature for 18 hours in the presence of H ₃ PO ₄ , and after workup and purification, compound (5) was obtained in a yield of 44% (601 mg). It was. The overall yield of compound (5) from compound (2) was 23%.

Next, the polysaccharide (6) and the compound (5) were reacted and coupled in a mixed solvent of MeOH and acetic acid at 40 to 50 ° C. for 5 hours, and then 2-picoline borane was added, The reaction was carried out at 6 ° C. for 6 hours for reduction and post-treatment. This reaction mixture was reacted for 15 hours at 40 ° C. in a mixed solvent of CH ₂ Cl ₂ and MeOH, PS-benzaldehyde, which is an amine scavenger, to obtain a sugar compound (7) after post-treatment and purification. It was. The sugar compound (7) was subjected to a reduction reaction using a 10% -Pd / C catalyst in a mixed solvent of MeOH and 1N hydrochloric acid under a hydrogen atmosphere, followed by filtration. Furthermore, neutralization reaction was performed for 30 minutes at room temperature using NaOAc, and the sugar compound (8) was obtained after purification. The sugar compound (8) was obtained in 20 mg. The overall yield of the saccharide compound (8) from the polysaccharide (6) was 50%.

  2.0 mg of the obtained sugar compound (8) was reacted with 1 equivalent of Sulfosuccinimidyl-6- (biotinamido) hexanoate (manufactured by PIERCE) in DMF in the presence of triethylamine for 4.5 hours. After purification, a sugar ligand (1) in which the terminal amino group was biotinylated was obtained in a yield of 32% (a yield of 0.9 mg). The reaction was carried out in a nitrogen atmosphere except for the reduction reaction with Pd / C.

The compounds (3) to (5) are purified by silica gel chromatography, and the sugar ligand (1), sugar compound (7) and polysaccharide (8) are purified by gel filtration (Sephadex TMLH-20, manufactured by Amersham Bioscience). I did it. The compounds (3) to (5) are identified by ¹ H NMR and ¹³ C NMR, and the sugar ligand (1), sugar compound (7) and (polysaccharide 8) are identified by ¹ H NMR and mass spectrometry. It was.

[Production of biological material complex using sugar ligand]
In the same manner as in Example 3, a succinimide group was introduced on the surface of the SPR sensor chip on which no grating was formed (this was used as an activated gold substrate).
Streptavidin (manufactured by WAKO) was dissolved in 100 mM carbonate buffer pH 9.0 to prepare solutions of 3.0, 1.0, 0.5, 0.25, 0.13% by weight.
Polymer A was dissolved in MilliQ water to prepare a solution of 3.0, 1.0, 0.5, 0.25, 0.13% by weight.
The polymer solution of the same concentration and the streptavidin solution were mixed at a ratio (volume ratio) of 1:10.

3 μL of the polymer A: streptavidin mixed solution was spotted on the activated gold substrate. After 30 minutes at room temperature, the substrate was immersed in 1M ethanolamine (SIGMA) aqueous solution pH 8.5 to block unreacted succinimide groups.
Further, after immersing the gold substrate in a 1 wt% BSA (manufactured by SIGMA) aqueous solution (pH 7.4 in PBS) for 30 minutes, the substrate was washed with MilliQ water and dried with nitrogen.

A 3 μL 1 mM aqueous solution of a sugar ligand was spotted on the biological material structure formed of the polymer A and streptavidin formed by the spot, and the sugar ligand was introduced by allowing to stand at room temperature for 30 minutes.
Thereafter, it was washed with MilliQ water and dried with nitrogen. Thereby, the biological material complex (sugar ligand biological material complex substrate) of the present invention using the specific substance as a sugar ligand was produced.

[Detection of lectin by immobilized sugar ligand]
The sugar-ligand biological material complex substrate prepared above is immersed in 10 μg / mL FITC-SSA (manufactured by J Oil Mills, Inc., a fluorescently labeled lectin used as an active substance) for 30 minutes at room temperature, and sugar-lectin The reaction was performed. After completion of the reaction, it was washed with MilliQ water and dried with nitrogen. Finally, detection was performed and evaluated using a fluorescence measuring apparatus FX PRO PLUS (manufactured by BioRAD). The result is shown in FIG.

As a result, it was found that the fluorescence intensity was improved as the weight percent of streptavidin was increased. As the amount of streptavidin in the biological material structure increased, a large amount of lectin as an active substance could be bound by using the biological material complex of the present invention in which many sugar ligands were immobilized. It is shown that. In addition, when it produced on the conditions which do not contain a polymer, the fluorescence signal was not able to be detected.
As described above, it was confirmed that the biological material complex of the present invention functions effectively even when sugar is used as the specific substance.

  Biological substance complex of the present invention, biological substance complex carrier, target substance purification method, affinity chromatography container, separation chip, target substance analysis method, target substance analysis separation apparatus and sensor chip The present invention can be used in any industrial field, but is particularly suitable for use in fields such as medical treatment, diagnosis, food analysis, and biological analysis. As a specific example, it can be used for analysis tools such as affinity purification or pharmaceutical action in which non-specific adsorption is suppressed with a small amount of sample. Further, for example, a biological material carrier having a biological material complex formed on the surface of a flow channel or the like, and by flowing a mixed solution to be separated and purified through the flow channel, an automated affinity purification apparatus or pharmaceutical action Etc. can be easily produced.

(A), (b) is a figure which shows typically the example of the structure of a biological material structure, in order to demonstrate one Embodiment of this invention. (A), (b) is a figure for demonstrating one Embodiment of this invention, (a) is a figure which shows a biological material and the compound for a coupling | bonding typically, (b) is a schematic diagram of a particulate lump. FIG. It is sectional drawing which shows typically the container for affinity chromatography as one Embodiment of this invention. It is a figure which shows typically the outline | summary of the affinity chromatography apparatus as one Embodiment of this invention. It is a figure which shows typically the outline | summary of the affinity chromatography apparatus as one Embodiment of this invention. (A), (b) is a graph showing the measurement result of the QCM measurement measured in Example 1 and Comparative Examples 1 and 2 of the present invention. (B) is an enlarged view of the main part of (a). It is a graph showing the measurement result of the SPR measurement measured in Example 2 and Comparative Example 3 of the present invention. It is a graph showing the measurement result of the SPR measurement measured in Reference Examples 1 and 2. It is a drawing substitute photograph showing the result of having observed the biological material complex in Example 3 of the present invention. It is a figure showing the power spectrum analysis result in Example 3 of this invention. In Example 5 of this invention, it is the figure which showed the reaction formula showing the procedure which synthesize | combines a sugar ligand (1). It is a graph showing the measurement result of Example 5 of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container body 2 Biological substance complex 3 Affinity chromatography container 10, 20 Affinity chromatography device 11 Tank 12 Pump 13 Autoinjector 14 Affinity separation chip 14A Substrate 14B Channel 14C Chip mounting part 15 Channel switching valves 16, 17 Recovery bottle 18 Control unit 19 Sample solution supply unit

Claims (25)

A biological material formed by binding a biological material and a compound capable of binding to the biological material and having a particulate mass having a particle size of 10 μm or less bonded to each other, and the specific material other than the biological material and the compound A biological material complex characterized in that is bound. The biological material complex according to claim 1, wherein a space is formed between the particulate lumps. The biological material complex according to claim 1, wherein a ratio of the weight of the biological material to the weight of the biological material complex is 0.1 or more. The biological material complex according to any one of claims 1 to 3, which has a diameter of 30 nm or more in a dry state. The biological material complex according to any one of claims 1 to 4, wherein at least one of the compounds has two or more functional groups capable of binding to the biological material. The biological material complex according to any one of claims 1 to 5, wherein the compound is uncharged. The biological material complex according to any one of claims 1 to 6, wherein the compound is miscible with water and miscible with at least one organic solvent. The biological material complex according to any one of claims 1 to 7, wherein the molecular weight of the compound is 1000 or more. The biological material complex according to any one of claims 1 to 8, wherein a diameter of the compound in a state of being mixed in a liquid is 1 nm or more. The biological material complex according to any one of claims 1 to 9, wherein the biological material is a biomolecule. The biological material complex according to claim 10, wherein the biomolecule is a protein. The specific substance is at least one selected from the group consisting of metal chelates, vitamins, sugars, glutathione, boronic acids, proteins, antigens, nucleic acids, bioactive substances, lipids, hormones, environmental hormones, and chelating groups. The biological material complex according to any one of claims 1 to 11, wherein the biological material complex is any one of claims 1 to 11. The biological substance complex according to any one of claims 1 to 12, wherein the specific substance and the biological substance are bonded via a hydrophilic molecule. The biological material complex according to claim 13, wherein the hydrophilic molecule contains ethylene oxide. A biological material complex-supporting body, wherein the biological material complex according to any one of claims 1 to 14 is fixed to a solid phase carrier. The biological material complex carrier according to claim 15, wherein the thickness of the biological material complex is 5 nm or more. The biological material complex according to any one of claims 1 to 14 is brought into contact with a sample liquid containing a target substance that can be adsorbed specifically to the specific substance,
Separating the biological material complex and the sample solution;
A method for purifying a target substance, wherein the target substance bound to the specific substance is released. A sample liquid containing a target substance that specifically interacts with the specific substance is circulated through the flow path holding the biological substance complex according to any one of claims 1 to 14.
A method for purifying a target substance, wherein a fraction containing the target substance is recovered from the eluate flowing out from the flow path. A container body capable of storing a fluid;
A container for affinity chromatography comprising the biological material complex according to any one of claims 1 to 14, which is held in the container body. A substrate formed with a flow path;
A separation chip comprising the biological material complex according to any one of claims 1 to 14 held in the flow path. 15. The biological material complex according to any one of claims 1 to 14, comprising a biological material complex using a specific substance capable of specifically adsorbing a substance having a specific structure, and a target substance. Contact the sample solution,
Separating the biological material complex and the sample solution;
A method for analyzing a target substance, comprising measuring the amount of the target substance adsorbed on the specific substance and analyzing the structure of the target substance. The biological material complex according to any one of claims 1 to 14, wherein the flow channel holds the biological material complex using a specific substance that can specifically interact with a substance having a specific structure. Circulate the sample solution containing the target substance,
A method for analyzing a target substance, wherein the structure of the target substance is analyzed by measuring the amount of the target substance in the fraction of the eluate flowing out from the flow path. The living body according to any one of claims 1 to 14, wherein a substrate on which a channel is formed and a specific substance that is held in the channel and can specifically interact with a substance having a specific structure are used. A separation chip comprising a substance complex;
A sample solution supply section for circulating a sample solution containing the target substance in the flow path of the separation chip;
A separation device for analyzing a target substance, comprising: a measuring unit that measures the amount of the target substance in a fraction of an eluate flowing out from the flow path. The living body according to any one of claims 1 to 14, wherein a substrate on which a flow path is formed and a specific substance that is held in the flow path and can specifically interact with a substance having a specific structure are used. A chip mounting portion for mounting a separation chip including the substance complex;
A sample liquid supply section for circulating a sample liquid containing a target substance in the flow path when the separation chip is mounted on the chip mounting section;
An apparatus for analyzing and separating a target substance, comprising: a measurement unit that measures the amount of the target substance in the fraction of the eluate flowing out from the flow path. A sensor chip comprising the biological material complex according to claim 1.